MXPA00009803A - Fusion proteins of mycobacterium tuberculosis - Google Patents

Abstract

The present invention relates to fusion proteins containing at least two Mycobacterium tuberculosis antigens. In particular, it relates to bi-fusion proteins which contain two individual M. tuberculosis antigens, tri-fusion proteins which contain three M. tuberculosis antigens, tetra-fusion proteins which contain four M. tuberculosis antigens, and penta-fusion proteins which contain five M. tuberculosis antigens, and methods for their use in the diagnosis, treatment and prevention of tuberculosis infection.

Description

FUSION PROTEINS ANTIGENS OF MYCOBACTERIUM TUBERCULOSIS, and its uses 1. INTRODUCTION The present invention relates to fusion proteins that contain at least Mycobacterium tuberculosis antigens. In particular, it refers to the bi-fusion proteins containing two individual M. tuberculosis antigens, to the tri-fusion proteins containing three M. tuberculosis antigens, to the tetra-fusion proteins containing four antigens of M. tuberculosis, and the penta-fusion proteins containing five antigens of M. tuberculosis, and the methods for their use in the diagnosis, treatment and prevention of tuberculosis infection. 2. BACKGROUND OF THE INVENTION Tuberculosis is a chronic infectious disease caused by infection with M. tuberculosis. It is an important disease in developing countries, as well as a growing problem in the developed areas of the world, with approximately 8 million new cases and 3 million deaths per year. Although the infection may be asymptomatic for a considerable time, the disease commonly manifests as an acute inflammation of the lungs, giving rise to fever and a non-productive cough. If left untreated, it leads to serious complications and commonly death. Although tuberculosis can usually be controlled using prolonged treatment with antibiotics, this treatment is not enough to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is fundamental, it is difficult to monitor the patient's behavior. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistance. To control the spread of tuberculosis, effective vaccination and accurate early diagnosis of the disease are of the utmost importance. Currently, vaccination with live bacteria is the most effective method to induce protective immunity. The mycobacterium that is most commonly used for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of M. bovis. However, the safety and effectiveness of BCG is a source of controversy and in some countries, such as the United States, the general public is not vaccinated with this agent. The diagnosis of tuberculosis is usually obtained using a skin test, which involves intradermal exposure to tuberculin PPD (purified protein derivative). Antigen-specific T cell responses give rise to measurable induration at the injection site for 48-72 hours after injection, indicating exposure to mycobacterial antigens. Sensitivity and specificity, however, have been a problem with this test, and individuals vaccinated with BCG can not be distinguished from infected individuals. Although it has been shown that macrophages act as the main effectors of immunity for M. tuberculosis, T cells are the predominant inducers of such immunity. The main role of T cells in protection against infection by M. tuberculosis is illustrated by the frequent presence of M. tuberculosis in patients with Acquired Immunodeficiency Syndrome, due to the depletion of CD4 + T cells associated with infection by immunodeficiency virus human (HIV) CD4 + T cells reactive for mycobacterium have been shown to be potent producers of interferon gamma (IFN-α), which in turn has been shown to activate the anti-mycobacterial effects of macrophages in mice. Although the function of IFN-? in humans it is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, alone or in combination with IFN-? or tumor necrosis factor a, activates human macrophages to inhibit M. tuberculosis infection. further, it is known that the IFN-? stimulates human macrophages to make 1, 25-dihydroxy-vitamin D3. Similarly, interleukin-12 (IL-12) has been shown to play a role in the stimulation of resistance for M. tuberculosis infection. For a review of the immunology of infection by M. tuberculosis see, Chan and Kaufmann, 1994, Tuberculosis: Pa thogenesis, Protection and control, Bloom (ed.), ASM Press, Washington, DC. Accordingly, there is a need for improved vaccines and improved methods for diagnosis, prevention and treatment of tuberculosis. 3. COMPENDIUM OF THE INVENTION The present invention relates to the fusion proteins of M. tuberculosis antigens. In particular, it refers to fusion polypeptides containing 2 or more M. tuberculosis antigens, the polynucleotides encoding these polypeptides, methods for using the polypeptides and polynucleotides in the diagnosis, treatment and prevention of M. tuberculosis infection. The present invention is based, in part, on the discovery of the inventors that polypeptides containing 2 to 5 M. tuberculosis coding sequences produce recombinant fusion proteins that retain the immunogenicity and antigenicity of their individual components. The fusion proteins described herein induce a response of T cells and B cells, when measured by the proliferation of T cells, cytokine production and production of antibodies. In addition, a fusion protein was used as an immunogen with adjuvants in vivo to produce cell-mediated and humoral immunity for M. tuberculosis. In addition, a fusion protein was prepared by a fusion construct and used in a vaccine formulation with an adjuvant to produce long-term protection in animals against the development of tuberculosis. The fusion protein was a more effective immunogen than a mixture of its individual protein components. In a specific embodiment of the invention, the isolated or purified M. tuberculosis polypeptides of the invention can be formulated as pharmaceutical compositions for administration to an individual for the prevention and / or treatment of M. tuberculosis infection. The immunogenicity of the fusion protein can be improved by the inclusion of an adjuvant. In another aspect of the invention, the isolated or purified polypeptides are used to produce recombinant recombinant fusion polypeptides in vitro. Otherwise, the polypeptides can be administered directly to an individual as DNA vaccines to elicit the expression of the antigen in an individual, and the subsequent induction of an anti-M immune response. tuberculosis. It is also an object of the invention that the polypeptides are used in in vitro assays to detect humoral antibodies or cell-mediated immunity against M. tuberculosis for the diagnosis of infection or monitoring of the progress of the disease. In addition, it is possible to use the polypeptides as an in vivo diagnostic agent in the form of an intradermal test. Otherwise, the polypeptides can be used as immunogens to generate anti-M antibodies. tuberculosis in a non-human animal. The antibodies can be used to detect the antigens chosen in vivo and in vi tro.

Four . BRIEF DESCRIPTION OF THE DRAWINGS Figure IA and IB. The nucleotide sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of the tri-fusion protein Ral2-TbH9-Ra35 (designated tb32A). Figure 2: Nucleotide sequence (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) of the tri-fusion protein Erdl4-DPV-MTI (called Mtb39A). Figure 3A and 3D: The nucleotide sequence (SEQ ID NO: 5) and the amino acid sequence (SEQ ID NO: 6) of the tri-fusion protein TbRa3-38KD-Tb38-l. Figure 4A and 4D: The nucleotide sequence (SEQ ID NO: 7) and the amino acid sequence (SEQ ID NO: 8) of the bifusion protein TbH9-Tb38-l. Figure 5A and 5J: The nucleotide sequence (SEQ ID NO: 9) and the amino acid sequence (SEQ ID NO: 10) of the tetrafusion protein TbRa3-38KD-Tb38-l-DPEP (de-labeled TbF-2) . Figure 6A and 6B: The nucleotide sequence (SEQ ID NO: 11) and the amino acid sequence (SEQ ID NO: 12) of the pentafusion protein Erdl4-DPV-MTI-MSL-MTCC2 (designated Mtb88f). Figure 7A and 7B: The nucleotide sequence (SEQ ID NO: 13) and the amino acid sequence (SEQ ID NO: 14) of the protein of tetra-fusion Erdl4-DPV-TI-MSL (termed Mtb46f). Figure 8A and 8B: The nucleotide sequence (SEQ ID NO: 15) and the amino acid sequence (SEQ ID NO: 16) of the DPV-TI-SL-TCC2 tetrafusion protein (termed Mtb71f). Figure 9A and 9B: The nucleotide sequence (SEQ ID NO: 17) and the amino acid sequence (SEQ ID NO: 18) of the trimer fusion protein DPV-MTI-MSL (called Mtb31f). Figure 10A and 10B: The nucleotide sequence (SEQ ID NO: 19) and the amino acid sequence (SEQ ID NO: 20) of the trimer fusion protein TbH9-DPV-MTI (referred to as Mtbdlf). Figure HA and 11B: The nucleotide sequence (SEQ ID NO: 21) and the amino acid sequence (SEQ ID NO: 22) of the trimeric protein Erdl4-DPV-MTI (called Mtb36f). Figure 12A and 12B: The nucleotide sequence (SEQ ID NO: 23) and the amino acid sequence (SEQ ID NO: 24) of the Diffusion protein TbH9-Ra35 (designated Mtb59f). Figure 13A and 13B: The nucleotide sequence (SEQ ID NO: 25) and the amino acid sequence (SEQ ID NO: 26) of the Diffusion protein Ral2-DPPD (designated Mtb24). Figure 14A-14F: Responses of the proliferation of T cells of 6 PPD + individuals when stimulated with two fusion proteins and their individual components. Figure 15A-15F: Production of IFN-? of 6 PPD + individuals when stimulated with two fusion proteins and their individual components. Figure 16A-16F: Proliferation of T cells from mice immunized from a fusion protein or its individual components and an adjuvant. Figure 17: IFN-α production of mice immunized with a fusion protein or its individual components and an adjuvant. Figure 18: IL-4 production of mice immunized with a fusion protein or its individual components and an adjuvant. Figure 19A-19F: Serum antibody concentrations of mice immunized with a fusion protein or its individual components and an adjuvant. Figure 20A-20C: Survival of guinea pigs after an aerosol challenge of M. tuberculosis. The fusion proteins Mtb31A and Mtb39A were formulated in adjuvant SBASlc (20A), SBAS2 (20B) ó SBAS7 (20C), and used as an immunogen in guinea pigs. Figure 21A and 21B: Stimulation of the proliferation and production of IFN-? in TbH9-specific T cells by the fusion protein TbH9-Tb38-l. Figure 22A and 22B: Stimulation of the proliferation and production of IFN-? in Tb38-1-specific T cells by fusion protein TbH9-Tb38-l. Figure 23A and 23B: Stimulation of the proliferation and production of IFN-? in T cells that previously showed antigen response TbH-9 and Tb38-1 by means of the fusion protein TbH9-Tb38-l. 5 . DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antigens useful for the treatment and prevention of tuberculosis, the polynucleotides encoding these antigens and the methods for their use. The antigens of the present invention are fusion polypeptides of M. tuberculosis antigens and variants thereof. More specifically, the antigens of the present invention consist of at least two M. tuberculosis polypeptides that are fused to a larger fusion peptide molecule. The antigens of the present invention may furthermore consist of other components designed to improve the immunogenicity of the antigens or to improve these antigens in other aspects, for example, the isolation of these antigens by the addition of an extension of histidine residues. at one end of the antigen. . 1 SPECIFIC ANTIGENS OF M. tuberculosis The antigens of the present invention are exemplified in Figures IA to 13B, including homologs and variants of these antigens. These antigens can be modified, for example, by the addition of peptide linker sequences as described below. These linker peptides may be inserted between one or more polypeptides that make up each of the fusion proteins presented in Figures IA to 13B. Other antigens of the present invention are the antigens that are described in Figures IA to 13B that have been linked to a known antigen of M. tuberculosis, such as the 38 kD antigen (SEQ ID NO: 27) already described ( Andersen and Hansen, 1989, Infect Immun. 57: 2481-2488; Genbank Accession No. M30046). . 2 IMMUNOGENICITY TESTS The antigens described herein, and the immunogenic portions thereof, have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce proliferation and / or cytokine production (ie, production of interferon-γ and / or interleukin-12) in T cells, NK cells, B cells and / or macrophages from an individual. immune to M. tuberculosis. The selection of the cell type for use in the evaluation of an immunogenic response to an antigen will depend on the desired response. For example, the production of interleukin-12 is more easily evaluated using preparations containing B cells and / or macrophages. An immune individual for M. tuberculosis is one that is considered resistant to the development of tuberculosis by virtue of being mounted an effective T cell response for M. tuberculosis (ie, substantially free of disease symptoms). These individuals can be identified based on a strongly positive response (i.e., greater than about 10 mm in diameter of induration) for the intradermal skin test for tuberculosis proteins (PPD) and an absence of signs or symptoms of tuberculosis diseases. tuberculosis. T cells, NK cells, B cells and macrophages from individuals immune to M. tuberculosis can be prepared using methods known to those skilled in the art. For example, it is possible to employ a PBMC preparation (ie, peripheral blood mononuclear cells) without further separation of the component cells. PBMC can generally be prepared, for example, using density centrifugation through "FICOLL" (Winthrop Laboratories, NY). T cells for use in the assays described herein can also be purified directly from PBMCs. Otherwise, an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins can be employed. These T cell clones can be generated, for example, by culturing PBMCs from individuals immune to M. tuberculosis with mycobacterial proteins for a period of 2 to 4 weeks. This allows the expansion of only the T cells specific for the mycobacterial protein, giving rise to a line composed of only these cells. These cells can then be cloned and tested with individual proteins, using methods known to those skilled in the art, to more precisely define the specificity of the individual T cells. In general, antigens that test positive in assays for proliferation and / or cytokine production (ie, production of interferon-α and / or interleukin-12) performed using T cells, NK cells, B cells and / or macrophages from of an immune individual for M. tuberculosis are considered immunogenic. These assays can be performed, for example, using the representative procedures described below. Immunogenic portions of these antigens can be identified using similar assays, and can be present without the polypeptides described herein. The ability of a polypeptide (e.g., an immunogenic antigen, or a portion or other variant thereof) to induce cell proliferation is evaluated by contacting the cells (e.g., T cells and / or NK cells) with the polypeptide and measuring the proliferation of cells. In general, the amount of polypeptide sufficient for the evaluation of about 10 cells ranges from about 10 ng / ml to about 100 μg / ml and, preferably, about 10 μg / ml. Incubation of the polypeptide with the cells is usually carried out at 37 ° C for about six days. After incubation with the polypeptide, the cells are assayed for a proliferative response, which can be evaluated by methods known to those skilled in the art, such as by exposing the cells to a radiolabeled thymidine pulse and measuring the incorporation of the mark on cellular DNA. In general, a polypeptide that results in at least a threefold increase in proliferation above the background (i.e., the proliferation observed for cells cultured without polypeptide) is considered capable of inducing proliferation.

The ability of a polypeptide to stimulate the production of interferon-? and / or interleukin-12 in cells can be evaluated by contacting the cells with the polypeptide and measuring the level of interferon-? or interleukin-12 produced by the cells. In general, the amount of polypeptide that is sufficient for the evaluation of about 10 cells ranges from about 10 ng / ml to about 100 μg / ml and, preferably, is about 10 μg / ml. The polypeptide can be, but is not necessarily, immobilized on a solid support, such as a biodegradable bead or microsphere, such as those described in U.S. Patent Nos. 4,897,268 and 5,075,109. Incubation of the polypeptide with the cells is normally carried out at 37 ° C for about six days. After incubation with the polypeptide, the cells are assayed for interferon-? and / or interleukin-12 (one or more subunits thereof), which can be evaluated by methods known to those skilled in the art, such as an enzyme-linked immunosorbent assay (ELISA), or, in the case of the P70 subunit of IL-12, a bioassay such as the assay measuring the proliferation of T cells. In general, a polypeptide that originates interferon-α production. per ml of cultivated supernatant (containing 104-105 T cells per ml) is considered capable of stimulating the production of interferon-? A polypeptide that stimulates the production of at least 10 mg / ml of the P70 subunit of IL-12 and / or at least 100 pg / ml of the P40 subunit of IL-12 by 105 macrophages or B cells (or by 3 x 105 PBMC) is considered capable of stimulating the production of IL-12. In general, immunogenic antigens are those antigens that stimulate the proliferation and / or production of cytokine (ie, the production of interferon-α and / or interleukin-12) in T cells,) NK cells, B cells and / or macrophages obtained from at least approximately 25% of individuals immune to M. tuberculosis. Among these immunogenic agents, polypeptides having superior therapeutic properties can be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for whom a response is observed. In addition, antigens that have superior therapeutic properties will not stimulate the proliferation and / or production of cytokine in vitro in cells from more than about 25% of individuals that are not immune to M. tuberculosis, thereby eliminating the responses that do not occur. they must specifically to cells sensitive to M. tuberculosis. Those antigens that induce a response in a high percentage of preparations of T cells, NK cells, B cells and / or macrophages from individuals immune to M. tuberculosis, (with a low incidence of responses in the cellular preparations of other individuals) have superior therapeutic properties. Antigens with superior therapeutic properties can also be identified based on their ability to decrease the severity of infection by M. tuberculosis, in experimental animals, when administered as a vaccine. Suitable vaccine preparations for use in experimental animals are described in detail below. It is possible to determine efficacy based on the ability of the antigen to offer at least about 50% reduction in bacterial numbers and / or at least about 40% decrease in mortality after experimental infection. Convenient experimental animals include mice, guinea pigs and primates. . 3 ISOLATION OF CODING SEQUENCES The present invention also relates to nucleic acid molecules that encode M. tuberculosis fusion polypeptides. In a specific embodiment by way of example in section 6, infra, 13 fusion coding sequences of M. tuberculosis were constructed. In accordance with the invention, any nucleotide sequence encoding the amino acid sequence of the fusion protein can be used to generate recombinant molecules that direct the expression of the coding sequence. To clone full length coding sequences or homologous variants to generate the fusion polynucleotides, labeled TNA probes designed from any portion of the nucleotide sequences or their complement described herein can be used to detect a genomic DNA or cDNA library made from different strains of M. tuberculosis to identify the coding sequence of each individual component. Isolation of the coding sequences can also be performed by polymerase chain reactions (PCR) using two combinations of degenerate oligonucleotide primers designed based on the coding sequences described herein. The invention also relates to isolated or purified polynucleotides complementary to the nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25, and the polynucleotides that selectively hybridize to these complementary sequences. In a preferred embodiment, a polynucleotide that hybridizes to the sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25, or its complementary sequence under conditions of low severity and encodes a protein that retains the immunogenicity of the fusion proteins of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 also they provide By way of example and not as limitation, exemplary conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Nati. Acad, Sci. USA 78; 6789-6792): filters containing DNA are pretreated for 6 hours at 40 ° C in a solution containing 35% formamide, 5X SSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP; Ficol 0.1%, BSA 1% and 500 μg / ml of denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: PVP 0.02%, Ficol 0.02%, BSA 0.2%, 100 μg / ml of salmon sperm DNA, dextran sulphate 10% (w / v) and 5- 20 x 106 cpm of the probe labeled with 32P are used. The filters are incubated in the hybridization mixture for 18-20 h at 40 ° C and then washed for 1.5 h at 55 ° C in a solution containing 2 x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and SDS 0.1%. The washing solution is replaced with fresh solution and incubated an additional 1.5 hours at 60 ° C. The filters are dried and exposed to autoradiography. If necessary, the filters are washed a third time at 65-68 ° C and re-exposed for film. Other conditions of low severity that can be used are well known in the art (eg, as used for cross-species hybridizations). In another preferred embodiment, a polynucleotide that hybridizes to the coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 or its complementary sequence under conditions of high severity and encodes a protein that retains the immunogenicity of the fusion proteins of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26 are also provided . For example, and not as a limitation, exemplary conditions of high stringency are as follows: prehybridization of filters containing DNA is performed for 8 hours or overnight at 65 ° C in a buffer solution composed of 6 x SSC, Tris- 50 mM HCl (pH 7.5), 1 mM EDTA, 0.02% Fich 0.02% PVP, 0.02% BSA, and 500 μg / ml denatured salmon sperm DNA. The filters are hybridized for 48 h at 65 ° C in a prehybridization mixture containing 100 μg / ml of denatured salmon sperm DNA and 5-20 x 106 cpm of the 32 P-labeled probe. The filters are washed at 37 ° C for 1 hour in a solution containing 2 x SSC, 0.01% PVP, 0.01% Ficol and 0.01% BSA. This is followed by a wash in 0.1 x SSC at 50 ° C for 45 minutes before autoradiography. Other conditions of high stringency that may be used are well known in the art. In yet another preferred embodiment, a polynucleotide that hybridizes to the coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23 and 25 or its complementary sequence under the conditions of moderate severity and encodes a protein encoding the immunogenicity of the fusion proteins of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26 are also provided. Exemplary conditions of moderate severity are as follows: filters containing DNA are pretreated for 6 hours at 55 ° C in a solution containing 6 x SSC, 5X Denhart's solution, 0.5% SDS and 100 μg / ml sperm DNA of denatured salmon. The hybridizations are carried out in the same solution and 5-20 x 106 cpm of the probe labeled with 72P is used. The filters are incubated in the hybridization mixture for 18-20 hours at 55 ° C and then washed twice for 30 minutes at 60 ° C in a solution containing 1 x SSC and 0.1% SDS. The filters are dried by absorption and put to autoradiography. Other conditions of moderate severity that can be used are well known in the art. The filters are washed at 37 ° C for 1 hour in a solution containing 2 x SSC, 0.1% SDS. . 4 POLYPEPTIDES CODED BY CODING SEQUENCES According to the invention, a polynucleotide of the invention that codes for a fusion protein, fragments of this or functional equivalents thereof can be used to generate recombinant nucleic acid molecules that direct the expression of the fusion protein, fragments thereof or functional equivalents thereof, in suitable host cells. The fusion polypeptide products encoded by these polynucleotides can be altered by molecular manipulation of the coding sequence. Due to the inherent generation of genetic code, other DNA sequences that encode practically the same amino acid sequence or a functionally equivalent one, can be used in the practice of the invention for the expression of the fusion polypeptides. These DNA sequences include those that are capable of hybridizing to the coding sequences or their complements described herein under the conditions of low, moderate or high severity described in sections 5.3, supra. Altered nucleotide sequences that can be used according to the invention include deletions, additions or substitutions of different nucleotide residues giving rise to a sequence encoding the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of the amino acid residues, which gives rise to a silent change thus producing an antigenic epitope with equivalent functionality. These conservative amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the unfriendly nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine, histidine and arginine; amino acids with uncharged polar groups having similar hydrophilicity values include the following: glycine, asparagine, glutamine, serine, threonine and tyrosine; and amino acids with non-polar head groups include alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine and tryptophan. The nucleotide sequences of the invention can be manipulated to alter the sequence encoding the fusion protein for different purposes, including but not limited to, alterations that encode the processing and expression of the gene product. For example, it is possible to introduce mutations using techniques that are well known, for example, site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, and so on. In an alternative embodiment of the invention, the coding sequence of a fusion protein can be synthesized in whole or in part, using well-known chemical methods. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7: 215-233; Crea and Horn, 180, Nuc Acids Res 9 (10): 2331; Matteucci and Caruthers, 1980, Tetrahedron Letter 21: 719; and Chow and Kempe, 1981, Nuc Acids Res. 9 (12): 2807-2817. Otherwise, the polypeptide itself can be produced using chemical methods to synthesize an amino acid sequence in its entirety or in parts, for example, the peptides can be synthesized by solid phase techniques. Dissociated from the resin and purified by preparative high-performance liquid chromatography (see, Creighton, 1983, Proteins Structures and Molecular Principles, W. H. Freeman and Co., N. Y. pp. 50-60). The composition of the synthetic polypeptides can be confirmed by analysis or sequencing of the amino acids (for example, the Edman degradation procedure, see Creighton, 1983, Proteins, Structures and Molecular Principles, WH Freeman and Co, NY pp. 34-49) . In addition, the coding sequence of a fusion protein can be mutated in vi tro in vivo to create and / or destroy the translation, initiation and / or termination sequences, or to create variations in the coding regions and / or form new sites of restriction endonuclease or destroy the pre-existing, to facilitate further modification in vi tro. It is possible to use any known mutagenesis technique, including, but not limited to, chemical mutagenesis, site-directed mutagenesis in vi tro (Hutchinson, C, et al., 1978, J. Biol. Chem. 253: 6551), the use of ® linkers (Pharmacia), and the like. It is important that the manipulations destroy the immunogenicity of the fusion polypeptides. In addition, non-classical amino acids or analogs of chemical amino acids can be introduced as a substitution or addition in the sequence. Non-classical amino acids include, but are not limited to, the D isomers of the common amino acids, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid,? -Abu, e-Ahx, 6-aminohexanoic acid , Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ß-alanine, fluoro-amino acids, amino acids designers such as ß-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids and amino acid analogs in general. In addition, the amino acid can be D (dextrorotatory) or L (Levorotatory). In a specific embodiment, the coding sequences of each antigen in the fusion protein are joined at their amino or carboxyl terminal through a peptide bond in any order. Otherwise, it is possible to employ a peptide linker sequence to separate the individual polypeptides that make up a fusion polypeptide at a sufficient distance to ensure that each polypeptide is doubled in its secondary and tertiary structure which maximizes its antigenic efficacy to prevent and treat tuberculosis. This peptide linker sequence is incorporated into the fusion protein using standard, well-known techniques. Suitable peptide linker sequences can be chosen based on the following factors: (1) their ability to adopt an extended, flexible conformation; (2) its inability to adopt a secondary structure that can interact with the functional epitopes in the primary and secondary polypeptides; and (3) the lack of hydrophobic or charged residues that can interact with the functional epitopes of the polypeptide. The preferred peptide linker sequences contain the Gly, Asn and Ser residues. Other almost neutral amino acids such as Thr and Ala can also be used in the linker sequence. The amino acid sequences that can be used as linkers include those that are described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Nati Acad. Sci USA 83: 8258-8262, 1986, U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. The linker sequence can be from 1 to about 50 amino acids in length. Peptide sequences are not necessary when the first and second polypeptides have non-essential n-terminal amino acid regions that can be used to separate the functional domains and prevent spherical interference. For example, the antigens in a fusion protein can be connected by a flexible polylinker such as Gly-Cys-Gly or Gly-Gly-Gly-Gly-Ser repeated 1 to 3 times (Bird et al., 1988, Science 242: 423-426; Chaudhary et al., 1990, Proc. Nati, Acad. Sci. USA 87; 1066-1070). In one embodiment, this protein is produced by recombinant expression of a nucleic acid encoding the protein. This fusion product can be prepared by ligating the nucleic acid sequences coding for the desired amino acid sequences to each other by methods known in the art, in the proper coding framework, and expressing the product by methods known in the art. Otherwise, this product can be elaborated by protein synthesis techniques, for example, by the use of a peptide synthesizer. The coding sequences for other molecules for cytokine or an adjuvant can also be added to the fusion polynucleotide. . 5 PRODUCTION OF FUSION PROTEINS To produce a M. tuberculosis fusion protein of the invention, the nucleotide sequence encoding the protein, or a functional equivalent, is inserted into a suitable expression vector, ie, a vector containing the necessary elements for the transcription and translation of the inserted coding sequence. Host cells or cell lines transfected or transformed with the recombinant expression vectors can be used for a variety of purposes. These include, but are not limited to, the large-scale production of the fusion protein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the fusion coding sequence and the appropriate transcription / translation control signals. These methods include recombinant DNA techniques, synthesis techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, NY and Ausubel, et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley. Interscience, NY). RNA capable of encoding a polypeptide can also be chemically synthesized (Gait, ed., 1984, Oligonucleotide Synthesis, IRL Press, Oxford). 5.5.1. EXPRESSION SYSTEMS It is possible to use a variety of host / expression vector systems to express a coding sequence for the fusion protein. These include, but are not limited to, micro-organisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, expression vectors plasmid DNA or cosmid DNA containing a coding sequence; yeast (eg, Saccharomycdes, Pichia) transformed with recombinant yeast expression vectors containing a coding sequence; insect cell systems infected with recombinant virus expression vectors (eg, baculovirus), containing a coding sequence; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transfected with recombinant plasmid expression vectors (eg, Ti plasmid) containing a coding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells). The expression elements of these systems vary in their concentration or intensity and specificities. Depending on the host / vector system used, any of a number of convenient transcription and translation elements, including constitutive and inducible promoters in the expression vector, can be used. for example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage γ, plac, ptrp, ptac (hybrid ptrp-lac promoter, cytomegalovirus promoter) and the like can be used; when cloning in insect cell systems, it is possible to use promoters such as the baculovirus polyhedron promoter; when cloning into plant cell systems, promoters obtained from plant cell genome (eg, heat shock promoters, the promoter for the small subunit of RUBISCO, the promoter for the chlorophyll a / b binding protein) or plant viruses (for example, the CaMV 35S RNA promoter, the TMV coat protein promoter) can be used, when cloning into mammalian cell systems, it is possible to use the promoters obtained from the mammalian cell genome (eg, the metallothionein promoter) or mammalian virus (eg, the adenovirus late promoter, the 7.5 K promoter of the vaccinia virus); When cell lines containing multiple copies of a sequence encoding the antigen are generated it is possible to use vectors based on SV40, BPV and EBV with the appropriate selectable marker. Bacterial systems are preferred for the expression of M. tuberculosis antigens. For in vivo delivery, a bacterium such as bacillus-calmette-guerrin can be engineered to express a fusion polypeptide of the invention on its cell surface. A number of other bacterial expression vectors may conveniently be selected depending on the intended use for the expressed products, for example, when large quantities of the fusion protein are to be produced for formulation of pharmaceutical compositions, vectors that direct expression may be desirable. of high concentrations of fusion protein products that are easily purified. These vectors include, but are not limited to, the E. coli expression vector pUR278 (Rfuther et al., 1983, EMBO J. 2: 1791), in which a coding sequence can be ligated into the vector in the frame with the lacZ coding region so that a hybrid protein is produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids, Res. 13: 3101-3109, Van Heeke and Schuster, 1989, J. Biol. Chem. 264: 5503-5509); and similar. It is also possible to use the pGEX vectors to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, these fusion proteins are soluble and can be easily purified from cells used by absorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease dissociation sites so that the cloned fusion polypeptide of interest can be released from the GST portion. . 5.2. PURIFICATION OF PROTEINS once a recombinant protein is expressed, it can be identified by assays based on the physical or functional properties of the product, including radioactive labeling of the product followed by gel electrophoresis, radioimmunoassay, ELISA, bioassays, etc. Once the encoded protein is identified, it can be isolated and purified by standard methods including chromatography (eg, high performance liquid chromatography, ion exchange, affinity and column chromatography by size), centrifugation, differential solubility or by any of the other normal techniques for protein purification. Actual conditions will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. The functional properties can be evaluated using any of the convenient assays such as antibody binding, induction of T cell proliferation, stimulation of cytokine production such as IL-2, IL-4 and IFN-α. For the practice of the present invention, it is preferred that each fusion protein be at least 80% purified from other proteins. It is more preferred that it be at least 90% purified. For in vivo administration, it is preferred that the proteins be greater than 95% purified. F . 6. USES OF THE FUSION PROTEIN CODING SEQUENCE The coding sequence of the fusion protein of the invention can be used to encode a protein product for use as an immunogen to induce and / or improve immune responses to M. tuberculosis. . Further, This coding sequence can be linked to a coding sequence of another molecule such as cytokine or an adjuvant. These polynucleotides can be used in vivo as a DNA vaccine (U.S. Patent Nos. 5,589,466; 5,679,647; 5,703,055). In this modality of In another embodiment of the invention, the polypeptide expresses its encoded protein in a container to directly induce an immune response. The polypeptide can be injected into a natural individual to initiate an immune response to its encoded product, or administered to an infected individual or immunized to improve secondary immune responses. In a preferred embodiment, a therapeutic composition consists of a sequence encoding the fusion protein or fragments thereof that are part of an expression vector. In particular, such a polynucleotide contains a The promoter operably linked to the coding region, wherein the promoter is inducible or constitutive, and optionally tissue-specific, in another embodiment, a polynucleotide contains a coding sequence flanked by regions that favor homologous recombination at a desired site in the genome, thus providing intrachromosomal expression of the coding sequence (Koller and Smithires, 1989, Proc Nati Acad Sci USA 86: 8932-8935; Zijlstra et al., 1989, Nature 342: 435-438). The delivery of the nucleic acid to an individual can be direct, in which case the individual is directly exposed to the nucleic acid or vector carrying the nucleic acid or indirectly, in which case the cells are first transformed with the nucleic acid in vitro then transplanted to the nucleic acid. individual. These two approaches are known, respectively, as in vivo or ex vivo gene transfer. In a specific embodiment, the nucleic acid is administered directly in vivo, where it is expressed to produce the encoded fusion protein product. This can be achieved by any of the various methods known in the art, for example, by building it as part of a suitable nucleic acid expression vector or by administering it to make it intracellular, for example, by infection using a defective or attenuating retroviral vector or other vector viral (see, U.S. Patent No. 4,920,286), or by direct injection of naked DNA, or by the use of microparticle bombardment (e.g., a gene gun); Biolistic, Dupont) or coating with lipids or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles or microcapsules (US Pat. Nos. 5,407,609; 5,853,363; 5,814,344 and 5,820,863), or by administering it in linkage to a peptide that is known enters the nucleus, administering it in ligand to a ligand subject to endocytosis mediated by the receptor (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 34429-4432) which can be used for cell types chosen that specifically express the receivers, et cetera. In another embodiment, the nucleic acid-ligand complex can be formed in which the ligand contains a fusogenic viral peptide for the disruption of endosomes, allowing the nucleic acid to avoid lysosomal degradation. In still another embodiment, the nucleic acid can be directed in vivo for specific uptake and expression of the cells, by choosing a specific receptor (see, for example, PCT publications WSO 92/07180 dated April 16, 1992; / 22635 of December 31, 1992; WO 92/20316 dated November 26, 1992; WO 93/14188 of July 22, 1993; WO 93/20211 of October 14, 1993). Otherwise, the nucleic acid can be introduced intracellularly and incorporated into host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc Nati Acad Sci USA 86: 8932-8935; Zijlstra et al., 1989, Nature 342: 435-438). In a specific embodiment, it is possible to use a viral vector as a retroviral vector (see Miller et al., 1993, Meth, Enzymol, 217: 581-599). Retroviral vectors have been modified for the deletion of retroviral sequences that are not necessary for packaging the viral genome and integration into the DNA of the host cell. A coding sequence of the fusion is cloned into the vector, which facilitates delivery of the nucleic acid into a container. More details about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6: 291-302, which describes the use of a retroviral vector to deliver the mdr 1 gene with hematopoietic primordial cells to make the primordial cells more resistant. to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93: 644-651; Kiern et al., 1994, Blood 83: 1467-1473; Salmons and Gubzberg, 1993, Human Gene Therapy 4: 129-141; and Grossman and Wilson, 1993, Curr. Opin. In Genetics and Devel. 3: 110-114.

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for the supply of genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium where they cause moderate disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Adeno-associated viruses (AAV) have also been proposed for use in gene transfer in vivo, (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med 204: 289-300.) Other approach includes transferring a construct to the cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection or viral infection.The method of transfer usually involves the transfer of a selectable marker to the cells.The cells are then placed under selection to isolate those cells that have captured and are expressing the transferred gene.These cells are then delivered to an individual.In this modality the nucleic acid is introduced into a cell before the in vivo administration of the resulting recombinant cell.This introduction can be done by any method known in the art, which includes but is not limited to transfection, electroporation, microinjection, infection with a vec viral or bacteriophage, containing nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, micro-cell-mediated gene transfer, spheroplast fusion, and so on. Numerous techniques are known for the introduction of foreign genes into cells (see, for example, Loeffer and Behr, 1993, Meth, Enzymol, 217: 599-618, Cohen et al., 1993, Meth. Enzymol, 217: 618-644). Cline, 1985, Pharmac Ther 29: 62-92) and can be used in accordance with the present invention. The polynucleotides of the invention can also be used in the diagnosis of tuberculosis for the detection of polynucleotide sequences specific for M. tuberculosis in a patient. Such detection can be achieved, for example, by isolating polynucleotides from a biological sample obtained from a patient suspected of being infected with the bacterium. With the isolation of the polynucleotides from the biological sample, a labeled polynucleotide of the invention that is complementary to one or more of the polynucleotides, will be allowed to hybridize to the polynucleotides in the biological sample using the nucleic acid hybridization techniques known to the skilled artisan. in the technique. For example, such hybridization can be carried out in solution or with a counterpart of the hybridization on a solid support. . 7 THERAPEUTIC AND PROPHYLACTIC USES OF FUSION PROTEIN Purified or partially purified fusion proteins or fragments thereof can be formulated as a vaccine or therapeutic composition. Such a composition may include adjuvants to improve immune responses. In addition, these proteins may additionally be suspended in an oily emulsion or cause a slower release of the proteins in vivo with injection. The optimum proportions of each component in the formulation can be determined by techniques well known to the experts. Any of a variety of adjuvants can be employed in the vaccines of this invention to improve the immune response. Most of the adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A Bordatella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are available commercially and include, for example, incomplete Freund's adjuvant and Freund's complete adjuvant (Difco Laboratories) and Merck's adjuvant 65 (Merck and Company, Inc. Rahway, NJ) other convenient adjuvants include alum, biodegradable micro spheres, monophosphoryl lipid quilA, SBASIlc, SBAS2 (Ling et al., 1997, Vaccine 15: 1562-1567), SBAS7 and Al (OH) 3. In vaccines of the present invention, it is preferred that the adjuvant induces an immune response containing Thl aspects. The adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3-de-0-acylated monophosphoryl lipid A (3D-MLP) together with an aluminum salt. An improved system includes the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of 3D-MLP and saponin QS 21 as described in WO 94/00153, or a less reactogenic composition where QS 21 is depleted with cholesterol as described in WO 96/33739. Previous experiments have demonstrated a clear synergistic effect of the combinations of 3D MLP and QS 21 in the induction of humoral and Thl-type cellular immune responses. A particularly potent adjuvant formation including QS 21, 3D-MLP and tocopherol is an oil-in-water emulsification as described in WO 95/17210 and is a preferred formulation. The formulations containing an antigen of the present invention may be administered to an individual per se or in the form of a pharmaceutical or therapeutic composition. The pharmaceutical compositions containing the proteins can be manufactured by the traditional processes of mixing, dissolving granulation, dragee manufacture, levigation, emulsification, encapsulation or lyophilization. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate the processing of the polypeptides in the preparations that can be used in pharmaceutical forms. The proper formulation depends on the chosen route of administration. For topical administration, the proteins can be formulated as solutions, gels, ointments, cream suspensions, etc., as is well known in the art. Systemic formulations include those designed for administration by include, for example, subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal, as well as those designed for transdermal, transmucosal, oral or pulmonary administration. For injection, the proteins can be formulated in aqueous solutions, preferably in physiologically compatible buffer solutions such as Hank's solution, Ringer's solution or physiological saline buffer. The solution may contain agents for the formulation, such as suspending, stabilizing and / or dispersing agents. Otherwise, the proteins may be in powder form for reconstitution with a convenient vehicle, eg, with pyrogen-free water, sterile before use. For transmucosal administration, it is possible to use in the formulation the appropriate penetrants for the barrier that is going to be permeated. These penetrants are generally known in the art. For oral administration, a composition can be easily formulated by combining the proteins with pharmaceutically acceptable carriers well known in the art. These carriers allow the proteins to be formulated as tablets, pills, dragees, capsules, liquids, syrup gels, slurries, suspensions and the like, for oral ingestion by an individual to be treated. For oral formulations, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol.; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, it is possible to add disintegrating agents such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate.

If desired, the solid dosage forms can be sugar coated or enteric coated using standard techniques. For oral liquid preparations such as, for example, elixir suspensions and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, and the like. In addition, it is possible to add flavoring agents, preservatives, coloring agents and the like. For buccal administration, the proteins can take the form of tablets, dragees, etc. formulated in conventional manner. For administration by inhalation, proteins for use in accordance with the present invention are conveniently supplied in the form of an aerosol spray from pressurized containers or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, dichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other convenient gas. In the case of pressurized aerosol, the dosage unit can be determined by providing a valve to supply a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mixture of the proteins and a convenient powder base such as lactose or starch.

The proteins can also be formulated in rectal, vaginal compositions such as suppositories or retention enemas, for example, containing the bases for traditional suppositories such as cocoa butter or other glycerides. In addition to the formulations described above, the proteins can also be formulated as a depot preparation, such long-acting formulations can be administered by implantation (eg, subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the proteins can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Otherwise, it is possible to employ other pharmaceutical delivery systems. Liposomes and emulsions are well known examples of delivery vehicles that can be used to deliver an antigen. Certain organic solvents such as dimethyl sulfoxide can also be used, although usually with a higher toxicity cost. The fusion proteins can also be encapsulated in microspheres (U.S. Patent Nos. 5,407,609, 5,853,763, 5,814,344 and 5,820,883). In addition, the proteins can be delivered using prolonged release systems, such as semipermeable matrices of solid polymers containing the therapeutic or vaccination agent. Various materials have been established for prolonged release and are well known to those skilled in the art. The extended-release capsules may, depending on their chemical nature, release the proteins for a few weeks to more than 100 days. Depending on the chemical nature and the biological stability of the reagent, it is possible to employ additional strategies for the stabilization of the protein. The determination of an effective amount of the fusion protein to induce an immune response in an individual is within the capabilities of one skilled in the art, especially in light of the detailed description that is provided herein. An effective dose can be estimated initially from in vitro tests. For example, it is possible to formulate a dose in animal models to obtain an induction of an immune response using well-known techniques. A person with ordinary skill in the art can easily optimize for administration in humans based on animal data. The amount of the dose and the interval can be adjusted individually. For example, when used as a vaccine, the polypeptides and / or polynucleotides of the invention may be administered in about 1 to 3 doses over a period of 1-36 weeks. Preferably, 3 doses are administered at intervals of 3-4 months, and booster vaccines can be given periodically thereafter. Alternate protocols may be suitable for individual patients. A convenient dose is an amount of polypeptide or DNA which, when administered as already described, is capable of raising an immune response in an immunized patient sufficient to protect the patient from M. tuberculosis infection for at least 1-2 years. In general, the amount of polypeptide present in a dose (or produced in itself by the DNA in a dose) ranges from about lpg to about 100 mg per host kg, usually from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 μg. The convenient dose range will vary with the size of the patient, but will usually range from about 0.1 ml to about 5 ml. . 8 DIAGNOSTIC USES OF FUSION PROTEIN The fusion polypeptides of the invention are useful in the diagnosis of tuberculosis infection in vi tro and in vivo. The ability of a polypeptide of the invention to induce cell proliferation or cytokine production can be assayed by the methods described in Section 5.2, supra. In another aspect, this invention provides methods for using one or more of the fusion polypeptides to diagnose tuberculosis using an in vivo skin test. When used herein, a skin test is any test that is performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as swelling, redness or dermatitis) is measured after intradermal injection of one or more polypeptides as already described. Such injection can be done using any convenient device sufficient to contact the polypeptide with skin cells of the patient, such as for example a tuberculin syringe or 1 ml syringe. Preferably, the reaction is measured at least about 48 hours after injection, more preferably about 48 hours to about 72 hours after injection. The DDT reaction is a cell-mediated immune response, which is greater in patients who have been previously exposed to the test antigen (i.e., the immunogenic portion of the polypeptide being used, or a variant thereof). The answer can be measured visually, using a rule. In general, a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection, which may or may not be manifested as an active disease. The fusion polypeptides of this invention are preferably formulated for use in a skin test, as pharmaceutical compositions containing a polypeptide and an acceptable carrier for physiological use. These compositions usually contain one or more of the above polypeptides in an amount ranging from about 1 μg to about 100 μg, preferably from about 10 μg to about 50 μg in a volume of 0.1 ml. Preferably, the carrier used in such pharmaceutical compositions is a saline solution with suitable preservatives, such as phenol and / or Tween 80®.

In another aspect, the present invention provides methods for using the polypeptides to diagnose tuberculosis. In this regard, methods for detecting M. tuberculosis infection in a biological sample are provided using the fusion polypeptides alone or in combination. As used herein, a "biological sample" is any sample that contains antibodies obtained from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is blood, serum or plasma obtained from a patient or a blood supply. The polypeptide (s) are used in an assay, as described below, to determine the presence or absence of antibodies to the polypeptide (s) in the sample relative to a predetermined cutoff value. The presence of these antibodies indicates prior sensitization to mycobacterial antigens that may be indicative of tuberculosis. In embodiments in which more than one fusion polypeptide is employed, the polypeptides used are preferably complementary (ie, a component polypeptide will tend to detect infection in samples where the infection would not be detected by another component polypeptide). Complementary polypeptides can generally be identified using each polypeptide individually to evaluate serum samples obtained from a series of patients who are known to be infected with M. tuberculosis. After determining which samples prove positive (as described below) with each polypeptideit is possible to formulate combinations of two or more fusion polypeptides that are capable of detecting infection in most, or all, of the samples reviewed. These polypeptides are complementary. Approximately 25-30% of the sera of individuals infected with tuberculosis are negative for antibodies to any individual protein. Therefore, the complementary polypeptides can be used in combination to improve the sensitivity of a diagnostic test. There are different assay formats known to those skilled in the art to use one or more polypeptides in order to detect antibodies in a sample. See, for example Harlow and La, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incorporated herein by reference. In a preferred embodiment, the assay includes the use of immobilized polypeptide on a solid support to bind to and withdraw the antibody from the sample. The bound antibody can then be detected using a reagent for detection containing a reporter group. Suitable reagents for detection include antibodies that bind to the antibody / polypeptide complex and release the polypeptide labeled with a reporter group (e.g., in a semi-competitive assay). Otherwise, it is possible to use a competitive assay, in which an antibody that binds to the polypeptides is labeled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The degree to which the components of the sample inhibit the binding of the labeled antibody to the polypeptide is indicative of the reactivity of the sample with the immobilized polypeptide. The solid support can be any solid material known to those skilled in the art to which the antigen can be attached. For example, the solid support can be a test well in a microtiter plate or a nitrocellulose membrane or other convenient one. Otherwise, the support may be a bead or disc, such as glass, fiberglass, latex or plastic material, such as polystyrene or polyvinyl chloride. The support can also be a magnetic particle or a fiber optic detector, such as those described, for example in U.S. Patent No. 5,359,681. The polypeptides can be attached to the solid support using a variety of techniques known to those skilled in the art. In the context of the present invention, the term "attached" refers to non-covalent association, such as adsorption, and covalent binding (which may be a direct link between the antigen and functional groups on the support or may be a linkage by means of a crosslinking agent). Bonding by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption can be achieved by contacting the polypeptide in a convenient buffer solution with the solid support for a sufficient amount of time. The contact time varies with the temperature, but it is usually between one hour and one day. In general, the contact in a well of a microtiter plate, of plastic (such as polystyrene or polyvinyl chloride) with an amount of polypeptide in the range from about 10 ng to about 1 μg, and preferably about 100 nm, it is enough to bind an adequate amount of the antigen. The covalent attachment of the polypeptide to a solid support can generally be obtained by first reacting the support with a bifunctional reagent that will react with the support and a functional group, such as a hydroxyl or amino group, on the polypeptide. For example, the polypeptide can be attached to supports having a suitable polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen in the polypeptide (see, for example, Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13). In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). This assay can be performed by first contacting a fusion polypeptide antigen that has been immobilized on a solid support, usually the well of a microtiter plate, with the sample, so that the antibodies for the polypeptide within the sample are let them bind to the immobilized polypeptide. The unbound sample is then removed from the immobilized polypeptide and a detection reagent capable of binding to the immobilized antibody / polypeptide complex is added. The amount of reagent for detection remaining bound to the solid support is then determined using a method suitable for the specific detection reagent. More specifically, once the polypeptide is immobilized on the support as described above, the remaining protein binding sites on the support are usually blocked. It is possible to employ any convenient blocking agent known to those skilled in the art, such as bovine serum albumin or Tween 20®.

(Sigma, Chemical Co., St. Louis, MO). Then, the immobilized polypeptide is incubated with the sample, and the antibody is allowed to bind to the antigen. The sample can be diluted with a convenient diluent, such as phosphate-buffered saline (PBS) before incubation. In general, a suitable contact time is that time which is sufficient to detect the presence of the antibody within a sample infected with M. tuberculosis. Preferably, the contact time is sufficient to obtain a degree of binding that is at least 95% of that achieved in the balance between bound and unbound antibody. Those skilled in the art will realize that the time necessary to achieve equilibrium can be easily determined by testing the degree of binding occurring during a given time. At room temperature, an incubation time of approximately 30 minutes will generally be sufficient. Then, the unbound sample can be removed by washing the solid support with a suitable buffer, such as PBS with a content of 0.1% Tween 20®. The reagent for detection can then be added in solid support. A suitable reagent for detection is any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a number of means known to those skilled in the art. Preferably, the detection reagent contains a binding agent (e.g., protein A, protein G, lecithin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, biotin, and colloidal particles, such as colloidal gold and colloidal selenium. The conjugation of the binding agent to the reporter group can be obtained using the normal methods known to those skilled in the art. The common binding agentles can also be purchased conjugated to a variety of reporter groups from different commercial sources (eg, Zymed Laboratores, San Francisco, CA, and Pierce, Rockford, IL). The reagent for detection is then incubated with the entire immobilized antibody-polypeptide for a sufficient amount of time to detect the bound antibody. An adequate amount of time can usually be determined from the manufacturer's instructions or by testing the degree of bonding that occurs over a period of time. The reagent for unbound detection is then removed and the detection reagent bound is detected using the reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation or autoradiographic methods are generally adequate. The spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin, coupled to a different reporter group (usually a radioactive or fluorescent group or an enzyme). Enzymatic reporter groups can generally be detected by the addition of substrate (generally, for a specific time) followed by spectroscopic analysis or another of the reaction products. To determine the presence or absence of anti-Ai antibodies. In the sample, the detected signal of the reporter group that remains attached to the solid support is generally compared to a signal corresponding to a predetermined cutoff value. In a preferred embodiment, the cut-off value is the average signal obtained when the immobilized agent is incubated with samples from an uninfected patient. In general, a sample that generates a signal that is three standard deviations per enzyme of the predetermined cutoff value is considered positive for tuberculosis. In an alternative preferred embodiment, the cut-off value is determined using a Receiver Operator Curve according to the method of Sackett et al. 1985, Clinical Epidemiology: A Basic Science for Clinical Medicine, Litte Brown and Co., pp. 106-107. In summary, in this modality, the cut-off value can be determined from a graph of pairs of real positive rates (ie, sensitivity) and false positive rates (100% specificity) that correspond to each possible cut-off value for the result of the diagnostic test. The cutoff value on the graph that is closest to the upper left corner (that is, the value that encloses the largest area) is the most accurate cutoff value, and a sample that generates a signal greater than the value of Cut determined by this method can be considered positive. Otherwise, the cut-off value can be shifted to the left along the graph, to minimize a false positive rate, or to the right, to minimize the false negative rate. In general, a sample that generates a signal greater than the cut-off value determined by this method is considered positive for tuberculosis. In a related embodiment, the assay is performed in a fluid test or rapid strip format, where the antigen is immobilized on a membrane, such as nitrocellulose. In the through-flow test, antibodies within the sample bind to the immobilized polypeptide as the sample passes through the membrane. A reagent for detection (eg, protein A-colloidal gold) is then bound to the antibody-polypeptide complex as the solution containing the reagent for detection flows through the membrane. The detection of the reagent for bound detection can then be performed as already described. In the strip test format, one end of the membrane to which the polypeptide binds is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing the reagent for detection and the area of the immobilized polypeptide. The concentration of the reagent for detection in the polypeptide indicates the presence of anti-Ai antibodies. tuberculosis in the sample. Usually, the concentration of the reagent for detection in this site generates a pattern, such as a line, that can be read visually. The absence of this pattern indicates a negative result. In general, the amount of polypeptide immobilized on the membrane is selected to generate a visually perceptible pattern when the biological sample contains a concentration of antibodies that would be sufficient to generate a positive signal in an ELISA, as already described. Preferably, the amount of polypeptide immobilized on the membrane ranges from about 5 ng to about 1 μg, and most preferably from about 50 ng to about 500 ng. These samples can usually be made with a very small amount (for example, a drop) of the patient's blood or serum. The invention having been described, offers the following examples as an illustration and not as a limitation. 6. EXAMPLE: THE PROTEINS OF FUSION OF ANTIGENS FOR M. TUBERCULOSIS, CONSERVE THE IMMUNOGENICITY OF THE INDIVIDUAL COMPONENTS 6.1 MATERIALS AND METHODS 6.1.1 CONSTRUCTION OF THE FUSION PROTEINS The coding sequences of the antigens for Ai. tuberculosis were modified by PCR to facilitate their fusion and subsequent expression of the fusion protein. DNA amplification was performed using 10 μl of 10X ufP buffer, 2 μl of 10 mM dNTP, 2 μl each of the PCR primers at a concentration of 10 μM, 81.5 μl of water, 1.5 μl of ufP DNA polymerase [ sic] (Stratagene, La Jolla, CA) and 1 μl of DNA at a concentration of 70 ng / μl (for the TbRa3 antigen) or 50 ng / μl (for the 38 kD and Tb38-1 antigens). For the TbRa3 antigen, denaturation at 94 ° C was performed for 2 minutes, followed by 40 cycles of 96 ° C for 15 seconds and 72 ° C for one minute, and finally 72 ° C for 4 minutes. For the 38 kD antigen, denaturation at 96 ° C was performed for 2 minutes, followed by 40 cycles of 96 ° C for 30 seconds, 68 ° C for 15 seconds and 72 ° C for 3 minutes, and finally 72 ° C. C for 4 minutes. For the Tb38-1 antigen, denaturation at 94 ° C for 2 minutes was followed by 10 cycles of 96 ° C for 15 seconds, 68 ° C for 15 seconds and 72 ° C for 1.5 minutes, 30 cycles of 96 ° C for 15 seconds, 64 ° C for 15 seconds and 72 ° C for 1.5, and finally 72 ° C for 4 minutes. After digestion with a restriction endonuclease to produce the desired cohesive or blunt ends, a polypeptide specific for each fusion polypeptide was ligated into an expression plasmid. Each resulting plasmid contained the coding sequence for the individual antigens of each fusion polypeptide. The expression vectors used were pET-12b and pT7AL2lL1.

Three coding sequences for the Ral2, TbH9 and Ra35 antigens were ligated to encode a fusion protein (SEQ ID NO: 1 and 2) (Figure IA and 2B). Three other coding sequences for Erdl4, DPV and MTI antigens were ligated to encode a second fusion protein (SEQ ID NO: 3 and 4) (Figure 2). Three coding sequences for the TbRa3, 38 kD and Tb38-1 antigens were ligated to encode a fusion protein (SEQ ID NO: 5 and 6) (Figure 3a-3D). Two coding sequences for the TbH9 and Tb38-1 antigens were ligated to encode a fusion protein (SEQ ID NO: 7 and 8) (Figure 4A-4D). Four coding sequences for the TbRa3, 38kD, Tb38-1 and DPEP antigens were ligated to encode a fusion protein (SEQ ID NO: 9 and 10) (Figure 5A-5J). Five coding sequences for the Erdl4, DPV, MTI, MSL and MTCC2 antigens were ligated to encode a fusion protein (SEQ ID NO: 11 and 12) (Figure 6A and 6B). Four coding sequences for the Erdl4, DPV, MTI and MSL antigens were ligated to encode a fusion protein (SEQ ID NO: 13 and 14) (Figure 7A and 7B). Four coding sequences for the DPV, MTI, MSL and MTCC2 antigens were ligated to encode a fusion protein (SEQ ID NO: 15 and 16) (Figure 8A and 8B). Three coding sequences for the DPV, MTI and MSL antigens were ligated to encode a fusion protein (SEQ ID NO: 17 and 18) (Figure 9A and 9B).

Three coding sequences for the antigens TbH9, DPV and MTI were ligated to encode a fusion protein (SEQ ID NO: 19 and 20) (Figure 10A and 10B). Three coding sequences for the Erdl4, DPV and MTI antigens were ligated to encode a fusion protein (SEQ ID NO: 21 and 22) (Figure HA and 11B). Two coding sequences for the TbH9 and Ra35 antigens were ligated to encode a fusion protein (SEQ ID NO: 23 and 24) (Figure 12A and 12B). Two coding sequences for the Ral2 and DPPD antigens were ligated to encode a fusion protein (SEQ ID NO: 25 and 26) (Figure 13A and 13B). Recombinant proteins were expressed in E. coli with 6 histidine residues in the amino terminal portion using the plasmid vector pET (pET-14b) and an expression system RNA polymerase T7 (Novagen, Madison, Wl). Strain BL21 of E. coli (DE3) pLysE (Novagen) was used for high expression level. The recombinant fusion proteins (His-Tag) were purified from the soluble supernatant or the insoluble inclusion body of 500 ml of IPTG batch cultures induced by affinity chromatography using the QIAexpress Ni-NTA Agarose one-step matrix (QIAGEN, Chatsworth, CA) in the presence of 8M urea. Briefly, 20 ml of the BL21 overnight saturated culture containing the pET construct were added in 500 ml of 2xYT medium containing 50 μg / ml ampicillin and 30 μg / ml chloramphenicol, grown at 37 ° C with shaking. Bacterial cultures were induced with 2 mM IPTG at an OD 560 of 0.3, and grown for an additional 3 hours (OD = 1.3 to 1.9). Cells were harvested from 500 ml of batch cultures by centrifugation and resuspended in 20 ml of binding buffer (0.1 M sodium phosphate, pH 8.0; 10 mM Tris-HCl, pH 8.0) containing 2 mM PMSF and 20 μg / ml of leupeptin plus one complete tablet of the protease inhibitor (Boehringer Mannheim) per 25 ml. E. coli was used by freezing-thawing followed by brief sonification, then centrifuged at 12 k rpm [sic] for 30 minutes until the inclusion bodies were packed. The inclusion bodies were washed three times in 1% CHAPS in 10 mM Tris-HCl (pH 8.0). This step greatly reduced the degree of contaminating LPS. The inclusion body was finally solubilized in 20 ml of the binding buffer containing 8M urea, or the 8M urea was added directly into the soluble supernatant. The recombinant fusion proteins with His-Tag residues were bound in batches to Ni-NTA agarose resin (5 ml of resin per 500 ml of inductions) by rolling at room temperature for one hour and the complex passed over a column. The flow was passed twice over the same column and the column was washed three times with 30 ml each of washing buffer (0.1 M sodium phosphate and 10 mM Tris-HCl, pH 6.3) also containing 8M urea. The binding protein was eluted with 30 ml of 150 mM imidazole in wash buffer and 5 ml fractions were collected. The fractions containing the recombinant fusion protein were combined, dialyzed against 10 mM Tris-HCl (pH 8.0) once again bound to the Ni-NTA matrix, eluted and dialysed in 10 mM Tris-HCl (pH 7.8). The product of the recombinant protein varies from 25-150 mg per liter of bacterial culture induced with more than 98% purity. The recombinant proteins were tested for endotoxin contamination using the Limulus assay (BioWhittaker) and were shown to contain < 10 E. U. Img. 6. 1.2 T-CELL PROLIFERATION TEST The purified fusion polypeptides were tested for the ability to induce T cell proliferation in peripheral blood mononuclear cell preparations (PBMC). PBMC preparations of positive donors in skin PPD test and whose T cells were shown to proliferate in response to PPD and crude soluble M. tuberculosis proteins were cultured in RPMI 1640 supplemented with 10% combined human serum and 50 μg / ml of gentamicin. The purified polypeptides were added in duplicate at concentrations of 0.5 to 10 μg / ml. After 6 days of culture in 96-well round bottom plates, in a volume of 200 μl, 50 μl of medium was removed from each well for the determination of IFN-α concentrations, as described below in the Section 6.1.3. The plates were then pulsed with 1 μCi / well of tritiated thymidine for another 18 hours. Harvested and determined the tritium captured using a gas scintillation counter. The fractions observed in the cells cultured in the medium alone were considered positive. 6. 1.3 INTERFERON TEST-? The spleens of mice were separated in aseptic environment and suspensions of individual cells were prepared in complete RMPI followed by lysis of the erythrocytes. 100 μl of cells (2 x 10"5 cells [sic]) were plated per well in 96-well flat bottom microtiter plates, cultures were stimulated with the indicated recombinant proteins for 24 hours and the supernatant was assayed for IFN The concentrations of IFN-α in the supernatant were analyzed by sandwich ELISA, using pairs of antibodies and the available procedures of PharMingen.The standard curves were generated using recombinant mouse cytokines.The ELISA plates (Corning) were coated with 50 ml / well (1 μg / ml, in bicarbonate buffer for the 0.1 M coating, pH 9.6) of a cytokine capture mAb (rat anti-mouse IFN- (Pharmingen, Cat # 18181D)) and incubated for 4 hours at room temperature The contents of the plates were shaken and blocked with PBS 0.05% Tween, 1.0% BSA (200 μl / well) overnight at 4 ° C and washed with 6X in PBS-0.1 Tween. % .The standards (mouse IFN-?) And the samples After the supernatant diluted in PBS-Tween 0.05%, 0.1% BSA were then added for 2 hours at room temperature. The plates were washed as in the previous case and then incubated for 2 hours at room temperature with 100 μl / well of a second Ab (rat IFN-? Biotin to [sic] (Cat. # 18112D; Pharmingen) at 0.5 μg / ml diluted in PBS-0.05% Tween, 0.1% BSA After washing, the plates were incubated with 100 μl / well of streptavidin-HRP (Zymed) in a 1: 2500 dilution in PBS-0.05% Tween, 0.1% BSA ambient temperature for one hour The plates were washed one last time and developed with 100 μl / well of TMB substrate (3, 3 ', 5,5'-tetramethylbenzidine, Kirkegaard and Perry, Gaithersburg, (MD) and the reaction was stopped after color development, with H2S0, 50 μl / well Absorbances (OD) were determined at 450 nm using 570 nm as the reference wavelength and the cytokine concentration was evaluated using the standard curve. 6. 2 RESULTS 6.2.1 IMMUNE RESPONSES INDUCED BY TRIFUSION PROTEINS Three coding sequences for Ai antigens. tuberculosis were inserted into an expression vector for the production of a fusion protein. The antigens designated Ral2, TbH9 and Ra35 were produced as a recombinant fusion protein (Figure IA and IB). The Erdl4, DPV and MTI antigens were produced as a second fusion protein (Figure 2). The two fusion proteins were affinity purified for use in in vitro and in vivo assays. The two fusion proteins were tested for their ability to stimulate T cell responses from 6 PPD + individuals. When the proliferation of T cells was measured, both fusion proteins exhibited a similar reactivity pattern as their individual components (Figure 14A-14F). A similar result was obtained when the production of IFN-α was measured. (Figure 15A-15F). For example, individual D160 responded to the TbH9 and MTI antigens individually. Individual D160 also responded to the fusion proteins containing these antigens (Figure 14B and 15B). In contrast, in D160 no response of T cells to other antigens was observed individually. Another individual, D201, who did not react with the Erdl4, DPV or MTI antigens individually, was also not sensitive to the fusion protein containing these antigens. It should be noted that when the responses of the T cells to the individual components of the two fusion proteins were not particularly strong, the fusion proteins stimulated responses that were equal to or greater than those induced by the individual antigens in most cases . The trifunctional protein Ral2-TbH9-Ra35 was also tested as an immunogen in vivo. In these experiments, the fusion protein was injected into the legs of mice for immunization. Each group of three mice received the protein in a different adjuvant formulation: SBASlc, SBAS2 (Ling et al., 1997, Vaccine 15: 1562-1567), SBAS7 and AL (OH) 3 [sic]. After two subcutaneous immunizations at three-week intervals, the animals were sacrificed one week later, and their draining lymph nodes were harvested for use as responder cells in T-cell proliferation and cytokine production assays. Without taking into account the adjuvant that was used in the immunization, the strong T cell proliferation responses were induced against TbH9 when this was used as an individual antigen (Figure 16A). Weaker responses were induced against Ra35 and Ral2 (Figure 16B and 16C). When the Ral2-TbH9-Ra35 fusion protein was used as an immunogen, a response similar to that observed against the individual components was observed. When the cytokine production was measured, the adjuvants SBASlc and SBAS2 produced IFN-α responses.

(Figure 17) and IL-4 (Figure 8) similar. However, the combination of SBAS7 and aluminum hydroxide produced the IFN-α responses. stronger and the lowest level of IL-4 production for the three antigens. With respect to the humoral antibody response in vivo, Figure 19A-19F shows that the fusion protein developed antigen-specific IgGi and IgG2a responses when used with any of the three adjuvants. In addition, C57BL / 6 mice were immunized with a combination of two expression constructs each containing the coding sequence Ral2-TbH9-Ra35 (Mtb32A) or Erdl4-DPV-MTI (Mtb39A) as DNA vaccines. The immunized animals showed significant protection against tuberculosis with a subsequent aerosol challenge of living bacteria. Based on these results, a fusion construct of the Mtb32A and Mtb39A coding sequences was prepared, and its encoded product was tested in a long-term protection model in guinea pigs. In these studies, the guinea pigs were immunized with a single recombinant fusion protein or a mixture of Mtb32A and Mtb39A proteins in formulations containing adjuvant. Figure 20A-20C shows that guinea pigs immunized with the fusion protein in SBASlc or SBAS2 were better protected against the development of tuberculosis with the subsequent challenge, compared to animals immunized with the two antigens in a mixture in the same adjuvant formulation . The fusion proteins in the SBAS2 formulation produced the greatest protection in the animals. Thus, the fusion proteins of different Ai antigens. Tuberculosis can be used as more effective immunogens in vaccine formulations than a mixture of individual components. 6. 2.2 IMMUNE RESPONSES INDUCED BY BIFUSION PROTEINS A bifusion protein containing the TbH9 and Tb38-1 antigens without a hinge sequence was produced by the recombinant methods. The ability of the TbG9-Tb38-l fusion protein to induce T cell proliferation and IFN-α production It was examined. PBMC of three donors were employed: previously it had been shown that a donor responds to TbH9 but not to Tb38-1 (donor 131); one had responded to Tb38-1 but not to TbH9 (donor 184); and one had responded to both antigens (donor 201). The results of these studies demonstrate the functional activity of both antigens in the fusion protein (Figures 21A and 21B, 22A and 22B) and 23A and 23B). 6. 2.3 A TETRAFUSION PROTEIN IN REACTION WITH PATIENT SUITS WITH TUBERCULOSIS A fusion protein containing the antigen TbRa3, 38KD [sic], Tb38-1 and DPEP was produced by the recombinant methods. The reactivity of this tetrafusion protein known as TbF-2 with sera from patients infected with Ai. tuberculosis was examined by ELISA. The results of these studies (Table 1) demonstrate that the four antigens function independently in the fusion protein. One skilled in the art will realize that the order of the individual antigens within each fusion protein can change and that comparable activity would be expected as long as each of the epitopes is still functionally available. In addition, truncated forms of proteins containing active epitopes can be used in the construction of fusion proteins. The present invention is not limited in scope by the exemplified embodiments that are proposed as illustrations of the individual aspects of the invention, and any of the clones, nucleotides or amino acid sequences that are functionally equivalent are within the scope of the invention. In fact, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the aforementioned description and the accompanying drawings. These modifications are proposed to fall within the scope of the appended claims. It should also be understood that all sizes of the base pairs provided for the nucleotides are approximate and are used for description purposes. All publications mentioned herein are incorporated by reference in their entirety.

TABLE 1 REACTIVITY OF THE FUSION PROTEIN TbF-2 WITH TB AND NORMAL SUEROS

Claims (13)

1. A purified polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24, the amino acid sequence may optionally contain one or more conservative amino acid substitutions.

2. A purified polypeptide encoded by a polypeptide that hybridizes under moderately severe conditions to a second polypeptide that is complementary to a nucleotide sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8 , 10, 12, 14, 16, 18, 20, 22 and 24, the amino acid sequence induces an immune response for Ai. tuberculosis.

3. The polypeptide of claim 2, which is a soluble polypeptide.

4. The polypeptide of claim 2, which is produced by a recombinant DNA method.

5. The polypeptide of claim 2, which is produced by a chemical synthesis method.

6. The polypeptide of claim 2, which induces an antibody response.

7. The polypeptide of claim 2, which induces a T cell response.

8. The polypeptide of claim 2, which is fused to a second heterologous polypeptide.

9. A method for preventing tuberculosis is to administer to an individual an effective amount of the polypeptide of claim 1.

10. A method for preventing tuberculosis is to administer to an individual an effective amount of the polypeptide of claim 2.

11. One method of preventing tuberculosis is to administer to an individual an effective amount of a polypeptide encoding the polypeptide of claim 2.

12. A pharmaceutical composition containing the polypeptide of claim 2.

13. A pharmaceutical composition containing a polynucleotide. encoding the polypeptide of claim 2.