Fusion proteins of mycobacterium tuberculosis

1. Field of the Invention

The present invention provides an immunogenic polypeptide, polynucleotide, polypeptide, pharmaceutical composition, expression vector, vaccine composition, polypeptide for use in treating or preventing Mycobacterium tuberculosis infections, polynucleotide for use in treating or preventing Mycobacterium tuberculosis infections, and a method of producing the polypeptide.

2. State of the art

Tuberculosis is a chronic inflammatory disease caused by infection with M. tuberculosis. It is one of the most important diseases in developing countries as well as an increasing problem in developed regions of the world; approximately 8 million new cases and 3 million deaths from tuberculosis are reported annually. Although the infection may go undiagnosed for an extended period of time, the disease usually presents as acute pneumonia, with fever and a non-productive cough. If left untreated, it usually leads to serious complications and death.

Although tuberculosis can generally be controlled with long-term antibiotic therapy, such treatment is insufficient to prevent the spread of the disease. Infected people may be asymptomatic for a while and infect other people. In addition, while adherence is critical, it is difficult to monitor patient behavior. Some patients do not complete the entire treatment cycle, which may lead to treatment failure and the development of drug resistance.

Effective vaccination and accurate early diagnosis are essential to control the spread of TB. Currently, the most effective method of inducing protective immunity is vaccination with live microorganisms. The most common Mycobacterium used for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of M. bovis. However, the safety and efficacy of BCG are questionable and in some countries, such as the United States, the general public is not vaccinated with this strain.

Tuberculosis is usually diagnosed by a skin test involving the intradermal injection of PPD (purified protein derivative) tuberculin. Antigen-specific T cell responses produce measurable induration at the injection site 48-72 hours after injection, indicating exposure to Mycobacterial antigens. However, sensitivity and specificity are an issue with this test, and it is not possible to distinguish between BCG vaccinated and infected individuals.

Although macrophages have been shown to be the main effectors of resistance to M. tuberculosis, T cells are the main inducers of resistance. Evidence of the key role of T cells in protecting against M. tuberculosis infection is the high prevalence of M. tuberculosis in patients with acquired immune deficiency syndrome (AIDS). ), associated with a decrease in the number of CD4 lymphocytes⁺ caused by human immunodeficiency virus (HIV) infection. Mycobacterium-responsive CD4 + T cells have been shown to be potent inducers of interferon gamma (IFN-γ), which in turn has been shown to trigger macrophage action against Mycobacterium in mice. Although the role of IFN-γ in humans is less obvious, studies have shown that 1,25-dihydroxyvitamin D3, alone or in combination with IFN-γ or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis. Furthermore, IFN-γ is known to stimulate human macrophages to produce 1,25-dihydroxyvitamin D3. Similarly, interleukin-12 (IL-12) has been shown to play a role in stimulating resistance to M. tuberculosis infection. A review of the immunology of M. tuberculosis infection can be found in Chan and Kaufmann, 1994, Tuberculosis: Pathogenesis, Protection and Control, Bloom Ed., ASM Press, Washington, DC

Accordingly, there is a need for improved vaccines and improved methods for the diagnosis, prevention and treatment of tuberculosis.

The invention relates to an immunogenic polypeptide having a sequence of amino acid residues 8 through 729 in SEQ ID No. 2, optionally containing one conservative amino acid substitution.

Preferably, such an immunogenic polypeptide according to the invention is the sequence of amino acid residues 8 to 729 in SEQ ID No. 2, optionally containing one conservative amino acid substitution.

Also preferably, the immunogenic polypeptide may comprise the amino acid sequence of SEQ ID No. 2, which optionally comprises one conservative amino acid substitution. It may also preferably contain the sequence of amino acid residues 8 to 792 in SEQ ID No. 2, or the amino acid sequence of SEQ ID No. 2. More preferably, it is the amino acid sequence of SEQ ID No. 2.

The invention also provides a polynucleotide that encodes a polypeptide as defined above in all aspects disclosed herein.

The invention also provides a polynucleotide comprising a first nucleotide sequence which hybridizes under moderate hybridization conditions to a second polynucleotide sequence which is complementary to a nucleotide sequence encoding a sequence of amino acid residues 8 to 729 in SEQ ID NO: 2, said polynucleotide comprising the first nucleotide sequence encoding a polypeptide that is functionally equivalent to the sequence of amino acid residues 8 to 729 in SEQ ID NO: 2, in attempts to produce T cell proliferation or interferon gamma.

Preferably, the polynucleotide comprises a first nucleotide sequence that hybridizes under highly stringent hybridization conditions to a second polynucleotide sequence that is complementary to a nucleotide sequence encoding a sequence of amino acid residues 8 to 729 in SEQ ID NO: 2, said polynucleotide comprising the first nucleotide sequence encoding the polynucleotide which is functionally equivalent to the sequence of amino acid residues 8 to 729 in SEQ ID NO: 2, in the production of T cell proliferation or interferon gamma.

The invention also relates to a polypeptide encoded by a polynucleotide according to the invention as defined above. Preferably, the polypeptide can be produced by recombinant DNA methods, optionally, also preferably by chemical synthesis methods. The polypeptide encoded by the polynucleotide of the invention preferably induces a T cell response.

The polypeptide encoded by the polynucleotide of the invention may preferably be fused to a second heterologous polypeptide.

The invention also relates to a pharmaceutical composition, characterized in that it comprises a polypeptide as defined above.

The invention also relates to a pharmaceutical composition comprising the polynucleotide above.

The invention also relates to an expression vector, characterized in that it comprises a polynucleotide as defined above. Preferably, the expression vector may be a viral vector.

The invention also relates to a vaccine composition that comprises the polypeptides as defined above and an adjuvant.

The invention also relates to a vaccine composition that comprises the polynucleotides as defined above.

The invention also relates to any polypeptide as defined above for use in the treatment or prevention of M. Tuberculosis infections.

The invention also discloses a polynucleotide as defined above for use in the treatment or prevention of M. Tuberculosis infections.

The invention also relates to a method of producing the polypeptides as defined above, which comprises recombinantly expressing a polynucleotide as defined above.

The present invention is based in part on the authors' finding that polynucleotides that contain two to five M. tuberculosis coding sequences produce recombinant fusion proteins that retain the immunogenicity and antigenicity of their individual components. The fusion proteins of the present invention described herein induce both T and B lymphocyte responses, which can be measured by T cell proliferation, cytokine production, and antibody production. In addition, an adjuvanted fusion protein has been used as an immunogen in vivo to induce both cellular and humoral immunity against M. tuberculosis. In addition, a fusion protein construct was produced for use in the preparation of an adjuvanted vaccine to obtain long-term protection of animals against the development of tuberculosis. The fusion protein was more effective than the mixture of its individual protein components.

The immunogenic M. tuberculosis polypeptide of the present invention can be formulated into pharmaceutical compositions for administration to a patient to prevent and / or treat a M. tuberculosis infection. The immunogenicity of the fusion protein can be enhanced by adding an adjuvant.

According to another aspect of the invention, the polynucleotides of the invention are used to produce recombinant fusion polypeptide antigens in vitro.

Polynucleotides can be administered directly to a patient as DNA vaccines in order to express the antigen in the patient's body, and as a result, to elicit an immune response against M. tuberculosis.

PL 202 844 B1

It is also possible to use the polypeptides of the invention for the preparation of in vitro assays for the detection of humoral antibodies or cellular immunity against M. tuberculosis to recognize an infection or to monitor the progress of a disease. In addition, the immunogenic polypeptides of the invention can be used as an in vitro diagnostic agent in the form of an intradermal skin test. Additionally, these polypeptides can be used as immunogens for the production of antibodies to M. tuberculosis in the body of an animal (non-human). The antibodies can be used to detect target antigens in vivo and in vitro.

4. Brief description of the drawings

Figures 1A and 1B: Nucleotide sequence (SEQ ID No. 1) and amino acid sequence (SEQ ID No. 2) of the Ra12-TbH9-Ra35 trifusion protein (designated as Mtb32A)

Figure 2: Nucleotide sequence (SEQ ID No. 3) and amino acid sequence (SEQ ID No. 4) of the Erd14-DPV-MTI trifusion protein (designated as Mtb3 9A)

Fig. 3A-3D: Nucleotide sequence (SEQ ID No. 5) and amino acid sequence (SEQ ID No. 6) of the trifusion protein TbRa3-38kD-Tb38-1

Fig. 4A-4D: Nucleotide sequence (SEQ ID No. 7) and amino acid sequence (SEQ ID No. 8) of the bifusion protein TbH9-Tb38-1

Figures 5A-5J: Nucleotide sequence (SEQ ID No. 9) and amino acid sequence (SEQ ID No. 10) of the tetrafusion protein TbRa3-38kD-Tb38-1DPEP (designated as TbF-2)

Figures 6A and 6B: Nucleotide sequence (SEQ ID No. 11) and amino acid sequence (SEQ ID No. 12) of the Erd14-DPV-MTI-MSL-MTCC2 pentafusion protein (designated as Mtb88f)

Figures 7A and 7B: Nucleotide sequence (SEQ ID No. 13) and amino acid sequence (SEQ ID No. 14) of the Erd14-DPV-MTI-MSL tetrafusion protein (designated as Mtb46f)

Figures 8A and 8B: Nucleotide sequence (SEQ ID No. 15) and amino acid sequence (SEQ ID No. 16) of the DPV-MTI-MSL-MTCC2 tetrafusion protein (designated as Mtb71f)

Figures 9A and 9B: Nucleotide sequence (SEQ ID No. 17) and amino acid sequence (SEQ ID No. 18) of the DPV-MTI-MSL trifusion protein (designated as Mtb31f)

Figures 10A and 10B: Nucleotide sequence (SEQ ID No. 19) and amino acid sequence (SEQ ID No. 20) of the TbH9-DPV-MTI trifusion protein (designated as Mtb61f)

Figures 11A and 11B: Nucleotide sequence (SEQ ID No. 21) and amino acid sequence (SEQ ID No. 22) of the Erd14-DPV-MTI trifusion protein (designated as Mtb36f)

Figures 12A and 12B: Nucleotide sequence (SEQ ID No. 23) and amino acid sequence (SEQ ID No. 24) of the bifusion protein TbH9-Ra35 (designated as Mtb59f)

Figures 13A and 13B: Nucleotide sequence (SEQ ID No. 25) and amino acid sequence (SEQ ID No. 26) of the Ra12-DPPD bifusion protein (designated as Mtb24)

Figures 14A-14F: T cell proliferative responses of six PPD + individuals after stimulation with two fusion proteins and their separate components

Figures 15A-15F: IFN-γ production in six PPD + individuals after stimulation with two fusion proteins and their separate components

Figures 16A-16F: T cell proliferation of mice immunized with the fusion protein or its separate components and adjuvant

Figure 17: IFN-γ production of mice immunized with the fusion protein or its separate components and adjuvant

Figure 18: Production of IL-4 in mice immunized with the fusion protein or its separate components and adjuvant

Figures 19A-19F: Serum antibody concentration of mice immunized with the fusion protein or its separate components and adjuvant

Figures 20A-20C: Survival of guinea pigs after exposure to M. tuberculosis by aerosol. Prior to bacterial challenge in guinea pigs, the Mtb32A and Mtb39A fusion proteins were used as an immunogen in adjuvant formulation with SBAS1c (20A), SBAS2 (20B) or SBAS7 (20C). The positive control is BCG.

Figures 21A and 21B: Stimulation of proliferation and IFN-γ production in TbH9-specific T cells by the TbH9-Tb38-1 fusion protein.

Figures 22A and 22B: Stimulation of proliferation and IFN-γ production in Tb38-1-specific T cells by the TbH9-Tb38-1 fusion protein.

Figures 23A and 23B: Stimulation of proliferation and IFN-γ production in T cells previously shown to be responsive to both TbH-9 and Tb38-1 antigens by the TbH9-Tb38-1 fusion protein.

PL 202 844 B1

5. Detailed Description of the Invention

The present invention provides immunogenic polypeptides which are antigens useful in the treatment and prevention of tuberculosis, polynucleotides encoding such antigens, and methods of use thereof. The antigens of the present invention are fusion polypeptides of M. tuberculosis antigens and variants thereof. More specifically, these antigens comprise at least two M. tuberculosis polypeptides fused to a larger fusion polypeptide molecule. The antigens of the present invention may further include other components designed to enhance the immunogenicity of the antigens or to improve the antigens in other aspects, for example, isolating the antigens by adding extensions from histidine residues at either end of the antigen.

5.1. M. tuberculosis specific antigens

The antigens of the present invention are shown in Figures 1A-13B, including homologues and variants of these antigens. These antigens can be modified, for example, by adding peptide linker sequences as described below. These linker peptides can be incorporated between one or more of the polypeptides that make up each of the fusion proteins shown in Figs. 1A-13B. Other antigens of the present invention are those described in Figures 1A-13B linked to a known M. tuberculosis, such as the previously described 38 kD antigen (SEQ ID No. 27) (Andersen and Hansen, 1989, Infect. Immun. 57: 2481-2488; Genbank acquisition no. M30046).

5.2. Immunogenicity tests

The antigens of the present invention, and their immunogenic portions, are capable of eliciting an immunogenic reaction. More specifically, the antigens have the ability to induce proliferation and / or cytokine production (i.e., interferon-γ and / or interleukin 12 production) in T cells, NK cells, B cells and / or macrophages derived from a M. tuberculosis resistant individual. The choice of the type of cell to be used in the evaluation of an immunogenic response will depend on the reaction being tested. For example, the production of interleukin 12 is most easily assessed using preparations containing B cells and / or macrophages. A M. tuberculosis-resistant individual is one that is believed to be resistant to the development of tuberculosis by having successfully responded with T cells against M. tuberculosis (i.e., essentially symptom-free). Such individuals can be identified by the strong positive (i.e., sclerosis greater than 10 mm in diameter) skin test for tuberculosis proteins (PPD) and the absence of any signs and symptoms of tuberculosis. T cells, NK cells, B cells, and macrophages derived from a M. tuberculosis resistant individual can be produced by methods known to those skilled in the art. For example, a preparation of PBMC (i.e., peripheral blood mononuclear cells) can be used without further separation of the component cells. PBMCs can generally be prepared, for example, using density centrifugation via "FICOLL" (Winthrop Laboratories, NY). T cells for assays of the present invention can also be purified directly from PBMCs. Alternatively, an enriched T cell line against Mycobacterium proteins, or T cell clones reactive to individual Mycobacterium proteins can be used. Such T cell clones can be generated, for example, by culturing PBMCs from M. tuberculosis resistant individuals with Mycobacterium proteins for 2-4 weeks. This allows only the expansion of T cells specific for Mycobacterium proteins, resulting in a lineage composed only of such cells. These cells can then be cloned and tested with individual proteins using methods known to those skilled in the art to more accurately define the specificity of individual T cells. 12), made with T lymphocytes, NK cells, B lymphocytes and / or macrophages derived from M. tuberculosis is believed to be immunogenic. Such tests can be performed, for example, using the exemplary procedures described below. Immunogenic portions of such antigens can be identified using similar assays, and may be present in the polypeptides described below.

The ability of a polypeptide (e.g., an immunogenic antigen, or a portion or other variant thereof) to induce cell proliferation is assessed by bringing cells (e.g., T cells and / or NK cells) into contact with the polypeptide and measuring cell proliferation. In general, an amount of polypeptide sufficient to evaluate about 10⁵ of cells, it is from about 10 ng / ml to about 100 µg / ml, and is preferably about 10 µg / ml. Typically incubation of the polypeptide with the cells is carried out at 37 ° C for about six days. After incubation with the polypeptide, cells are tested for a proliferative response that can be assessed by methods known to those skilled in the art, for example, by exposing the cells to a pulse of radiolabeled thymidine and measuring the incorporation of the label into the cell DNA.

PL 202 844 B1

In general, a polypeptide that causes at least a threefold increase in proliferation above background level (i.e., the proliferation seen with cells cultured without the polypeptide) is considered capable of inducing proliferation. The ability of a polypeptide to stimulate the production of interferon γ and / or interleukin 12 can be assessed by bringing the cells into contact with the polypeptide and measuring the level of interferon γ or interleukin 12 produced by the cells. Generally, an amount of polypeptide that is sufficient to judge approximately

10⁵ of cells, is from about 10 ng / ml to about 100 µg / ml, and preferably, is about 10 µg / ml. The polypeptide may be, but not necessarily, immobilized on a solid support such as a bead or a biodegradable microsphere, such as those described in US Patent Nos. 4,897,268 and 5,075,109. Incubation of the polypeptide with the cells is typically conducted at 37 ° C for about six days. After incubation with the polypeptide, cells are tested for interferon γ and / or interleukin 12 (or one or more of its subunits), which can be assessed by methods known to those skilled in the art, such as an enzyme-linked immunosorbent assay (ELISA), or, in the case of the IL- P70 subunit. 12, a bioassay such as an assay that measures T cell proliferation. Generally, a polypeptide that produces at least 50 pg of gamma interferon per ml of culture supernatant (containing 10⁴-10⁵cells per ml) is believed to be capable of stimulating the production of interferon-y. A polypeptide that stimulates the production of at least 10 pg / ml of the IL-12 P70 subunit and / or at least 100 pg / ml of the IL-12 P40 subunit, per 10⁵ macrophages or B lymphocytes (or at 3x10⁵ PBMC) are believed to be capable of stimulating IL-12 production.

Generally, immunogenic antigens are those antigens that promote the proliferation and / or production of cytokines (i.e., interferon γ and / or interleukin 12) in T lymphocytes, NK cells, B lymphocytes, and / or macrophages from at least about 25% of subjects resistant to M. tuberculosis. Among these immunogenic antigens, polypeptides with favorable therapeutic properties can be distinguished based on the magnitude of the response in the above assays and on the percentage of individuals for whom a response is observed. In addition, antigens with favorable therapeutic properties will not promote proliferation or in vitro cytokine production in cells from more than 25% of M. tuberculosis, which eliminates responses not specifically caused by M. tuberculosis responsive cells. Antigens that stimulate a response in a high percentage of T-lymphocyte, NK-cell, B-cell, and / or macrophage preparations from M. tuberculosis-resistant individuals (with low response frequency in cell preparations from other individuals) have favorable therapeutic properties.

Antigens with favorable therapeutic properties can also be identified by their ability to reduce the severity of a M. tuberculosis infection in experimental animals when administered as a vaccine. Preferred vaccine formulations for use in experimental animals are described in detail below. Efficacy can be determined based on the ability of the antigen to provide at least about 50% reduction in bacterial counts and at least about 40% reduction in mortality following experimental infection. Preferred experimental animals include mice, guinea pigs, and primates.

5.3. Isolation of coding sequences

The present invention also provides nucleic acid molecules that encode M. tuberculosis fusion polypeptides. In the specific embodiments exemplified in Example 6 below, thirteen M. tuberculosis fusion coding sequences were built. According to the present invention, any nucleotide sequence that encodes the amino acid sequence of the fusion protein can be used to generate recombinant molecules that direct the expression of the encoding sequence.

To clone full-length coding sequences or homologous variants to generate fusion polynucleotides, labeled DNA probes made from any part of the nucleotide sequences or their complements of the present invention can be used to screen a genomic or cDNA library made up of different strains of M. tuberculosis in to identify the coding sequence of each component. Isolation of coding sequences can be performed by polymerase chain reaction (PCR) using two pools of degenerate oligonucleotide primers prepared from the coding sequences of the present invention.

The invention also discloses 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 polynucleotides selectively hybridizing to such complement sequences. In a preferred embodiment, a polynucleotide hybridizing to the sequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 is also provided.

PL 202 844 B1 and 25 or its complementary sequence under stringent hybridization conditions and encoding a protein that retains the immunogenicity of fusion proteins of SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26. As an example, the following low stringency hybridization conditions can be cited (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78: 6789-6792): DNA-containing filters 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, 0.1% Ficoll, 1% BSA and 500 pg / ml denatured salmon sperm DNA. Hybridization is carried out using the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg / ml salmon sperm DNA, 10% (w / v) dextran sulfate are used and 5-20 X 10⁶ cpm of the labeled probe ³²P. Filters are incubated in the hybridization mixture for 18-20 h at 40 ° C, and then rinsed for 1.5 h at 55 ° C in a solution containing 2xSSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS. The washing solution is replaced with fresh solution and incubated for an additional one and a half hours at 60 ° C. Filters are blotted dry and exposed to autoradiography. If necessary, the filters are rinsed a third time at 65-68 ° C and then exposed to the film again. Other mild hybridization conditions that can be used are known in the art (e.g., as used for interspecies hybridization).

Also disclosed is 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 sequences under stringent hybridization conditions and encodes a protein that retains immunogenicity of the fusion proteins of SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26. As an example, the following stringent hybridization conditions can be given: pre-hybridization of filters containing DNA is carried out from 8 hours to time overnight at 65 ° C in a buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500pg / ml denatured salmon sperm DNA. The filters are hybridized for 48 hours at 65 ° C in a pre-hybridization mixture containing 100 pg / ml denatured salmon sperm DNA and 5-20 X 10⁶ cpm of the labeled probe ³²P. Filters were washed at 37 ° C for 1 h in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. Thereafter, washing is performed in 0.1 X SSC at 50 ° C for 45 minutes before autoradiography. Other stringent hybridization conditions that can be used are known in the art.

In another preferred embodiment, a polynucleotide is provided which hybridizes to the coding sequence of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25, or to a sequence complementary to it under moderate hybridization conditions and encodes a protein that preserves the immunogenicity of the fusion protein of SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26. Exemplary average hybridization conditions are as follows: DNA-containing filters are pretreated for 6 h at 55 ° C in a solution containing 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100pg / ml denatured salmon sperm DNA. Hybridization is carried out in the hybridization mixture for 18-20 h at 55 ° C and then washed twice for 30 minutes at 60 ° C in a solution containing 1 X SSC and 0.1% SDS. Filters are blotted dry and exposed to autoradiography. Other average hybridization conditions that can be used are known in the art. The filters were washed at 37 ° C for 1 h in a solution containing 2 X SSC, 0.1% SDS.

5.4. Polypeptides encoded by coding sequences

In accordance with the present invention, a polynucleotide of the present invention encoding a fusion protein, fragments thereof, or functional equivalents thereof, can be used to produce recombinant nucleic acid molecules that direct the expression of the fusion protein, fragments thereof, or functional equivalents in suitable host cells. The fusion polypeptide products encoded by such polynucleotides can be modified by molecular manipulation of the coding sequence.

Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the present invention to express the fusion polypeptides. Such DNA sequences include those that are capable of hybridizing to the coding sequences or their complements described in the present invention under the low, medium, or high stringency hybridization conditions described in Section 5.3 above.

Modifications to the nucleotide sequences that can be used in the present invention include deletions, additions, or substitutions of different nucleotide residues that result in sequences encoding the same or a functionally equivalent gene product. The gene product itself

The PL 202 844 B1 may contain deletions, additions, or substitutions of amino acid residues that result in a silent change, thus resulting in a functionally equivalent antigenic epitope. Such conservative amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues in question. 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 start groups of similar hydrophilicity include glycine, asparagine, glutamine, serine, threonine, and tyrosine, and amino acids with nonpolar start groups include alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, and tryptophan.

The nucleotide sequences of the present invention can be generated to alter the coding sequence of a fusion protein for a variety of purposes, including, but not limited to, alterations that modify the processing and expression of the gene product. For example, mutations can be introduced by methods known in the art such as site directed mutagenesis to introduce new restriction sites, to alter the pattern of glycosylation, phosphorylation, etc.

In another embodiment of the present invention, the coding sequence of the fusion protein may be synthesized, in whole or in part, by known chemical methods. See for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Cheese. 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. Alternatively, the polypeptide itself can be produced using chemical methods to produce all or part of the amino acid sequence. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (See Creighton, 1983, Proteins Structures And Molecular Principles, WH Freeman and Co., NY, pp. 50-60). The composition of the synthetic polypeptides can be confirmed by amino acid analysis or sequencing (for example, the Edman degradation method, see Creighton, 1983, Proteins, Structures and Molecular Principles, WH Freeman and Co., NY, pp. 34-49).

In addition, the fusion protein coding sequence may be mutated in vitro or in vivo to create and / or remove translational, initiation and / or termination sequences, or to create variants in the coding regions and / or to create new restriction endonuclease sites or to destroy preexisting sites. to facilitate further in vitro modifications. Any mutagenesis technique known in the art can be used, including, but not limited to, chemical mutagenesis, site-directed mutagenesis in vitro (Hutchinson, C. et al., 1978, J. Biol. Chem. 253: 6551), the use of TAB connectors^® (Pharmacia) and the like. It is important that the manipulations do not destroy the immunogenicity of the fusion polypeptides.

In addition, non-classical amino acids or chemical analogs of the amino acids can be introduced as substitutions or additions to the sequence. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, α-aminobutyric acid, 4-aminobutyric acid, Aby, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminobutyric acid, 2- aminopropionic, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, formed amino acids such as β-methylacids such as β-methylacids Na-methylamino acids and amino acid analogues in general. Furthermore, the amino acid can be D (right-handed) or L (left-handed).

In a specific embodiment, the coding sequences of each antigen in the fusion protein are joined at their amino or carboxy termini via a peptide bond in any order. Alternatively, a peptide linker sequence may be used to separate the individual polypeptides that make up the fusion polypeptide by a distance sufficient to ensure that each polypeptide assembles into a secondary and tertiary structure that maximizes its antigenic efficacy in preventing and treating tuberculosis. Such a peptide linker sequence is incorporated into the fusion protein using standard methods known in the art. Preferred peptide linker sequences may be selected based on the following factors (1) their ability to adopt a flexible extended conformation (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides and (3) the absence of hydrophobic or charged residues that would react with functional epitopes of the polypeptide. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other nearly neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences which can advantageously be used as linkers include those described

By Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262,186; United States Patent No. 4,935,233 and United States Patent No. 4,751,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide sequences are not necessary since the first and second polypeptides have a region of non-essential N-terminal amino acids that can be used to separate functional domains and prevent steric interference. For example, the antigens in the fusion protein can be linked via a flexible polylinker such as GlyCys-Gly or Gly-Gly-Gly-Gly-Ser, repeated 1-3 times (Bird et al., 1988, Science 242: 423-426; Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070).

In one embodiment, such a protein is produced by recombinant expression of a nucleic acid that encodes the protein. Such a fusion product can be produced by ligating together the appropriate nucleic acid sequences encoding the desired amino acid sequences by methods known in the art in an appropriate coding frame, and expressing the product by methods known in the art. Alternatively, such a product may be produced by protein synthesis techniques, for example by the use of a peptide synthesizer. Coding sequences for other molecules, such as a cytokine or adjuvant, may also be added to the fusion polynucleotide.

5.5. Production of fusion proteins

To produce a M. tuberculosis fusion protein according to the present invention, a nucleotide sequence encoding the protein, or a functional equivalent thereof, is inserted into an appropriate expression vector, i.e., a vector containing the elements necessary for transcription and translation of the inserted coding sequence. Host cells or cell lines transfected or transformed with recombinant expression vectors can be used for a variety of purposes. These include, but are not limited to, large-scale production of the fusion protein.

Methods known to those skilled in the art can be used to produce expression vectors containing the fusion coding sequence and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA methods, synthetic techniques, and in vivo recombination / genetic recombination. (See for example, the methods described by 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). It is also possible to chemically produce RNA capable of encoding a polypeptide (Gait, 1984, Oligonucleoide Synthesis, IRL Press, Oxford).

5.5.1. Expression systems

A variety of expression vector host systems can be used to express the fusion protein coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the coding sequence, yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the coding sequence, insect cell systems, infected with recombinant viral expression vectors (e.g. baculovirus), containing the coding sequence, plant cell systems, infected with recombinant viral expression vectors (e.g. cauliflower mosaic virus - CaMV; tobacco mosaic virus - TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing coding sequence or mammalian cell systems (e.g. COS, CHO, BHK, cells 293, 3T3). The expression elements of these systems vary in strength and properties.

Depending on the host / vector system used, any of a number of advantageous transcription and translation elements may be used in the expression vector, including constitutive and inducible promoters. For example, when cloning in bacterial systems, inducible promoters such as pL bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter; cytomegalovirus promoter) and the like; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g. heat shock promoters, RUBISCO small subunit promoter, α / β chlorophyll binding protein promoter) or from plant viruses (e.g. CaMV 35S RNA promoter; TMV shell protein promoter) can be used ); when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g. adenoviral late promoter, vaccinia 7.5K promoter); SV-40, BPV and EBV based vectors with an appropriate selectable tag can be used to generate cell lines containing multiple copies of the antigenic coding sequence.

PL 202 844 B1

Bacterial systems are preferred for the expression of M. tuberculosis antigens. For in vivo use, a bacterium such as Bacillus Calmette-Guerin can be produced to express the fusion polypeptide of the present invention on the cell surface. Other bacterial expression systems may advantageously be selected depending on the intended use of the expressed products. For example, when a large amount of fusion protein is to be produced for the production of pharmaceutical compositions, vectors directing the expression of high levels of fusion protein products that are readily purified may be advantageous. Such vectors include, without limitation, the E. coli pUR278 expression vector (Inouye and Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 264: 5503-5509) and the like. PGEX vectors can also be used to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins. Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are engineered to contain thrombin or factor Xa protease cleavage sites such that the preferred cloned fusion polypeptide can be released from the GST particle.

5.5.2. Purification of proteins

Once the recombinant protein is expressed, it can be identified by tests based on the physical or functional properties of the product, including radioactive labeling of the product and subsequent analysis by gel electrophoresis, radioimmunoassay, ELISA, bioassays, etc.

Once the encoded protein has been identified, it can be isolated and purified by standard methods including chromatography (e.g., high performance liquid, ion exchange, affinity, and size difference column chromatography), centrifugation, differential solubility, or any other standard protein purification method. Actual conditions will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art. Functional properties can be assessed using any suitable assay, such as antibody binding, induction of T cell proliferation, stimulation of the production of cytokines such as IL2, IL-4 and IFN-γ. It is preferred for the practice of the present invention that each fusion protein is at least 80% purified from other proteins. It is more preferred that it is at least 90% pure. For in vivo administration, it is preferred that the proteins are greater than 95% purified.

5.6. Use of a fusion protein coding sequence

The sequence encoding the fusion protein of the present invention can be used to encode a protein product for use as an immunogen to induce and / or enhance an immune response to M. tuberculosis. Furthermore, such a coding sequence may be ligated to the coding sequence of another molecule, such as a cytokine or an adjuvant. Such polynucleotides can be used in vivo as DNA vaccines (US Pat. Nos. 5,589,466, 5679647, 5703055). In this embodiment of the invention, the polynucleotide expresses the protein it encodes in the recipient organism to directly stimulate an immune response. A polynucleotide can be injected into an unimmunized patient to initiate an immune response to the product encoded by that polynucleotide, or administered to an infected or immunized patient to enhance secondary immune responses.

In a preferred embodiment, the therapeutic composition comprises a fusion protein coding sequence or fragments thereof that are part of an expression vector. In particular, such a polynucleotide comprises a promoter operably linked to the coding region, the promoter being inducible or constitutive, and optionally tissue specific. In another embodiment, the polynucleotide comprises a coding sequence flanked by regions favoring homologous recombination at a desired site in the genome, thereby ensuring intra-chromosomal expression of the coding sequence (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA, 86: 8932-8935; Zijlstra and et al. 1989, Nature 342: 435-438).

Administration of the nucleic acid to the patient can be direct - in this case the patient is exposed directly to the nucleic acid or vector containing the nucleic acid, or indirect - in which case the cells are first transformed with nucleic acid in vitro and then implanted into the patient. These two methods are known as in vivo and ex vivo gene transfer, respectively.

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 done by any of a number of methods known in the art, for example, by making it part of an appropriate nucleic acid expression vector and administering it to become intracellular on

For example, by infection using a defective or attenuated retroviral vector or other viral vector (see U.S. Patent No. 4,980,286) or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., gene guns; Bioloistic, Dupont) or coating with lipids or cell surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules (US Patent Nos. 5407609, 5853763, 5814344, and 5820883), or by administering it in combination with a known peptide into the nucleus by administration in combination with a receptor-mediated endocytic ligand (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), which can be used with target cell types expressing the receptors specifically and the like. In another embodiment, a nucleic acid ligand complex can be prepared in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, thus avoiding lysosomal breakdown of the nucleic acid. In yet another embodiment, a nucleic acid can be targeted in vivo to cell-specific reuptake and expression by targeting a specific receptor (see for example, PCT Publication WO 92/06180 published April 16, 1992; WO 92/22635 published December 23, 1992; WO 92/20316 published November 26, 1992; WO 93/14188 filed July 22, 1993 and WO 93/20221 filed October 14, 1993). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into host cell DNA for expression by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935; Zijlstra et al., Nature 342: 435 -438).

In a specific embodiment, a viral vector, such as a retroviral vector, may be used (see Miller et al., 1993, Meth. Enzymol. 217: 581-599). Retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging the viral genome and incorporation into host cell DNA. The fusion coding sequence is cloned into the vector, which facilitates delivery of the recipient nucleic acid. More details on 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 mdrl gene to hematopoietic stem cells to render the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors for gene therapy are: Clowers et al., 1994, J. Clin. Invest. 93: 644-651; Kiem et al., 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 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 particularly attractive vehicles for delivering genes to the epithelium of the respiratory tract. Adenoviruses naturally infect the epithelium of the respiratory tract, where they cause mild disease. Other targets of 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. It has also been proposed to use adeno-associated virus (AAV) for in vivo gene transfer (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 2 04: 289-300).

Another technique involves transferring structure into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Typically, the method of transfer comprises the transfer of a selectable marker to the cells. The cells are then placed under selection conditions to isolate those cells that have taken up and expressed the transferred gene. These cells are then administered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to the in vivo administration of the resulting recombinant cell. Such introduction may be performed by any method known in the art, including, without limitation, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing nucleic acid sequences, cell fusions, chromosomal gene transfer, microcell gene transfer, spheroplast fusion, etc. Numerous methods of introducing foreign genes into cells are known in the art (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217: 599-618; Cohen et al., 1993, Meth. Enzymol. 217: 618644; Cline, 1985, Pharmac. Ther. 29: 69-92) and can be used according to the present invention.

The polynucleotides of the present invention can also be used in the diagnosis of tuberculosis to detect M. tuberculosis specific polynucleotide sequences in a patient. Such detection can be made, for example, by isolating polynucleotides from a biological sample obtained from a patient suspected of being infected with the bacterium. After isolating the polynucleotides from the biological sample, the labeled polynucleotide of the present invention is allowed to hybridize.

Complementary to one or more polynucleotides to polynucleotides in a biological sample, using nucleic acid hybridization techniques known to those skilled in the art. Such hybridization can be carried out, for example, in solution or with the use of a hybridization partner on a solid support.

5.7. Therapeutic and prophylactic use of the fusion protein

Purified or partially purified fusion proteins or fragments thereof can be prepared as vaccines or a therapeutic composition. Such compositions may contain adjuvants to enhance immune responses. Moreover, such proteins can be further suspended in the oil emulsion to cause a slower release of the proteins in vivo after injection. The optimal ratio of each ingredient in the formulation can be determined by techniques known to those skilled in the art.

Any of a variety of adjuvants can be used in the vaccines of the present invention to enhance the immune response. Most adjuvants contain a substance intended to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific immune stimulant, such as lipid A, Bordetella pertussis, or Mycobacterium tuberculosis. Suitable adjuvants are commercially available and include, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories) and Merck's Adjuvant (Merck and Company, Inc., Rahway, NJ). Other preferred adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A, quil A, SBAS1c, SBAS2 (Ling et al., 1997, Vaccine 15: 1562-1567), SBAS7, and Al (OH) 3.

In the vaccines of the present invention, it is preferred that the adjuvant induces an immune response that includes Th1 aspects. Preferred adjuvants include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MLP) together with an aluminum salt. The enhanced system comprises the combination of monophosphoryl lipid A and a saponin derivative, especially the combination of 3D-MLP and QS21 saponin, as described in WO 94/00153, or a less reactogenic composition in which QS21 is rapidly cooled with cholesterol as described in WO 96/33739 . Previous experiments have demonstrated a clear synergistic effect of the combination of 3D-MLP and QS21 in inducing both humoral and Th1-type immune responses. A particularly potent adjuvant, including QS21, 3D-MLP and tocopherol in an oil-in-water emulsion, is described in WO 95/17210, and is the preferred formulation.

Formulations containing the antigen of the present invention can be administered to the patient per se or in the form of a pharmaceutical or therapeutic composition. Protein-containing pharmaceutical preparations can be made by means of conventional mixing, dissolving, granulating, dragee-making, wet milling and sedimentation fractionation, emulsifying, encapsulating, trapping or lyophilizing processes. Pharmaceutical compositions can be prepared in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate processing of the polypeptides into preparations which can be used pharmaceutically. The appropriate preparation depends on the chosen route of administration.

For topical administration, the proteins can be formulated as solutions, gels, ointments, creams, suspensions etc. known in the art.

Preparations for systemic effect include preparations intended for administration by injection, for example, subcutaneously, intravenously, intramuscularly, intrathecal or intraperitoneally, as well as preparations intended for transdermal, transmucosal, oral or pulmonary administration.

For injection, the proteins may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline. The solution may contain production enhancing agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the proteins may be formulated as a powder for reconstitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrier in question are used in the formulation. Such penetrants are generally known.

For oral administration, the composition can be readily prepared by combining the proteins with pharmaceutically acceptable carriers known in the art. These carriers make it possible to prepare preparations in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions and the like from proteins, for oral administration to the treated patient. Suitable excipients for oral solid preparations such as powders, capsules, and tablets include fillers such as

Sugars, e.g. lactose, sucrose, mannitol and sorbitol; cellulose derivatives such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidone (PVP);

granulating agents and binding agents. If preferred, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

If preferred, solid dosage forms may be sugar coated or enteric coated using standard techniques.

Oral liquid preparations, such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, and the like. In addition, flavoring, preservatives, dyes and the like may be added.

For buccal administration, the proteins may be formulated into tablets, lozenges etc. in a conventional manner.

For administration by inhalation, the proteins for use in the present invention are preferably administered in the form of a pressurized aerosol or nebulizer using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other preferred gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing it with a valve for delivering a metered dose . Capsules and cartridges, e.g. of gelatin, for use in an inhaler may be prepared containing a protein mixture powder and a suitable powder base such as lactose or starch.

The proteins may also be formulated into rectal or vaginal compositions, suppositories or enemas, containing, for example, conventional suppository bases such as cocoa butter or other glycerides.

In addition to the preparations described above, the proteins can also be produced in the form of sustained release preparations. Such long-acting preparations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. For example, the proteins may be formulated with suitable polymeric or hydrophobic substances (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, e.g. in the form of a poorly soluble salt.

Instead, other pharmaceutical delivery systems can be used. Examples of well-known drug delivery vehicles are liposomes and emulsions, which can be used to deliver the antigen. Certain organic solvents such as dimethylsulfoxide can also be used, although usually at the cost of greater toxicity. Fusion proteins can also be encapsulated in microspheres (US Patent Nos. 5,407,609, 5,853,763, 5,814,344, and 5,820,883). Additionally, the proteins can be administered using a sustained release system, such as semipermeable matrices of solid polymers containing a drug or a vaccine. Various substances are known for the preparation of sustained release preparations. The prolonged-release capsules may, depending on their chemical nature, release proteins over a period of several weeks to over 100 days. Depending on the chemical nature and biological stability of the reagent, other methods of protein stabilization may be used.

It is within the skill of the art to determine the amount of fusion protein effective to elicit an immune response in a patient, particularly in light of the present detailed description.

The effective dose can be estimated initially from in vitro tests. For example, a dose can be formulated in animal models to induce an immune response using methods known in the art. The skilled artisan will readily optimize the use in humans depending on the animal data. The doses and the interval between them can be set individually. For example, when used as a vaccine, the polypeptides and polynucleotides of the present invention may be administered in about 1-3 doses over a period of 1 to 36 weeks. Preferably, 3 doses are administered approximately 3-4 months apart, followed by a periodic booster dose. Other administration regimens may be appropriate for some patients. A preferred dose is that amount of polypeptide or DNA which, when administered as described above, is capable of eliciting an immune response in an immunized patient sufficient to protect the patient from infection with M. tuberculosis for at least 1-2 years. Generally, the amount of polypeptide present in a dose (or generated in situ by the DNA contained in the dose) is from about 1 µg to about 100 mg per kg of host body weight, typically from about 10 µg to about 1 mg, and preferably from about 100 µg to about 100 µg to about 100 mg. about

PL 202 844 B1 pg. The preferred dose range will depend on the patient's weight and height, but will typically be from 0.1 ml to about 5 ml.

5.8. Diagnostic application of a fusion protein

The fusion polypeptides of the present invention are advantageous in the diagnosis of tuberculosis infection in vitro and in vivo. The ability of a polypeptide of the present invention to induce proliferation or cytokine production can be tested by the methods described in Section 5.2 above.

In another aspect, the present invention provides methods of using one or more fusion polypeptides in the diagnosis of tuberculosis using an in vivo skin test. As used herein, a "skin test" is any test performed directly on a patient that measures a delayed-type hypersensitivity (DTH) reaction (such as swelling, redness, or dermatitis) following intradermal injection of one or more polypeptides as described herein. above. Such injection may be made using any suitable instrument sufficient to bring the polypeptide into contact with the skin cells of the patient, such as, for example, a tuberculin syringe or a 1 ml syringe. Preferably, the reaction is measured at least 48 hours post injection, more preferably from about 48 to about 72 hours post injection.

The DTH reaction is a cellular type immune response, greater in patients previously exposed to the test antigen (i.e., to the immunogenic portion of the polypeptide used, or a variant thereof). The response can be measured visually using a ruler. Generally, a reaction greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is considered positive, indicative of tuberculosis infection, which may or may not manifest as an active disease.

The fusion polypeptides of the present invention are preferably prepared for use as a skin test in the form of pharmaceutical compositions comprising the polypeptide and a physiologically acceptable carrier. Such compositions typically contain one or more of the above polypeptides in an amount of 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 physiological saline with suitable preservatives, such as phenol and / or Tween 80 ™.

In another aspect, the present invention relates to methods of using the polypeptides for the diagnosis of tuberculosis. In this aspect, methods are provided for detecting a M. tuberculosis infection in a biological sample using fusion polypeptides, alone or in combination. As used herein, a "biological sample" is any antibody-containing sample taken from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample taken from a patient or from a blood container. The polypeptide (s) are used in an assay as described below to determine the presence or absence of antibodies against the polypeptide (s) in a sample relative to a predetermined cut-off value. The presence of such antibodies indicates prior sensitization with Mycobacterium antigens, which may indicate tuberculosis.

In embodiments where more than one fusion polypeptide is used, the polypeptides used are preferably complementary (i.e., one constituent polypeptide will tend to detect infection in samples where infection would not be detected by the other constituent polypeptide). Complementary polypeptides can usually be identified by using each polypeptide separately to evaluate serum samples obtained from a series of patients known to be infected with M. tuberculosis. After determining which samples are positive (as described below) for each polypeptide, formulations combining the two or more fusion polypeptides capable of detecting infection in most or all of the test samples can be made. Such polypeptides are complementary. About 25-30% of sera from people infected with tuberculosis are negative for antibodies to individual proteins. Complementary polypeptides can therefore be used in combination to improve the sensitivity of a diagnostic test.

There are many assays known to those skilled in the art of using one or more polypeptides to detect antibodies in a sample. See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; this publication is incorporated herein by reference. In a preferred embodiment, the assay comprises using a polypeptide immobilized on a solid support to bind and remove the antibody from the sample. The bound antibody can then be detected using a detection reagent that contains a reporter group. Preferred detection reagents include antibodies that bind to the antibody / polypeptide complex and to free polypeptides labeled with a reporter group (e.g., in a semi-competitive assay). Alternatively, a competition assay may be used in which the antibody that binds to the polypeptide labels

This is a reporter group and is allowed to bind to the immobilized antigen after the antigen is incubated with the sample. The extent of inhibition of the labeled antibody binding to the polypeptide by the sample components 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 may be a test well in a microtiter plate or a nitrocellulose film or other film of interest. Instead, the substrate may be a ball or disc, fiberglass, latex, or a plastic material such as polystyrene or polyvinyl chloride. The substrate may also be a magnetic particle or an optical fiber sensor as described, for example, in US Patent 5359681.

The polypeptides can be bound to a solid support by a variety of methods known to those skilled in the art. In the context of the present invention, the term "bound" means both a non-covalent bond, such as adsorption, and a covalent bond (which may be a direct bond between the antigen and functional groups on the support or may be a linkage via a cross-linking agent). Binding 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 preferred buffer, with the solid support for a preferred time. The contact time varies with the temperature, but is typically from about 1 hour to about a day. In general, contacting a well of a plastic microtiter plate (e.g., polystyrene or polyvinyl chloride) with a polypeptide in an amount of about 10 ng to about 1 µg, preferably, about 100 ng, is sufficient to bind the appropriate amount of antigen.

Covalent binding of a polypeptide to a solid support can generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the polypeptide. For example, a polypeptide can be linked to substrates having a suitable polymer coating using a benzoquinone or by condensation of an aldehyde group on the substrate with an amino group and active hydrogen on the polypeptide (see for example, Pierce Immunotechnology Catalog and Handbook, 1991, pp. A12-A13).

In some embodiments, the assay is an Immunosorbent Enzyme Assay (ELISA). This assay can be performed by first contacting the fusion polypeptide antigen, immobilized on a solid support, usually a well of a microtiter plate, with the sample such that antibodies to the polypeptide in the sample are allowed to bind to the immobilized polypeptide. The unbound sample is then removed from the immobilized polypeptide, and a reaction reagent capable of binding the immobilized antibody-polypeptide complex is added. The amount of detection reagent that remains free from the solid support is then determined using a method appropriate to 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 typically blocked. Any suitable blocking agent known to those skilled in the art can be used, such as bovine serum albumin or Tween 20 ™ (Sigma Chemical Co., St. Louis, MO). The immobilized polypeptide is then incubated with the sample, and the antibody is allowed to bind to the antigen. The sample may be diluted with a suitable diluent such as phosphate buffered saline (PBS) prior to incubation. In general, an appropriate contact time is sufficient time to detect the presence of the antibody in a sample infected with M. tuberculosis. Preferably, the contact time is sufficient to achieve a binding level of at least 95% of that achieved at equilibrium between bound and unbound antibody. One skilled in the art will recognize that the time required to reach equilibrium can be readily determined by the level of binding occurring at any given time. Typically, an incubation time of around 30 minutes is sufficient at room temperature.

Unbound sample can then be removed by washing the solid support with an appropriate buffer, for example PBS containing 0.1% Tween 20 ™. The detection reagent can then be added to the solid support. A suitable detection reagent is any compound that binds to the immobilized antibody-polypeptide complex which can then be detected by a variety of methods known to those skilled in the art. Preferably, the detection reagent comprises a binding agent (e.g. protein A, protein G, lectin or free antigen) coupled to a reporter group. Preferred reporter groups include enzymes (e.g. horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, biotin, and colloidal particles such as colloidal gold and selenium. Coupling the binding agent with the reporte group16

Preparation can be achieved by standard methods known to those skilled in the art. Most common binders can also be purchased coupled to a variety of reporter groups from a variety of commercial sources (e.g., Zymed Laboratories, San Francisco, CA, and Pierce, Rockford, IL).

The detection reagent is then incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound antibody. The appropriate time can usually be determined by the manufacturer's recommendations or by testing the level of bonding over time. Then, the unbound detection reagent is removed and the bound detection reagent is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. In general, scintillation counting or autoradiographic methods are suitable for radioactive groups. Spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin, coupled to another reporter group (usually a radioactive or fluorescent group, or to an enzyme). Enzymatic reporter groups can usually be detected by adding a substrate (usually for a specified period of time) followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of anti-M. tuberculosis antibodies in a sample, the signal detected from the reporter group that remains bound to the solid support is usually compared to the signal corresponding to a predetermined cut-off value. In one preferred embodiment, the cut-off value is the average signal obtained after incubating the immobilized antigen with samples from uninfected patients. In general, a positive result for tuberculosis is considered to be when the signal produced by the sample is more than three standard deviations above the specified cut-off value. In another preferred embodiment, the cut-off value is determined using a receiver operating curve using the method of Sackett et al., 1985, Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., pp. 106-107. Briefly, in this embodiment, the cut-off value can be determined from a pair of true positive (i.e., sensitivity) and false positive (100% -specific) frequency pairs corresponding to each possible cut-off value for the diagnostic test result. The cut-off value in the graph that is closest to the upper-left corner (i.e. the value containing the largest area) is the most accurate cut-off value, and a sample producing a signal higher than the cut-off value determined by this method can be considered positive. Instead, the cut-off value can be shifted to the left along the plot to minimize the false-positive rate or to the right to minimize the false-compliant rate. In general, a sample producing a signal above the cut-off determined by this method is considered positive for tuberculosis.

In a related embodiment, a rapid flow test or a strip test is performed in which the antigen is immobilized on a membrane such as nitrocellulose. In a flow-through test, antibodies in the sample bind to the immobilized polypeptide as the sample passes through the membrane. The detection reagent (e.g., protein A - colloidal gold) then binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. Then, detection of the bound detection reagent can be performed as described above. In the strip test, one end of the membrane to which the polypeptide is bound is immersed in the solution containing the sample. The sample migrates along the membrane through the region containing the detection reagent and into the region of the immobilized polypeptide. The concentration of the detection reagent on the polypeptide indicates the presence of anti-M. tuberculosis antibodies in the sample. Typically, the concentration of detection reagent at this location produces a standard, such as a line, that can be read visually. The absence of such a pattern indicates a negative test result. In general, the amount of polypeptide immobilized on the membrane is selected to produce a visually distinguishable standard when the biological sample contains antibodies at a level sufficient to elicit a positive signal in the ELISA, as discussed above. Preferably, the amount of polypeptide immobilized on the membrane is from 5 ng to about 1 µg and greater, preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount (e.g., one drop) of the patient's serum or blood.

The description of the invention is followed by examples intended for the purpose of illustration only, and not to limit the invention.

6. Example: M. tuberculosis antigen fusion proteins retain the immunogenicity of the individual components

6.1. Material and methods

6.1.1. Production of fusion proteins

PL 202 844 B1

The coding sequences of the M. tuberculosis antigens were modified by PCR to facilitate their fusion and subsequent expression of the fusion protein. DNA was amplified using 10 μl of 10X Pfu buffer, 2 μl of 10 mM dNTP, 2 μl of each PCR primer at a concentration of 10 μM, 81.5 μl of water, 1.5 μl of Pfu DNA polymerase (Stratagene, La Jolla, CA) and 1 µl of DNA at a concentration of 70 ng / l (for the TbRa3 antigen) or 50 ng / l (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 sec. and 72 ° C for 1 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 sec, 68 ° C for 15 sec. and 72 ° C for 3 minutes, and finally 72 ° C for 4 minutes. For the Tb38-1 antigen, after denaturation at 94 ° C for 2 minutes, 10 cycles of 96 ° C for 15 seconds, 68 ° C for 15 seconds and 72 ° C for 1.5 minutes were run, 30 cycles of 96 ° C for 15 seconds. 15 sec., 64 ° C for 15 sec. and 72 ° C for 1.5, and finally 72 ° C for 4 minutes.

Following restriction endonuclease digestion to obtain the desired sticky or blunt ends, a polynucleotide 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 pT7L2IL1.

To encode one fusion protein (SEQ ID Nos. 1 and 2) (Fig. 1A and 2B), three coding sequences for the antigens Ra12, TbH9 and Ra35 were ligated. The other three coding sequences for the antigens Erd14, DPV and MTI were ligated to encode a second fusion protein (SEQ ID Nos. 3 and 4) (Figure 2). Three coding sequences for the Tbra3, 38 kD and Tb38-1 antigens were ligated to encode one fusion protein (SEQ ID Nos. 5 and 6) (Figures 3A-3D). Two coding sequences for the antigens TbH9 and Tb38-1 were ligated to encode one fusion protein (SEQ ID Nos. 7 and 8) (Figures 4A-4D). Four coding sequences for the TbRa3, 38 kD, Tb38-1, and DPEP antigens were ligated to encode one fusion protein (SEQ ID Nos. 9 and 10) (Figures 5A-5J). Five coding sequences for the antigens Erd14, DPV, MTI, MSL and MTCC2 were ligated to encode one fusion protein (SEQ ID Nos. 11 and 12) (Figures 6A and 6B). Four coding sequences for the antigens Erd14, DPV, MTI and MSL were ligated to encode one fusion protein (SEQ ID Nos. 13 and 14) (Figures 7A and 7B). Four coding sequences for the DPV, MTI, MSL and MTCC2 antigens were ligated to encode one fusion protein (SEQ ID Nos. 15 and 16) (Figures 8A and 8B). The three coding sequences for the DPV, MTI and MSL antigens were ligated to encode one fusion protein (SEQ ID Nos. 17 and 18) (Figures 9A and 9B). The three coding sequences for the antigens TbH9, DPV and MTI were ligated to encode one fusion protein (SEQ ID Nos. 19 and 20) (Figures 10A and 10B). The three coding sequences for the antigens Erd14, DPV and MTI were ligated to encode one fusion protein (SEQ ID Nos. 21 and 22) (Figures 11A and 11B). Two coding sequences for the antigens TbH9 and Ra35 were ligated to encode one fusion protein (SEQ ID Nos. 23 and 24) (Figures 12A and 12B). Two coding sequences for the Ra12 and DPPO antigens were ligated to encode one fusion protein (SEQ ID Nos. 25 and 26) (Figures 13A and 13B).

Recombinant proteins were expressed in E. coli with six histidine residues at the amino terminal end using the pET plasmid vector (pET-17b) and the T7 RNA polymerase expression system (Novagen, Madison, WI). E. coli BL21 (DE3) pLysE (Novagen) strain was used for high level expression. Recombinant (His-Tag) fusion proteins were purified from the soluble supernatant or insoluble inclusion body of 500 ml IPTG-induced cultures by affinity chromatography using one step of QIAexpress Ni-NTA Agarose (QIAGEN, Chatsworth, CA) in the presence of 8M urea. Briefly, 20 ml of an overnight saturated culture of BL21 containing the pET structure was added to 500 ml of 2xYT medium containing 50 µl / ml ampicillin and 34 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 another 3 h (OD 1.3-1.9). Cells were harvested from 500 ml of culture 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 mM PMSF and 20 g / ml leupeptin plus one tablet of total protease inhibitor (Boehringer Mannheim) per 25 ml. The E. coli was lysed by lyophilization followed by brief sonication then centrifuged at 12k rpm. for 30 minutes to obtain granulation of inclusion bodies.

Inclusion bodies were washed three times with 1% CHAPS in 10 mM Tris-HCl (pH 8.0). This step significantly reduced LPS contamination. The inclusion body was finally dissolved in 20 ml of binding buffer containing 8M urea or 8M urea and added directly to the soluble supernatant. Recombinant fusion proteins with His-Tag residues were collectively bound to Ni-NTA agarose resin (5 ml of resin per 500 ml of induction) by rocking at room temperature for 1 h and the complex was passed through the column. The flow was performed twice on the same column

And the column was washed three times with 30 ml of each of the washing buffers (0.1 M sodium phosphate and 10 mM Tris-HCl, pH 6.3), also containing 8 M urea. Bound protein was eluted with 30 ml of 150 mM imidazole in washing buffer and 5 ml fractions were collected. Fractions containing each recombinant fusion protein were pooled, dialyzed against 10 mM Tris-HCl (pH 8.0) bound back to the Ni-NTA matrix, eluted and dialyzed in 10 mM Tris-HCl (pH 7.8). The yield of the recombinant protein varies from 25 to 150 mg per liter of induced bacterial culture with a purity of more than 98%. Recombinant proteins were tested for endotoxin contamination using the Limulus assay (BioWhittaker) and were shown to contain <10 EUImg.

6.1.2. T cell proliferation test

Purified fusion polypeptides were tested for the ability to induce proliferation of T lymphocytes in peripheral blood mononuclear cell (PBMC) preparations. PBMCs from donors known to be positive by PPD skin test and for which T cells were shown to proliferate in response to PPD and crude M. tuberculosis soluble proteins were cultured in RPMI 1640 supplemented with 10% human serum from multiple donors and 50 gl / ml gentamicin. Purified polypeptides were added in duplicate at a concentration of 0.5 to 10 µg / ml. After six days of culturing in 200 µl in 96-well round-bottom plates, 50 µl of medium was removed from each well for determination of IFN-γ levels as described below in section 6.1.3. The plates were then pulsed with 1 gCi / well of tritiated thymidine for a further 18 hours, harvested and the tritium uptake was determined using a gas scintillation counter. The proliferation fractions in both copies more than three times the proliferation observed in cells grown in medium alone were considered positive.

6.1.3. The interferon γ test

Spleens of mice were removed under sterile conditions and single cell suspensions were prepared in complete RPMI after red blood cell lysis. 100 g of seed (2x10^-5 cells) per well in a 96-well flat bottom microtiter plate. Cultures were stimulated with the indicated recombinant proteins for 24 h and the supernatant was tested for IFN-γ.

The level of IFN? In the supernatant was analyzed by sandwich ELISA using antibody pairs methods available from PharMingen. Standard curves were generated using recombinant murine cytokines. ELISA plates (Corning) were coated with 50 µl / well (1 gg / ml, in 0.1 M bicarbonate coating buffer, pH 9.6) Cytokine Capture mAb (rat IFN-γ directed against mouse antigens (PharMingen, Cat # 18181D) and incubated for 4 h at room temperature. The plate contents were shaken and blocked with PBS-0.05% Tween, 1.0% BSA (200 µl / well) overnight at 4 ° C and washed up to 6X in PBS-0.1% Tween. Standard (mouse IFN-γ) and supernatant samples, diluted in PBS-0.05% Tween, 0.1% BSA, were then added for h at room temperature. The plates were washed as above and then incubated for 2 hours at room temperature with 100 µl / well of a second Ab (biotinylated rat anti-mouse IFN-γ antibody (Cat # 18112D, PharMingen) at 0.5 gg / ml, diluted in PBS-0.05% Tween, 0.1% BSA After washing, the plates were incubated with 100 µl / well streptavidin-HRP (Zymed) at a 1: 2500 dilution in PBS-0.05% Tween, 0.1% BSA at room for 1 h. The plates were washed one last time and developed with 100 g / well TMB substrate (3,3 ', 5,5'-tetramethylbenzidine, Kirkegaard and Perry, Gaithersburg, MD) and the reaction stopped after color development with H 2 SO 4, 50 g / well. The absorbance (OD) at 450 nm was determined using 570 nm as the reference wavelength and the cytokine concentration was determined using the standard curve.

6.2. Results

6.2.1. Immunological reactions induced by trifusion proteins

Three M. tuberculosis coding sequences were inserted into an expression vector to generate a fusion protein. The antigens, designated Ra12, TbH9, and Ra35, were produced as one recombinant fusion protein (Figures 1A and 1B). The Erd14, DPV and MTI antigens were generated as a second fusion protein (Figure 2). Both fusion proteins underwent affinity purification for use in in vitro and in vivo assays.

Both fusion proteins were tested for their ability to stimulate T-cell responses in six PPD subjects⁺. When measuring T cell proliferation, both fusion proteins showed a similar pattern of reactivity to their individual components (Figures 14A-14F). A similar result was obtained when measuring IFN-γ production (Figures 15A-15F). For example, patient D160 responded to the TbH9 and MTI antigens separately. Patient D160 also responded to fusion proteins containing these antigens

(Figs. 14B and 15B). In contrast, in D160 no T cell responses to other antigens were observed separately. Another patient, D201, which did not respond to the Erd14, DPV or MTI antigens separately, also did not respond to the fusion protein containing these antigens. It should be noted that while T lymphocyte responses to the individual components of both fusion proteins were not particularly strong, the fusion proteins stimulated responses equal to or greater than those induced by the individual antigens in most cases.

The trifusion protein Ral2-TbH9-Ra35 has also been tested as an in vivo immunogen. In these experiments, the fusion protein was injected into the footpads of mice for immunization. Each group of three mice received the protein in a different adjuvant formulation: SBAS1c, SBAS2 (Ling et al., 1997, Vaccine 15: 1562-1567), SBAS7 and Al (OH) 3. After two subcutaneous immunizations three weeks apart, the animals were sacrificed one week later and their draining lymph nodes were harvested for use as responsive cells in the T cell proliferation and cytokine production assays.

Regardless of which adjuvant was used for immunization, strong T cell proliferation against TbH9 was induced when used as a single antigen (Fig. 16A). Weaker reactions were induced against Ra35 and Ra12 (Figures 16B and 16C). When using the Ra12TbH9-Ra35 fusion protein as an immunogen, a reaction similar to that against single components was observed.

When measuring cytokine production, SBASIc and SBAS2 adjuvants produced similar IFNγ (Figure 17) and IL-4 (Figure 18) responses. However, the combination of SBAS7 and aluminum hydroxide gave the strongest IFN-γ reaction and the lowest level of IL-4 production for all three antigens. With regard to the humoral antibody response in vivo, Figures 19A-19F show that the fusion protein elicited antigen-specific responses, both IgG1 and IgG2, when used with any of the three adjuvants.

Additionally, C57B1 / 6 mice were immunized with a combination of two expression structures each containing the Ra12-TbH9-Ra35 (Mtb32A) or Erd14-DPV-MTI (Mtb39A) coding sequence as DNA vaccines. Immunized animals showed significant protection against tuberculosis after subsequent aerosol exposure to live bacteria. Based on these results, a fusion structure of the Mtb32A and Mtb39A coding sequences was generated, and the product encoded by them was tested in a long-term protection model in guinea pigs. In these studies, guinea pigs were immunized with a single recombinant fusion protein or a mixture of Mtb32A and Mtb39A proteins in adjuvant-containing formulations. Fig. 20A-20C show that guinea pigs immunized with the fusion protein in SBAS1c or SBAS2 were better protected against the development of tuberculosis after post-exposure compared to animals immunized with the two antigens in a mixture with the same adjuvant formulation. The fusion proteins in the SBAS2 preparation provided the animals with the best protection. Thus, the fusion proteins of the various M. tuberculosis can be used as immunogens in vaccine preparations more effectively than a mixture of the individual components.

6.2.2. Immunological reactions induced by bifusion proteins

A bifusion fusion protein containing the TbH9 and Tb38-1 antigens without a hinge sequence was produced by recombinant methods. The ability of the TbH9-Tb38-1 fusion protein to induce T cell proliferation and IFN-γ production was investigated. Three donors were used for PBMC: one donor had a prior reaction to TbH9 but not to Tb38-1 (donor 131); another showed a response to Tb38-1 but not to TbH9 (donor 184) and yet another showed a response 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. The tetrafusion protein reacted with the sera of tuberculosis patients

A fusion protein containing the TbRa3, 38KD, Tb38-1 and DPEP antigen was produced by recombinant means. The reactivity of this tetrafusion protein, designated as TbF-2, with sera from patients infected with M. tuberculosis was tested by ELISA method. The results of these studies (Table 1) show that all four antigens act independently in the fusion protein.

One skilled in the art will appreciate that the order of the individual antigens in each fusion protein can be varied and that comparable activity can be expected, provided that each epitope remains functionally available. In addition, truncated forms of proteins containing active epitopes can be used to form fusion proteins.

PL 202 844 B1

Table 1

TbF-2 fusion protein reactivity with tuberculosis (TB) patient serum and normal serum (PR)

<td>No serum</td><td>State</td><td>TbF OD450</td><td>State</td><td>TbF-2 OD450</td><td>State</td><td colspan="4">Reactivity in ELISA</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>38 kD</td><td>TbRa3</td><td>Tb38-1</td><td>DPEP</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>B931-40</td><td>TB</td><td> 0.57</td><td> +</td><td> 0.321</td><td> +</td><td> -</td><td> +</td><td> -</td><td> +</td>

<td>B931-41</td><td>TB</td><td> 0.601</td><td> +</td><td> 0.396</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td>B931-109</td><td>TB</td><td> 0.494</td><td> +</td><td> 0.404</td><td> +</td><td> +</td><td> +</td><td> ±±</td><td> -</td>

<td>B931-132</td><td>TB</td><td> 1.502</td><td> +</td><td> 1.292</td><td> +</td><td> +</td><td> +</td><td> +</td><td> ±±</td>

<td> 5004</td><td>TB</td><td> 1.806</td><td> +</td><td> 1.666</td><td> +</td><td> ±±</td><td> ±±</td><td> +</td><td> -</td>

<td> 15004</td><td>TB</td><td> 2.862</td><td> +</td><td> 2.468</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td> 39004</td><td>TB</td><td> 2.443</td><td> +</td><td> 1.722</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td> 68004</td><td>TB</td><td> 2.871</td><td> +</td><td> 2.575</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td> 99004</td><td>TB</td><td> 0.691</td><td> +</td><td> 0.971</td><td> +</td><td> -</td><td> ±±</td><td> +</td><td> -</td>

<td> 107004</td><td>TB</td><td> 0.875</td><td> +</td><td> 0.732</td><td> +</td><td> -</td><td> ±±</td><td> +</td><td> -</td>

<td> 92004</td><td>TB</td><td> 1.632</td><td> +</td><td> 1.394</td><td> +</td><td> +</td><td> ±±</td><td> ±±</td><td> -</td>

<td> 97004</td><td>TB</td><td> 1.491</td><td> +</td><td> 1.979</td><td> +</td><td> +</td><td> ±±</td><td> -</td><td> +</td>

<td> 118004</td><td>TB</td><td> 3.182</td><td> +</td><td> 3.045</td><td> +</td><td> +</td><td> ±±</td><td> -</td><td> -</td>

<td> 173004</td><td>TB</td><td> 3.644</td><td> +</td><td> 3.578</td><td> +</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td> 175004</td><td>TB</td><td> 3.332</td><td> +</td><td> 2.916</td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td>

<td> 274004</td><td>TB</td><td> 3.696</td><td> +</td><td> 3.716</td><td> +</td><td> -</td><td> +</td><td> -</td><td> +</td>

<td> 276004</td><td>TB</td><td> 3.243</td><td> +</td><td> 2.56</td><td> +</td><td> -</td><td> -</td><td> +</td><td> -</td>

<td> 282004</td><td>TB</td><td> 1.249</td><td> +</td><td> 1.234</td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td> 289004</td><td>TB</td><td> 1.373</td><td> +</td><td> 1.17</td><td> +</td><td> -</td><td> +</td><td> -</td><td> -</td>

<td> 308004</td><td>TB</td><td> 3.708</td><td> +</td><td> 3.355</td><td> +</td><td> -</td><td> -</td><td> +</td><td> -</td>

<td> 314004</td><td>TB</td><td> 1.663</td><td> +</td><td> 1.399</td><td> +</td><td> -</td><td> -</td><td> +</td><td> -</td>

<td> 317004</td><td>TB</td><td> 1.163</td><td> +</td><td> 0.92</td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td> 312004</td><td>TB</td><td> 1.709</td><td> +</td><td> 1.453</td><td> +</td><td> -</td><td> +</td><td> -</td><td> -</td>

<td> 380004</td><td>TB</td><td> 0.238</td><td> -</td><td> 0.461</td><td> +</td><td> -</td><td> ±±</td><td> -</td><td> +</td>

<td> 451004</td><td>TB</td><td> 0.18</td><td> -</td><td> 0.2</td><td> -</td><td> -</td><td> -</td><td> -</td><td> ±±</td>

<td> 478004</td><td>TB</td><td> 0.188</td><td> -</td><td> 0.469</td><td> +</td><td> -</td><td> -</td><td> -</td><td> ±±</td>

<td> 410004</td><td>TB</td><td> 0.384</td><td> +</td><td> 2.392</td><td> +</td><td> ±±</td><td> -</td><td> -</td><td> +</td>

<td> 411004</td><td>TB</td><td> 0.306</td><td> +</td><td> 0.874</td><td> +</td><td> -</td><td> +</td><td> -</td><td> +</td>

<td> 421004</td><td>TB</td><td> 0.357</td><td> +</td><td> 1.456</td><td> +</td><td> -</td><td> +</td><td> -</td><td> +</td>

<td> 528004</td><td>TB</td><td> 0.047</td><td> -</td><td> 0.196</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td>

PL 202 844 B1 cont. table 1

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>A6-87</td><td>PR</td><td> 0.094</td><td> -</td><td> 0.063</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-88</td><td>PR</td><td> 0.214</td><td> -</td><td> 0.19</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-89</td><td>PR</td><td> 0.248</td><td> -</td><td> 0.125</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-90</td><td>PR</td><td> 0.179</td><td> -</td><td> 0.206</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-91</td><td>PR</td><td> 0.135</td><td> -</td><td> 0.151</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-92</td><td>PR</td><td> 0.064</td><td> -</td><td> 0.097</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-93</td><td>PR</td><td> 0.072</td><td> -</td><td> 0.098</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-94</td><td>PR</td><td> 0.072</td><td> -</td><td> 0.064</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-95</td><td>PR</td><td> 0.125</td><td> -</td><td> 0.159</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>A6-96</td><td>PR</td><td> 0.121</td><td> -</td><td> 0.12</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Cutting off</td><td></td><td> 0.284</td><td></td><td> 0.266</td><td></td><td></td><td></td><td></td><td></td>

Patent claims