Abstract:
A genetically modified strain of  M. tuberculosis  or  Mycobacterium bovis  BCG is provided, wherein the genetically modified strain comprises at least one modified sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, or both, having at least one mutation at T2, Q4, F8, A14, L28, L29, W43, G45, Y51, Q55, Q56, N66, M83, V90, M93, or F94 in SEQ ID NO:1; or at least one mutation at Q3, F7 A13, L27, W42, G44, Y50, Q54, N65, N67, M82, V89, M92, or F93 in SEQ ID NO:2, or a deletion at the terminal end of less than 20 amino acids. In a preferred embodiment, the mutation is at least one of T2H, Q4L, F8I, AI4R, L28A, L29S, W43R, G45T, Q55I, Q56A, N66I, N67A, M83I, V90R, M93T, or F94Q in SEQ ID NO:1, and Q3L, F7I, A13R, L27A, L28S, W42R, Q44T, Q54I, N65I, M82I, V89R, M92T, and F93Q in SEQ ID NO:2. Similarly, the genetically modified strain may also secrete ESAT-6 with a hexa-histidine tag, tetra-cysteine tag, or FLAG-tag, a GFP-fusion, or a short truncation at the C-terminal end of less than 20 amino acids.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is based on and claims the benefit of U.S. Provisional Application No. 60/692,561, filed Jun. 22, 2005. The entire disclosure of this application is relied upon and incorporated by reference herein. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to functional analysis of ESAT-6, the dominant T-cell antigen of  Mycobacterium tuberculosis , and to its key residues involved in secretion, complex-formation, virulence and immunogenicity. 
     BACKGROUND OF THE INVENTION 
     Several attenuated or avirulent members of the  Mycobacterium tuberculosis  complex, like  Mycobacterium bovis  BCG (BCG), the vole  bacillus. Mycobacterium microti , or the dassie  bacillus , are deleted for an overlapping portion of the genome, known as region of difference 1 (RD1) (1-3). This segment is localized close to the origin of replication (4) in all fully virulent members of the complex (5) and harbors genes esxA, coding for the 6 kDa early secreted antigenic target (ESAT-6), and esxB, encoding the 10 kDa culture filtrate protein (CFP-10). The region was recently shown to be required for full virulence of  M. tuberculosis  (6-8), and when integrated into BCG, improved the ability of the recombinant BCG strain to protect against dissemination of tuberculosis in the mouse- and the guinea pig model (9). 
     Independent but complementary studies revealed that ESAT-6 and CFP-10 are secreted via the ESAT-6 system-1 (ESX-1), a dedicated secretion apparatus encoded by genes flanking esxA and esxB in the extended RD1 region (9-11). Among the proteins predicted to be involved in this process are a member of the AAA-family of ATPases (Rv3868), which may perform chaperone-like functions by assisting in the assembly and disassembly of protein complexes, and several putative membrane proteins with 1, 3 or 11 transmembrane domains (Rv3869, Rv3870, Rv3877), or ATP binding sites (Rv3871), which could be involved in forming a transmembrane channel for the translocation of the effector molecules. 
     Although several publications have recently addressed the function of this secretion system (7, 9-14), there is a need in the art for information relating to the effector proteins. 
     SUMMARY OF THE INVENTION 
     This invention aids in fulfilling the needs in the art by providing a genetically modified strain of  Mycobacterium  of  Mycobacterium tuberculosis  complex and preferably  M. tuberculosis  or  Mycobacterium bovis  BCG, wherein the genetically modified strain produces at least one polypeptide comprising modified sequence SEQ ID NO: I, SEQ ID NO: 2, or both, having at least one mutation at T2, Q4, F8, A14, L28, L29, W43, G45, Y51, Q55, Q56, N66, M83, V90, M93, or F94 in SEQ ID NO:1, or a deletion at the C-terminal end of less than 20 amino acids; or at least one mutation at 03, F7, A13, L27, W42, G44, Y50, Q54, N65, N67, M82, V89, M92, or F93 in SEQ ID NO:2, or a deletion at the terminal end of less than 20 amino acids. In a preferred embodiment, the mutation is at least one of T2H, Q4L, F8I, A14R, L28A, L29S, W43R, G45T, Q55I, Q56A, N66I, N67A, M83I, V90R, M93T, or F94Q in SEQ ID NO:1, and Q3L, F7I, A13R, L27A, L28S, W42R, G44T, Q541, N65I, M82I, V89R, M92T, and F93Q in SEQ ID NO:2, and the deletion is from amino acid 84 to amino acid 95. The genetically modified strain can further comprise a hexa-histidine tag, a tetra-cysteine tag, a FLAG-tag, or a GFP polypeptide. 
     SEQ ID NO: 1 is the amino acid sequence shown in  FIG. 1A  for  M. tuberculosis.    
     SEQ ID NO: 2 is the amino acid sequence shown in  FIG. 1A  for  M. leprae.    
     The locations of the mutations in SEQ ID NO: 1 and SEQ ID NO: 2 are shown by the numbered residues below the sequences in  FIG. 1A . This invention concerns a genetically modified strain of  Mycobacterium  of  Mycobacterium tuberculosis  complex and preferably  M. tuberculosis  or  Mycobacterium bovis  BCG, wherein the genetically modified strain produces at least one modified polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2, or both, having a deletion of the extreme C-terminal end, outside the helical region of the polypeptide. Preferably the deleted amino acids are the last 12 C-terminal residues. 
     This invention provides a genetically modified strain of  Mycobacterium tuberculosis  complex, wherein the genetically modified strain produces a recombinant polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2 or esxB and a small tag. The small tag can be chosen from among hexa-histidine tag, tetra-cysteine tag, and FLAG tag. 
     A genetically modified strain of  Mycobacterium tuberculosis  complex is provided, wherein the genetically modified strain produces a recombinant polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2 or esxB and a small tag, and wherein the genetically modified strain induce an IFN-γ response to ESAT-6 and/or CFP-10 antigens. 
     A genetically modified strain of  Mycobacterium tuberculosis  complex is provided, wherein the genetically modified strain produces a recombinant polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2, or esxB fused to a reporter polypeptide, such as GFP. 
     This invention also provides deposits of biological material. 
     In addition, this invention provides a method of modulating an immune response in a host, wherein method comprises administering to the host a genetically modified strain of the invention in an amount sufficient to modulate in the host an immune response to infection by at least one pathogen selected from  M. tuberculosis  and  M. leprae . In a preferred embodiment of the invention the immune response is a protective immune response against at least one of the pathogens. 
     Further, this invention provides a purified composition consisting essentially of early secreted antigenic target protein of about 6 kDa (ESAT-6) complexed with culture filtrate protein of about 10 kDa (CFP-10) of  Mycobacterium tuberculosis . The purified complex can comprise a detectable tag, such as hexa-histidine. 
     A purified antibody that immunologically reacts with the complex of the invention is also provided. The antibody can be a polyclonal antibody or a monoclonal antibody. 
     An immunogenic composition of the invention comprises the genetically modified strain and a pharmaceutically acceptable carrier therefor. The composition can be a vaccine. 
     In addition, this invention provides a modified sequence comprising SEQ ID NO: I, SEQ ID NO: 2, or both, having at least one mutation at T2, Q4, F8, A14, L28, L29, W43, G45, Y51, Q55, Q56, N66, M83, V90, M93, or F94 in SEQ ID NO:1; or at least one mutation at Q3, F7, A13, L27, W42, G44, Y50, Q54, N65, N67, M82, V89, M92, or F93 in SEQ ID NO:2, or a deletion at the terminal end of less than 20 amino acids. In a preferred embodiment, the mutation is at least one of T2H, Q4L, F8I, AI4R, L28A, L29S, W43R, G45T, Q55I, Q56A, N66I, N67A, M83I, V90R, M93T, or F94Q in SEQ ID NO:1, and Q3L, F7I, A13R, L27A, L28S, W42R, G44T, Q54I, N65I, M82I, V89R, M92T, and F93Q in SEQ ID NO:2, or a deletion at the C-terminal end of less than 20 amino acids. Nucleic acid molecules encoding these modified sequences are also within the scope of this invention. 
     In addition, this invention provides a vector comprising a nucleic acid molecule of the invention and a host cell comprising the vector. In a preferred embodiment, the host cell is  Mycobacterium bovis  BCG. 
     In addition, this invention provides a vector coding for a polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2, or both or SEQ ID NO: 1, SEQ ID NO: 2 or both having mutations as described above, and further comprising a detectable tag, such as hexa-histidine (HHHHH) (SEQ ID NO: 71), Tetra cysteine-tag (FLNCCPGCCMEP) (SEQ ID NO: 72), or FLAG (DYKDDDDK) (SEQ ID NO: 73). A host cell containing the vector is also provided. 
     This invention further provides a method for screening a large range of chemical substances that may inhibit the ESX-1 secretion system, wherein the method comprises the steps of:
         a) contacting the chemical substance to screen with a culture of a genetically modified strain;   b) detecting presence of ESAT-6 and/or CFP-10 in the supernatant of the culture by immunological tools (antibodies), and   c) determining the inhibitor activity of the chemical substance on the ESX-1 secretion system when ESAT-6 and/or CFP-10 are not detected in the supernatant or detected in a lesser quantity than without the chemical substance.       

     A fluorescent antibody can be used for detecting ESAT-6 and/or CFP-10 in the supernatant of the culture. 
     A therapeutical method of the invention that relies on Th1 mediated immunity comprises administering a genetically modified strain of the invention or a composition of the invention. 
     The therapeutical method can be used to treat bladder cancer or asthma. 
     This invention provides method for screening mutations in SEQ ID NO: 1 or SEQ ID NO: 2 that inhibit the formation of complex ESAT-6-CFP-10, wherein the method comprises the following steps:
         a) mutagenesis of a genetically modified strain of the invention,   b) culture of the mutagenized strain obtained at step a) and recovering the culture supernatant,   c) affinity purification of the recombinant polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2 or esxB and a small tag,   d) immunologically detecting the presence of ESAT-6 and CFP-10 in the affinity purification eluate of step c), and   e) determining that the mutation is a mutation that inhibits the formation of the complex ESAT-6-CFP-10 if the two polypeptides ESAT-6 and CFP-10 are not both detected in the affinity purification eluate.       

     The genetically modified strain thus obtained by the method of is also provided. 
     This invention includes the use of at least one polypeptide of the invention as antigen in a diagnostic kit for detecting a  Mycobacterium  infection and discriminating between an infection caused by  Mycobacterium tuberculosis  and infections caused by environmental pathogenic mycobacterial strains secreting an ESAT-6 ortholog but not belonging to  M. tuberculosis  complex. The pathogenic mycobacterial strains to discriminate from  M. tuberculosis  can be  Mycobacterium kansasii, Mycobacterium smegmatis, Mycobacterium marinum , and  Mycobacterium szulgai.    
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       This invention will be described in greater detail with reference to the drawings in which: 
         FIG. 1 . A) Comparison of ESAT-6 from  M. tuberculosis  (SEQ ID NO: 1) and  M. leprae  (SEQ ID NO: 2). The arrows indicate amino acids that were individually mutated in cosmid pRD1-2F9. B) Immunoblot analysis of the 13 different H37RvΔRD1::2F9EsxA mutant strains, showing the cytosol-membrane (CM) and the supernatant (S) fraction using a monoclonal anti-ESAT-6 antibody. Note that mutation F8I may have changed the recognition site for the monoclonal anti-ESAT-6 antibody. C) Schematic representation of the secondary structure of ESAT-6 (19), and localization of mutations that were studied for their impact on virulence in SCID mice and immunogenicity in C57BL/6 mice. 
         FIG. 2 . Model of the ESAT-6/CFP-10 complex, with CFP-10 in white and ESAT-6 in blue. The mutations and their effect are indicated by a color code: red: enhanced virulence abolished; green: enhanced virulence retained. However, note that different degrees of enhanced in vivo growth rates in the various strains were detectable (Table 1). 
         FIG. 3 . Co-purification of ESAT-6 and CFP-10 from culture supernatants and cytosolic fractions of strains BCG::RD1-2F9, BCG::2F9-EsxA-HIS (ESAT-6-HIS) and BCG::2F9-HIS-EsxB (HIS-CFP-10) using Ni-NTA agarose mini columns A) Coomassie stained gel. Arrows indicate the presence of CFP-10 (EsxB) (upper) and ESAT-6-HIS (lower) bands. B) Western blotting analysis using either monoclonal anti-ESAT-6 antibodies or polyclonal anti-CFP-10 antibodies after purification of native (RD1-2F9) or hexahistidine-tagged proteins (ESAT-6-HIS, HIS-CFP-10) present in culture filtrate or cell lysate from recombinant BCG strains. C) Western blotting analysis using either monoclonal anti-ESAT-6 antibodies or polyclonal anti-CFP-10 antibodies after purification of native (RD1-2F9) or hexahistidine-tagged and W43R mutated ESAT-6 (EsxA-W43R-HIS) proteins present in culture filtrate of recombinant BCG. 
         FIG. 4 . In vivo growth of different BCG recombinants in mice. A) colony forming units (CFU) in lungs (L) and spleens (S) four weeks after intravenous infection of SCID mice with strains, BCG::2F9-HIS-EsxA (HIS-ESAT-6), BCG::2F9-EsxA-HIS (ESAT-6-HIS), BCG::RD1-2F9 (ESAT-6), H37RvΔRD1::2F9 or BCG vector control (pYUB412). B) CFU in lungs and spleens 4 weeks after aerosol infection of C57BL/6 mice (initial dose: 100 CFU+/−20) with BCG::2F9-HIS-EsxA (HIS-ESAT-6), BCG::2F9-EsxA-HIS (ESAT-6-HIS), BCG::RD1-2F9 (ESAT-6),  M. tuberculosis  H37Rv and BCG vector control (PYUB412). C) CFU in lungs (L) and spleens (S) 4 weeks after intranasal infection of C57BL/6 mice (initial dose: 10 5  CFU). D) IFN-γ production by splenocytes in response to different antigens after vaccination of C57BL/6 mice with BCG::2F9-HIS-EsxA (HIS-ESAT-6), BCG::2F9-EsxA-HIS (ESAT-6-HIS), BCG::RD1-2F9 (ESAT-6), or BCG::pYUB412 (vector only). 
         FIG. 5 . Schematic representation of the strategy, which was used to obtain recombinant BCG and  M. tuberculosis  strains that express and secret genetically modified ESAT-6, for example, ESAT-6 with a C-terminal histidine tag (BCG::EsxA-HIS). Figure discloses SEQ ID NO: 67 and SEQ ID NO: 68 coding SEQ ID NO: 69, respectively, in order of appearance. 6×His tag disclosed as SEQ ID NO: 70. 
         FIG. 6 . A) Probability plots for the formation of the coiled-coil structures within ESAT-6 and CFP-10 obtained by the use of the Multicoil program. B) Putative organization of the ESAT-6 and CFP-10 helical strands, showing residues that potentially contribute to the characteristic abcdefg seven-residue repeat. 
         FIG. 7 . (A) Immunoblot analysis of different cell fractions of BCG::2F9-HIS-EsxA (HIS-ESAT-6), BCG::2F9-EsxA-HIS (ESAT-6-HIS), BCG::RD1-2F9, and BCG, using a monoclonal anti-ESAT-6 antibody or polyclonal antibodies against Histidine tags, CFP-10 or PPE68. Note the size-difference between the native ESAT-6 and histidine tagged ESAT-6. (B) Immunoblot analysis of different cell fractions of BCG::2F9-HIS-EsxA (HIS-ESAT-6) and BCG::2F9-EsxA-HIS (ESAT-6-HIS) showing the cytosol-membrane (CM)-, the cell wall (CW)- and the supernatant (S)-fraction using an monoclonal ESAT-6 antibody or an anti-HIS tag antibody. Note that for construct BCG::2F9-HIS-EsxA an anti-HIS antibody binds to a large fragment that is retained in the cytosolic and membrane fraction, whereas the protein which reacts with the monoclonal ESAT-6 antibody has the usual size of ESAT-6 and does not react with the HIS-antibody, suggesting cleavage of the N-terminal HIS-tag. (C) Immunoblot analysis of different cell fractions of BCG::RD1-2F9 and BCG::RD1-2F9-esxAA84-95 using a monoclonal anti-ESAT-6 antibody or polyclonal antibodies against CFP-10 or PPE68. Note the size-difference between the native ESAT-6 and ESAT-6 deleted for the last 12 amino acids. (D) Immunoblot analysis of the cytosol-membrane (CM)-, the cell wall (CW)- and the supernatant (S)-fraction from strain BCG::2F9-EsxA-EsxR and BCG::RD1-2F9. (E) Immunoblot of the supernatant from BCG::2F9-EsxA-GFP and BCG::RD1-2F9 and using monoclonal anti-ESAT-6 antibodies. (F) Immunofluorescence microscope image of bone marrow derived murine macrophages that have ingested BCG::2F9-EsxA-GFP. (G) Immunoblot analysis of cytosol-membrane (CM)- and the supernatant (S)— fraction of BCG::RD1-2F9EsxA-Tetra-Cysteine-tag using a monoclonal anti-ESAT-6 antibody or polyclonal antibodies against CFP-10. Similar results were obtained for BCG::RD1-2F9EsxA-FLAG-tag (data not shown). 
         FIG. 8 . The results of virulence testing of recombinant BCG strains are depicted. Specifically, C57BL/6 mice were subjected to aerosol infections with the recombinant strains. Bacterial loads 30 days post-infection are shown in the Figure. 
         FIGS. 9(A) and 9(B) . The results of a first round of a protection study of recombinant BCG strains are shown. Colony Forming Units (CFU) of  M. tuberculosis  (Beijing) after challenge of C57BL/6 mice vaccinated with different recombinant BCG strains are reported.  FIG. 9(A)  depicts CFU in the lungs in the first round of the protection study.  FIG. 9(B)  depicts CFU in the spleen in the first round of the protection study. 
         FIGS. 10(A) and 10(B) . The results of a second round of a protection study of recombinant BCG strains are shown. Once again, colony forming units (CFU) of  M. tuberculosis  (Beijing) after challenge of C57BL/6 mice vaccinated with different recombinant BCG strains are reported.  FIG. 10(A)  depicts CFU in the lungs in the second round of the protection study.  FIG. 10(B)  depicts CFU in the spleen in the second round of the protection study. 
         FIGS. 11(A) ,  11 (B), and  11 (C). The protective potential of recombinant strain BCG-RD1-ESAT-6-HIS (synonymous to BCG::2F9-EsxA-HIS) was evaluated in a guinea pig model. After vaccination with BCG or saline (non-vaccinated control) or BCG::RD1-2F9-EsxA-HIS (synonymous to BCG::RD1-ESAT-6-HIS), the animals were challenged with a  M. tuberculosis  H37Rv. Weight (in grams) was monitored as shown on the Y axis in  FIGS. 11(A) ,  11 (B), and  11 (C) 
         FIG. 12  is the overview of the guinea pig vaccination-challenge experiment. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     ESAT-6 and CFP-10, which have been shown to form a 1:1 complex in vitro (15), belong to a large family of small proteins identified in Gram positive bacteria. Proteins of this family have a size of around 100 amino acids and are characterized by a conserved motif Trp-X-Gly (WXG) (16). To further elucidate the structure—function relationship in members of the ESAT-6, or WXG, family, a strategy has been developed to study variants of ESAT-6 in its natural bacterial host. The residues to be modified were selected according to their potential importance for secretion, immunogenicity and virulence. Among them were several amino acids that are highly conserved among ESAT-6 orthologs from different bacterial species. Other regions of interest were the amino- and carboxy termini of the protein. It was also of interest to identify modifications that would allow specific binding of biologically active ESAT-6 to selected matrices, and modifications that may be used to localize ESAT-6 in cell biological experiments. This invention relates to the behavior of these mutant strains in immunogenicity and virulence tests relative to control strains and show how the modifications allowed new features of ESAT-6 and the corresponding secretion system to be discovered. 
     The combined efforts of this invention using bioinformatics, protein engineering, as well as immunological and virulence studies made it possible to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity. The invention has clearly shown that it is indeed ESAT-6, in combination with CFP-10 that mediates ESX-1-associated virulence. This invention demonstrates that mutation of only one or two selected residues in ESAT-6 can abolish the virulent phenotype of the recombinant  M. tuberculosis  strain and links this phenomenon in part to the destruction of the α-helical/coiled-coil motifs present in ESAT-6 and its protein partner CFP-10. 
     The sequences of coiled-coils in proteins consist of heptad repeats denoted “abcdefg”, harboring two characteristic hydrophobic positions at sites “a” and “d” of an α-helix (23). Amino acids in other positions, in particular in “e” and “g”, are often polar residues important for the water solubility of the protein complex and the specificity of interactions between neighboring helices at different pH levels ( FIG. 6 ). It has been shown that short stretches of 30 to 40 residues may be sufficient to account for such interaction of proteins (24). It is interesting to note that many proteins involved in type III secretion systems of Gram-negative bacteria contain coiled-coil motifs, which enable or stabilize protein-protein interactions (25). In the case of ESAT-6 and CFP-10 the presence of multimeric, but not dimeric coiled-coil motifs ( FIG. 6A ), supposes that three or four helices may be involved in such an interaction. As described in the Examples section, the effects of the mutations introduced into ESAT-6 on virulence of recombinant strains can be rationalized from this model. The data confirm and extend predictions by Pallen, who described a large family of small proteins from Gram positive bacteria, including ESAT-6 and CFP-10 from  M. tuberculosis  or EsxA/B from  Staphylococcus aureus  (26), which are all characterized by the WXG motif and have a size of around 100 residues (16). The W43 residue could have a functional role in interacting with other proteins. However, in this model it is turned inward (on an “a” position) ( FIG. 2 ) and helical in the published NMR results (19). It could, therefore, be important for structural reasons, interacting with another conserved hydrophobic amino acid, Y51 in ESAT-6. 
     Also, the almost perfect conservation of a tryptophan at this position in the whole protein family points to a structural role rather than a purely functional role. This hypothesis is supported by the observation that EsxA-W43R-HIS secreted by recombinant BCG did not retain CFP-10 by Ni 2+  affinity chromatography ( FIG. 3C ). The finding that replacement of a single residue severely disturbs binding to CFP-10 emphasizes the specificity of the ESAT-6/CFP-10 interaction, and may explain why ESAT-6 did not bind to other members of the Esx protein family (21, 22). 
     The neighboring mutation G45T seems to be important for the folding of ESAT-6, as glycine shows more flexibility and can adopt different conformations than other amino acids. It is, therefore, often found in short loops or turns. 
     Another attenuating mutation, L28A/L29S, can be explained by the prediction that L29 is at a “a” position, critical for the stability of the coiled-coil structure and also affecting secretion. Finally, two mutations showing attenuating effects, V90R, and F94Q are both in the extreme C-terminal end, outside the helical region. As the extreme C-terminus of ESAT-6 represents a floppy, structurally not well-defined region of the protein (19), it seems that mutation of residues at the C-terminus may inhibit a specific interaction of ESAT-6 with host proteins rather than affecting the structure or stability of the protein itself. It was recently shown that the C-terminus of ESAT-6 was apparently not involved in the protein interaction with CFP-10 (22), as is also suggested by this model. Moreover, based on this model, amino acids that are important for the correct function of CFP-10 can now be predicted and experimentally verified. 
     Research on ESAT-6 and the ESAT-6 system-1 has become an important topic for studies on the pathogenesis and immunogenicity of  M. tuberculosis  infections due to the outstanding role of ESAT-6 in these processes. However, to date many groups have used ESAT-6 molecules expressed by  E. coli , and there are several examples in the literature that recombinant proteins may show characteristics that differ from the naturally occurring mycobacterial proteins (27). For this reason, one of the goals of this invention was to establish an experimental system in the  M. tuberculosis  complex that allows ESAT-6 to be studied while retaining the characteristics of the naturally occurring molecule. 
     In this respect, the C-terminal HIS construct of ESAT-6 was particularly interesting, as it fulfilled all these expected features. In contrast to predictions based on  E. coli -expressed ESAT-6 (15), proteolytic cleavage of the 11 C-terminal residues did not occur for ESAT-6 expressed by BCG and  M. tuberculosis . So far, in all ESAT-6 constructs that contained C-terminal tags or fusions there was observed a size shift, and no detection of molecules that had lost their tag ( FIG. 7 ). The results obtained with co-purification assays, indicating that CFP-10 was eluted together with the HIS-tagged ESAT-6 protein from a Ni 2+  affinity column after passage of supernatant from recombinant BCG is the first proof that indeed, in a biologically relevant, non-denatured state, ESAT-6 and CFP-10 form a protein complex even after secretion. As the BCG::2F9-EsxA-HIS strain retained intermediate virulence and specific immunogenicity, short tags added at the C-terminus of the ESAT-6 protein do not seem to alter greatly the biological activity of ESAT-6 produced by recombinant BCG. With this knowledge, it is now possible to create recombinant BCG strains secreting ESAT-6 with small C-terminal tags, like BCG::2F9-EsxA-Tetra-cysteine-tag ( FIG. 7G ) or BCG::2F9-EsxA-FLAG-tag, applicable to cell biological experiments that are needed to investigate targeting and trafficking of ESAT-6 in macrophages or dendritic cells. 
     Certain isoforms of the ESAT-6 molecule from  M. tuberculosis , separated by 2-DE, were identified as carrying a post translational modification, namely, an acetylation of the threonine residue at position 2, and it was observed that only the unacetylated form of ESAT-6 interacted with CFP-10 (28). Acetylation affects diverse protein functions like enzyme activity, stability and protein-protein interactions (29), but the role of the acetylation of ESAT-6 during infection with  M. tuberculosis  is not known. This was one of the reasons why a mutant strain H37RvΔRD1::2F9EsxA-T2H, which had the threonine replaced by histidine, was constructed. However, as the mutant strain secreted ESAT-6 normally and retained the enhanced in vivo growth in SCID mice, it was concluded that this particular post-translational modification of the T2 residue in ESAT-6 does not have a major effect upon its function. 
     The observation that mutations in ESAT-6, which diminish or abolish virulence, also inhibit ESAT-6 specific T-cell responses in mice clearly demonstrates that optimal recognition of ESAT-6 by the immune system not only requires secretion of the antigen but also needs a biologically active form of ESAT-6. The finding that a mutation at the C-terminus oblates immunogenicity upon stimulation with the N-terminal ESAT-6 peptide 1-20 opens future studies on antigen processing and innate and/or adaptive immune responses. The immune recognition may depend on proper trafficking of ESAT-6, and this process possibly requires interaction of ESAT-6 with—as yet unknown—host proteins. 
     In a more practical perspective, the use of the ESAT-6-HIS constructs expressed by BCG opens up new ways to identify such proteins. Furthermore, ESAT-6-HIS, EsxA-Tetra-cysteine-tag, EsxA-FLAG-tag and/or ESAT-6-GFP constructs should enable high-throughput screening procedures to be established, to identify potential inhibitors of the ESAT-6 system-1, which in turn could lead to the identification of new anti-tuberculous drugs. As another application, certain strains secreting mutated or tagged ESAT-6 with intermediate virulence are promising candidates for new, more effective vaccines that combine lower virulence, than the previously tested BCG::RD1-2F9 strain (12), with the beneficial effects of strong T-cell responses against the immunodominant proteins ESAT-6 and CFP-10 (9). 
     Expression Systems 
     The present invention provides recombinant cloning and expression vectors containing DNA, as well as host cell containing the recombinant vectors. Expression vectors comprising DNA may be used to prepare the polypeptides or fragments of the invention encoded by the DNA. A method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture. The skilled artisan will recognize that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is membrane-bound or a soluble form that is secreted from the host cell. 
     Any suitable expression system may be employed. The vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector. 
     In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell. 
     Suitable host cells for expression of polypeptides include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al.  Cloning Vectors: A Laboratory Manual , Elsevier, N.Y., (1985). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from DNA constructs disclosed herein. 
     Prokaryotic Systems 
     Prokaryotes include gram-negative or gram-positive organisms. Suitable prokaryotic host cells for transformation include, for example,  E. coli, Bacillus subtilis, Salmonella typhimurium , and various other species within the genera  Pseudomonas, Streptomyces , and  Staphylococcus . The use of  Mycobacterium  host cells, such as  M. bovis  BCG,  M. tuberculosis, M. microti , and  M. smegmatis , is preferred. The use in  M. smegmatis  is particularly preferred. In a prokaryotic host cell, such as  E. coli , a polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide. 
     Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. An appropriate promoter and a DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA). 
     Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Chang et al.,  Nature  275:615, 1978; and Goeddel et al.,  Nature  281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,  Nucl. Acids Res.  8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,  Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage 8P L  promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the 8P L  promoter include plasmid pHUB2 (resident in  E. coli  strain JMB9, ATCC 37092) and pPLc28 (resident in  E. coli  RR1, ATCC 53082). 
     DNA may be cloned in-frame into the multiple cloning site of an ordinary bacterial expression vector. Ideally the vector would contain an inducible promoter upstream of the cloning site, such that addition of an inducer leads to high-level production of the recombinant protein at a time of the investigator&#39;s choosing. For some proteins, expression levels may be boosted by incorporation of codons encoding a fusion partner (such as hexahistidine) between the promoter and the gene of interest. The resulting “expression plasmid” may be propagated in a variety of strains of  E. coli.    
     For expression of the recombinant polypeptide, the bacterial cells are propagated in growth medium until reaching a pre-determined optical density. Expression of the recombinant polypeptide is then induced, e.g. by addition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activates expression of polypeptides from plasmids containing a lac operator/promoter. After induction (typically for 1-4 hours), the cells are harvested by pelleting in a centrifuge, e.g. at 5,000×G for 20 minutes at 4° C. 
     For recovery of the expressed polypeptide, the pelleted cells may be resuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl and then passed two or three times through a French press. Most highly expressed recombinant polypeptides form insoluble aggregates known as inclusion bodies. Inclusion bodies can be purified away from the soluble polypeptides by pelleting in a centrifuge at 5,000×G for 20 minutes, 4° C. The inclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/1% Triton X-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTTf. Any material that cannot be dissolved is removed by centrifugation (10,000×G for 20 minutes, 20° C.). The polypeptide of interest will, in most cases, be the most abundant polypeptide in the resulting clarified supernatant. This polypeptide may be “refolded” into the active conformation by dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCl 2 /5 mM Zn(OAc) 2 /1 mM GSSG/0.1 mM GSH. After refolding, purification can be carried out by a variety of chromatographic methods, such as ion exchange or gel filtration. In some protocols, initial purification may be carried out before refolding. As an example, hexahistidine-tagged fusion proteins may be partially purified on immobilized Nickel. 
     While the preceding purification and refolding procedure assumes that the polypeptide is best recovered from inclusion bodies, those skilled in the art of polypeptide purification will appreciate that many recombinant proteins are best purified out of the soluble fraction of cell lysates. In these cases, refolding is often not required, and purification by standard chromatographic methods can be carried out directly. 
     Yeast Systems 
     Alternatively, the polypeptides may be expressed in yeast host cells, preferably from the  Saccharomyces  genus (e.g.,  S. cerevisiae ). Other genera of yeast, such as  Pichia  or  Kluyveromyces , may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al.,  J. Biol. Chem.  255:2073, 1980) or other glycolytic enzymes (Hess et al.,  J. Adv. Enzyme Reg.  7:149, 1968; and Holland et al.,  Biochem.  17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. ( J. Biol. Chem.  258:2674, 1982) and Beier et al. ( Nature  300:724, 1982). Shuttle vectors replicable in both yeast and  E. coli  may be constructed by inserting DNA sequences from pBR322 for selection and replication in  E. coli  (Amp r  gene and origin of replication) into the above-described yeast vectors. 
     The yeast α-factor leader sequence may be employed to direct secretion of the polypeptide. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al.,  Cell  30:933, 1982 and Bitter et al.,  Proc. Natl. Acad. Sci. USA  81:5330, 1984. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene. 
     Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al.,  Proc. Natl. Acad. Sci. USA  75:1929, 1978. The Hinnen et al. protocol selects for Trp +  transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 mg/ml adenine, and 20 mg/ml uracil. 
     Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a “rich” medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium. 
     Mammalian or Insect Systems 
     Mammalian or insect host cell culture systems also may be employed to express recombinant polypeptides. Bacculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers,  Bio/Technology  6:47 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,  Cell  23175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. ( EMBO J.  10: 2821, 1991). 
     Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J.,  Large Scale Mammalian Cell Culture,  1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipid reagent, can be used to transfect cells (Feigner et al.,  Proc. Natl. Acad. Sci. USA  84:7413-7417, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. ( Molecular Cloning: A Laboratory Manual,  2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al.,  Meth. in Enzymology  185:487-511, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin,  Proc. Natl. Acad. Sci. USA  77:4216-4220, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds. 
     Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al.,  Nature  273:113, 1978; Kaufman,  Meth. in Enzymology,  1990). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included. 
     Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al.,  Animal Cell Technology,  1997, pp. 529-534 and PCT Application WO 97/25420) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al.,  J. Biol. Chem.  257:13475-13491, 1982). The internal ribosome entry site (IRES) sequences of viral origin allow dicistronic mRNAs to be translated efficiently (Oh and Samow,  Current Opinion in Genetics and Development  3:295-300, 1993; Ramesh et al.,  Nucleic Acids Research  24:2697-2700, 1996). Expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (e.g. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman,  Meth. in Enzymology,  1990). Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by Mosser et al.,  Biotechniques  22:150-161, 1997, and p2A51 described by Morris et al.,  Animal Cell Technology,  1997, pp. 529-534. 
     A useful high expression vector, pCAVNOT, has been described by Mosley et al.,  Cell  59:335-348, 1989. Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg ( Mol. Cell. Biol.  3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. ( Mol. Immunol.  23:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al.,  Nature  312:768, 1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982, incorporated by reference herein. In yet another alternative, the vectors can be derived from retroviruses. 
     Additional useful expression vectors, pFLAG® and pDC311, can also be used. FLAG® technology is centered on the fusion of a low molecular weight (1 kD), hydrophilic, FLAG® marker peptide to the N-terminus of a recombinant protein expressed by pFLAG® expression vectors. pDC311 is another specialized vector used for expressing proteins in CHO cells. pDC311 is characterized by a bicistronic sequence containing the gene of interest and a dihydrofolate reductase (DHFR) gene with an internal ribosome binding site for DHFR translation, an expression augmenting sequence element (EASE), the human CMV promoter, a tripartite leader sequence, and a polyadenylation site. 
     Regarding signal peptides that may be employed, a heterologous signal peptide or leader sequence may be used, if desired. The choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced. To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al.,  Nature  312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846. 
     Purification 
     The invention also includes methods of isolating and purifying the polypeptides and fragments thereof. An isolated and purified polypeptide according to the invention can be produced by recombinant expression systems as described above or purified from naturally occurring cells. The polypeptide can be substantially purified, as indicated by a single protein band upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). One process for producing polypeptides comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes the polypeptide under conditions sufficient to promote expression of polypeptide. The polypeptide is then recovered from culture medium or cell extracts, depending upon the expression system employed. 
     In one preferred embodiment, the purification of recombinant polypeptides or fragments can be accomplished using fusions of polypeptides or fragments of the invention to another polypeptide to aid in the purification of polypeptides or fragments of the invention. Such fusion partners can include a poly-His or other antigenic identification peptides. 
     With respect to any type of host cell, as is known to the skilled artisan, procedures for purifying a recombinant polypeptide or fragment will vary according to such factors as the type of host cells employed and whether or not the recombinant polypeptide or fragment is secreted into the culture medium. 
     In general, the recombinant polypeptide or fragment can be isolated from the host cells if not secreted, or from the medium or supernatant if soluble and secreted, followed by one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification or size exclusion chromatography steps. As to specific ways to accomplish these steps, the culture medium first can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In addition, a chromatofocusing step can be employed. Alternatively, a hydrophobic interaction chromatography step can be employed. Suitable matrices can be phenyl or octyl moieties bound to resins. In addition, affinity chromatography with a matrix which selectively binds the recombinant protein can be employed. Examples of such resins employed are lectin columns, dye columns, and metal-chelating columns. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups) can be employed to further purify the polypeptides. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide an isolated and purified recombinant polypeptide. 
     Recombinant polypeptide produced in bacterial culture is usually isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. 
     Transformed yeast host cells are preferably employed to express a secreted polypeptide in order to simplify purification. Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. ( J. Chromatog.  296:171, 1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column. 
     It is also possible to utilize an affinity column comprising a polypeptide-binding protein of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention. 
     The desired degree of purity depends on the intended use of the protein. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides are purified such that no protein bands corresponding to other proteins are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide may be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-PAGE. The protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography. 
     The polypeptide or nucleic acid molecule is preferably 50%, 75%, 80%, 85%, 90%, 95%, or 98% pure, most preferably more than 99% pure. 
     Antibodies 
     Antibodies that are immunoreactive with the polypeptides and complexes of the invention are provided herein. Such antibodies specifically bind to the polypeptides and complexes via the antigen-binding sites of the antibody (as opposed to non-specific binding). Thus, the complexes and polypeptides, fragments, variants, fusion proteins, etc., as set forth above may be employed as immunogens in producing antibodies immunoreactive therewith. More specifically, the polypeptides, fragment, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies. 
     These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers,  Immuno Biology  3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers,  Immuno Biology  2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes may be identified by any of the methods known in the art. 
     Thus, one aspect of the present invention relates to the antigenic epitopes of the polypeptides and complexes of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in detail below. Additionally, epitopes from the polypeptides and complexes of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology. 
     As to the antibodies that can be elicited by the epitopes of the polypeptides and complexes of the invention, whether the epitopes have been isolated or remain part of the polypeptides and complexes, both polyclonal and monoclonal antibodies may be prepared by conventional techniques as described below. 
     In this aspect of the invention, polypeptides and complexes can be utilized to prepare antibodies that specifically bind to polypeptides. The term “antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F(ab′)2 and Fab fragments, as well as any recombinantly produced binding partners. Antibodies are defined to be specifically binding if they bind polypeptides with a K a  of greater than or equal to about 10 7  M −1 . Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al.,  Ann. N.Y Acad. Sci.,  51:660 (1949). 
     Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures that are well known in the art. In general, a purified polypeptide or a peptide based on the amino acid sequence of polypeptide that is appropriately conjugated is administered to the host animal typically through parenteral injection. The immunogenicity of polypeptides and complexes can be enhanced through the use of an adjuvant, for example, Freund&#39;s complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to polypeptides and complexes. Examples of various assays useful for such determination include those described in  Antibodies: A Laboratory Manual , Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and 4,486,530. 
     Monoclonal antibodies can be readily prepared using well known procedures. See, for example, the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas:  A New Dimension in Biological Analyses , Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980. Briefly, the host animals, such as mice, are injected intraperitoneally at least once and preferably at least twice at about 3 week intervals with isolated and purified polypeptides or conjugated polypeptides, optionally in the presence of adjuvant. Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of polypeptides or conjugated polypeptides. Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as  125 I-labeled polypeptide, is added to each well followed by incubation. Positive wells can be subsequently detected by autoradiography. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia). 
     The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., “Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”,  Strategies in Molecular Biology  3:1-9 (1990), which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al.,  Biotechnology,  7:394 (1989). 
     Antigen-binding fragments of such antibodies, which may be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab′) 2  fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided. 
     The monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. ( Nature  332:323, 1988), Liu et al. ( PNAS  84:3439, 1987), Larrick et al. ( Bio/Technology  7:934, 1989), and Winter and Harris ( TIPS  14:139, May, 1993). Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incorporated by reference herein. 
     The invention provides immunogenic compositions, and more particularly, protective compositions for use in vaccines. The compositions can comprise the modified polypeptides of the invention. These compositions can also be employed as immunotherapy or vaccination drug therapy by administering the compositions to an animal, preferably a mammal. Effective quantities of the compositions of the invention can be administered to the mammal. The mammal that is treated by the method of the invention can be, but is not limited to, a dog, cat, cow, sheep, pig, goat, horse, monkey, or preferably, a human. 
     The mutant strains of the invention can be formulated as a pharmaceutical composition and administered in a variety of dosage forms adapted to the chosen route of administration, including, but not limited to, orally or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes. Administration via the respiratory tract, such as by inhalation, can also be employed. 
     Thus, the mutant strains can be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. It can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient&#39;s diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations can be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active agent in such therapeutically useful compositions is such that an effective dosage level will be obtained. 
     The tablets, troches, pills, capsules, and the like can also contain the following: binders, such as gum tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, fructose, lactose or aspartame or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring, such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices. 
     The active compound can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. 
     The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. 
     Sterile injectable solutions can be prepared by incorporating the active agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. 
     The ability of the compositions and vaccines of the invention to induce protective humoral immunity in a host can be enhanced by emulsification with an adjuvant, incorporating in a liposome, coupling to a suitable carrier, or by combinations of these techniques. In a preferred embodiment, the compositions of the invention can be administered with a conventional adjuvant, such as aluminum phosphate and aluminum hydroxide gel, in an amount sufficient to potentiate humoral or cell-mediated immune response in the host. Similarly, the compositions can be bound to lipid membranes or incorporated in lipid membranes to form liposomes. The use of nonpyrogenic lipids free of nucleic acids and other extraneous matter can be employed for this purpose. 
     The compositions and vaccines of the invention can be administered to the host in an amount sufficient to prevent or inhibit infection. In any event, the amount administered should be at least sufficient to protect the host, even though infection may not be entirely prevented. 
     An immunogenic response can be obtained by administering the compositions containing the modified polypeptides of the invention to the host in an amount of about 200 micrograms to about 1 mg, preferably about 500 micrograms per dose, by intramuscular injection in a subject. The dose depends upon whether the recipient is an infant, a child, an adolescent, or an adult, and also upon the health of the recipient. The composition of the invention can be administered together with a physiologically acceptable carrier. For example, a diluent, such as water or a saline solution, can be employed. 
     The immunization schedule will depend upon several factors, such as the susceptibility of the host to infection and the age of the host. A single dose of the composition of the invention can be administered to the host or a primary course of immunization can be followed in which several doses at intervals of time are administered. Subsequent doses used as boosters can be administered as needed following the primary course. 
     Another aspect of the invention provides a method of DNA vaccination. The method includes administering the expression vectors encoding at least one of the modified polypeptides of the invention, per se, with or without carrier molecules, to the subject. 
     Thus, the methods of treating include administering immunogenic compositions comprising the modified polypeptides, or compositions comprising nucleic acids encoding the modified polypeptides as well. Those of skill in the art are cognizant of the concept, application, and effectiveness of nucleic acid vaccines (e.g., DNA vaccines) and nucleic acid vaccine technology, as well as protein and polypeptide based technologies. The nucleic acid based technology allows the administration of nucleic acids encoding the modified polypeptides, naked or encapsulated, directly to tissues and cells without the need for production of encoded proteins prior to administration. The technology is based on the ability of these nucleic acids to be taken up by cells of the recipient cell or organism and expressed to produce an immunogenic determinant to which the recipient&#39;s immune system responds. Typically, the expressed antigens are displayed on the surface of cells that have taken up and expressed the nucleic acids, but expression and export of the encoded antigens into the circulatory system of the recipient individual is also within the scope of the present invention. Such nucleic acid vaccine technology includes, but is not limited to, delivery of expression vectors encoding the modified polypeptides. Although the technology is termed “vaccine”, it is equally applicable to immunogenic compositions that do not result in a protective response. Such non-protection inducing compositions and methods are encompassed within the present invention. Although it is within the present invention to deliver nucleic acids encoding the modified polypeptides and carrier molecules, the present invention also encompasses delivery of nucleic acids as part of larger or more complex compositions. 
     In yet another embodiment, the invention provides a method of screening for compounds that modulate the ESAT-6 secretion system 1. Chemical compounds can be tested for their activity to block the ESAT-6 secretion machine, by monitoring fluorescence intensity of the supernatant of a culture from mycobacterial strains that have integrated modified versions of the RD1-2F9 cosmid, secreting tagged ESAT-6 molecules (e.g. ESAT-6-GFP, EsxA-tetra-cysteine tag, or EsxA-FLAG-tag, or ESAT-HIS using appropriate antibodies). Throughout this application, various publications are referenced in parentheses by number. Full citations for these references may be found at the end of the specification immediately preceding the sequence listings and the claims. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains. 
     The following strains were deposited at the Collection Nationale de Cultures de Microorganismes (C.N.C.M.), of Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris, Cedex 15, France, and assigned the following Accession Nos.:
         I-3461: mutation L28A/L29S in ESAT-6 gene   I-3463: mutation W43R in ESAT-6 gene   I-3460: mutation G45T in ESAT-6 gene   I-3462: mutation V90R in ESAT-6 gene   I-3459: mutation F94Q in ESAT-6 gene   I-3458: deletion 84-95 in ESAT-6 gene   I-3456: fusion ESAT-6 gene-HIS tag   I-3457: region RD1-2F9 deleted from ca. 4337 kb to ca. 4342 kb (2F9-delta rv3860-3866)   I-3464:2F9EsxA-mod=library of various mutations in ESAT-6 gene (T2H, Q4L, F8I, A14R, Q55I/Q56A, N66I/N67A, M83I, M93T) and various gene fusions His tag-CFP10, ESAT-6-GFP, ESAT-6-tetra C tag and ESAT-6-FLAG tag.   I-3624: an  E. coli  strain containing the cosmid RD1-2F9EsxA-M93T   I-3625: an  E. coli  strain containing the cosmid RD1-2F9-delta rv 3860-3866.       

     Biological sample 2F9EsxA-delta 84-95 is  Escherichia coli  DH10B containing the cosmid 2F9EsxA-delta 84-95. Mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NO: 3. 
     Biological sample 2F9EsxA-mod is a plasmid library (cosmid 2F9) with clones that have modifications in the EsxA (ESAT-6). The clones are easy to differentiate either by sequencing or by stringent hybridization. Mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NOS: 4-15. 
     Biological sample 2F9-delta rv 3860-3866 is  Escherichia coli  DH10B containing the cosmid 2F9-delta rv 3860-3866. It contains only the region from  M. tuberculosis  H37RV starting from position ca. 4342, 200 and higher. Insert of cosmid 2F9-Delta-Rv3860-3866 (size 26379 bp) corresponding to sequence in the genome of  M. tuberculosis  H37Rv from 4342239 bp to 4368616 bp is shown in SEQ ID NO: 65. 
     Biological sample 2F9-ESAT-6-His is  Escherichia coli  DH10B containing the cosmid 2F9-ESAT-6-His. Mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NO: 16. 
     Biological sample 2F9EsxA-F94Q is  Escherichia coli  DH10B contained in cosmid 2F9EsxA-F94Q. To form 2F9EsxA-F94Q, mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NO: 20. 
     Biological sample 2F9 EsxA-G4ST is  Escherichia coli  DH10B containing the cosmid 2F9 EsxA-G4ST. Mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NO: 17. 
     Biological sample 2F9 EsxA-W43R is  Escherichia coli  DH10B containing the cosmid 2F9 EsxA-W43R. Mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NO: 18. 
     Biological sample 2F9EsxA-L28A/L295 is  Escherichia coli  DH10B containing the cosmid 2F9-EsxA-L28A/L295. Mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NO: 19. 
     Biological sample 2F9EsxA-V90R is  Escherichia coli  DH10B containing the cosmid 2F9-EsxA-V90R. Mutations were introduced in the ESAT-6 sequence of  Mycobacterium tuberculosis  in the cosmid RD1-2F9. Nucleotide modifications are shown in boldface in SEQ ID NO: 66. 
     This invention will be described in greater detail in the following Examples. 
     Example 1 
     Methods 
     A. Genetic Constructs and Mycobacterial Strains. An aim of the present invention was to introduce extra sequences or base substitutions into previously used cosmid pRD1-2F9 (12) to obtain BCG and/or  M. tuberculosis  strains that produce and secrete genetically modified variants of ESAT-6. The exact procedure is shown in  FIG. 5  and is described above. The following primer sequences were used to introduce the various genetic modifications into pRD1-2F9. 
     
       
         
               
               
               
               
             
           
               
                 1F: 
                 GGAATTCCGTTAACACGCTTTTCGAGAG 
                 SEQ ID NO: 21 
                   
               
               
                   
               
               
                 2R: 
                 CGGGATCCACTAGTCATGTTTTTGCTCCGTTTC 
                 SEQ ID NO: 22 
               
               
                   
               
               
                 3F: 
                 GCTCTAGACTAGTCATCATCATCATCATCACGAGCAGCAGTGGAATTTCGC 
                 SEQ ID NO: 23 
               
               
                   
               
               
                 5R: 
                 CGGGATCCACTAGTCTAGTGATGATGATGATGATGTGCGAACATCCCAGTGACG 
                 SEQ ID NO: 24 
               
               
                   
               
               
                 7F: 
                 GCTCTAGACTAGTGGCAACGCCGAGTTCGCG 
                 SEQ ID NO: 25 
               
               
                   
               
               
                 8R: 
                 CCCAAGCTTGGCCGGAAGAGCTTGTCG 
                 SEQ ID NO: 26 
               
               
                   
               
               
                 12R: 
                 CGGGATCCACTAGTCTACTTTCTCTCTTTACC 
                 SEQ ID NO: 27 
               
               
                   
               
               
                 14F: 
                 GCTCTAGACTAGTATGCATCATCATCATCATCACGCAGAGATGAAGACCGATGCCG 
                 SEQ ID NO: 28 
               
               
                   
               
               
                 16R: 
                 CGGGATCCACTAGTTGCGAACATCCCAGTGACG 
                 SEQ ID NO: 29 
               
               
                   
               
               
                 17F: 
                 GCTCTAGACTAGTTAGGGCAACGCCGAGTTCGCG 
                 SEQ ID NO: 30 
               
               
                   
               
               
                 18F: 
                 GCTCTAGACTATGGTTCGGAGGCGTACC 
                 SEQ ID NO: 31 
               
               
                   
               
               
                 19R: 
                 CGGGATCCACTAGTGCCCCAGGCCGCTGCG 
                 SEQ ID NO: 32 
               
               
                   
               
               
                 22F: 
                 GCTCTAGAGGCGCCGGTATCGAGGCCGCGGC 
                 SEQ ID NO: 33 
               
               
                   
               
               
                 23R: 
                 CGGGATCCCGATCGATATTCCACTGCTGCTCTGT 
                 SEQ ID NO: 34 
               
               
                   
               
               
                 24F: 
                 GCTCTAGAATGCATGAGCAGCAGTGGAATTTCG 
                 SEQ ID NO: 35 
               
               
                   
               
               
                 25R: 
                 CGGGATCCCGATGCATGTTTTTGCTCCGTTTCTT 
                 SEQ ID NO: 36 
               
               
                   
               
               
                 27F: 
                 GCTCTAGAGGCGCCAAATGGGACGCCACGGGTAC 
                 SEQ ID NO: 37 
               
               
                   
               
               
                 28R: 
                 CGGGATCCATCGATGACACCCTGGTACGCCTC 
                 SEQ ID NO: 38 
               
               
                   
               
               
                 29R: 
                 CGGGATCCATCGATCAGCTCGGTAGCCGTGGCG 
                 SEQ ID NO: 39 
               
               
                   
               
               
                 30F: 
                 GCTCTAGAGCGCCGCGCTGCAGAACCTGGC 
                 SEQ ID NO: 40 
               
               
                   
               
               
                 31R: 
                 CGGGATCCATCGATTGCCTGACCGGCTTCGC 
                 SEQ ID NO: 41 
               
               
                   
               
               
                 32F: 
                 GCTCTAGAGGCGCCTCGACCGAAGGCAACGTC 
                 SEQ ID NO: 42 
               
               
                   
               
               
                 36F: 
                 GCTCTAGAGGCGGTAGCGGTTCGGAG 
                 SEQ ID NO: 43 
               
               
                   
               
               
                 37R: 
                 CGGGATCCGCTAGCCGCTGCGAGCTTGGTC 
                 SEQ ID NO: 44 
               
               
                   
               
               
                 38F: 
                 D3: GCTCTAGAGCTAGCGACGAGGGGAAGCAGTC 
                 SEQ ID NO: 45 
               
               
                   
               
               
                 39R: 
                 CGGGATCCGCTAGCGGAATGAATGGACGTGACATT 
                 SEQ ID NO: 46 
               
               
                   
               
               
                 40F: 
                 GCTCTAGAGCAAGCGCAATCCAGGGAA 
                 SEQ ID NO: 47 
               
               
                   
               
               
                 41R: 
                 CGGGATCCGCTAGCCTCGATTCCCGCGAAATT 
                 SEQ ID NO: 48 
               
               
                   
               
               
                 44R: 
                 GGAATTCTGTCATGTTTTTGCTCCG 
                 SEQ ID NO: 49 
               
               
                   
               
               
                 45F: 
                 GCTCTAGACAATTGCAGTGGAATTTCGCGGGT 
                 SEQ ID NO: 50 
               
               
                   
               
               
                 46R: 
                 CGGGATCCCAATTGCTCTGTCATGTTTTTG 
                 SEQ ID NO: 51 
               
               
                   
               
               
                 47F: 
                 GCTCTAGACAATTGTGGAATTTCGCGGGTA 
                 SEQ ID NO: 52 
               
               
                   
               
               
                 48R: 
                 CGGGATCCCGTACGGTTGCCTTCGGTCGAAGC 
                 SEQ ID NO: 53 
               
               
                   
               
               
                 49F: 
                 GCTCTAGACGTACGGGGATGTTCGCATAGGGC 
                 SEQ ID NO: 54 
               
               
                   
               
               
                 50R: 
                 GGGGTACCAGTGACGTTGCCTTCG 
                 SEQ ID NO: 55 
               
               
                   
               
               
                 51F: 
                 GGGGTACCTTCGCATAGGGCAACGCC 
                 SEQ ID NO: 56 
               
               
                   
               
               
                 52F: 
                 AAACTGCAGGCATAGGGCAACGCCG 
                 SEQ ID NO: 57 
               
               
                   
               
               
                 53R: 
                 CGGGATCCATGCATCCCAGTGACGTTGCC 
                 SEQ ID NO: 58 
               
               
                   
               
               
                 eGFP-F: 
                 GGACTAGTATGGTGAGCAAGGGCGAGGAGCTG 
                 SEQ ID NO: 59 
               
               
                   
               
               
                 eGFP-R: 
                 ACGGATCCTTATCTAGATCCGGTGGATCC 
                 SEQ ID NO: 60 
               
               
                   
               
               
                 ESXA-M1-F: 
                 GACTAGTCCCGGTGAACATACTCATGAC 
                 SEQ ID NO: 61 
               
               
                   
               
               
                 EsxA-M1-R: 
                 GACTAGTATACAGGCGTGGCACTTTCCTGC 
                 SEQ ID NO: 62 
               
               
                   
               
               
                 EsxR-F: 
                 GGACTAGTCAGATTATGTACAACTATCCGG 
                 SEQ ID NO: 63 
               
               
                   
               
               
                 EsxR-R: 
                 GGACTAGTGCCCCACTTGGCGG 
                 SEQ ID NO: 64 
               
             
          
         
       
     
     B. Bioinformatics. Selected sequences taken from the TubercuList server at the Institut Pasteur (http://genolist.pasteur.fr/TubercuList) were screened for the presence of putative coiled-coil motifs using the MultiCoil software available at the Massachusetts Institute of Technology (http://multicoil.lcs.mit.edu/cqi-bin/multicoil). Based on the coiled-coil prediction and available experimental evidence, we used the structure of the four helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor (17) to build a model of the ESAT-6 and CFP-10 complex. In this model, the amino acids in position “a” and “d” of the helices in the template were occupied as far as possible by hydrophobic amino acids. In agreement with reported NMR data (19) the two resulting helical hairpins for CFP-10 and ESAT-6 were put side-by-side and antiparallel, with the WXG motif at opposite ends of the complex structure. Structures were refined with a short molecular dynamics/simulated annealing protocol (18), restraining the experimentally known helical elements (Ala 8 to Gln 40 and Ala 46 to Gly 80 for CFP-10; Phe 8 to Trp 43 and Glu 49 to Ala 84 for ESAT-6)(19). 
     C. Protein Extraction, Column Purification, and Immunoblotting. Cell-free protein extracts were prepared from early log phase cultures of recombinant strains grown in 7H9 or Sauton&#39;s media and processed by standard procedures. Culture filtrates were concentrated using a Millipore filter with a 3 kDa cutoff. Western blots and antigen detection were performed as described previously, using anti-ESAT-6 monoclonal antibodies purchased from the Statens Serum Institute (Denmark) and in-house polyclonal antibodies directed against CFP-10 or PPE-68 (12). HIS-tagged proteins were purified using mini-columns containing 80 μl Ni 2+  NTA agarose. Cell lysates or culture filtrates were passed through the mini-columns then washed with phosphate buffered saline (PBS), (pH 7.3) with 300 mM NaCl and 20 mM Imidazole. Bound protein was eluted with PBS containing 300 mM NaCl and 250 mM Imidazole. Samples were precipitated with 10% TCA, dissolved in PBS and separated by SDS-PAGE (15%), before blotting onto a Nitrocellulose membrane. Monoclonal anti-ESAT-6, anti-HIS or polyclonal CFP-10 antibodies were used for immune-detection. 
     D. Virulence Studies and Immunological Assays. Virulence and immunological assays were done using SCID mice (IFFA CREDO, France) for intravenous infection and/or C57BL/6 mice for aerosol infection or subcutaneous vaccination as previously described (12-14). 
     Example 2 
     Mutation of Conserved Residues of ESAT-6 Identifies Biologically Important Amino Acids 
     Sequence alignment of ESAT-6 orthologs from  M. tuberculosis  and  M. leprae  revealed that 33 amino acids are conserved between the two ESAT-6 molecules ( FIG. 1A ), and 12 of these are also conserved in ESAT-6 like proteins from the phylogenetically more distant species  Corynebacterium diphtheriae . Fourteen residues scattered along the  M. tuberculosis  ESAT-6 molecule were selected for mutation in pRD1-2F9 and the resultant cosmids were integrated into a  M. tuberculosis  ARD1 strain (H37RvΔRD1)(7). Using our cloning strategy ( FIG. 5 ), recombinant  M. tuberculosis  strains that expressed ESAT-6 with one or two amino-acid replacements were obtained and screened for secretion of ESAT-6, immunogenicity and virulence (Table 1). 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Characteristics of recombinant  M. tuberculosis  strains 
               
               
                 carrying ESTA-6 mutation 
               
             
          
           
               
                   
                   
                 CFP-10 
                 ESAT-6 
                   
                   
                   
               
               
                   
                   
                 specific 
                 specific 
                 Ratio 
               
               
                 Strain 
                   
                 IFN-γ 
                 IFN-γ 
                 in 
                 Ratio 
               
               
                 H37RvΔRD1 
                 Primers 
                 responses 
                 responses 
                 lungs* 
                 in spleen* 
                 Virulence 
               
               
                   
               
             
          
           
               
                 ::pYUB412 
                 −control 
                 &lt; 
                 &lt; 
                 6.7 
                 23 
                 A 
               
               
                 ::2F9 
                 +control 
                 13–18 ng/ml 
                 12–19 ng/ml 
                 1500 
                 24444 
                 V 
               
               
                 ::2F9-EsxA-T2H 
                 1-25-24-8 
                    3 ng/ml 
                    4 ng/ml 
                 2000 
                 40000 
                 V 
               
               
                 ::2F9-EsxA-Q4L 
                 1-44-45-8 
                 nd 
                  1.5 ng/ml 
                 440 
                 130 
                 V 
               
               
                 ::2F9-EsxA-F8I 
                 1-23-22-8 
                 &lt; 
                 &lt; 
                 17 
                 1200 
                 V 
               
               
                 ::2F9-EsxA-A14R 
                 1-41-40-8 
                 &lt; 
                 &lt; 
                 21 
                 800 
                 V 
               
               
                 ::2F9-EsxA-L28A/L29S 
                 1-39-38-8 
                 &lt; 
                 &lt; 
                 1.7 
                 12 
                 A 
               
               
                 ::2F9-EsxA-W43R 
                 1-37-36-8 
                 &lt; 
                 &lt; 
                 2 
                 3 
                 A 
               
               
                 ::2F9-EsxA-G45T 
                 1-19-18-8 
                 &lt; 
                 &lt; 
                 1.1 
                 31 
                 A 
               
               
                 ::2F9-EsxA-Q55I/Q56A 
                 1-28-27-8 
                    5 ng/ml 
                    6 ng/ml 
                 53 
                 1400 
                 V 
               
               
                 ::2F9-EsxA-N66I/N67A 
                 1-30-29-8 
                 &lt; 
                 &lt; 
                 73 
                 620 
                 V 
               
               
                 ::2F9-EsxA-M83I 
                 1-32-31-8 
                 nd 
                 &lt; 
                 160 
                 350 
                 V 
               
               
                 ::2F9-EsxA-V90R 
                 1-49-48-8 
                 &lt; 
                 &lt; 
                 3 
                 15 
                 A 
               
               
                 ::2F9-EsxA-M93T 
                 1-51-50-8 
                 &lt; 
                 nd 
                 41 
                 210 
                 V 
               
               
                 ::2F9-EsxA-F94Q 
                 1-52-53-8 
                 &lt; 
                 &lt; 
                 38 
                 34 
                 A 
               
               
                   
               
               
                 Table 1. ESAT-6-specific T-cell immunity in C57BL/6 mice immunized with recombinant H37RvDRD1 strains. Concentration of IFN-γ in culture supernatants of splenocytes stimulated ESAT-6: 1–20 peptide. Efficient immunization and specificity of IFN-γ secretion by splenocytes were validated in all cases by stimulation with PPD, as positive control, or MalE: 100–114 peptide, as negative controls, respectively. 
               
               
                 &lt; means signal was below detection. 
               
               
                 *Virulence in SCID mice, ratio of CFU in spleens or lungs three to four weeks post infection compared to day 0, 
               
               
                 V = virulent (enhanced in vivo growth), 
               
               
                 A = attenuated (similar to pYUB vector control), 
               
               
                 nd = not determined, 
               
             
          
         
       
     
     Among the 13 mutants tested, eight showed different degrees of enhanced in vivo growth after three to four weeks of infection in SCID mice, as witnessed by splenomegaly and high bacterial counts in lungs and spleen, whereas five mutant strains showed characteristics similar to the H37RvΔRD1::YUB412 vector control ( FIG. 1C , Table 1). For most samples, the variant ESAT-6 proteins and CFP-10 were found in the culture supernatant ( FIG. 1B ). We had previously noticed that an enhanced in vivo growth phenotype was only observed when ESAT-6 was secreted. Thus, these findings suggest that loss of virulence in four of the five attenuated mutants is not caused by defective secretion, but rather due to potential loss of interaction with protein partners or putative receptors. For further analysis of these results we used bioinformatic tools. Most interestingly, when we screened ESAT-6 and CFP-10 for the presence of coiled-coil motives using the MultiCoil program (20), a high probability of multimeric coiled-coil formation was found ( FIG. 6 ). 
     These observations together with the results from the virulence assay of mutant strains and structural data from the available literature (15, 19, 21), made it possible for us to build a model of the ESAT-6-CFP-10 protein complex, which is crucial for the interpretation of the observed effects of ESAT-6 mutations on virulence. As shown in  FIG. 2 , mutations that abolish virulence concern residue L29, important for the stability of the hairpin/coiled-coil structure of the molecule, residues W43 and G45 that constitute the loop and contain the conserved WXG motif, as well as residues V90 and F94 in the extreme C-terminal tail. Similarly, M93T, also located in the extreme C-terminus, showed an almost attenuated phenotype. The other tested mutants (T2H, Q4L, F8I, A14R, Q55I/Q56A, N66I/N67A, M83I) did not abolish the virulence of the recombinant  M. tuberculosis  strains in SCID mice as shown by splenomegaly and different degrees of enhanced in vivo growth rates (Table 1). These mutations either affect residues in the N- or C-terminal regions (T2H, Q4L, M83I), are predicted to be at the outer surface of the four-helical bundle structure (A14R, Q56A, N66I, N67A), or are conservative in the sense that the mutated residue can stabilise the structure in a similar way: F8I replaces an aromatic with a hydrophobic residue, Q55I replaces a polar residue in the interior with a hydrophobic residue. In our model, Q55 is mostly surrounded by hydrophobic amino acids, and its replacement by a hydrophobic residue could probably be accommodated by small rearrangements in the structure. 
     Together, these results suggest that correct ESAT-6/CFP-10 complex formation is an important requirement for full virulence of  M. tuberculosis . In order to obtain experimental evidence of the protein-protein interaction involved in this process, recombinant BCG strains that expressed and secreted hexahistidine (HIS) tagged ESAT-6 and CFP-10 molecules were genetically engineered. 
     Example 3 
     Tagged ESAT-6 and CFP-10 Molecules in BCG Allow Co-Purification of Protein Partners 
     When concentrated culture filtrates of recombinant BCG expressing ESAT-6-HIS at the C-terminus were applied to a Ni 2+  affinity column, two bands of low molecular weight were visible after elution ( FIG. 3A ). As shown in  FIG. 3B , western blot analyses identified the upper band as CFP-10, and the lower band as ESAT-6-HIS. The two molecules, overexpressed as recombinant proteins by  Escherichia coli  have previously been shown to interact, forming a 1:1 protein complex in in vitro experiments (15, 22). This invention confirms these observations and further demonstrates that in mycobacteria, ESAT-6 and CFP-10 are indeed present as a protein complex. This is the first experimental evidence that these two molecules form a tight complex even after being secreted by their proper secretion machinery. 
     The same binding behavior was also observed using samples of the cytosolic fraction of BCG::2F9-EsxA-HIS ( FIG. 3B ), indicating that the two molecules interact already shortly after synthesis. The specificity of this interaction is shown by the results obtained with a C-terminal HIS-tagged ESAT-6 W43R BCG mutant. Most interestingly, for this strain ESAT-6 and CFP-10 are found in the supernatant, but co-purification was no longer possible ( FIG. 3C ), suggesting that mutation of a single residue, W43R, was sufficient to reduce the affinity between ESAT-6 and CFP-10 in such a way that binding between the two antigens did not occur under the given pH conditions. This finding is also of interest for the interpretation of results obtained by virulence screening of mutant strains. As shown above, mutation W43R was associated with attenuation. 
     When CFP-10 carrying an N-terminal HIS tag was purified via Ni 2+  affinity chromatography, ESAT-6 was obtained after elution of bound HIS-CFP-10 ( FIGS. 3A , B), though the amounts of ESAT-6 co-purified with CFP-10 were lower. However, this experiment confirmed the strong interaction of these two molecules both in and outside the mycobacterial cell and the utility of the attached HIS-tag for purification of proteins produced by slow growing mycobacteria. Like for the ESAT-6-W43R mutant, a double band was observed for the HIS-tagged molecule, induced by the tag. 
     In contrast to the results obtained with the C-terminal HIS tagged ESAT-6, an N-terminal HIS tag for ESAT-6 was not effective for purification of the protein from BCG by Ni 2+  affinity chromatography (data not shown) as the N-terminal HIS tag had been removed or cleaved from the ESAT-6 molecule in the cytosol, possibly by the action of an endopeptidase ( FIG. 7B ). Furthermore, ESAT-6 carrying the N-terminal HIS tag did not induce ESAT-6 and CFP-10 specific IFN-γ production by splenocytes of infected mice upon restimulation with the antigen, despite being secreted ( FIG. 4 ). From these experiments we concluded that ESAT-6 with the C-terminal HIS tag resembles native ESAT-6, produced by  M. tuberculosis , more closely than the N-terminal HIS construct. 
     Example 4 
     Larger ESAT-6 Fusion Proteins are Difficult to Transport Outside the Mycobacterial Cell 
     As shown by using hexahistidine tags, modified ESAT-6 proteins containing more amino-acids than the wildtype protein are still secreted by the ESAT-6 system-1. In order to evaluate whether this secretion machinery could be used to export larger fusion proteins, which may be of importance for the development of new recombinant vaccines, we constructed various ESAT-6 protein fusions and tested if they were exported. One of them, ESAT-6 fused at its C-terminus with EsxR (Rv3019c, SwissProt Accession Number: P64093) (EsxA-EsxR) was secreted into the supernatant in sufficient amounts to be detected by Western blotting ( FIG. 7D ). For this construct, CFP-10 was also found in the supernatant. However, due to the larger size and different structure, much of the fusion protein was retained in the cell wall fraction, where wildtype ESAT-6 was not present ( FIG. 7D ) 
     Another recombinant protein, ESAT-6 fused to the ESAT-6 orthologue of  M. leprae  (MLEsxA, SwissProt Accession Number: Q50206), was strongly expressed, but only very little amounts of the protein were detected in the culture supernatant, suggesting that the structural conformation of this fusion protein did not match the requirements of the secretion machinery. As with EsxA-EsxR, the EsxA-MLEsxA fusion protein was present in the cell wall fraction (data not shown). Also constructed was an ESAT-6-C-terminal GFP fusion. There was obtained a recombinant BCG characterized by green fluorescent colonies. Western Blot analysis of the culture supernatant of this recombinant BCG strain showed a large fusion protein that reacted with the ESAT-6 monoclonal antibody and infection of native macrophages revealed that the ESAT-6-GFP-fusion protein was expressed in the engulfed bacteria ( FIGS. 7E , F). However, this ESAT-6-GFP fusion protein, that is several times larger than ESAT-6 alone, did not induce ESAT-6 specific T-cell responses in C57BL/6 mice, and did not increase virulence in SCID mice (data not shown), indicating that ESAT-6 fused to GFP had lost its biological function. For this reason, constructs that only contain small tags are better suited to preserve the biological function of the ESAT-6 molecule and still allow specific purification and detection procedures to be employed. In addition to HIS-tag constructs, EsxA-Tetra-cysteine-tag ( FIG. 7G ) and/or EsxA-FLAG-tag constructs (data not shown) are useful constructs. 
     Example 5 
     Analysis of IFN-γ Responses and Virulence Defines Three Groups of Mutant Strains 
     To monitor the biological effects of the various genetic modifications of ESAT-6, intravenous infection of SCID mice and recording of IFN-γ responses from splenocytes of immuno-competent C57BL/6 mice were used. 
     It was first observed that the tested strains that induced an IFN-γ response to ESAT-6 were also found to cause splenomegaly and enhanced in vivo growth in SCID mice. However, variation in virulence occurred, as for some strains (e.g. BCG::2F9-EsxA-HIS) growth in SCID mice was less pronounced (Table 1,  FIG. 4A ). A few selected strains were also tested in immunocompetent C57BL/6 mice by aerosol infection. This approach revealed that strain BCG::2F9-EsxA-HIS Was able to multiply in the lungs of these mice but to a lesser degree than the BCG::RD1-2F9 control and the BCG::2F9-HIS-EsxA ( FIG. 4B ). As such, the fusion of a HIS tag to the C-terminal end of ESAT-6 (BCG::2F9-EsxA-HIS) resulted in intermediate virulence. The strain, however, retained its capacity to induce IFN-γ responses upon restimulation with ESAT-6 peptides or proteins in C57BL/6 mice ( FIG. 4D ). 
     In contrast, the opposite was not always true, as mutants of the second group were virulent in the SCID mouse model, but did not elicit ESAT-6 specific T-cell responses, probably due to changes in the T-cell epitope or conformational changes, possibly interfering with protein processing. Examples of such strains are BCG::2F9-HIS-EsxA, H37RvΔRD1::2F9EsxA-F8I or H37RvΔRD1::2F9EsxA-A14R (Table 1). 
     The third group of strains was the one, in which mutation of ESAT-6 abolished both, virulence and immunogenicity. This phenomenon was observed for several strains (H37RvΔRD1::2F9EsxA-L28A/L29S, H37RvΔRD1::2F9EsxA-W43R, H37RvΔRD1::2F9EsxA-G45T, and H37RvΔRD1::2F9EsxA-V90R, H37RvΔRD1::2F9EsxA-F94Q). Among these were constructs that had ESAT-6 mutated at the extreme C-terminus (H37RvΔRD1::2F9EsxA-V90R, H37RvΔRD1::2F9EsxA-F94Q). Similar results were obtained with a recombinant BCG strain (BCG::RD1-2F9-esxAA84-95) secreting a C-terminally truncated ESAT-6 protein ( FIG. 7C ), which did not induce ESX-1 related enhanced virulence nor ESAT-6 specific T-cell responses compared to BCG::RD1-2F9 strain (data not shown). 
     Example 6 
     Table 2 is a comparison of ESX-1 specific T-cell immunity (i.e. immunity against ESAT-6 and CFP-10 antigens) and virulence obtained with  M. microti  recombinants containing modified pRD1-2F9 cosmids. Concentration of IFN-γ in culture supernatants of splenocytes that were stimulated with ESAT-6 1-20 peptide, recombinant ESAT6 protein (rESAT-6) or recombinant CFP10 protein (rCFP-10). In all cases efficient immunisation and specificity of IFN-γ secretion by splenocytes was validated with purified protein derivative (PPD) and MalE:100-114 peptide as positive and negative controls respectively. Results are of at least 2 independent experiments with three mice and are expressed as mean values of duplicate cuture wells. SD values are within 10%. &lt; indicates that the signal was below detection limit (1 ng/ml). Ratio of colony-forming units (CFU) at day 30 relative to initial dose recovered in the lungs and spleen of severe combined immunodeficient mice (SCID) after intravenous infection with 10 6  CFU. 
     
       
         
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   
                   
                 Virulence in SCID mice 
                   
               
               
                   
                 Immunogenicity 
                 Ratio of CFU at day 
                 Attenuated/ 
               
               
                 Recombinant 
                 IFNγ (ng/ml) 
                 30 relative to day 0 
                 Virulent 
               
             
          
           
               
                 strain 
                 ESAT-6: 1-20 
                 r-ESAT-6 
                 r-CFP-10 
                 Lungs 
                 Spleen 
                 phenotype 
               
               
                   
               
             
          
           
               
                 
                   M. microti 
                 
                   
                   
                   
                   
                   
                   
               
               
                 pYUB412 
                 21 
                 &lt; 
                 &lt; 
                 4 
                 2 
                 A 
               
               
                 pRD1-2F9 
                 16 
                 20 
                 17 
                 100 
                 80 
                 V 
               
               
                 Δrv3860-64 
                 22 
                 17 
                 16 
                 94 
                 26 
                 V 
               
               
                 Δrv3860-66 
                 12 
                 12 
                 10 
                 14 
                 2 
                 A 
               
               
                   
               
             
          
         
       
     
       M. microti  strains containing variants of pRD1-2F9, lacking either rv3860-rv3864 or rv3860-rv3866, clearly secreted ESAT-6 and CFP-10 (Δrv3860-66 shown in  FIG. 5A ). 
     Moreover  M. microti  Δrv3860-66 retained antigen specific T-cell immunogenicity (Table 2) but without showing enhanced in vivo growth in SCID mice. Loss of rv3865 and part of rv3866 did not affect ESAT-6 secretion or immunogenicity, but led to attenuation. This suggests that Rv3865 and/or Rv3866 may represent virulence factors independent of ESAT-6 and CFP-10. 
     In summary, proteins of the 6 kDa (early secreted antigenic target) ESAT-6 secretion system-1 of  Mycobacterium tuberculosis  are not only strongly involved in the anti-mycobacterial Th1-host immune response, but are also key players for virulence. In this invention, protein engineering together with bioinformatic, immunological, and virulence analyses made it possible to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in  Mycobacterium . Mutation of the Trp-X-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses and virulence. Replacement of conserved Leucine residues interfered with secretion, coiled-coil formation and virulence, whereas certain mutations at the extreme C-terminus did not affect secretion, but caused attenuation, possibly due to altered ESAT-6 targeting or trafficking. In contrast, mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6/CFP-10 complex showed much less effects. 
     Construction of recombinant BCG expressing ESAT-6 with a C-terminal tag made it possible to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction in and outside of the mycobacterial cell. These strains induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active in spite of the tag. Together with previous NMR data, the results of this invention have allowed a biologically relevant model of the ESAT-6/CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis. 
     Example 7 
     Recombinant BCG strains were constructed that carry a modified version of the RD1-2F9 cosmid, previously used for integration of the modified RD1 region into  M. tuberculosis  Delta RD1 (see Brodin et al., J. Biol. Chemistry, 2005, 280:33953-59.) To obtain the different variants of the pRD1-2F9 cosmid, the previously described procedures were used. 
     Specifically, 250 to 300 microliters of electrocompetent cells of BCG Pasteur 1173P2, which were obtained by washing and concentrating 7 to 10 days old BCG cultures, were mixed with 5 to 10 microliters of concentrated and dialized miniprep DNA of the modified RD1-2F9 cosmids, and subjected to electroporation as previously described (according to standard procedures described in Pym et al., Mol. Microbiol., 46:709-717, 2002). 
     The following cosmids were electroporated into BCG and further tested:
         BCG-RD1-ESAT-6-L28A/L29S   BCG-RD1-ESAT-6-W43R   BCG-RD1-ESAT-6-G45T   BCG-RD1-ESAT-6-V90R   BCG-RD1-ESAT-6-M93T   BCG-RD1-ESAT-6-HIS.       

     For aerosol infections of C57BL/6 mice with recombinant BCG strains, 5 ml of a briefly sonicated strain suspension of 5×10 6  recombinant BCG bacteria was nebulized into containers containing groups of 12 mice, resulting in an average infection dose in the lung of about 10 to 100 CFU. Mice were left for 3 to 4 weeks, and sacrificed. For the first experiment, enumeration of bacteria on plates was rendered difficult due to contamination and dilution/plating problems. Nevertheless, parts of the infected lungs from these experiments, which were fixed in formaldehyde solution, were subjected to histological examination that permitted classification of the samples according to the lesions as follows:
         BCG-RD1-ESAT-6-W43R: Few lesions, congestion minima, some macrophages;   BCG-RD1-ESAT-6-L28A/L29S: Some granuloma + to ++, lymphocytes and perivascular histiocytes;   BCG-RD1-ESAT-6-G45T: Granuloma +/−, some lymphocytes and histiocytes;   BCG-RD1-ESAT-6-V90R: Some infiltrates without real granuloma, minimal lesions;   BCG-RD1-ESAT-6-M93T: Peribronchic infiltrates and some granuloma; and   BCG-RD1-2F9: Important granuloma +++, typical for a mycobacterial infection, spumous macrophages, +++ lymphocytes.       

     Example 8 
     In a second round of experiments, using an aerosol infection of C57BV6 mice with the same dose as in Example 7, the following strains were tested:
         BCG-vector control,   BCG-RD1-ESAT-6-W43R,   BCG-RD1-ESAT-6-L28A/L29S,   BCG-RD1-ESAT-6-HIS,   BCG-RD1-ESAT-6-M93T, and   BCG-RD1-2F9.
 
Enumeration of the bacterial load after 30 days gave the values shown in  FIG. 8 .
       

     Examples 7 and 8 show that the recombinant BCG-RD1-ESAT-6-modified strains are less virulent than the BCG::RD1-2F9 control strain, showing at least 1 log less bacteria in the lungs after 30 days than the control. For BCG::RD1-L28A/L29S, no colonies were detected on the corresponding plates for the lung samples obtained at day 30. 
     Example 9 
     The six recombinant vaccine strains identified in Example 6 were tested in two rounds of mouse vaccination and challenge studies, using ca. 10 5  bacteria as subcutaneous inoculum. Two months after vaccination, mice were infected by aerosol (nebulization) with either  M. tuberculosis  strain “Beijing” or H37Rv. Four weeks after the challenge, mice were sacrificed and organs homogenized before being plated out for enumeration, obtaining the results shown in  FIGS. 9 and 10 . 
     First round of protection study of recombinant BCG strains in C57BL/6 mice is shown in  FIGS. 10(A) and 10(B) . The Y axis of the two graphs shows the bacterial charges in CFU in lungs and spleen, respectively. 
     Second round of the protection study of recombinant BCG strains in C57BL/6 mice is shown in  FIGS. 11(A) and 11(B) . Once again, the Y axis of the two graphs shows the bacterial charges in CFU in lungs and spleen, respectively. 
     The two rounds of mouse protection experiments have shown that some of the recombinant BCG::RD1 vaccine strains that express modified ESAT-6 exhibit a better protective efficacy against  M. tuberculosis  that the BCG vector control strains. For example, BCG::RD1-M93T showed better protection in lungs and spleens in both experiments. For BCG::RD1-ESAT-6-HIS, better protection than the BCG control was observed in the lungs (both experiments) and in the spleen in the second experiment. BCG::RD1-L28A/L29S was only tested once and showed improved protection relative to the control. BCG::RD1-W43R and BCG::RD1-G45T showed intermediate efficacy, while BCG::RD1-V90R and BCG::RD1-PPE68ko (containing an intact esxA gene, but having a truncation in the neighboring ppe68 gene) did not show better efficacy than the BCG control strain. 
     As the BCG::RD1-ESAT-6-HIS strain showed one of the best protective efficacies in the lungs, this strain was further used in a guinea pig vaccination experiment, detailed in Example 10 below. 
     Example 10 
     The protective potential of recombinant strain BCG-RD1-ESAT-6-HIS (synonymous to BCG::2F9-EsxA-HIS) was evaluated in the guinea pig model. The conditions of the experiments were very similar to the ones described in Pym et al., Nature Medicine, 2003. In brief, after vaccination with BCG or saline (non-vaccinated control) or BCG::RD1-2F9-EsxA-HIS (synonymous to BCG::RD1-ESAT-6-HIS), the animals were challenged 10 weeks post vaccination with an aerosol of  M. tuberculosis  H37Rv resulting in 500 colony forming units (CFU) in the lungs of the animals. After the challenge, the weight (in grams) was monitored as shown on the Y axis in  FIG. 11 . Animals that showed a weekly weight loss due to tuberculosis of more than 20% were sacrificed. 
     The non-vaccinated (saline control) guinea pigs had to be all sacrificed within the first 70 days post challenge due to severe tuberculosis indicated by strong weight loss, as shown on the Y axis in grams in  FIG. 11(A) . The numbers in the box correspond to the identification names of the various guinea pigs that were tested. 
     From the 8 BCG-Danish vaccinated guinea pigs, 4 (595A, 464B NC60688, 4075, and 4F6F) had already been sacrificed due to severe tuberculosis indicated by strong weight loss, as shown on the Y axis in grams in  FIG. 11(B) . 
     Five of six guinea pigs vaccinated with the recombinant BCG::RD1-ESAT-6-His were still alive at day 150. Only one guinea pig (60707 BK) had to be sacrificed due to weight loss.  FIG. 11(C) . 
       FIG. 12  shows the overview of the guinea pig vaccination-challenge experiment, showing that the group of animals vaccinated with BCG::RD1-ESAT-6-His (BCG::2F9-EsxA-His) was best protected, as there was only one guinea pig lost at day 150 post challenge. Apart from the names BCG::2F9-EsxA-His, BCG Danish, and the saline control, the remaining names in the box correspond to the other participants of the experiment. 
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