EP1463526A2 - Früherkennung einer mycobakteriellen erkrankung unter verwendung von peptiden - Google Patents

Früherkennung einer mycobakteriellen erkrankung unter verwendung von peptiden

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Publication number
EP1463526A2
EP1463526A2 EP02759228A EP02759228A EP1463526A2 EP 1463526 A2 EP1463526 A2 EP 1463526A2 EP 02759228 A EP02759228 A EP 02759228A EP 02759228 A EP02759228 A EP 02759228A EP 1463526 A2 EP1463526 A2 EP 1463526A2
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EP
European Patent Office
Prior art keywords
protein
antigens
seq
kda
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02759228A
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English (en)
French (fr)
Other versions
EP1463526A4 (de
Inventor
Suman Laal
Susan Zolla-Pazner
John T. Belisle
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Colorado State University Research Foundation
New York University NYU
Colorado State University
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Colorado State University Research Foundation
New York University NYU
Colorado State University
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Application filed by Colorado State University Research Foundation, New York University NYU, Colorado State University filed Critical Colorado State University Research Foundation
Publication of EP1463526A2 publication Critical patent/EP1463526A2/de
Publication of EP1463526A4 publication Critical patent/EP1463526A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention in the fields of microbiology and medicine relates to methods for rapid early detection of mycobacterial disease in humans based on the presence of antibodies to particular "early" mycobacterial protein antigens, and reactive epitopes thereof, which have not been previously recognized for this purpose.
  • Assay of such antibodies on selected mycobacterial proteins, peptides thereof, or fusion polypeptides (peptide multimers, polyproteins) permits diagnosis of TB earlier than has been heretofore possible.
  • a surrogate marker for screening populations at risk for TB in particular subjects infected with human immunodeficiency virus (HIN).
  • HIN human immunodeficiency virus
  • M. tuberculosis Mycobacterium tuberculosis
  • Mtb Mycobacterium tuberculosis
  • High risk populations are also found in the United States, primarily intravenous drug users, homeless people, prison inmates and residents of slum areas (Fitzgerald, IM et al, 1991, Chest 100:191-200; Graham et al, supra; Friedman, L ⁇ et al, 1996, New Engl J. Med. 334:828-833) as well as household contacts of TB patients.
  • discovery of additional surrogate markers for early detection and prompt treatment of active, subclinical TB in such high risk populations is urgently required.
  • Antibody responses in TB have been studied for several decades primarily for the purpose of developing serodiagnostic assays. Although some seroreactive antigens/epitopes have been identified, interest in antibody responses to Mtb has waned because of the lack of progress in simple detection of corresponding antibodies. Studies using crude antigen preparations revealed that healthy individuals possess antibodies that cross-react with several mycobacterial antigens presumably elicited by exposure to commensal and environmental bacteria and vaccinations (Bardana, EJ et al, 1973, Clin. Exp. Immunol. 13:65-11; Das, S et al, 1992, Clin. Exp. Immunol.
  • MPB denotes a protein purified from M. bovis BCG followed by a number denoting its relative mobility in 7.7% polyacrylamide gels at a pH of 9.5.
  • MPT denotes a protein isolated from Mtb.
  • Mtb proteins which include a complex of 3 proteins termed antigens 85 A, 85B and 85C (also known as the "85 complex” or "85cx") (Wiker, H.G. et al, 1992, Scand. J. Immunol. 36:301-319; Wiker, H.G. et al, 1992, Microbiol. Rev. 56:648-661).
  • the corresponding components of Mtb are also actively secreted.
  • the 85 complex is considered the major secreted protein constituent of mycobacterial culture fluids though it is also found in association with the bacterial surface. In most SDS- polyacrylamide gel electrophoresis (SDS-PAGE) analyses, 85A and 85C are not properly resolved, whereas isoelectric focusing resolves three distinct bands.
  • EIA enzyme immunoassay
  • the N-terminal sequence of MPT51 showed 72% homology with the sequence of the Ag 85 components (when P at position 2 is aligned with P at position 7 of the three Ag 85 components.
  • Studies of TB patients showed that assays of antibodies to the Ag 85 complex had a sensitivity of about 50%.
  • the Ag 85 components are highly cross- reactive so that positive responses are expected (and found) in healthy controls, particularly in geographic areas of high exposure to atypical mycobacteria. The different degree of specificity is thus highly dependent on the kind of control subjects used. It is noteworthy that traditional BCG vaccination does not appear to induce a significant antibody response, though it is interesting that antibodies to mycobacterial antigens increased when anti-TB chemotherapy was initiated.
  • Mtb 50/55 kDa antigen secreted glycoproteins.
  • the ⁇ -terminal sequences and total amino acid content of these proteins were very similar. 2D gel electrophoresis showed at least seven different components in the Mtb 50/55 kDa antigen.
  • the ⁇ -terminus of the Mtb Al kDa antigen known as MPT32 was very similar to the ⁇ -termini of the 50/55 kDa - and the 45-47 kDa proteins. The authors speculated about a diagnostic potential for these antigens based on these observation However, the potential of this antigen as an early diagnostic agent for TB was neither analyzed nor even suggested.
  • Urnovitz HB et al (Lancet Dec 11 1993, 342:1458-9), discovered that 7 individuals who were negative for HIV-1 antibody in a licensed serum EIA were positive in a urine EIA and western blot (WB).
  • Connell JA et al JMed Virol 1993, 41:159-64, described a rapid, simple, and robust IgG-capture enzyme-linked immunosorbent assay (GACELISA) suitable for the detection of anti-HJN 1 and 2 antibodies in saliva and urine.
  • GACELISA IgG-capture enzyme-linked immunosorbent assay
  • HBs hepatitis B surface antigen
  • HBc hepatitis B core antigen
  • CMN CMN and HTV
  • HBs hepatitis B surface antigen
  • HBc hepatitis B core antigen
  • CMN CMN and HTV
  • the present inventors have systematically analyzed the reactivity of sera and urine from TB patients with antigens from Mtb to delineate the major targets of human antibody responses which occur relatively early in the progression of the infection to disease. They observed that initial immunoadsorption of patient sera with E. coli antigens successfully reduced interference by cross-reactive antibodies, thus allowing a new approach to serological studies.
  • the immunoadsorbed sera allowed identification of a number of antigens of Mtb that are recognized by antibodies in a large proportion of patients, and during earlier stages of disease progression. These antigens are therefore useful tools in methods of diagnosing TB.
  • Prominent among these antigens is a high molecular weight secreted protein of 88kDa or 85kDa (depending on conditions as will be described below). This protein is termed “the 88 kDa protein” and, as discovered later, as the product of the glcB gene, is also termed GlcB (see below).
  • the present invention In addition to its utility for early diagnosis of mycobacterial disease in a subject prior to the development of radiographic or bacteriological evidence of the disease, the present invention also provides for the first time a surrogate marker that can be used in an inexpensive screening method in individuals at heightened risk for developing TB.
  • This utility was discovered by applying the approach described herein to analyze antibody responses of HTV-infected TB patients (HIN/TB) to the secreted antigens of Mtb during different stages of disease progression.
  • HIN/TB HTV-infected TB patients
  • a majority of the HIN/TB patients had detectable antibodies to the secreted antigens of Mtb for months, even years, prior to the clinical manifestation of active tuberculous disease. These patients are termed "HTV/pre-TB".
  • HIN/TB patients compared to the TB patients not infected with HIN (designated "non-HIN/TB"), HIN/TB patients had significantly lower levels of antibodies which showed specificity for a restricted repertoire of Mtb antigens.
  • Antibodies to the 88 kDa antigen mentioned above were present in about 75% of the HIN/pre-TB sera patients who eventually developed clinical TB. HIV/TB patients who failed to develop anti- t ⁇ antibodies did not differ in their lymphocyte profiles from those that were antibody-positive.
  • the present invention is directed to an antigenic composition useful for early detection of M. tuberculosis disease or infection or for immunizing a subject against M. tuberculosis infection, comprising
  • ELA APDEIREEVDNNC (SEQ ID NO: 110); (4) HRRRREFKARAAEKPAPSDRAG (SEQ ID NO: 111); (5) ARDELQAQIDK HRRR (SEQ ID NO: 112); (6) LNRDRNYTAPGGGQ (SEQ ID NO: 113); (7) GAPQLGRWKWHDPWV (SEQ ID NO: 114) (8) VGNLRIARVLYDF (SEQ ID NO: 117); (9) QAQIDKWHRRRVI (SEQ ID NO: 126); (10) WHRRRVIEPIDMD (SEQ ID NO: 127); (11)
  • IEPIDMDAYRQFL (SEQ ID NO: 128); (12) ITTTAGPQLVVPV (SEQ ID NO:134); (13) PQLVVPVLNARFA (SEQ ID NO:135); (14) VLNARFALNAANA (SEQ ID NO: 136); (15) ALNAANARWGSLY (SEQ ID NO: 137); (16) AR GSLYDALYGT (SEQ ID 0:138); (17) SVLLINHGLHIEI (SEQ ID NO: 154); (18) HGLHIEILIDPES (SEQ ID NO: 155); (19) GGQFTLPGRSLMF
  • composition is not the full length protein having the sequence SEQ ID NO: 106 or SEQ ID NO: 107;
  • the above antigenic composition is a fusion polypeptide that includes: (a) one or more of the peptides (l)-(23) or the variants, linked to (b) one or more proteins selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO: 106, SEQ ID NO:
  • an antigenic composition as above which is:
  • peptide multimer reacts with an antibody specific for the GlcB or MPT51 protein.
  • the invention is also directed to an antigenic composition as above which is a recombinant peptide multimer having the formula:
  • P 1 and P 2 may be the same or different and each occurrence of P x in the 1 -Gly 2 structure may be a different peptide or variant from its adjacent neighbor;
  • An antigenic composition useful for early detection of M. tuberculosis disease or infection may comprise one or more peptides in a mixture or linked in a peptide multimer or fusion protein, which one or more peptides are derived from or have a sequence corresponding to a fragment of an early M. tuberculosis antigen which antigen is characterized as being (i) reactive with antibodies found in tuberculosis patients who are in a stage of disease prior to the onset of sputum smear-positivity and cavitary pulmonary lesions, and
  • a preferred method for the early detection of mycobacterial disease or infection in a subject comprises assaying a biological fluid sample from a subject suspected of having active TB for the presence of antibodies specific for the above peptide or variant, fusion protein or peptide multimer, wherein the presence of the antibodies is indicative of the presence of the disease or infection.
  • the biological fluid sample is preferably taken from a subject having symptoms of active tuberculosis, but before the onset of symptoms identifiable as advanced tuberculosis that is distinguished by (a) smear positivity of sputum or other pulmonary associated fluid for acid-fast bacilli, (b) cavitary puhnonary lesions, or both (a) and (b).
  • the method includes, before the assaying, the step of obtaining the biological fluid sample from the subject
  • the above method preferably includes, prior to the assaying step, removing from the sample antibodies specific for cross-reactive epitopes or antigens between proteins present in M. tuberculosis and in other bacterial genera, for example, by immunoadsorption of the sample with
  • the above method may further comprise assaying the sample for the presence of antibodies specific for one or more additional early antigens of M. tuberculosis selected from the group consisting of:
  • the preferred subject in the above methods is a human, such as human infected with HIV-1 or at high risk for tuberculosis.
  • the biological fluid sample is serum, urine or saliva.
  • 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 invention is also directed to a kit useful for early detection of M. tuberculosis disease, the kit comprising:
  • the kit may also comprise one or more early antigens of M. tuberculosis, for example, an antigens is selected from the group consisting of:
  • Figure 1 shows the reactivity of sera from TB neg HTV neg PPD + controls( O ); TB neg HTV nes
  • LAM-free culture filtrate proteins (LFCFP) of Mtb H Rv, before and after adsorption with E. coli lysate. Values are individuals OD's with the mean shown as a horizontal bar.
  • Figure 2 shows reactivity of sera from non-HIN, PPD skin test positive (PPD + ) healthy controls (non-HIN/PPD), non-HIN TB patients (non-HIN/TB) and HIN-infected TB patients (HIN/pre-TB, HIN/at-TB and HIN/post-TB) with total LFCFP of Mtb.
  • the cut-off was determined by the mean optical density (OD) ⁇ 3 standard deviations, obtained with the healthy control sera.
  • Figure 3 is a graph showing reactivity of sera from advanced (black bars) and early (gray bars) TB patients to M. tuberculosis LFCFP, purified Ag85C or three fractions (13, 10 and 15) enriched for three early antigens (shown in parentheses below the fraction designation).
  • Figure 4 is a graph showing reactivity of sera from advanced (black bars) and early (gray bars) TB patients to Mtb LFCFP, purified Ag85C or purified MPT32.
  • Figure 5 is a graph showing reactivity of urine and sera from late TB patients with LFCF and MP32 protein. Results are presented as percent samples that are positive.
  • the present invention provides a diagnostic immunoassay method to detect and/or quantitate antibodies specific for mycobacterial antigens, in particular, antibodies developing early in the progression of M. tuberculosis infection to disease and before clinical manifestations of that disease.
  • the best antigen available prior to this invention for serodiagnosis of TB was the 38 kDa secreted protein also known as Ag 78 (see above).
  • the present invention permits detection of serological reactivity in subject who lack detectable antibodies to this 38 kDa antigen.
  • the immunoassay method is based upon the present inventors' discovery that certain Mtb antigens induce in humans an earlier response than do other antigens which elicit antibodies only after the disease is already clinically advanced. In HIN-infected subjects with dysfunctional immune systems, antibodies to some of these antigens are detectable long before TB is clinically manifest. Five secreted proteins have been identified as early antigens with diagnostic value.
  • a preferred early antigen is a 88kDa secreted protein of Mtb GlcB, preferably enriched or semipurified (at least 50% pure) or highly purified (at least 95% pure, preferably at least 99% pure).
  • epitope-bearing peptides from GlcB and from MPT51 that are reactive with TB sera and which are used in early diagnosis in the form of peptides (single peptide or mixtures), peptide multimers (synthetic or recombinant) comprising one or more different epitope-bearing peptides, or fusion polyproteins that comprise at least two full length early antigen proteins and may include additional epitopes based on peptides of the same or other Mtb proteins.
  • the present method is further based on the inventors' conception of the importance of first removing antibodies specific for cross-reactive antigens (which are not Mtb-specific) prior to analyzing the antigenic reactivity and specificity of serum from patients infected with Mtb on crude or semipurified antigenic preparations.
  • purified antigens are provided or epitope-specific competitive EIAs are established based on this invention (see, for example, Wilkins, E. et al, 1991, Eur. J. Clin. Microbiol. Infect. Dis. 20:559-563)
  • the need for such prior absorption steps should be obviated.
  • 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 defined in terms of the stage of development of TB. Early and late (or advanced) TB are defined in the table below.
  • a subject with early TB is asymptomatic or, more typically, has one or more "constitutional symptoms" (e.g., fever, cough, weight loss), hi early TB, Mtb bacilli are too few to be detectable as acid-fast bacilli in smears of sputum or other body fluid, 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, i.e., 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 without cavitation.
  • substitutional symptoms e.g., fever, cough, weight loss
  • Mtb bacilli are too few to be detectable as acid-fast bacilli in smears of sputum or other body fluid, primarily those fluids associated with the lungs (such as
  • any antibody present in such early stages is termed an “early antibody” and any Mtb antigen recognized by such antibodies is termed an “early antigen.”
  • 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.
  • the term “late” or “advanced” is characterized in that the subject has frank clinical disease and more advanced cavitary lesions in the lungs, h 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.
  • an antibody that first appears after the onset of diagnostic clinical and other characterizing symptoms is a late antibody, and an antigen recognized by a late antibody (but not by an early antibody) is a late antigen.
  • 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.
  • the present immunoassay 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 reagent which includes one or more Mtb early antigens. They may be combined as mixtures or as polyproteins or peptide multimers based on units of epitope-bearing peptide. The binding of antibodies in the sample to the mycobacterial antigen(s) is then detected.
  • biological fluid 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.
  • the mycobacterial antigenic composition or preparation of the present invention maybe one or a combination of isolated proteins or peptides of aM tuberculosis secreted protein. As stated above, the combination may be produced as a mixture or as a polyprotein or peptide multimer.
  • the antigen composition may be a substantially purified or recombinantly produced preparation of one or more M. tuberculosis proteins or epitope-bearing peptides thereof.
  • the antigen composition may be a partially purified or substantially pure preparation containing one or more M tuberculosis epitopes which are capable of being bound by antibodies of a subject with TB. Such epitopes may be in the form of peptide fragments of the early antigen proteins or other "functional derivatives" of M. tuberculosis proteins or peptides as described below.
  • a 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 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 fragment thereof.
  • a variant peptide may be conveniently prepared by direct chemical synthesis or by recombinant means.
  • a "chemical derivative” of the antigenic protein or peptide contains additional chemical moieties not normally part of the native protein (or of a peptide fragment).
  • 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.
  • Mtb proteins or glycoproteins, identified in culture filtrates of Mtb, are the preferred early Mtb antigens (or sources of antigenic peptides) of the present invention. Thus, although these proteins are considered to be secreted proteins, they 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 as follows: (1) 88 kDa protein (GlcB)
  • This protein was discovered by the present inventors as an Mtb secreted protein having a molecular mass of 88 kDa and an isoelectric point of about pH 5.2 when isolated from the culture filtrate. This protein migrated at a molecular mass range of 82 - 85 kDa in one co- inventor's laboratory (or 88kDa in another co-inventor's laboratory) and a pi range of 5.12 - 5.19. This protein was originally thought to react with both mAb IT-42 and mAb IT-57, but it was later found that a second proteins in this MW range, the catalase/peroxidase (katG gene producfjwas reactive with those mAbs.
  • Th 88 kDa protein is a major antigenic component of Fraction 15 (Example I) and Fraction 14 (Example U).
  • This protein corresponds to the protein spot designated Ref. No. 124 in 2D gels now shown here (see US Patent 6,245,331 and WO 98/29132 (published 09 July 1998) both incorporated by reference in their entirety; see also Tables 4 and 6, below).
  • a single protein is intended (although different isoforms may be found to exist).
  • Example IN the sequence of this protein was identified by the present inventors based on amino acid composition in relation to the Mtb genomic sequence obtained after the filing date of the present inventors' priority application Serial No. US 60/034,003, filed 31 December 1996.
  • This protein is the product of the Mtb glcB gene which encodes the malate synthase enzyme and is termed the GlcB protein.
  • This protein has the amino acid sequence (SEQ ID NO: 106) as shown below:
  • Mtb ⁇ l which they concluded may be useful for the diagnosis of TB, especially for patients coinfected with HIN.
  • Recombinant Mtb81 tested by ELISA detected antibodies in 25/27 TB patients (92%) seropositive for HIN as well as in 38/67 individuals (57%) who were TB positive alone.
  • Antigen 85C This is an Mtb secreted protein having an apparent molecular weight of about 31 kDa and an isoelectric point of about pH 5.17. This protein is reactive with mAb IT-49 and has also been designated MPT45. Ag85C corresponds to the protein spot designated Ref. No. 119 in Table 4 or Table 6.
  • MPT51 Mtb secreted protein having an apparent molecular weight of about 31 kDa and an isoelectric point of about pH 5.17. This protein is reactive with mAb IT-49 and has also been designated MPT45. Ag85C corresponds to the protein spot designated Ref. No. 119 in Table 4 or Table 6.
  • This Mtb secreted protein has an apparent molecular mass of about 27kDa and an isoelectric point of about 5.91 and the amino acid sequence SEQ ID NO:107.
  • GenBank sequence includes the full length gene so that the amino acid sequence includes a 33 residue signal sequence that is cleaved from the protein before it is secreted.
  • the final protein product is SEQ ID NO:107 as shown.
  • MPT51 is reactive with mAb IT-52. This protein corresponds to the protein spot designated Ref. No. 170 in 2D gels not shown here (summarized in Table 4 and Table 6).
  • This glycoprotein has an apparent molecular mass (as a doublet peak) of 38 and 42 kDa (42/45 kDa according to Espitia et al.(supra)) and an isoelectric point of about pH 4.51. It is reactive with a polyclonal anti-MPT 32 antiserum. This protein is a major antigenic component of Fraction 13 (see Examples). MPT32 corresponds to the protein spot designated Ref. No. 14 in Table 4 or Table 6.
  • 49 kDa protein One additional protein, termed the “49 kDa protein,” has an apparent molecular mass of about 49 kDa and an isoelectric point of about pH 5.1. This protein reacts with mAb IT-58 and corresponds to a spot identified as Ref. No. 82 in Table 4 or Table 6.
  • the present invention also provides peptides of GlcB and of MPT51, early antigenic Mtb proteins. Such peptides are also useful as diagnostic and vaccine compositions. As shown in Examples IX, preferred peptides that were predicted and indeed shown to react with TB sera include, but are not limited to
  • GAPQLGRWKWHDPWV which corresponds to MPT51 residues 167-181 (SEQ ID NO: 114);
  • LRIARVLYDF (SEQ ID NO: 117); (9) QAQIDKWHRRRNI (SEQ ID NO: 126); (10) WHRRRVIEPIDMD (SEQ ID NO: 127);
  • ARWGSLYDALYGT SEQ ID NO: 138
  • FVRNVGHLMTNDA (SEQ ID NO: 172); (21) DRWFTNTGFLDR (SEQ ID NO: 191);
  • a peptide which includes an antibody epitopes should have at least about 5 amino acids.
  • a T cell epitope is preferably between about 10 and 15 amino acids.
  • the present invention includes peptides having between about 5 and 30 residues, having the native sequences of the Mtb early antigenic proteins or being homologues, substitution variants, addition variants or deletion variants thereof.
  • the peptide When the peptide is to be administered to a subject, particularly for the vaccine embodiments of this invention, the peptide may be capped at its N and C termini with an acyl (abbreviated “Ac”) -and an amido (abbreviated “Am”) group, respectively, for example acetyl (CH 3 CO-) at the N terminus and amido (-NH 2 ) at the C terminus.
  • Ac acyl
  • Am amido
  • N-terminal capping functions preferably in a linkage to the tenninal amino group, is contemplated, for example: formyl; alkanoyl, having from 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl; alkenoyl, having from 1 to 10 carbon atoms, such as hex-3-enoyl; alkynoyl, having from 1 to 10 carbon atoms, such as hex-5-ynoyl; aroyl, such as benzoyl or 1-naphthoyl; heteroaroyl, such as 3-pyrroyl or 4-quinoloyl; alkylsulfonyl, such as methanesulfonyl; arylsulfonyl, such as benzenesulfonyl or sulfanilyl; heteroarylsulfonyl, such as pyridine-4-sulfonyl; substituted alkanoyl
  • the C-terminal capping function can either be in an amide or ester bond with the terminal carboxyl.
  • Capping functions that provide for an amide bond are designated as NR R 2 wherein R and R may be independently drawn from the following group: hydrogen; alkyl, preferably having from 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl; alkenyl, preferably having from 1 to 10 carbon atoms, such as prop-2-enyl; alkynyl, preferably having from 1 to 10 carbon atoms, such as prop-2-ynyl; substituted alkyl having from 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, halogenoalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylal
  • Capping functions that provide for an ester bond are designated as OR, wherein R may be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy.
  • the peptides of the invention may be prepared using recombinant DNA technology. However, some of the shorter peptides may be prepared using solid-phase synthesis, such as that generally described by Merrifield, J. Amer. Chem. Soc, 55:2149-54 (1963), although other equivalent chemical syntheses known in the art are also useful. Solid-phase peptide synthesis may be initiated from the C-terminus of the peptide by coupling a protected ⁇ -amino acid to a suitable resin.
  • Such a starting material can be prepared by attaching an ⁇ -amino-protected amino acid by an ester linkage to a chloromethylated resin or to a hydroxymethyl resin, or by an amide bond to a BHA resin or MBHA resin.
  • Such methods well-known in the art, are disclosed, for example, in U.S. 5,994,309 (issued 11/30/1999) which is incorporated by reference in its entirety.
  • peptides in which at least one amino acid residue and preferably, only one, has been removed and a different residue inserted in its place compared to the native Mtb sequence.
  • the types of substitutions which maybe made in the peptide molecule of the present invention are conservative substitutions, which are typically exchanges within one of the following groups: 1. Small aliphatic, nonpolar or slightly polar residues: e.g. , Ala, Ser, Thr, Gly;
  • Polar, negatively charged residues and their amides e.g., Asp, Asn, Glu, Gin;
  • Polar, positively charged residues e.g., His, Arg, Lys;
  • substitutions that are less conservative, such as between, rather than within, the above groups (or two other amino acid groups not shown above), which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Preferred substitutions according to the present invention are those that do not produce radical changes in the characteristics of the peptide molecule.
  • Addition variants of the present Mtb peptides preferably include from 1-4 amino acids, but may include as many as X amino acids, added either at the N-terminus, the C-terminus or both.
  • Amino acids that are added to the basic peptide unit are ones that permit the peptide to maintain its biological reactivity in accordance with this invention, namely antigenicity (recognition by antibodies or T lymphocytes) or immunogenicity in the case of vaccine embodiments.
  • preferred variants are those that have increased stability and/or immunogenicity.
  • stability is increased by introducing one or more Cys residues into strategic positions , where the formation of disulfide bonds between two Cys residues increases stability.
  • Another approach is based on introduction of residues that form ⁇ helices at sites that do not impede the peptide immunologic activity, for example at the N- and C- termini.
  • n residues as many as (n-5) amino acids ' maybe substituted, provided that the characteristic immunoreactivity with early Mtb antibodies is not lost.
  • Lysinyl and amino terminal residues may be derivatized with succinic or other carboxylic acid anhydrides. Derivatization with a cyclic carboxylic anhydride has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia.
  • the present invention also includes longer peptides or polypeptides in which a sequence of an Mtb early antigenic peptide or a substitution or addition variant thereof, or a chemical derivative thereof, is repeated from two to about 100 times, with or without intervening spacers or linkers.
  • Such molecules are termed in the art, interchangeably, multimers, concatemers or multiepitope polyproteins and will be referred to herein primarily as peptide multimers. When produced recombinantly, they are also considered to be fusion polypeptides or fusion proteins.
  • peptide multimers may be built from any of the antigenic peptides or variants described herein.
  • a peptide multimer may comprise different combinations of peptide monomers (either from the native sequence or variants thereof).
  • a multimer may include several sequential repeats of a first peptide, followed by one or more repeats of a second peptide, etc.
  • Such multimeric peptides can be made by chemical synthesis of individual peptides, recombinant DNA techniques or a combination, e.g., chemical linkage of recombinantly produced multimers.
  • the multimers When produced by chemical synthesis, the multimers preferably have from 2-12 repeats, more preferably 2-8 repeats of the core peptide sequence, and the total number of amino acids in the multimer should not exceed about 110 residues (or their equivalents, when including linkers or spacers).
  • a preferred synthetic chemical peptide multimer has the formula
  • a preferred synthetic chemical peptide multimer has the formula
  • P and P 2 are Mtb peptides or addition variants of these peptides, wherein
  • P 1 and P 2 may be the same or different; moreover, each occurrence of P 1 in the multimer may be a different peptide (or variant) from its adjacent neighbor;
  • a preferred recombinantly produced peptide multimer has the formula:
  • P and P is preferably selected from any one of the followings SEQ ID NO's: 108- 114; 117;126-128, 134-138, 154, 155, 170, 172, 191, 216, and 217.
  • the multimer is optionally capped at its N- and C-termini
  • multimers may be built from any of the peptides or variants described herein. Although it is preferred that the addition variant monomeric units of the multimer have the biological activity described above, that is not necessary as long as the multimer to which they contribute has the activity.
  • the present invention includes as fusion polypeptide which may comprise a linear multimer of two or more repeats of the above peptide monomers linked end to end, directly or with a linker sequences present between the monomer repeats and further fused to another polypeptide sequence which permits or enhances the activity of the antigenic peptides in accordance with this invention.
  • the present multimers and fusion polypeptides may therefore include more than one epitope from the same or different Mtb proteins that do not occur together, i.e., in a contiguous structure, in a native Mtb protein.
  • polyproteins or fusion proteins which combine longer polypeptides, even full length Mtb proteins such as GlcB, MPT51 and other Mtb early antigens described herein in various combinations, such as a fusion of GlcB and MPT51 or these two with another one or more early antigenic protein.
  • Mtb proteins such as GlcB, MPT51 and other Mtb early antigens described herein in various combinations, such as a fusion of GlcB and MPT51 or these two with another one or more early antigenic protein.
  • These full length proteins may be combined in polyproteins with shorter epitope-bearing Mtb peptides or variants thereof or with peptide multimers (homo- or heteromultimers.
  • Such fusion proteins optionally includes spacers or linkers between some or all of the individual protein or peptide units.
  • Peptides and multimers may be chemically conjugated to form multimers and larger aggregates.
  • Preferred conjugated multimers include Cys and are made by forming disulfide bonds between the -SH groups of these residues, resulting in branched chains as well as straight chain peptides or polypeptides.
  • the present multimers and fusion polypeptides may include linkers that are cleavable by an enzyme.
  • Preferred enzymes are a matrix metalloprotealse, urokinase, a cathepsin, plasmin or thrombin.
  • a preferred linker is a peptide having the sequence VPRGSD (SEQ ID NO:l 15) or DDKDWH (SEQ ID NO:238).
  • peptides may be combined in the form of fusion polypeptides that comprise one or more repeats of a single peptide or mixtures of such peptides fused to other proteins, e.g., carrier molecules or other proteins which would enhance their immunogenicity when used as vaccine compositions.
  • Additional compositions within the scope of this invention are the foregoing peptides, multimers or fusion polypeptides immobilized to a solid support or carrier for use in immunoassays.
  • solid phase support is intended any support capable of binding antigen or antibodies.
  • Supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, polyvinylidene difluoride, agaroses such as Sepharose®, and magnetic beads.
  • the support material may have virtually any possible structural configuration so long as the immobilized peptide or polypeptide is capable of binding to its target molecule, e.g., antibody.
  • the support configuration can include microparticles, beads, porous and impermeable strips and membranes, the interior surface of a reaction vessel such as a test tube or a microtiter plate, the external surface of a rod, and the like.
  • a reaction vessel such as a test tube or a microtiter plate
  • the external surface of a rod and the like.
  • kits of the present invention may include one or more of the various peptide compositions described herein.
  • the mycobacterial antigen composition is brought in contact with, and allowed to bind to, a solid support or carrier, such as nitrocellulose or polystyrene, allowing the antigen composition to adsorb and become immobilized to the solid support.
  • a solid support or carrier such as nitrocellulose or polystyrene
  • 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.
  • second antibody an anti-immunoglobulin 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” maybe specific for epitopes characteristic of a particular human immunoglobulin isotype, for example IgM, IgG ⁇ IgG 2a , IgA and the like, thus permitting identification of the isotype or isotypes of antibodies in the sample which are specific for the mycobacterial antigen.
  • the second antibody may be specific for an idiotype of the ant-Mtb antibody of the sample.
  • binding partners for detection of the sample antibody other known binding partners for human immunoglobulins may be used.
  • staphylococcal immunoglobulin binding proteins the best know of which is protein A.
  • 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 H 3 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)).
  • 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 mycobacterial antigen reagent, which may be detectably labeled. Bound antigen is then measured by measuring the immobilized detectable label. If the mycobacterial 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.
  • solid phase support any support capable of binding a proteinaceous antigen or antibody molecules or other binding partners according to the present invention.
  • 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.
  • 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.
  • 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.
  • a preferred type of immunoassay to detect an antibody specific for a mycobacterial antigen is an enzyme-linked immunosorbent assay (ELISA) or more generically termed an enzyme immunoassay (EIA).
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • 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, ⁇ -galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, ⁇ - 5-steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detectable label may be a radiolabel, and the assay termed a radioimmunoassay (RIA), as is well known in the art. See, for example, Yalow, R. et al, Nature 184:1648 (1959); Work, T.S., et al, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, NY, 1978, incorporated by reference herein.
  • 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 125 1, 131 L 35 S,
  • fluorophore it is also possible to label the antigen or antibody reagents with a fluorophore.
  • fluorescently labeled antibody 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.
  • fluorophores are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine or fluorescence-emitting metals such as Eu 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.
  • a chemiluminescent- tagged antibody or antigen is then determined by detecting the luminescence that arises during the course of a chemical reaction.
  • useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • 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 maybe 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.
  • 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.
  • 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.
  • agglutination assays both direct and indirect, which are well known in the art.
  • the agglutination of particles containing the antigen 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, US 4,308,026; Gupta et al, J. Immunol. Meth. 80:177-187 (1985); Castelan et al, J. Clin. Pathol.
  • the present invention provides methods to detect and enumerate cells secreting an antibody specific for a mycobacterial antigen.
  • a sample containing lymphocytes such as peripheral blood lymphocytes
  • 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.
  • antibodies 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.
  • the sample containing antibody-secreting cells is added to a surface coated with an antigen-bearing reagent, for example, a mycobacterial 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.
  • an antigen-bearing reagent for example, a mycobacterial antigen alone or conjugated to bovine serum albumin
  • the present invention is also directed to a kit or reagent system useful for practicing the methods described herein.
  • 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.
  • kits for determining the presence of anti-Mtb early antibodies may contain one or more early Mtb antigens, either in immobilizable form or already immobilized to a solid support, and a detectably labeled binding partner capable of recognizing the sample anti-Mtb early antibody to be detected, for example, a labeled anti-human Ig or anti-human Fab antibody.
  • a kit for determining the presence of an early Mtb antigen may contain an immobilizable or immobilized "capture” antibody which reacts with one epitope of an early 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 maybe 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 also provides an approach to the identification, isolation and characterization of early Mtb antigens.
  • an adsorbed patient serum or pool of sera containing antibody for one or more antigens can be used in initial stages of antigen preparation and purification, as well as in the process of cloning of a protein antigen.
  • This antiserum can be further adsorbed with an Mtb or other mycobacterial preparation to render it functionally monospecific or oligospecific.
  • This "enriched" antiserum can be used along with standard biochemical purification techniques to assay for the presence of the antigen it recognizes in fractions obtained during the purification process.
  • the antiserum can also be used in immobilized form as an immunoadsorbent in affinity purification of the antigen in accordance with standard methods in the art. hi addition, the antiserum can be used in an expression cloning method to detect the presence of the antigen in bacterial colonies or phage plaques where the antigen is expressed.
  • the antigen can be used to immunize animals to prepare high titer antisera or, preferably, to obtain a mAb specific for that antigen.
  • an animal antiserum or mAb can be employed advantageously in place of the patient antiserum or in combination with a test body fluid sample in a competition immunoassay.
  • the antiserum or mAb can be used for antigen production or purification, or in an immunoassay for detecting the antigen, for example as a binding partner (either the capture antibody or the detection antibody) in a sandwich immunoassay.
  • the present invention provides an immunoassay for detecting the presence of an Mtb early antigen in a body fluid or in a bacterial culture grown from a body fluid of a subject suspected of being infected with Mtb.
  • a sensitive immunoassay such as a direct sandwich EIA or a competitive EIA can detect an Mtb protein (early antigen) in picogram amounts.
  • a competitive assay allows detection of specific epitopes of the Mtb antigen without the necessity of starting with a purified antigen preparation. Such assays permits detection of Mtb in the patient sample at an earlier time than standard bacteriological analysis (i.e., appearance of colonies on agar).
  • This method therefore provides a basis for clinical decisions to initiate therapy after several hours or days if the antigen can be detected in a body fluid. In any case, this is a major advantage over the conventional two to four (or more) weeks commonly needed to grow out Mtb organisms from a patient sample. The earlier the stage of the infection, the lower would be the titer of Mtb in a given body fluid, and the greater would be the advantage of the present assay over conventional diagnosis.
  • a number of immunoassays for various Mtb antigens are known in the art and can serve as the basis for development of assays for the early antigens of the present invention (Wilkins et al, supra; Verbon, 1994, supra; Benjamin, RG et al, 1984, J. Med. Micro.
  • test antigen preparation for example an Mtb culture supernatant or extract is added to the immobilized antibody.
  • a second "detection" antibody such as a murine mAb specific for the same antigen or preferably for a different epitope of the same protein, allowed to bind in the presence of a fixed amount of a mAb, preferably of murine origin, specific for the epitope of interest.
  • the detection mAb maybe enzyme-conjugated.
  • a second step reagent such as an enzyme-labeled antibody specific for murine immunoglobulin may be used for detection of antigen which has become immobilized.
  • 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 mycobacterial 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).
  • 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, PK et al, 1989, Med. Microbiol. Immunol. 275:229-233; Radhakrishnan, VV et al, 1992, J. Med. Microbiol. 35:128-131). Methods for detection of immune complexes are well-known in the art. Complexes maybe dissociated under acid conditions and the resultant antigens and antibodies detected by immunoassay. See, for example, Bollinger, RC 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.
  • Mtb early antigens as described herein are preferably produced using recombinant methods. See Example TV.
  • Conventional bacterial expression systems utilize Gram negative bacteria such as E. coli or Salmonella species. However, it is believed that such systems are not ideally suited for production of Mtb antigens (Burlein, JE, In: Tuberculosis: Pathogenesis, Protection and Control, B. Bloom, ed., Amer Soc Microbiol, Washington, DC, 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.
  • 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.
  • 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-mycobacterial antibodies in urine from TB patients with early disease (non-cavitary, smear negative TB patients) and from HTV-infected TB patients; (2) determine the profile of specific mycobacterial 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.
  • Serum and urine samples from non-cavitary and/or smear negative, culture positive TB patients and from HlV-infected TB patients are obtained Cohorts comprising PPD-positive and PPD-negative healthy individuals, non-tuberculous HIN-infected individuals, or close contacts of TB patients can serve as negative controls.
  • the reactivity of the serum samples with culture filtrate proteins of M. tuberculosis, and the purified antigens (MPT 32, Ag 85C and the 88 kDa, 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.
  • E. coli lysates (suspended at 500 ⁇ g/ml) are coated onto wells of ⁇ LISA plates (Immulon 2, Dynex, Chantilly, NA.) 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 ⁇ LISA assays.
  • the antigen-antibody binding is allowed to proceed for 90 min at 37°C.
  • the plates are washed 6 times with PBS-Tween 20 (0.05%) and 50 ⁇ l/well of alkaline phosphatase-conjugated goat anti-human IgG (Zymed, CA), diluted 1 :2000 in PBS/Tween 20 is added.
  • the plates are washed 6 times with Tris buffered saline (50 mM Tris, 150 mM ⁇ aCl) 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 ⁇ l of 0.3M H 2 SO 4 .
  • the reactivity of the urine samples with the various antigens is determined initially with undiluted urine samples as described above.
  • results obtained by the present inventors showed that the optimal concentration of the culture filtrate protein preparation is 125 ⁇ l/well of 4 ⁇ g/ml suspension, and for MPT 32 is 125 ⁇ l/well of 2 ⁇ g/ml.
  • the urine is left overnight in the antigen coated wells: However, if urine antibody titers of smear-negative and HiN-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.
  • the present inventors Based on this map, the present inventors generated a 2-D map of the antigens that are recognized by the early serum antibodies (from smear-negative patients), antibodies from advanced, smear-positive HIV-uninfected TB patients, and from HIV-infected TB patients
  • Screening permits the determination of whether additional antigens besides the MPT 32, Ag 85C and the 88 kDa protein are to be included in the assay for its optimization. It is preferred to determine if the anti-MPT 51 antibodies are well represented in the urine since this protein is highly recognized by serum antibodies during both early and late stages of TB, and its identity and sequence are known.
  • Culture filtrate antigens of Mtb are fractionated on 2-D gels and transferred to obtain 2-D blots as described below. Briefly, 70 ⁇ g of culture filtrate proteins are resuspended in 30 ⁇ l of isoelectric focusing sample buffer (e.g., 9M urea, 2% NP-40, 5% ⁇ -mercaptoethanol, and 5% ampholytes at pH 3-10 or pH 4 - 6.5).
  • the ampholytes used in the Examples below from Pharmacia are designated "PharmalytesTM" and are co-polymers of glycine, glycylglycine, amines and epichlorhydrin.
  • the tube gels are soaked in sample transfer buffer for 30 min and then electrophoresed in the second dimension by using a 15% SDS-polyacrylamide gel. This is carried out at 20 mA per gel for 0.3 hrs followed by 30 mA per gel for 1.8 hrs.
  • the separated proteins are then transferred for subsequent immunoblotting.
  • the 2-D western blots are washed with PBS, and blocked for 2-2.5 hr. with 5% BSA. After washing the blots again, they are exposed to the individual urine samples (undiluted, or concentrated) overnight with shaking. After subsequent washing with PBS-Tween, the blots are exposed to alkaline-phosphatase conjugated anti-human IgG, and then to the appropriate substrate.
  • the antigens that are reactive with the urine samples are identified on the basis of the 2-D maps already generated.
  • Antigens that are strongly recognized by the urine antibodies, as well as by serum antibodies, are candidates for inclusion in the preferred diagnostic assay.
  • Preferred antigens are the 88 kDa protein GlcB described above or is MPT 51 and epitopes thereof, such those present in the various peptides described above. DNA encoding these proteins or fragments or variants thereof are cloned and expressed.
  • the Mtb culture filtrate preparation contains >100 different proteins (205 protein spots), and most of the proteins in the 49-76 kDa range are expressed in low abundance in the culture medium (Sonnenberg et al. , supra). This may be a result of growing Mtb is in minimal medium to obtain these proteins, to avoid difficulties associated with the proteins of enriched media (BSA, casein digests, etc.). If the immunoreactive protein is well- expressed in the culture filtrate, and reasonably isolated on the gel, it can be excised from the PVDF blot and sequenced. Since the entire genomic sequence of Mtb is known , the peptide sequence is used to identify the protein with complete precision.
  • the nucleotide sequence of the gene (i.e., open reading frame) encoding that protein then becomes the basis for PCR amplification of the relevant DNA from genomic DNA, followed by cloning into an expression vector. Since many of the culture filtrate proteins are present in small quantities, an alternative, possibly more reliable, approach would utilize the urine antibodies to immunoscreen an expression library of Mtbs to obtain the gene(s) encoding the relevant protein(s). These approaches may be used, for example, to clone the MPT 51 gene or to identify the immunoreactive proteins in the 49-76 kDa region. For expression of MPT 51, the shuttle vector pW16 is preferred; this vector has an E.
  • This vector can be used for expression in E. coli or in M. smegmatis. Since mycobacterial proteins expressed in E. coli host often show poorer immunological reactivity than the same proteins expressed in the mycobacterial host, it would be preferred to express the antigen inM. smegmatis. The methods for expression of genes in mycobacterial hosts are well described (Gaora, PO et al, 1997, Med. Principles Pract. 6:91).
  • PCR amplification of the target gene using primers that contain restriction sites to generate in-frame fusions is performed.
  • the PCR product is purified, and digested with the appropriate restriction enzymes and purified again.
  • the vector D ⁇ A is cut with the appropriate restriction enzymes and purified.
  • the PCR product and the vector are ligated, electroporated into DH5 ⁇ , and grown in the presence of hygromycin overnight.
  • Several antibiotic-resistant colonies are grown in a small volume of medium, and the plasmid D ⁇ A isolated by miniprep. The size of the insert is checked in these colonies. Inserts from one or more colonies are sequenced.
  • the bacteria are grown shaking in 7H9 medium till they reach an absorbance of 0.8-1.0.
  • the bacteria are harvested, washed twice with ice cold water, once with ice-cold 10% glycerol and suspended in the same.
  • An aliquot of the cells are electroporated with the plasmid D ⁇ A from the colony whose insert was sequenced.
  • the electroporated cells are grown for 3-4 hrs in 7H9, and plated on antibiotic containing plates. Several resistant colonies are grown in minimal media for 48-72 hrs. The M.
  • smegmatis cell pellets are sonicated, the lysates fractionated by SDS-PAG ⁇ and the presence of the immunoreactive protein confirmed by reactivity with the antibody-containing urine samples.
  • Colonies which express the desired protein are expanded, and the His-tagged recombinant protein is purified using of commercially available ⁇ ickel-agarose columns (Qiagen).
  • the reactivity of the recombinant protein with the entire cohort of urine samples is evaluated by ⁇ LISA as described herein. Combinations of antigens, preferably of individual epitopes, that provide the best sensitivity and specificity are delineated.
  • an expression library of Mtb genomic DNA is screened with the antibody-positive urine samples. A pool of TB patient urine samples (which show strong reactivity on western blots with culture filtrate proteins of Mtb) from 10-15 TB patients is adsorbed against E. coli lysate, and used at an appropriate dilution to screen the library.
  • E. coli Y1090 infected with appropriate plaque forming units of the phage from the library are plated in top agar on LB plates. After 2.5 hrs at 42°C, isopropyl ⁇ -D thiogalactoside (IPTG) saturated nitrocellulose filters are overlaid on the top of the plates for 2.5 hrs at 37°C. The filters are removed, washed extensively, and exposed to the pooled urine overnight. After washing again, the filters are exposed to 1 : 1000 dilution of Alkaline
  • Phosphatase conjugated anti-human IgG followed by BCIP-NBT substrate.
  • the positive plaques are located on the original plates, excised and re-screened till purified.
  • the screening of the library by the urine antibodies can be expected to identify several proteins.
  • the cloned phages are used to establish lysogens in E. coli Y1089. Single colonies from lysogens are grown in LB medium at 32°C till an absorbance (at 600 nm) of 0.5 is obtained.
  • the lysogens are induced to express the recombinant proteins by raising the temperature to 45°C and addition of IPTG (10 mM). The cultures are grown for additional 1.5 hrs at 37°C to allow accumulation of the recombinant proteins, and the bacterial pellets are obtained.
  • the pellets are sonicated in small volume of PBS and the lysates fractionated on 10% SDS-PA gels and electroblotted onto nitrocellulose membranes.
  • the blots are probed with individual urine samples from 20-25 TB patients, and clones coding for strongly immuno-reactive proteins recognized by all or a vast majority of the urine samples are identified. Lysates from E. coli Y1089 alone or Y1089 lysogenized with lambda gtl 1 vector are used as controls.
  • DNA from the recombinant clones encoding strongly immunoreactive proteins is isolated by the commercial Wizard Lambda Preps DNA Purification system (Promega), digested with ⁇ coRl and the insert obtained.
  • the insert DNA from the clone(s) is subcloned into pG ⁇ M ⁇ X-1 vector (Promega) whose reading frame at the EcoRI cloning site is identical to lambda gtl 1.
  • Competent E. coli JM 109 cells are transformed with the recombinant plasmid (pGEMEX plus insert from the clone(s)).
  • Plasmid DNA is isolated using Wizard Plus Minipreps (Promega), and used for automated sequencing with primers from SP6 and T3 promoter specific primers flanking the multiple cloning site in the PGEMEX- 1 followed by 'primer- walking'.
  • the nucleotide sequence is used in similarity searches against the Mtb genomic sequence to identify the protein and to obtain the sequence of the whole gene. Once the protein has been identified and the sequence of the gene is known, cloning it for expression is done as was described above for the exemplary cloning of the MPT 51 gene.
  • the vaccine compositions and methods are designed to augment this immunity, and preferably, to induce it a stage wherein the bacterial infection can be prevented or curtailed.
  • the vaccine compositions are particularly useful in preventing Mtb infection in subjects at high risk for such an infection, as discussed above.
  • the vaccine compositions and methods are also applicable to veterinary uses.
  • this invention includes a vaccine composition for immunizing a subject against Mtb infection.
  • An Mtb early antigen preferably one of the four described herein in more detail or a peptide thereof, is prepared as the active ingredient in a vaccine composition.
  • These four proteins are (a) the 88 kDa protein having a pi of about 5.2 and SEQ ID NO: 106; (b) the protein characterized as Mtb antigen 85C; (c) the protein characterized as Mtb antigen MPT51 (SEQ ID NO: 107); and (d) the glycoprotein characterized as Mtb antigen MPT32.
  • the vaccine may also comprises one or more of the proteins described herein, peptides thereof or functional derivatives as described, or DNA encoding the protein, and a pharmaceutically acceptable vehicle or carrier.
  • Preferred peptides for use in a vaccine composition, alone, in combination, or in linear multimers, include the 23 peptide described above in the context of diagnostic compositions.
  • the vaccine comprises a fusion protein or peptide multimer which includes an Mtb early antigen, e.g., a full length protein and/or one or more of the above peptides, as described above.
  • the vaccine composition may further comprise an adjuvant or other immune stimulating agent.
  • an adjuvant or other immune stimulating agent for use in vaccines, the Mtb early antigen protein or epitope-bearing peptide thereof is preferably produced recombinantly, preferably in prokaryotic cells.
  • Full length proteins or longer epitope-bearing fragments of the Mtb early antigen proteins are preferred immunogens, in particular, those reactive with early antibodies. If a shorter epitope-bearing fragment, for example containing 20 amino acids or less, is the active ingredient of the vaccine, it is advantageous to couple the peptide to an immunogenic carrier to enhance its immunogenicity.
  • an immunogenic carrier to enhance its immunogenicity.
  • Such coupling techniques are well known in the art, and include standard chemical coupling techniques using linker moieties such as those available from Pierce Chemical Company, Rockford, Illinois.
  • Suitable carriers are proteins such as keyhole limpet hemocyanin (KLH), E. coli pilin protein k99, BSA, or rotavirus NP6 protein.
  • Another vaccine embodiment is a peptide multimer or fusion protein which comprise the Mtb early antigen protein or an epitope-bearing peptide region fused linearly to an additional amino acid sequence. Because of the ease with which recombinant materials can be manipulated, multiple copies a selected epitope-bearing region may be included in a single fusion protein molecule. Alternatively, several different epitope-bearing regions can be "mixed and matched" in a single multimer or fusion protein.
  • the active ingredient such, preferably a recombinant product, is preferably administered as a protein or peptide vaccine, i another embodiment, the vaccine is in the form of a strain of bacteria (preferably a known "vaccine strain") which has been genetically transformed to express the protein or epitope-bearing peptide.
  • a strain of bacteria preferably a known "vaccine strain”
  • Salmonella dublin live vaccine strain SL5928 aroA148fliC(i)::InlO and S. typhimurium LB5000 hsdSB121 leu-3121 Newton S. M. et al, Science 1989, 244: 70
  • a Salmonella strain expressing the Mtb protein or fragment of this invention may be constructed using known methods.
  • a plasmid encoding the protein or peptide may first be selected in an appropriate host, e.g., E. coli strain MCI 061.
  • the purified plasmid is then introduced into S. typhimurium strain LB5000 so that the plasmid DNA is be properly modified for introduction into Salmonella vaccine strains.
  • Plasmid DNA isolated from LB5000 is introduced into, e.g., S. dublin strain SL5928 by electroporation. Expression of the Mtb protein or fragment encoded by the plasmid in SL5928 can be verified by Western blots of bacterial lysates and antibodies specific for the relevant antigen or epitope.
  • the active ingredient, or mixture of active ingredients, in protein or peptide vaccine composition is formulated conventionally using methods well-known for formulation of such vaccines.
  • the active ingredient is generally dissolved or suspended in an acceptable carrier such as phosphate buffered saline.
  • Vaccine compositions may include an immunostimulant or adjuvant such as complete or incomplete Freund's adjuvant, aluminum hydroxide, liposomes, beads such as latex or gold beads, ISCOMs, and the like.
  • an immunostimulant or adjuvant such as complete or incomplete Freund's adjuvant, aluminum hydroxide, liposomes, beads such as latex or gold beads, ISCOMs, and the like.
  • 0.5 ml of Freund's complete adjuvant or a synthetic adjuvant with less undesirable side effects is used for intramuscular or subcutaneous injections, preferably for all initial immunizations; this can be followed with Freund's incomplete adjuvant for booster injections.
  • General methods to prepare vaccines are described in Remington 's Pharmaceutical
  • Liposomes are pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the active protein is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension.
  • the hydrophobic layer, or lipidic layer generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • Adjuvants, including liposomes are discussed in the following references, incorporated herein by reference:
  • the vaccine compositions preferably contain (1) an effective amount of the active ingredient, that is, the protein or peptide together with (2) a suitable amount of a carrier molecule or, optionally a carrier vehicle, and, if desired, (3) preservatives, buffers, and the like.
  • an effective amount of the active ingredient that is, the protein or peptide together with (2) a suitable amount of a carrier molecule or, optionally a carrier vehicle, and, if desired, (3) preservatives, buffers, and the like.
  • the immunogenically effective amounts of the proteins or peptides of the invention must be determined empirically. Factors to be considered include the immunogenicity of the native peptide, whether or not the peptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and the route of administration for the composition, i.e., intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be administered. Such factors are known in the vaccine art, and it is well within the skill of the immunologists to make such determinations without undue experimentation.
  • the vaccines are administered as is generally understood in the art. Ordinarily, systemic administration is by injection; however, other effective means of administration are known. With suitable formulation, peptide vaccines may be administered across the mucus membrane using penetrants such as bile salts or fusidic acids in combination, usually, with a surfactant. Transcutaneous administration of peptides is also known. Oral formulations can also be used. Dosage levels depend on the mode of administration, the nature of the subject, and the nature of carrier/adjuvant formulation. Preferably, an effective amount of the protein or peptide is between about 0.01 ⁇ g/kg -1 mg/kg body weight.
  • Subjects may be immunized systemically by injection or orally by feeding, e.g., in the case of vaccine strains of bacteria, 10 -10 bacteria on one or multiple occasions.
  • multiple administrations of the vaccine in a standard immunization protocol are used, as is standard in the art.
  • the vaccines can be administered at approximately two to six week intervals, preferably monthly, for a period of from one to four inoculations in order to provide protection.
  • Vaccination with the vaccine composition will result in an immune response, either or both of an antibody response and a cell-mediated response, , which will block one or more steps in the Mtb bacterium's infective cycle, preferably the steps of binding to and entry into host cells in which it grows.
  • the study population included 58 HIV neg individuals with confirmed pulmonary TB. Of these, 16 were individuals attending the Infectious Disease Clinic at the Veterans Affairs Medical Center, New York. All patients were Mtb culture-positive, 9/16 patients were smear- negative, 14/16 showed minimal to no radiological lesions, and all were bled either prior to, or within 1-2 weeks of initiation of chemotherapy for TB. Eight sera were obtained from Leonid Heifitz and Lory Powell (National Jewish Center, Denver, CO). An additional 20 sera were provided by J.M. Phadtare ( Grant Medical College, Bombay, India). Fourteen serum samples obtained from Lala Ram Sarup Tuberculosis Hospital, Mehrauli, New Delhi, India were provided by S. Singh. A majority of these 42 patients were smear-positive, had radiological appearance of moderate to advanced pulmonary lesions and were bled 4-24 weeks after initiation of chemotherapy. The control populations consisted of the following groups:
  • Group (b) subjects were included because TB has emerged as a major opportunistic disease in the HTV-infected population.
  • the antigen preparations were total cellular sonicate (CS), total culture filtrate (CF), lipoarabinomannan (LAM), LAM-free culture filtrate proteins (LFCFP), whole cell walls (CW),
  • SDS-soluble cell wall proteins SDS-soluble cell wall proteins (SCWP), and cell wall core (CWC), all isolated from Mtb H 37 Rv.
  • CS was obtained from Mtb grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) for 2-3 weeks.
  • the bacilli were harvested by centrifugation at 1000 rpm for 30 min and the pellet resuspended in phosphate buffered saline (PBS) containing PMSF, EDTA and DTT at a final concentration of ImM each.
  • PBS phosphate buffered saline
  • the suspension was frozen in liquid nitrogen and thawed (several times) to weaken the cell walls, followed by sonication for 20 min at 4°C.
  • the sonicate was centrifuged for 10 min at 10,000 rpm and the supernatant collected.
  • Mtb was grown to mid-logarithmic phase (14 days) in glycerol-alanine-salts medium.
  • the cells were removed by filtration through a 0.22 ⁇ m membrane, and the culture supernatant was concentrated by ultrafiltration using an Amicon apparatus (Beverly, MA) with a 10,000 MW cut-off membrane.
  • the concentrated material (CF) was dialyzed against 100 mM ammonium bicarbonate and dried by lyophilization.
  • CF was suspended (7mg/ml) in a buffer containing 50mM Tris HCI (pH 7.4), and 150mM NaCl, following which 20% Triton X-114 was added to obtain a final concentration of 4%.
  • the suspension was allowed to rock overnight at 4°.
  • a biphasic partition was set up by warming the 4% Triton X-114 suspension to 37° for 40 minutes, followed by centrifugation at 12,000 x g.
  • the aqueous phase was re-extracted twice with 4% Triton X-114 to ensure complete removal of the lipoarabinomannan, lipomannan (LM) and phosphatidyl-inositol- mannoside (PPM).
  • LM lipomannan
  • PPM phosphatidyl-inositol- mannoside
  • the final aqueous phase was precipitated with 10 volumes of cold acetone, and the pellet washed several times with cold acetone to remove residual Triton X-114.
  • the LAM-free aqueous phase CFPs were suspended in 100 mM ammonium bicarbonate, aliquoted and dried by lyophilization.
  • LAM, LM and PIM were extracted from whole cells by mechanical lysis of the bacilli in PBS (pH 7.4) containing 4% Triton-X 114 in a Bead Beater (Biospec Products, Bartelsville, OK). Unbroken cells and cell wall material were removed by centrifugation at 12000g, 4° for 15 min. The supernatant was collected and a biphasic partition set up. The detergent phase was obtained, back-extracted several times with cold PBS and the macromolecules in the final detergent phase were precipitated with 10 volumes of cold acetone. The precipitate was collected by centrifugation and allowed to air dry. This material (which contained the lipoglycans) was suspended in PBS and residual proteins were removed by extraction with PBS- saturated phenol.
  • aqueous phase was collected and, after dialyses against distilled water, the lipoglycans were lyophilized.
  • LAM was further purified away from LM and PIM by size exclusion chromatography as previously described (Chatteriee, D. et al, 1992, J. Biol. Chem. 269:66222,-66233).
  • Mtb cells were inactivated by isothermal killing at 80° for 1 h and suspended at a concentration of 0.5g cells/ml, in a buffer containing PBS, pH 7.4, 4% Triton X- 114, PMSF, pepstatin, EDTA, and DNase.
  • the cells were disrupted in a Bead Beater using 0.1 mm Zirconia beads.
  • the lysed cells were first centrifuged at 3000 x g for 5 min to remove unbroken cells followed by centrifugation at 27,000 x g, 4° for 20 min.
  • the resulting pellet was washed three times with cold PBS at room temperature. This final pellet was termed the CW.
  • the SCWP were obtained by washing the CW twice with 2% SDS in PBS, pH 7.4 at room temperature.
  • the tightly associated proteins were isolated by extracting the CW pellet three times with 2% SDS in PBS, pH 7.4, at 55°.
  • the 55°, 2% SDS extract was recovered, and the SDS was removed by using an Extracti Gel column (Pierce, Rockford, EL). The eluate from the column was dialyzed against twice-distilled H 2 O, aliquoted and dried by lyophilization.
  • the CWC mycolyl-arabinogalactan-peptidoglycan complex
  • the SDS- insoluble material obtained after extraction of the SCWP was suspended in PBS, 1% SDS, 0. lmg/ml proteinase K and incubated for 20h at 50°.
  • the insoluble material was pelleted by centrifugation, washed twice with 2% SDS at 95° for lh and collected by centrifugation. This was washed several times with water and 80% acetone to remove SDS.
  • Fractionation of LFCFP by size was performed by using a preparative SDS-PAGE system (model 491 Prep cell, Bio-Rad, Hercules, CA).
  • CFP (20-25 mg) was loaded directly onto a 30ml 10% preparative polyacrylamide tube gel containing a 6% stacking gel, that was poured in a casting tube with a 37mm internal diameter.
  • the running buffer used consisted of 25mM Tris, pH 8.3, 192mM glycine, 0.1% SDS.
  • the proteins were separated by electrophoresis using an increasing wattage gradient of 8W for 3.13h, 12W for 2.5h, and finally 20W for 11. lh.
  • Proteins were eluted from the bottom of the tube gel with a constant flow of 5mM sodium phosphate, pH 6.8. The initial 65ml of eluant were collected as the void volume, after which 80 fractions of 4.2 ml were collected at a rate of 0.4ml/min. Individual fractions were assayed by one dimensional SDS-PAGE and were pooled accordingly. SDS was removed from the pooled concentrated fractions by elution through an Extracti-Gel (Pierce) column. The pooled fractions were dried and stored frozen until testing. Adsorption of sera with E. coli sonicate
  • E. coli Y1090
  • Luria-Bertani medium Overnight cultures of E. coli (Y1090) grown in Luria-Bertani medium were centrifuged to obtain bacterial pellets that were treated as described for the Mtb sonicate, except that sonication was performed for 30 sec.
  • BSA bovine serum albumin
  • HTV was inactivated by addition of Triton X-100 (1% final concentration) to each serum sample, followed by heating at 55° for 60 min.
  • Samples from non-HIN infected individuals were treated in the same manner to maintain consistency in sample preparation.
  • Serum from each individual (20 ⁇ l) was diluted to 200 ⁇ l in PBS/Tween 20 (0.05%) in a 96-well tissue culture plate.
  • the diluted serum samples were transfereed to the E. coli-coated, blocked ELISA plate by using a multichannel pipetter.
  • the sera samples were exposed to the bound E. coli antigens for 90 min after which they were transferred to another ELISA plate that had been coated with E. coli and blocked as above.
  • the serum samples were exposed to 8 cycles of adsorption with E.
  • coli antigens following which they were transferred to a 96-well tissue culture plate where sodium azide (ImM final concentration) was added to each well. This protocol allows rapid and efficient processing of small volumes of multiple samples. Adsorbed serum samples were used within one week.
  • the antigen-antibody binding was allowed to proceed for 90 min at 37°, following which the plates were washed 6 times with PBST. Fifty ⁇ l of alkaline phosphatase-conjugated goat anti-human IgG (Zymed, CA), diluted 1 :2000 (in the same diluent as the serum samples) were added to each well. After 60 min the plates were washed 6 times with Tris-buffered saline (50mM Tris, 150mM ⁇ aCl) and the Gibco BRL Amplification System (Life Technologies, Gaithersburg, MD) used for development of color. The plates were read at 490 nm after stopping the reaction with 50 ⁇ l of 0.3M H 2 SO 4 . The optimal antigen and antibody concentrations for each antigen were determined by checkerboard titration with limited numbers of control and non-TB sera prior to performing the ELISA with the total serum panel.
  • the ELISA with each of the sized fractions generated by preparative polyacrylamide gel electrophoresis was performed as described as above, except that antigen was coated at 2 ⁇ g/ml and the sera were tested at a final dilution of 1 :200. Forty-two TB sera and 44 non-TB controls (16 PPD + ; 7 HTV neg , PPD neg ; and 21 HrV ⁇ , asymptomatic individuals) were included in these assays.
  • the "IT" designations are World Health Organization standards for its collection of anti- Mtb antibodies.
  • the alternative names of the mAbs, the antigens they recognize and the laboratory of origin are provided in Engers, H. et al. , 1986, Infect. Immun. 51:118-720;
  • the second antibody was an alkaline phosphatase-conjugated rabbit anti-mouse IgG or goat anti-rabbit IgG (1:2000, Sigma Immunochemicals) added in a volume of 50 ⁇ l/well. SDS-PAGE and immunoblotting
  • the cutoff for positivity in all ELISA assays was set to be the mean absorption or optical density (OD) ⁇ 3 standard deviations (SD) of the control group.
  • the Wilcoxon signed rank test for paired samples was used to compare reactivity of sera pre- and post-adsorption.
  • the SD of the above two groups were compared by using the F test.
  • the reactivity of TB sera with LFCFP was compared to the reactivity with the other antigen preparations by using McNemar's paired test.
  • the Graphpad Instat program was used for all statistical analyses.
  • Antibodies reactive with LFCFP were detectable in 25/42 (60%) of the unadsorbed TB sera ( Figure 1).
  • anti-mycobacterial antibodies were detectable in 4/17 (24%) additional, previously negative sera, raising the sensitivity to 69% ( Figure 1).
  • a Wilcoxon signed rank test comparing the preadsorbed and post adsorbed sera.
  • b F test comparing the standard deviations of the preadsorbed and post adsorbed sera.
  • NS not significant.
  • Antibodies to an 88kDa Secreted Antigen of M. tuberculosis Serve as a Surrogate Marker of Pre-clinical TB in HIV-infected Subjects
  • HTV/TB HTV-infected individuals attending the Infectious Disease Clinic at the V. A. Medical Center, New York, who developed or presented with TB (HIV/TB) during the last several years. A total of 259 serum samples were available from these individuals. Of these samples: (a) 136 were obtained from 38 patients on several occasions prior to manifestation of clinical TB ("HTV/pre-TB");
  • non-HIV TB patients Sera from 20 non-HIV TB patients (non-HTV/TB), 19 of whom were smear-positive, and all of whom showed radiological evidence of moderate to advanced cavitary disease, were included as positive controls. Sera from 19 non-HTV/ PPD skin test-positive individuals were included as negative controls.
  • the study included (i) sera from 35 HTV-infected, asymptomatic individuals, with CD4 cell counts >800 and (ii) 48 serum samples from 16 HTV-infected subjects whose blood cultures were positive for Mycobacterium avium-intracellulare ("HIV/MAI"). Of these, 28 HIV/MAI serum samples were obtained during the months preceding advent of MAI bacteremia.
  • LAM-free culture filtrate proteins were prepared as described in Example I. This antigen mixture was subsequently fractionated based on the molecular weight of the proteins using a BioRad 491 Prep Cell (Hercules, CA) with a 30 ml 10% preparative polyacrylamide tube gel containing a 6% stacking gel as above. Fractions were pooled according to molecular weights (as determined by SDS- PAGE) and dried. The LFCFP and the sized fractions thereof, were resolved on 10% SDS-PA mini gel and transferred onto a nitrocellulose membrane prior to probing with sera.
  • LAM-free culture filtrate proteins LAM-free culture filtrate proteins
  • the second antibody used was alkaline-phosphatase conjugated rabbit anti- human IgG and the substrate was BCIP/NBT (Kirkegaard and Perry Laboratories, Gaithersburg, MD). All sera were adsorbed with E. coli lysates prior to use in ⁇ LISA assays. Adsorptions and ⁇ LISAs were performed as described in Example I.
  • the specificity of the anti-Mtb antibody responses in the HTV/TB patients was evaluated.
  • Anti-Mtb antibodies were tested in multiple sera from 6 antibody- positive, 3 antibody-negative HIN/TB patients, and 3 HIN/MAI patients. All 6 antibody- positive individuals had circulating antibodies for different intervals during the years preceding the clinical manifestation of TB. One of the six patients developed anti-Mtb antibodies about 1.5 yr before clinical diagnosis of TB, and another about 4.5 yr prior to that time. The remaining 4 patients had circulating antibodies for the preceding 5-6 yr. In contrast, similar samples from 3 antibody-negative HTV/TB patients and 3 HTV/MAI bacteremia patients were consistently negative.
  • LFCFP were probed with sera from nine ELISA "1" HTV/TB (two HTV/at-TB, seven HTV/pre-TB) and three non-HTV/TB patients. These results were compared to the antibody reactivity of six HIN- + asymptomatic controls and five non-HIN/PPD + healthy controls (ELISA neg ). As described in Example I, all sera (healthy and disease) reacted with antigens of 65kDa and 30-32kDa. The sera from non-HTV/TB patients reacted with multiple antigens (approximately 20) ranging in size from about 26kDa to about 115kDa.
  • the LFCFP material was fractionated into 14 overlapping fractions based on molecular weight. Identification of fractions containing strongly seroreactive proteins was achieved by probing Western blots with pooled sera from six ELISA 4" non-HTV/TB or six HTV/TB patients. Besides the 65 kDa and 30-32 kDa antigens which were previously shown (Example I) to be reactive with all sera (healthy and disease), the non-HTV/TB serum pool reacted primarily with antigens with molecular weights above 30-32 kDa in fractions 6-14.
  • HTV/TB patients recognize only a subset. For example, antibodies to the 38 kDa antigen are not found in HTV/TB, whereas antibodies to antigens in fraction 10-fraction 14, and in particular to the 88 kDa antigen are maintained despite HTV infection.
  • HTV/PPD + control sera as cutoff 16/20 (80%) non-HTV/TB sera had antibodies to the total LFCFP.
  • Example I the non-HTV/TB patients who were reactive with the unfractionated LFCFP were also reactive with the antigens in fraction 14 in this study
  • the reactivity of HTV/TB sera with the unfractionated LFCFP and antigens in fraction 14 was also analyzed by comparing HTV/pre-TB and HTV/at-TB groups. Thirty-one percent of the HTV/at-TB were reactive with the total LFCFP, as were 55% of the HTV/pre-TB sera, hi contrast, 66% of the HTV/at-TB, and 74% of the HTV/pre-TB sera had antibodies which bound fraction 14 antigens.
  • Example I shows that the 88 kDa antigen (GlcB) (present in Fraction 15 in that study, but present in Fraction 14 in the study of Example U) is one of the secreted antigens of Mtb that elicits antibodies during early stages of disease progression (in non-HTV TB patients).
  • the detection of anti-88 kDa antibodies in the high risk HTV-infected population can serve as a diagnostic test, and the antibody as a surrogate marker, for identifying individuals with active pre-clinical TB.
  • PPD 4" Fritzgerald J.M. et al.
  • HIN patients who are anergic to PPD are large, ranging from 33% in Zaire to over 90% in Brazil, and ranges from 43% in early HTV infection to 100% in advanced HTV disease (Raviglione et al, 1992, supra). Delayed hypersensitivity skin test reactivity is known to be unstable in HTV individuals.
  • the A-60 antigen used by some investigators provides poor sensitivity and poor specificity even in the non- HTV/TB patients, a group known to have higher antibody levels (Charpin D et al, Am Rev Respir Dis 1990, 242:380-384; Qadri, S. et al, Can J Microbiol 1991, 35:804-806).
  • Anti-mycobacterial antibodies in seemingly antibody-negative patients may be circulating in the form of immune complexes with the antigens, thereby obscuring the presence of antibody in the assay used. That this may occur in at least a proportion of the patients is suggested by the increased frequency of antibodies detected in HTV/post-TB sera.
  • the present results suggest that patients with persistently circulating antibodies to the
  • Mtb 88 kDa antigen, GlcB may benefit from preventive anti-TB therapy, as has been found to be the case with PPD 4" HTV-infected individuals (Shafer, et al, supra; Pape, J.W. et al, Lancet 1993, 342:268-272).
  • the patients in the present inventors' cohort were chosen on the basis of clinical confirmation of TB. Their PPD reactivity is not known.
  • the length of time from a positive PPD skin test to the development of clinical disease ranges from 1-7 years in HIN- infected individuals (Selwyn et al, supra; Huebner et al, supra. There is no parameter which assists in determining the most appropriate time and duration of prophylactic anti-TB therapy.
  • CFPs culture filtrate proteins
  • Culture filtrates include not only actively secreted proteins but also somatic molecules that are released into the medium during replication or by autolysis .
  • the protein profile of the culture filtrate is highly dependent on cultivation time. Further, the medium used and the means of incubation (static vs. shaking) may also impact on the profile of CFP .
  • the protocol used for CFP preparation a clear understanding of the protein composition of this fraction is difficult to obtain from the current literature.
  • the present inventors have combined 2-D PAGE, western blot analysis, N-terminal amino acid sequencing and liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) to develop a detailed map of culture filtrate proteins and have obtained the partial amino acid sequences for five previously undefined, relatively abundant proteins within this fraction which are found to be useful as early antigens for serodiagnosis of TB.
  • LC-MS-MS liquid chromatography-mass spectrometry-mass spectrometry
  • Mtb strains H37Rv (ATCC 27294) and H37Ra (ATCC 25177) were obtained from American Type Culture Collection (Rockville, MD).
  • Mtb strain Erdman (TMC 107) was obtained from the Trudeau Mycobacterial Collection. Initially, each Mtb strain was inoculated from a 1 ml frozen stock into 10 ml of glycerol alanine salts (GAS) media; three such cultures were prepared for each strain. After incubation at 37°C for 14 days with gentle agitation each 10 ml culture was passed two more times increasing the volume of media by ten times for each pass. The resulting one liter cultures were termed pass number four.
  • GAS glycerol alanine salts
  • mAb A3h4 was obtained from Drs. P.K. Das and A. Rambukana, University of Amsterdam, Amsterdam, The Netherlands and mAbs F 126-2 and HYB 76-8 were obtained from Dr. A.Kolk, Royal Tropical Institute, Amsterdam, The Netherlands, and Dr. I. Rosenkrands, Statens Seruminstitut, Copenhagen, Denmark, respectively. All other mAbs were supplied through the WHO Monoclonal Antibody Bank then maintained by Dr. T. Shinnick, CDC, Atlanta, Georgia. Anti-MPT63 polyclonal serum was provided by Dr. H. Wiker, University of Oslo, Norway. Dr. S. Nagai provided polyclonal sera specific for MPT 32, MPT 35, MPT 46, MPT 53, and MPT 57.
  • 2-D PAGE separation of proteins was achieved by the method of O'Farrell with minor modifications. Specifically, 70 ⁇ g of CFP was dried and suspended in 30 ⁇ l of isoelectric focusing (TEF) sample buffer (9M urea, 2% NP-40, 5% ⁇ -mercaptoethanol, and 5% ampholytes pH 3 - 10 (Pharmacia Biotech, Piscataway, NJ)), and incubated for 3 h at 20°C. An aliquot of 25 ⁇ g of protein was applied to a 6% polyacrylamide TEF tube gel (1.5 mm by 6.5 cm) containing 5% Pharmalytes pH 3 - 10 and 4 - 6.5 in a ratio of 1 :4.
  • TEF isoelectric focusing
  • the proteins were focused for 3 h at 1 kN using 10 mM H 3 PO 4 and 20 mM NaOH as the catholyte and anolyte, respectively.
  • the tube gels were subsequently imbibed in sample transfer buffer for 30 min and placed on a preparative SDS-polyacrylamide gel (7.5 x 10 cm x 1.5 mm) containing a 6% stack over a 15% resolving gel.
  • Electrophoresis in the second dimension was carried out at 20 mA per gel for 0.3 h followed by 30 mA per gel for 1.8 h. Proteins were visualized by staining with silver nitrate.
  • Proteins subjected to 2-D or SDS-PAGE, were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH.) which were blocked with 0.1% bovine serum albumin in 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 80 (TBST). These membranes were incubated for 2 h with specific antibodies diluted with TBST to the proper working concentrations (Table 2). After washing, the membranes were incubated for 1 h with goat anti- mouse or -rabbit alkaline phosphatase-conjugated antibody (Sigma) diluted in TBST. The substrates nitro-blue-tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate (BCTP) were used for color development.
  • BCTP 5-bromo-4-chloro-3-indoyl phosphate
  • the total protein population was conjugated to digoxigenin by incubating the membrane for one hour at room temperature in a solution of 0.05 M K 2 HPO 4 , pH 8.5 containing 0.3 ng/ml digoxigenin-3- O-methylcarbonyl- ⁇ -amino-caproic acid N-hydroxysuccinimide ester and 0.01 % Nonidet-P40.
  • the membranes were subsequently blocked with a solution of 3% bovine serum albumin in 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl (TBS) for 1 h followed by washing with TBS. Incubation with specific antibodies was performed as described, followed by incubation of the membranes with mouse anti-DIG-Fab fragments conjugated to alkaline phosphatase diluted 1 :2000 in TBS, for 1 h. The membranes were washed three times with TBS and probed with goat anti-mouse or -rabbit horse radish peroxidase-conjugated antibody.
  • TBS Tris-HCl, pH 7.5, 0.15 M NaCl
  • CFPs 200 ⁇ g were resolved by 2-D PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Milford, Mass.) by electroblotting at 50 V for 1 h, using CAPS buffer with 10% methanol. The membrane was stained with 0.1% Coomassie brilliant blue in 10% acetic acid and destained with a solution of 50% methanol and 10% acetic acid. Immobilized proteins were subjected to automated Edman degradation on a gas phase sequencer equipped with a continuous-flow reactor. The phenylthiohydantoin amino acid derivatives were identified by on-line reversed- phase chromatography as described previously. 7.
  • LC-MS-MS analysis 200 ⁇ g was resolved by 2-D PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Milford, Mass.) by electroblotting at 50 V for 1 h, using CAPS buffer with 10% methanol. The membrane was stained with 0.1% Coomassie brilliant blue in 10% acetic acid and destained with
  • CFPs were subjected to LC-MS-MS to determine the sequence of internal peptide fragments.
  • CFPs 200 mg were resolved by 2-D PAGE and the gel stained with 0.1% Coomassie brilliant blue and destained as described for proteins immobilized to PVDF membranes.
  • the protein of interest was excised from the gel, washed several times with distilled water to remove residual acetic acid and subjected to in-gel proteolytic digestion with trypsin.
  • Peptides were eluted from the acrylamide and separated by CI 8 capillary RP-HPLC.
  • the microcapillary RP-HPLC effluent was introduced directly into a Finnigan-MAT (San Jose, CA) TSQ-700 triple sector quadrupole mass spectrometer. Mass spectrometry and analysis of the data was performed as described by Blyn et al.. C. RESULTS
  • N-terminal amino acid sequencing of selected CFPs The N-terminal amino acid sequences or complete gene sequences and functions of several of the CFPs of Mtb, mapped with the available antibodies, are known. However, such information is lacking for the proteins that reacted with IT-42 IT-43, IT-44, IT-45, IT-51, IT-52, IT-53, IT-57, IT-59 and IT-69, as well as several dominant proteins not identified by these means. Of these, the most abundant proteins (IT-52, IT-57, IT 42, IT-58 and proteins labeled A- K) were selected and subjected to N-terminal amino acid sequencing (Table 3). Three of these proteins were found to conespond to previously defined products.
  • the N- terminal amino acid sequence of the protein labeled D was identical to that of Ag85 B and C. This result was unexpected given that the IT-49 mAb failed to detect this protein and N-terminal amino acid analysis confirmed that those proteins reacting with IT-49 were members of the Ag85 complex.
  • the protein labeled E had an N-terminal sequence identical to that of glutamine synthetase.
  • a third protein which reacted with IT-52 was found to be identical to MPT 51.
  • the protein labeled I possessed an N-terminal sequence with 72% identity to the amino terminus of an ⁇ -hydroxysteroid dehydrogenase from a Eubacterium species , and the protein labeled F was homologous to a deduced amino acid sequence for an open reading frame identified in the Mtb cosmid MTCY1 Al 1.
  • Examples I and H show that a high molecular weight fraction of CFP of Mtb reacted with a preponderance of sera from TB patients and that this fraction was distinguished from other native fractions in that it possessed the product reactive to mAb IT-57.
  • the protein cluster (including the 88 kDa protein GlcB) defined by IT-42 and IT-57 was excised from a 2-D polyacrylamide gel, digested with trypsin and the resulting peptides analyzed by LC- MS-MS.
  • a second product sequenced was a 25 kDa protein with a pi of 5.34. Its N-terminal sequence (XPVM/LVXPGXEXXQDN, [SEQ ID NO: 100]) showed homology to an internal fragment (DPNLVFPGMEIRQDN, [SEQ ID NO: 105]) corresponding to open reading frame 28c of the Mtb cosmid MTCYl Al 1. Analysis of that deduced sequence revealed a signal peptidase I consensus sequence (Ala-Xaa-Ala) and an apparent signal peptide preceding the N-terminus of the 25 kDa protein sequenced above N-terminal sequencing of selected CFPs identified three novel products:
  • the protein cluster which was recognized by mAbs IT-42 and IT-57 was a primary focus of this study. These proteins migrated at a molecular mass range of 82 - 85 kDa in one co- inventor's laboratory (or 88kDa in another co-inventor's laboratory) and a pi range of 5.12 - 5.19. Results described in Examples I, II and N referred to a CFP of approximately 88 kDa that reacted with 70% of sera from TB patients and demonstrated a specificity of 100%. Subsequent 2-D mapping coupled with 2-D western blot analysis showed these dominant antigens which induce early antibody responses in TB patients are the same as the proteins reactive with IT-57 and IT-42. As stated above, this antigen is referred to as the 88 kDa protein GlcB.
  • coli lysogenized with ⁇ gtl 1 (IT- 57) and the LFCFP were separated by SDS-PAG ⁇ polyacrylamide on 10% gels, transferred to nitrocellulose filters and probed with mAb IT-57.
  • the mAb IT-57 recognized an 88 kDa band in the LFCFP and in the lysate of E. coli lysogen of ⁇ gtl 1 (IT-57). No proteins in the lysate from the E. coli 1089 lysogenized with the wild type ⁇ gtl 1 reacted with the mAb
  • the katG gene was excised from pMD31 with the enzymes Kpnl and Xbal to yield an insert of 2.9 kb.
  • An insert of approximately 3.2 kb obtained after ⁇ coRI digestion of the DNA from ⁇ gtl 1 (IT-57) was used for hybridization with the katG gene.
  • the 3.2 kb insert from ⁇ gtl 1 (IT-57) hybridized with itself and with both the uncut pMD31 vector containing the katG gene and the katG insert DNA itself (2.9 kb). Therefore, the 88 kDa antigen that reacted with mAb IT-57 was in fact the catalase/peroxidase protein.
  • E. coli, the tG-negative BCG strain 35747 transformed with either the pMD31:Mtb katG or with the control pMD31 plasmid (vector control) were tested.
  • the LFCFPs, crude lysates from the lysogen ⁇ gtl 1 (IT-57), lysogenic E. coli 1089 infected with wild-type ⁇ gtl 1 , katG negative BCG strain containing pMD31 Mtb katG and the katG-negative BCG containing pMD31 were separated by SDS-PAG ⁇ polyacrylamide on 10% gels.
  • the fractionated proteins were transferred to nitrocellulose filters and probed with an anti-catalase/peroxidase polyclonal serum (obtained from Dr. Clifton Barry, Rocky Mountain Laboratories, NIAID, Hamilton, MT), mAb IT-57, mAb IT-42 and serum from an advanced TB patient.
  • the anti-catalase/peroxidase polyclonal serum and the mAb IT-57 reacted strongly with an 88 kDa antigen in the LFCFP, in the Mtb katG containing M. bovis BCG and in E. coli ⁇ gtl 1 (IT-57).
  • MAb IT-42 reacted with the same bands in the LFCFP and the Mtb katG BCG, but not with the 88 kDa protein expressed in E. coli.
  • the serum from the TB patient recognized an 88 kDa antigen in the lysates of the katG-negative BCG strain. This is evidence that the seroreactive 88 kDa antigen is a novel protein which has not been previously described.
  • the culture filtrate protein from a tetG-negative strain of Mtb was resolved as above by 2-D PAGE.
  • the protein spot ("Spot 1") corresponding to the sero-reactive 88kDa protein was cut out of the gel and subject to an in-gel digestion with trypsin.
  • the resulting tryptic peptides were extracted, applied to a C 18 RP-HPLC column, and eluted with an increasing concentration of acetonitrile.
  • the peptides eluted in this manner were introduced directly into a Finnigan LCQ Electrospray mass spectrometer. (See Materials and Methods above for further details.)
  • the molecular mass of each peptide was determined, as was the charge state, with a zoom-scan program.
  • Protein pi (from 3.0 to 10.0) All 0
  • Sample ID (comment): Magic Bullet digest Database searched: NCBInr.07.09.99 Molecular weight search (65000 - 97000 Da) selects 21170 entries. Full pi range: 324311 entries. Species search (MYCOBACTERIUM) selects 5990 entries. Combined molecular weight, pi and species searches select 333 entries. MS-Fit search selects 80 entries (results displayed for top 10 matches
  • the protein was identified as GlcB (Z78020) of Mtb, which is believed to be the enzyme malate synthase based on sequence homology to known proteins of other bacteria.
  • This protein has the Accession number CAB01465 on the NCBI Genbank database (based on Cole, S.T. et al, Nature 393:537-544 (1998), which describes the complete genome sequence of Mtb).
  • the sequence of this protein is SEQ ID NO: 106 as provided above.
  • the goal of this study was to determine the repertoire of antigens recognized by antibodies in TB patients in order to elucidate the human humoral response to Mtb and to evaluate the potential of these antigens as candidates for serodiagnosis. This was accomplished by immunoblotting Mtb H37Rv secreted antigens, which had been separated by 1- and 2- dimensional electrophoresis, with sera (E. co/ ⁇ -absorbed) from TB patients and healthy controls. Of the more than 200 secreted proteins of Mtb, only 26 elicited antibodies in TB patients.
  • the identity of several of these antigens was determined based on (a) their reactivity with murine mAbs, (b) N-terminal amino acid sequencing and (c) liquid chromatography-mass spectrometry( ⁇ xample HI). Twelve of these 26 antigens were recognized by sera from patients with early, non-cavitary TB and by patients with advanced cavitary TB. Of these twelve antigens, five, including the 88 kDa antigen (Example I), the MPT32 and Ag 85C, reacted strongly with sera from TB; the other two antigens have yet to be identified. The present invention is directed to the development of serodiagnostic assays (as described herein) employing these antigens that elicit antibodies in both early and advanced TB patients.
  • Serum samples from 33 HlV-negative individuals with confirmed pulmonary TB were included in the study. Twenty of these sera were provided by Dr. J. M. Phadtare (see Example I). Nineteen of these patients were smear-positive and all had radiological evidence of moderate to advanced cavitary lesions. All these patients were bled 4- 24 weeks after initiation of therapy.
  • Control groups Twenty-three HIV nes , TB nes , healthy individuals were included as controls. Sixteen of these were PPD + (skin test) and the remaining 7 were PPD neg .
  • Antigens Twenty-three HIV nes , TB nes , healthy individuals were included as controls. Sixteen of these were PPD + (skin test) and the remaining 7 were PPD neg .
  • Example I Culture filtrates from log phase Mtb H 3 Rv were used as the source of secreted antigens as described in Example I (LAM-free culture filtrate proteins or CFPs).
  • the LFCFP preparation contained over 200 proteins (Example m, supra).
  • Antigens were size fractionated by loading onto a preparative polyacrylamide tube gel, and proteins were separated by electrophoresis using an increasing wattage gradient (model 491 Prep Cell; Bio-Rad, Hercules, CA.). Fractions were collected, assayed by SDS-PAGE and pooled according to molecular weights. Contaminating SDS was removed as described above.
  • Example I Reactivity of each fraction with human sera and an extensive panel of murine mAbs to Mtb antigens are described in Example I. Immunoadsorption of sera against E. coli lysates was performed as described in Example I. All ELISA assays, described in Example I, were perfonned using sera previously immunoadsorbed on E. coli lysates.
  • the 1-D and 2-D blots were blocked with 3% BSA in phosphate buffered saline (PBS) for 2 hrs, and washed for 1 hr with PBS/Tween 2% (wash buffer). Individual lanes containing fractionated LFCFPs were exposed overnight at 4° to individual sera (diluted 1:100 with 1% BSA in PBS). The blots containing the 2-D fractionated LFCFPs were probed with four different serum pools comprised of individual sera whose reactivity with the above antigen preparations were previously determined by ELISA.
  • PBS phosphate buffered saline
  • n number of individuals in each group
  • Sera were grouped according to reactivity by ELISA with total LFCFPs, or the sized fraction containing the 38 kDa PstS or the 88 kDa seroreactive protein (Table 5).
  • Group I includes sera from 16 PPD + and 7 PPD neg healthy controls, none of whom were positive in ELISA with any of these antigen preparations.
  • Group II includes 9 TB patients who tested antibody negative with all three antigen preparations; five of these patients were smear-positive and had cavitary disease. The remaining four patients lacked cavitary lesions, but two of these four were smear-positive.
  • Group III includes thirteen patients with antibodies to both the LFCFPs and the fraction containing the 88 kDa antigen, but not the fraction containing the 38 kDa antigen. Five of these patients were smear-positive and had pulmonary cavitations. An additional four were smear-positive but lacked any cavitary lesions. The remaining four were smear negative and had no cavitations. Group TV included eleven patients, all of whom had antibodies to all three antigen preparations; 10/11 were smear-positive and all had radiological evidence of moderate to advanced cavitary disease.
  • the 30-32 and 65 kDa antigens were also recognized by sera of the 9 PPD + healthy controls, though only 3/9 sera in this group recognized the 26 kDa antigen, and one serum sample recognized an additional 68 kDa antigen.
  • Group JJ tuberculous sera were antibody negative with all 3 antigen preparations by
  • 2D-PAGE provides enhanced resolution of complex protein mixtures.
  • the LFCFPs preparation resolves into about 200 different proteins by this method.
  • a complete 2-D map of the total CFPs of Mtb is shown in US 6,245,331and WO 98/29132 (and discussed in Example HI).
  • 2D immunoblots of the fractionated LFCFPs were probed with serum pools corresponding to patient groups I-TV. The reactivity of each serum pool was compared with the reactivity of murine mAbs to identify the antigens recognized by TB patients' sera (Table 6).
  • the other two antigens reactive with all serum groups had molecular weights of 55 kDa (#114, 120) and 58 kDa (#86, 96, 105) and failed to react with the murine mAbs.
  • the former antigen has been identified as the glutamine synthetase by ⁇ - group analysis (Example HI, above). These antigens may correspond to the 65 kDa antigen that was reactive with the individual sera on 1-D blots.
  • a 26 kDa antigen (#19, 29) and a 46 kDa (#51) were reactive with the control sera (group I) and antibody positive TB sera (group IJJ and group IV), but failed to react with the antibody negative TB serum pool (group IJ).
  • the former antigen 26 kDa, #19, 29 was identified as MPT64 based on reactivity with the murine mAb IT- 67 and may be the 26 kDa antigen recognized by several control sera on 1-D blots
  • antigens correspond to the multiple bands in the 30 to 60 kDa region on the 1-D blots.
  • a 85 kDa protein (#113, 124, IT-42, IT-57) was reactive with this serum pool, but no antigens corresponding to the 74 and 76 kDa antigens seen on 1-D blots were discernible on the 2-D blot.
  • the 85 kDa antigen (#113, 124) on the 2-D immunoblots corresponds to the 88 kDa antigen GlcB (Example I and Example HI).
  • the serum pool from group IN TB patients recognized 11 of 12 antigens that were reactive with the group IJJ serum pool (except the 28 kDa antigen, #77; Table 6B).
  • the reactivity of the group IN serum pool however, with the 26 kDa (#170, MPT51), 31 kDa (#119, Ag 85C), 35 kDa (#66, PstS), 38/42 (#11, 14, MPT32), 49 kDa (#82; IT-58), 85 kDa (#113, 124) and the 104 kDa (#111) antigens, was stronger than with the group HI serum pool.
  • the group IV pool was reactive with all four isomers recognized by murine mAb IT- 23.
  • the group HI and IV serum pools Table 6B
  • the latter group also reacted with eight additional antigens (Table 6C).
  • the antigen with a molecular weight below 30 kDa was the 13/14 kDa protein (#23, 38, IT-12 and SA12, GroES).
  • this serum pool recognized four new antigens, with the same 31 kDa molecular weight but differing in their pi values: 31 kDa (#15, 16, 22, 25), 31 kDa (#62), 31 kDa (#57) and 31 kDa (#37), and a fifth antigen of 38 kDa (#32). Of these only the 31 kDa (#15, 16, 22, 25) was reactive with the mAb IT-44, while the remaining 4 antigens have not been previously described.
  • this pool reacted with a 66/72 kDa protein (#65, 79, mAb IT-40 and IT-41, DnaK), and an unidentified 79 kDa antigen (#78).
  • coli lysates eliminates the cross-reactive antibodies that have hindered the definition of seroreactive antigens, hi addition, the 2-D analysis and mapping of each antigen as described herein has allowed precise definition of antigens that appear to be critical for rational design of serodiagnosis and at least 5 secreted proteins as useful serodiagnostic agents.
  • Antibodies to one of these, the 88 kDa antigen GlcB, are present in 80% of the advanced and 50% of the early TB.
  • MPT32 has also been suggested to have serodiagnostic potential (Espitia et al, 1995, supra) but not as an "early" antigen.
  • the remaining 3 antigens, the 49 kDa (#82; IT-58), 31 kDa antigen (#119, Ag 85C), and the 26 kDa (#170, IT-52) have never been used for assessing seroreactivity in patients until the making of the present invention.
  • the present inventors' laboratories have changed the Reference Number designation of some of the spots on the 2D gels shown in US 6,245,331 (12 June 2001) and WO 98/29132 (published 09 July 1998) from a numeric to an alpha (letter) labeling.
  • the presence of one or more of these antigens, an epitope-bearing peptide thereof or a reactive variant of the peptide, in an immunodiagnostic preparation in combination with one or more of the five early antigens (or a peptide thereof) described above enhances the sensitivity of the diagnostic assay.
  • MPT64 26 kDa, #19, 29
  • group I Another protein currently being assessed as a serodiagnosis candidate is MPT64 (26 kDa, #19, 29) (Nerbon et al, 1993, supra) was reported to provide sensitivities of about 46% in active TB patients.
  • MPT64 26 kDa, #19, 29
  • group I the healthy controls
  • the early antigens identified herein may not be the only early antigens secreted during Mtb growth in vivo. These antigens may be the only ones that are distinguishable because of their strongly seroreactive epitopes.
  • Several antigens of Mtb were either up- or down-regulated when the organisms were grown intracellularly in macrophages. The present inventors propose that, in vivo, Mtb organisms produce only those proteins required for survival and growth under these particular conditions which may differ from the requirements during growth in culture media. It is noteworthy that several of the antigens that elicit antibodies relatively early in TB (based on reactivity with group HI sera), are implicated as having a role in pathogenesis in vivo.
  • Ag 85 A, Ag 85C and MPT51 all belong to the family of secreted proteins which bind to fibronectin (Wiker et al, 1992, Scand. J. Immunol, supra)).
  • MPT32 is homologous to a fibronectin-binding protein of leprae (Schorey, J.S. et al, 1995, Infect. Immun. 63:2652- 2657).
  • the present inventors have identified seroreactive antigens which are useful for diagnostic assays for TB patients who are relatively early in disease progression, view of the expected homology of these antigens with similar proteins in other mycobacterial species, species-specific epitopes should now be defined for serodiagnostic uses. If the absence of detectable antibodies (by ELISA) is due to the formation of immune complexes in vivo (Grange, supra), the present invention provides methods to identify such complexes containing these antibodies. hi view of the large number of antigens secreted by replicating Mtb in culture, it is significant that such a small number of antigens are reactive with TB patient antibodies.
  • any single antigen on the 2-D blots with pooled sera may represent reactivity with only some of the individual sera comprising the pool.
  • individual sera were assessed for antibodies to two of the antigens identified by the group JJI serum pool, Ag 85C and MPT32 which the present inventors had purified. Reactivities with the purified 38 kDa PstS antigen and the 88 kDa antigen GlcB (in fraction 15) was also tested.
  • HC healthy controls. HIV* patients with TB included those diagnosed before (pre) or at the time of (at) TB diagnosis. **Borderline values of OD
  • the combination of smear and ELISA could diagnose 50/54 (93%) of the TB patients.
  • the reactivity of the serum samples with the culture filtrate proteins of Mtb was evaluated by ELISA as described above.
  • ELISA plates were coated overnight with 125 ⁇ l of a 4 ⁇ g/ml suspension of the culture filtrate proteins of Mtb at 4°C. The next morning, the plates were washed with PBS, and 125 ⁇ l of urine were added to each well. After 90 min., the plates were washed with PBS-Tween, and the bound antibody detected by anti-human IgG-Alkaline Phosphatase conjugate, and the substrate for the enzyme.
  • Urine samples from the TB patients (tested undiluted) reacted with a similar profile of antigens, albeit less well.
  • the 88 kDa protein GlcB was recognized by antibodies in both urine samples, but the reactivity of the urine samples with MPT 32, Ag 85C and MPT 51 could not be ascertained on 1-D blots.
  • ELISA results showed that anti-MPT 32 antibodies are present in the urine. This suggests that the antibodies in the urine are directed against the same antigens as are those in the serum, although antibody titers are lower. Together, these results indicate that (1) Anti-mycobacterial antibodies are present in the urine of a significant proportion of smear positive (late)TB patients, thereby serving as the basis for a urine based diagnostic test for TB.
  • a urine or urine/serum based diagnostic test for TB is performed using the same antigens of Mtb described herein (as well as others) including full length proteins, polyproteins, peptides, and peptide multimers, that are identified as seroreactive.
  • proteins and peptides that are defined herein as being useful for serodiagnosis of TB or TB vaccines in humans can also be the basis of serodiagnostic assays and vaccines for TB in other mammals.
  • the present invention is useful in the veterinary medical setting as well.
  • the Hopp-Woods method was described in Hopp, TP & Woods, KR, Proc Natl Acad Sci USA, 1981, 75:3824-3828; Hopp & Woods, Mol Immunol, 1983 20:483-489).
  • the method locates protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant.
  • the prediction success rate depends on averaging group length, with hexapeptide averages yielding optimal results.
  • the method was originally developed using 12 proteins for which extensive immunochemical information was available and subsequently was used to predict antigenic determinants
  • the 1983 publication describes a computerized method for predicting the locations of protein antigenic determinants which requires only amino acid sequence information. This procedure was used to predict the major antigenic determinant of the hepatitis B surface antigen, and is suitable for use on personal computers (having been written in BASIC to make it available to investigators with limited computer experience and/or resources.
  • This document also demonstrated a means of locating multiple antigenic sites on a homologous series of proteins using influenza hemagglutinin. Another approach to analyze "flexibility" of the molecules is the Karplus-Stultz method
  • DLD dynamic ligand design
  • This package included a novel algorithm for the prediction of potential antigenic sites.
  • the software package offers a powerful tool to analyze an amino acid sequence for antigenic site analyses.
  • Results are shown in Table 10, below which records the peptides to which reactivity was observed. Reactivity is indicated as the fraction of TB sera that reacted positively (absorbance >2.5 standard deviations above the negative control sera). Sera were assayed against the peptides at multiple dilutions; optimal responses were usually observed at a 1/10 dilution, though 1/5 and 1/20 dilutions were also tested.
  • a custom library of 13-mer peptides, overlapping by 7 amino acids, and attached covalently to a pre-derivatized cellulose membrane was synthesized by Sigma Genosys in accordance with the present inventors' instructions.
  • the sequences of the 122 peptides, spanning the entire protein are shown in Table 11, below.
  • the membrane was used for colorimetric detection of immunoreactivity. After regeneration, the free immobilized peptides are "regenerated” and a second and subsequent antibody or antiserum is applied. Thus, different patterns are obtained on the identical peptide matrix and can be compared qualitatively and quantitatively.
  • the membrane was rinsed briefly in methanol, and washed thrice (10 min each) in Tris-buffered saline. The membrane was blocked overnight at room temperature with a casein-containing blocking agent provided by the Sigma Genosys. After blocking, the membrane was washed with Tris-buffered saline containing Tween-20 (0.05%) and exposed to 1:100 dilution of a serum pool prepared from sera of 6 patients with confirmed, smear-positive TB.
  • the overlapping peptide library was incubated with the pooled serum for 4 hrs, washed and probed with la :200 dilution of ⁇ -galactosidase-conjugated anti-human IgG. After this incubation the membrane was washed and exposed to the substrate for ⁇ -galactosidase (X-gal in N,N'-dimethyl formamide (DMF).
  • the membrane could be regenerated for reuse by washing extensively in deionized water (30 min, 3 changes), and then in DMF before stripping with buffer A (urea 48% w/v, SDS, 1% w/v, and ⁇ -mercaptoethanol, 1/1000 dilution of the neat reagent) and buffer B (50% ethanol /10% acetic acid (v/v)).
  • buffer A urea 48% w/v, SDS, 1% w/v, and ⁇ -mercaptoethanol, 1/1000 dilution of the neat reagent
  • buffer B 50% ethanol /10% acetic acid (v/v)
  • the membrane was probed with the ⁇ -galactosidase-conjugated anti-human IgG (without prior exposure to the human serum) to identify peptides that bind non-specifically to the secondary antibody (or enzyme). Based on the reactivity of the 122 overlapping peptides (SEQ ID NO:l 16-237) with the pooled sera, the following peptides were identified as being strongly immunogenic in TB patients: SEQ ID NO:117; SEQ ID NO:126; SEQ ID NO:127; SEQ ID NO:128; SEQ ID NO:134; SEQ ID NO:135;SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:138; SEQ ID NO:154; SEQ ID NO:155; SEQ ID NO:170; SEQ ID NO:172; SEQ ID NO:191; SEQ ID NO:216; and SEQ ID NO:217.
  • sequences represent peptides that comprise strongly recognized epitopes (2-5x more intense staining with TB serum pool vs. control. This is in addition to those epitopes identified earlier on the basis of computer algorithms and found to be immunogenic in patients (see Example IX).
  • the references cited above are all incorporated by reference herein, whether specifically incorporated or not.

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CN100415771C (zh) * 2006-06-06 2008-09-03 中国人民解放军第二军医大学 激发人体抗结核杆菌的保护性免疫反应的抗原表位及其用途
CN101298471B (zh) * 2006-06-06 2011-01-19 中国人民解放军第二军医大学 激发人体抗结核杆菌的保护性免疫反应的抗原表位及其用途
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