SA08290425B1 - Fusion Proteins of Mycobacterium Tuberculosis Antigens and Their Uses - Google Patents

Abstract

Abstract: The present invention relates to fusion proteins containing at least two mycobacterium tuberculosis antigens. In particular, the invention relates to binary fusion proteins containing two individual M. tuberculosis antigens, tri-fusion proteins containing three antigens against M. tuberculosis and tetra-fusion proteins containing four antigens against Mycobacterium tuberculosis and penta-fusion proteins that contain five antigens against M. tuberculosis antigens and methods for their use in the diagnosis, treatment and prevention of tuberculosis infection. Figure 1.

Description

Y-antigen fusion proteins against Mycobacterium tuberculosis and their uses

Fusion Proteins of Mycobacterium Tuberculosis Antigens and Their Uses Full Description Background of the invention This application is a partial application of Application No. 5760784, which was filed in the Kingdom

Ce Yeo [ON / 11 corresponding to AYEYT / 01 / 01 Saudi Arabia on date This application is a partial application of Application No. 4 1760728* which was filed in the Kingdom of Saudi Arabia © on 01 0 / 1 471 AH corresponding to ‎ ca Yeo [AY 11] The present invention relates to fusion proteins containing at least US of mycobacterium tuberculosis antigens. In particular the invention relates to binary fusion probiotics containing two independent M. tuberculosis antigens and tri- fusion proteins Ve containing three anti-M. tuberculosis antigens and tetra- fusion proteins fusion proteins, which contain four antigens against M. tuberculosis, and penta-fusion proteins, which contain five antigens against M. tuberculosis. tuberculosis antigens and methods for their use in the diagnosis, treatment and prevention of tuberculosis infection Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis, M. tuberculosis It is 0 a major disease in developing countries, in addition to being a growing problem in developed regions of the world, where about A million new cases are discovered as it dies? million each year though that might be Yay ¢

1 = - The infection is asymptomatic for a long period of time. In the most common way, the disease appears in the form of acute inflammation of the lungs, which results in fever and a nonproductive cough. If not treated, serious health problems and death are the typical outcome. ©0 Generally though this can be controlled with extended antibiotic therapy | extended antibiotic therapy; This treatment is not sufficient to prevent the spread of the disease. It is possible that individual disease states affected are asymptomatic; However, it is a carrier of infection; For some time ; add to that ; Although acquiescence to the prevailing treatment regimen is crucial; It is difficult to monitor the patient's behavior. Some patients do not complete the course of treatment; Which may lead to ineffective treatment 0 and the occurrence of resistance to the drug used. In order to control the spread of tuberculosis, effective vaccination and accurate early diagnosis of the disease is of paramount importance. currently ; Vaccination with live bacteria is one of the most effective methods of inducing protective immunity. The most common mycobacterium used for this purpose is Bacillus Clamite. Vo Guerin Bacillus Calmette-Guerin (BCG) ¢ A subspecies of Mbovis . Although the safety and effectiveness of BCG is a source of controversy and disagreement in some countries; such as USA; Which do not feed the public with this factor. It is common to make a diagnosis of tuberculosis using a skin pick; Which involves exposing the inner skin with tuberculin PPD (a protein-purified derivative of Hie). Antigen-specific 1 cell T responses result Yo Asal. Measurable stiffness measurable induration I have a position

d - injection after about 77-48 hours have passed; This indicates exposure to Mycobacterial antigens. However, sensitivity and specificity are issues associated with this test; There is no possibility to distinguish individuals vaccinated with BCG from infected individuals + while macrophages appeared to act as peripheral effectors of immunity to Mycobacterium tuberculosis (principal effectors of M. tuberculosis©); T cells are dominant inducers of this immunity. The essential role of 7 cells in protection against Mycobacterium tuberculosis infection is illustrated by the frequent occurrence of Mycobacterium tuberculosis in patients with acquired immunodeficiency syndrome as a result of the utilization of 7+004 cells associated with human immunodeficiency virus (HIV) infection. 7+4 cells showed bacterial interactors that they are effective products of “ll (IFN-Y) gamma-interferon 0 in turn 0” appears to release anti-mycobacterial effects of macrophages in mice in mice While the role of gamma-interferon in humans is less clear, studies have shown that: 03 1,25-dihydroxy-vitamin » either alone or in the form of a chemical union with gamma-interferon or tumor necrosis factor-Q tumor necrosis factor-alpha ¢ activates human macrophages to inhibit M0 . add to that ; 1770-7 under human macrophages is known to synthesize 1,25-dihydroxy-vitamin D3 + similarly “pure interleukin-12 (IL-12) plays in inducing resistance to M. tuberculosis infection. For immunology review My infection, Mycobacterium tuberculosis, Chan and Kaufmann aif + 1944 + Tuberculosis: Pathogenesis, Prevention, and Control « Bloom (Editor); ASM Press; Washington; District of Columbia 0

Therefore, there is a need for improved vaccinations and improved methods for diagnosing, preventing and treating tuberculosis. GENERAL DESCRIPTION OF THE INVENTION The present invention relates to fusion proteins 0 antigens against Mycobacterium tuberculosis 0 mycobacterium tuberculosis antigens © generally 0 It relates to fusion polypeptides containing OL or more of mycobacterium antigens tuberculosis; polynucleotides encoding these polypeptides and methods for using polypeptides and polynucleotides in diagnosis, treatment and prevention of mycobacterium tuberculosis treatment and prevention of M. tuberculosis infection The present invention depends 0 partly; The inventors discovered polynucleotides polynucleotides that contain a number of two to five coding sequences of Mycobacterium tuberculosis that produce recombinant fusion proteins that maintain immunogenicity and antigenicity of these independent compartments. The fusion proteins described here described in context both 7-cell and B-cell responses; According to what was described by Yo, cell proliferation 1, cytokine production, and antibody alias 0 production, moreover; A fusion protein is used as an immunogen with adjuvants in vivo to trigger SS of cell- Ae lially mediated humoral immunity to M. tuberculosis. In addition to that; A fusion protein is made by a fusion formulation and then used in the formulation of a vaccine with Yo adjuvant in order to prevent long-term protection in animals against the development of tuberculosis.

0 68 against the of tuberculosis fusion protein z gene =e is more effective immunogen than a mixture of the two proteins alone. In a specific embodiment of the invention; The isolated or purified M. tuberculosis polypeptides may be formulated as pharmaceutical formulations for administration to a test subject in the event of prevention and/or treatment of M. tuberculosis infection. The immunogenicity of the fusion protein may be enhanced by the inclusion of adjuvant 0 in another embodiment of the invention; Separated or purified polynucleotides are used to produce recombinant fusion polypeptide antigens in vitro 00. alternatively Polynucleotides may be administered directly to a test subject as DNA vaccines to induce the ability to genetically modify the antigen in the test subject; Subsequent induction of an immune response against Mycobacterium tuberculosis anti-M. tuberculosis immune response + It is also an objective of the present invention to use polypeptides in titrations in 1 test tubes for the purpose of detecting humoral antibodies or cell-mediated immunity against Mycobacterium tuberculosis to diagnose infection or monitor disease progression. additionally; Polypeptides may be used as an in vivo diagnostic agent in the form of a transdermal skin test. alternatively; Polypeptides may be used as immunogens to generate antibodies against Mycobacterium tuberculosis in a non-human animal. The antibodies can be used to detect Ye target antigens in vivo and in test tubes.

= For a brief explanation of the graphics, Figure 1 or above: a nucleotide sequence (with Sequence ID: 1) and an amino acid sequence (with Sequence ID: 7) of the SU - fusion protein tri- paras Mtb2A ) Ra35-TbH9-Ral2 fusion protein (form ?: nucleotide sequence (with sequence ID: ?) and an amino acid sequence (with sequence ID: 4) from tri- MTI- protein 0117-4 ( 1105398 designer ) tri-fusion fusion protein + form ?a-”d: nucleotide sequence (with sequence identification number: 0) and an amino acid sequence (with number: Sequence identification: 3) of the tri-fusion protein 101563-38 KD-Tb38-1 0 + form A-D: nucleotide sequence (Vr and sequence Amino acid sequence (SEQ ID: 8) of binary fusion protein 10119-1038-1. Figure 5a-d: nucleotide sequence (SEQ ID: 9) and Vo acid Amino (sequence identification number 01) of quaternary fusion protein 38KD-Tb38-1-DPEP 0 ThRa3 (abbreviated as 1515-2) 0 Figure 1a and ab: nucleotide sequence The nucleotide sequence (VY: amino acid sequence of the pentameric fusion protein Erd14-DPV-MSL-MTCC2) is abbreviated as Mtb8sf. .

This is a #A-LAB motif: the nucleotide sequence (SEQ ID: 1”) and the amino acid sequence (SEQ ID: ;1) of the quaternary fusion protein FRD14 -DPV-MTI-MSL (mt646F form +a-/b: nucleotide sequence (SEQ ID: 105) © and an amino acid (SEQ ID: 105) © 01) of a quaternary fusion protein DPV ~MTI-MSL-MTCC2 (1405718) 0 Fig. 4a-1b : nucleotide sequence (SEQ ID: 117 ) and the 1 amino acid sequence (sequence identification number: 18) of a fusion protein “NAAH: DPV-MTLMSL (abbreviated as 1415318) 0) 019 no. Sequence identification: ( nucleotide sequence Fig. 10!a-10b : nucleotide sequence 0 of a protein ) 010 : Sequence identification number ( amino acid sequence of amino 1 and acid sequence - ) 110617 abbreviated as a (referred to) TGHO-DPV-MTT tripartite fusion (71: nucleotide sequence ID and 11b: nucleotide sequence 11 isoform of the protein (YY sequence ID: amino acid) amino 1 sequence and acid sequence 0 (1415364) Erdl4 —DPV-MTI fusion 10 (YY) sequence identification number: nucleotide sequence Figure 11?-71b: nucleotide sequence Fusion protein (YE sequence identification number: ( amino acid sequence 0 ) 110598 1119-8235) abbreviated as fusion protein

Yay

_!sa fig. It is abbreviated as 111524). of test subjects + PPD upon induction with two fusion proteins and their independent parts Figure 11a-11b: cell proliferation of 7 mice immunized with fusion protein or its independent parts and substance Adjuvant 0 A 0 Fig. 0: IFN-Y production in mice immunized with a fusion protein or its independent parts and an adjuvant YA Fig. 4-11 production in mice immunized with a fusion protein Fusion protein or its independent parts and adjuvant 0 Fig. 513-114 : Antibody alias concentrations of hi-immune serums Fusion protein, its independent parts and adjuvant 0 adjuvant 01 Figure 10a-. Surviving Indian pigs after an aerosol test of Mycobacterium tuberculosis. The fusion proteins SBASIC(20A) mth629A mt632 A or SBAS2(20B) or SBAST(20C) are formulated and used as immunogens in Indian pigs before testing with bacteria immunogen the BCG positive control group. ‎ . In guinea pigs prior to challenge bacteria with 0 positive control

Yave

— 11 1 production of 1712-7 in cells Stimulation of proliferation Induction of proliferation: (YY) and fy) fig. TBHO-Tb38-1 The specificity of antigen 113119 by fusion protein 1 in IFN-Y cells and the production of Stimulation of proliferation Figure 7?A-7?B: Induction of replication. 10119-1638-1 specificity of L 1038-1 by fusion protein of T in IFN-y cells and production Stimulation of proliferation form ??A-"B: induce recurrences © and 1038-1 By protein 1611-9 antigens previously shown to respond to antigens TBHO-Tb38-1 fusion treatment Jud useful for the treatment and prevention of antigens disease The present invention relates to polynucleotides antigens; polynucleotides encoding antigens Antigens and prevention of tuberculosis 0 According to the invention, antigens and special methods for their use. More specifically; antigens against the present invention include a fusion of Mycobacterium tuberculosis that is fused to a fusion polypeptide molecule of less than greater than 10. The antigens of the present invention may additionally include ¢; the polypeptide molecule© of the antigens or To improve immunogenicity, other procedures designed to enhance the immunogenicity of these antigens in other manifestations, such as isolation of these antigens by adding the extended form of . I have a histidine tip. Residues. The tuberculosis specific antigens. Specific antigens for Mycobacterium tuberculosis - 1 m.

-- 11a through 11b; For example ; By adding linker peptide sequences as described above. Peptide sequences in which each of these polypeptides combines one or more of the fusion © fusion polypeptides found in Figures 1a and 1b can be inserted. Other antigens of the present invention a and 11b conjugated to a known antigen 1 are antigens described in Figures 717: previously described (with sequence identification number 38KD antigen alge of M. tuberculosis Like

VEAMYEA) Pages: ov. Immunity from infection ae 0910894 «Andersen and Hansen] Genband Accession No.M30046 + 0 immunogenicity assays The antigens described in the present context are distinguished; immunogenic proteins are among them; with the ability to induce an immunogenic response in portions thereof

AS antigens have the ability to induce 0 in a more specific way. response and/or interferon-y, i.e. cytokine production or 5 Stimulation of proliferation 0, 1116 cells, 8 cells, and/or 7 Wa macrophages derived from interleukin-12 macrophages An M. tuberculosis - from a person (immune) to Mycobacterium tuberculosis The choice of cell type to use in evaluating an immune individual interleukin response to an antigen will depend on the desired response. For example ; Most readily evaluated using preparations containing 13 cells and/or bacteriophages 12 0

- 117 A person who is immune to Mycobacterium tuberculosis is the person who is aware of . large macrophages (it is resistant to the development of tuberculosis by virtue of containing an effective amount of the 7-cell response to Mycobacterium tuberculosis, meaning; essentially free of symptoms of the disease). These subjects are distinguished based on tuberculosis proteins (strongly positive intradermal skin test response to tuberculosis proteins (mm diameter sclerosis) and absence of any signs or symptoms of disease 01 (i.e. more than about PPD©) and cells 3 and macrophages derived from NK-tuberculosis subjects.7 and 7 cells immunocompromised to Mycobacterium tuberculosis may be prepared using methods known to those of ordinary skill in the art, viz., blood mononuclear cells (PBMCS) for example A preparation may be used from peripheral cells (without additional separation of constituent cells. Generally 7131405; for example New York) can be prepared. Also 0 for example using density centrifugation of “710017” cells (Winfrop coefficient 0 0 PBMCs 7 cells may be purified for use in the titrations described in the present context directly from

TR dibs Sf phate lig aim Jail) jae T 4s a Alternatively ¢ may be used for independent mycobacterial proteins. These 7 cell clones may be formed by; For example, 1- From individuals with immunity to Mycobacterium tuberculosis using mycobacterial proteins for example PBMCs; culture defined with mycobacterial protein; leading to a T line; weeks. This allows only 5 of these cells to expand. Then clones of these cells may be formed and then tested with independent proteins 1; in ways known to those of ordinary skill in the field; In order to determine the quality of a cell more precisely. in general; that the antigens that were tested positive and | Or interferon-y 0 cytokine production in assays of bacteriophages and/or production of 8 and/or phagocytes Way NK conducted using interleukin-1 cells and interleukin-12 0 cells.

1” _ macrophages derived from a person with immunity to Mycobacterium tuberculosis are recognized as immunogens. Such calibrations may be made; For example ; with the representative procedures described below. The immunogenic fragments of these antigens may be distinguished using similar assays and may be shown in the range of polypeptides described in the present context. © The ability of a polypeptide (eg antigen or other fragment or variants thereof) to induce Stimulation of proliferation (408) is assessed by cell contact (eg Ja7 cells and/or NK cells). In general, the range of polypeptide quantity considered sufficient to assess about 10 m cells ranges from about 10 ng/mL to about 100 µg/mL and preferably estimated at approx. Incubation of the polypeptide with the cells at TV temperature °C for an estimated period of about six days Followed by incubation with the polypeptide 0 titration of the cells for the proliferative response, which may be assessed by methods known to those with ordinary skill in This field, such as exposing cells to a pulse of radiolabeled thymidine, and measuring the incorporation of label into cellular DNA. A polypeptide that leads to an estimated increase of at least threefold in the aforementioned background than proliferation (i.e., the proliferation observed for cells cultured without the polypeptide to be able to induce proliferation) The ability of the peptide to induce proliferation may be evaluated. Production of interferon-y and/or interleukin-12 in cells by contacting LOA with a peptide and then measuring the level of interferon-y or interleukin-12 produced in cells - 00 overall ¢ Tobi range of 002 µm is sufficient to evaluate about 10 cells from about 10

— 1 µg/10 µg/mL preferably about 100 ng/mL to about ; But he does not need that; On a rigid support ¢ such as a polypeptide bead or globule mL. 1,744,174 biodegradable minutes may be installed; As described in US Patent No. 07581504 with incubation of polypeptide 07581504 typically conducts polypeptide incubation with 1°C for six days »following incubation with 1% of cells at 1°C, 1°C, or more (interleukin-12 and/or interferon-y) titration of cells for "polypeptide which may be evaluated by known methods" one or more subunits thereof of its enzyme subunits - for those with normal skill in This domain ¢ as an enzyme-linked immunosorbent assay; a bioassay of 11-12 p70 Alla subunit or in a linked immunosorbent assay (ELISA) triggering polypeptide production as a cell proliferation assay1. In general, “Ve” 01-501 per ml culture supernatant (containing at least 0 pg of interferon-y on a polypeptide is considered “interferon-y 7 cells per ml) of Capable of inducing the production of; and/or at least 11-12 770 subunit pg/ml of at least subunit 01 production of macrophages "01 per" 1112 240 subunit pg/ml from subunit 0

IL- is capable of inducing the production of (PBMC “01*3) or per BLA Jf macrophages 06 12 Generally; immunogenic antigens are those antigens that induce the proliferation of T cells and/or Ie 12-SSFLA 1161) in interferon-y cells, meaning; cytokine production or production of at least 7 Yo derived from about alia large macrophages and/or 3 Wa phages (NK) immune individuals to Mtb. Among immunogenic antigens; PE 0 with superior therapeutic properties may be distinguished depending on the amount of responses Polypeptides Polypeptides

he — in the above calibrations and depending on the percentage of subjects for whom the response is observed. in addition to ; Antigens with superior therapeutic properties will not induce multiplication and/or cytokine production in test tubes in cells derived from no more than about Yo 7 individuals who are not immune to M. tuberculosis; 0 thereby removing responses that do not specifically result in Mtb-responsive cells. These response-inducing antigens are characterized by a high homozygous ratio of 1¢ NK cell preparations; 3-cell and/or macrophages from individuals with immunity to Mycobacterium tuberculosis (with a low incidence of responses in cell preparations from other subjects) with 0 superior therapeutic properties Antigens with therapeutic properties may also be distinguished Antigens with superior lly therapeutic properties on their ability to reduce the severity of infection with Mycobacterium tuberculosis in experimental animals ¢ When it was given in the form of a vaccine, vaccine preparations suitable for use on experimental animals are described. In detail below. Efficacy may be determined depending on the antigenic potential to provide at least a 7 50 reduction in bacterial population and/or at least 7 0 decrease in mortality following experimental infection. Suitable experimental animals include mice, guinea pigs, Lie and primates (order of mammals) 0?-0 isolation of coding sequences The present invention also relates to nucleic acid molecules acid molecules that encode multiple Ye fusion polypeptides of Mycobacterium tuberculosis. In a specific form in the manner of an example in a clip

11 - - 1 ¢ below; Thirteen fusion coding sequences of Mycobacterium tuberculosis are constructed. According to the present invention; Any nucleotide sequence that encodes an amino acid sequence of a fusion protein can be used to generate generate recombinant molecules that direct genetic modification of the coding sequence. © For the formation of clones of clone full-length coding sequences or homologous variants to generate the homologous variants to generate the polynucleotides 119108 probes (probes) encyclopedia of DNA from any part of the nucleotide sequences nucleotide sequences or their complements disclosed in the present context for the purpose of filtering a genomic dc gana Screen a genomic or cDNA made from multiple strains of M. tuberculosis 0 to characterize the coding sequence for each independent component. Separation of coding sequences may also be done by polymerase chain reactions (PCR) using two degenerate oligonucleotide primer pools designed on the basis of the coding sequences disclosed in the present context. The invention also relates to the separation or purification of polynucleotides of purified polynucleotides complementary Yeo sequences with sequence identification numbers 1, 1, #wu or gu 11, SY daw 1, 434 17, 77 and pe YO providing a polyteclotide polynucleotides that selectively hybridize to a sequence of sequence IDs 1, “F de Lao 1, AVY 11, 51, 17, 71” and Yo or their completed sequences under stringent JB conditions and encode a protein that retains immunogenicity of fusion proteins of sequence identification numbers: 1, ko or VT sh 0, 41, 1, 18, 40 77, 74, and YT by way of example but not limited to; be cases

- Y =; The Society's procedures, 0580 Shilo, Weinberg, representative, less stringent, are as follows (see also pp. 1797-7784): Attending: VA International Academy. United States of America Number of °C in solution containing 40 for 7 hours at temperature DNA containing filters

Tris-HCl Tris-HCl mmol Tris-HCl 00, 25907 and formamide / 6

Ficoll of 1-1 PVP of EDTA + .) mmol of © 5(V.0 pH pH (©)

Asse denatured salmon of clean salmon DNA μg/ml of 500 of 858, 7 and 1 in the same solution with the following modifications Hybridizations natural pages. Hybridizations are carried out μg/ml of 001, R 7 388 and Ficoll 7 slurry and J v.nY. W/V (and probe) 701 dextran sulfates are used. denatured salmon clean salmon DNA measured by MEK no cycle per minute Filters are incubated 2p labeled probe labeled 0 hours in 1-18 hours for hybridization mixture | At a temperature of 00 ° C at 1 ° C, and then washed for a period of 0 °C with a concentration of (Tris-HCI mmol of Tris-HCl) a solution containing 27 550 F, and the solution is replaced Washing SDS 7 11, 8078 and © mmol Vf pH pH 10°C Dry with fresh solution then incubate for an additional 50 minutes at 10° if necessary. autoradiography Filters are allowed to dry and then autoradiated at °C and re-exposed to the film TA Filters are washed three times at 0 to 10 °C Other less stringent conditions that may be used are well known in the field (eg; as The type employed for cross-species hybridizations is used for cross-species hybridizations).

CA - in another preferred form; A polynucleotide that hybridizes to the coding sequence is provided by the sequence identification numbers: 1, 50 7, 53 11, 1, 10 NUS 1 | and ?? and Yo or its complementary sequence under highly stringent conditions and encodes a protein that maintains immunogenicity to fusion proteins Sequence ID numbers: 7, 71, 4, 10, 17©, 14, 11, 118, 70 and 77, 4" and YT by way of example and not by limitation; highly rigorous representative conditions are as follows. Filters containing DNA are pre-hybridized for 1 hour overnight at 10 °C in a buffer solution of SSC * 6, 50 mmol of Tris-HCl (pH 7.9), 1 mmol of EDTA, SPVP/ “oY 1101777, 7+388, 5001 μg/ ml of DNA of 0 clean Algae denatured salmon normal traits EA filters were hybridized for 1 hour at 15 °C in a pre-hybridized mixture containing 100 μg/ml DNA of salmon sperm denatured salmon from normal transduced salmon and 3.101 x cycles per Ady of a labeled probe -0. Filters washed at TV °C for 1 hour in a solution containing 2X SSC 5 ct Ficoll Z 1 3PVP and .++ BSA Z. This is followed by washing at .4 x 880 at 0°C for 0£0 min prior to autoradiography. . Other conditions Ade rigor in which it may be used are well known in the art. Also in another preferred model; A polynucleotide that hybridizes to a coding sequence is provided from the sequence identification numbers: 1, “, #59, 11, #1, 1*, 71, 11, 71 Y., and ?? and Yo or its complementary sequence under moderately stringent conditions and encodes a protein that maintains immunogenicity to fusion proteins Sequence identification numbers: 7, 4, 4, 10, and 11

Yay

- 11 Medium stringent representative conditions YT, 17, 77, 74, 118, 111, 1 and are as follows. Exemplary conditions of moderate stringency filters are pretreated for 1 hour at 85°C in a solution including 1 μg/ml DNA containing Filters 001 μg/mL of 001 508 Jo and 10608718 solution And Dernhart's solution 6X SSC, normalized. Hybridization of a denatured salmon with a DNA© M32 labeled probe takes place one cycle per minute from a Vex 1 labeled probe. The same solution is used for the duration of the hybridization mixture in a hybridization mixture. Filters are incubated. 10 minutes at 0” hours at 86°C and then washed twice for 1-70 minutes. The filters are dried to dryness of 70.1 SDS and 1 x SCC in a solution containing Other moderately stringent conditions that autoradiography and 0° VY autoradiography may be used are well known in the field. Filters are washed at 8057 + .) and SSC 2 hours in . solution comprising sad epitopes Polypeptides encoded by the coding;- polypeptides encoded by /°0 coding sequences of the invention encoding the polynucleotide of the invention; La, Ve polytecolides or small portions may be used of it or functional equivalents thereof for the purpose of generating recombinant fusion protein molecules that direct the ability to genetically modify fusion nucleic acid molecules, small parts thereof, or functional equivalents thereof in appropriate host cells. 0 molecular manipulation of the coding sequence 0 molecular manipulation of the coding sequence

Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same sequences or a functionally equivalent amino acid sequence may be used; In the practical field of the invention for the genetic modification of fusion polypeptides. Such DNA sequences include those8 that are capable of hybridizing to the coding all sequences or their complements disclosed in the present context under the described low, medium or high stringency conditions. in segment 0/7; Same as the previous source. Variable nucleotide sequences which may be used according to the invention include deletions, additions or substitutions of different remaining nucleotide segments leading to a sequence encoding the same sequences or a functionally equivalent gene product. The gene product itself may contain deletions, additions, or substitutions of remaining amino acid segments; which lead to a dormant change thus producing an epitope Functionally equivalent antigen The mentioned protective amino acid substitutions can be made on the basis of similarity in polarity, charge, Ve, solubility, water repellency, hydrophobicity and/or dual pathogenicity of the residues existing. The negatively charged ¢ amino acids include for example; contains aspartic acid “glutamic acid.” Positively charged amino acids include histidine 3 lysine and its potential (amino acids with uncharged polar head groups that have similar affinity values for water include 1 The following: glycine tyrosine 5 ¢ threonine 5 + serine 5 « glutamine y « asparagine 0 ; It includes acids

: - 1 on amino acids with nonpolar head groups + tryptophan ¢ alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine The nucleotide sequences of the invention can be designed to alter the coding sequence of the incorporation protein for a polymer of the endings that include; For example but not limited to ; On the alterations that work to process and modify the genetic product. Transformations can, for example, be introduced using © methods well known in the art; such as place-oriented congenital change; To insert new enclosures;

Caruthers « partial; using chemical methods known in the field. Look ; For example, 0 Gel “Crea and Horn are attached. v; VAAL 6 Nucleic Acid Research Symposium and others; 1980 “Matteucci and Caruthers 7 (01) 4 Nucleic Acid Research; Its alternative is that the amino acid sequence polypeptide itself be produced using chemical methods to synthesize the polypeptide 0 amino acid sequence in whole or in part.For example, peptides can be synthesized by solid phase methods (0 broken from the resin; purified). By a preparative high-performance liquid chromatography process

Proteins Structures; Protein Structures and Molecular Basics 0 1487, Creighton « See .] 1-5. Pages 0 and his company » New York WH. Freeman 0 And Molecular Principles The structure of synthetic polypeptides can be confirmed by amino acid analysis or sequencing (on 0

1- For example; Edman's decomposition procedure » See Creighton; 0187; Protein structures and molecular basics « WH.

Freeman et al., NY, 0 pages (EAE) In addition, the coding sequence for the incorporation protein can be transformed outside or inside the body to create and/or destroy translation, initiation, sand/or termination sequences © / or from new restriction nuclease sites or the destruction of pre-existing ones to facilitate the process of modification outside the body It may include, but is not limited to, congenital alteration known in the field »and includes, for example Hutchinson, C.) et al. (Hutchinson, C.) et al. ¢ 074 « Journal of Biochemistry Yor : 18251 ] ; use of 1437 (Pharmacia) ¢ links and the like; It is important that the treatment does not lead to the destruction of the immune formation process for 1 peptides to incorporate multiple -fusion polypeptides. In addition, nonclassical amino acids or chemical amino acid analogs can be introduced as an alternative or Add to the sequence. Jos non-traditional amino acids; For example but not limited to ; On the Ve D-isomers of common amino acids: a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, €- Ahx, 6- amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, B -alanine, fluoro-amino

ET acids, designer amino acids such as B-methyl amino acids, Ca-methyl amino acids, methyl amino acids, and amino acid analogues in general. The amino acid may additionally be D- (dextrorotary) or B (levorotary). In one particular embodiment of the invention, the coding sequences of each antigen are connected to their insertion protein 0 by means of an amino- or carboxy- bond. terminus at the amino or carboxy terminus, in any order. In an alternative method, a peptide linker peptide sequence can be used to separate the single polypeptides forming the poly-integration peptide by a sufficient distance to ensure that each of the polypeptides has been folded into a binary and triploid structure; Which leads to an increase in its beneficiary efficiency to prevent and treat tuberculosis. Said peptide-binding sequence is incorporated into the fusion protein using 0 standard methods well known in the art. The selection of appropriate peptide-binding sequences can be made according to their inability to internalize (Y) their ability to internalize an extended flexible confirmation process (1) for the following factors a secondary structure that can interact with functional antigen-determining factors on the first multiple peptides and specifying the second alse; (1) lack of hydrophobic or charged residues that can interact with 017 antigen-functional polypeptides. Preferred peptide-binding sequences contain Vo and Thr Jie residues and can also be Use of other near neutral amino acids Ser, Asn, and in the linker sequence Amino acid sequences that can be used as linkers include Ala sequences disclosed in Maratia et al. Gene 460:41-79 1185 and Murphy

CAVIY=AYOA : AY United States of America « Proc. Natl et al. » Academic Science in . 5791176 and US Patent No. 497087 and US Patent No. » 11817 0

1 l and the length of the linker sequence can be from 1 to about 050 amino acids. Peptide sequences are not required when the first and second polypeptides are without non-essential N-terminal amino acid regions; Which can be used to separate functional areas and prevent stereoscopic overlap. can for example; that the © antigens are delivered into a fusion protein by a flexible polylinker such as Gly-Cys-Gly or Ser - Gly—Gly—Gly - and repeats of 1-? times (ay) et al. ¢ Natp.Acad.Scipro: +43 « USA 0101-01 AY ) . In one embodiment, such a protein is produced by recombinant genetic modification of protein-coding DNA. Said integration product can be made by joining the appropriate DNA sequences encoded by 0 amino acid sequences of interest to each other by methods known in the + domain in the correct coding framework; Genetic modification of the product by methods known in the field. Alternatively, Jie could be made of this protein by synthetic protein methods; For example ; using a peptide maker. Lad coding sequences for other molecules (cytokine Jie or an adjuvant 0¢) can be added to the fusion polynucleotide. 0 << production of fusion proteins until the Mycobacterium tuberculosis fusion protein of the invention is produced; A nucleotide sequence coding for a protein or its functional equivalent is inserted within a genetic modification vector ie; The carrier that contains the elements necessary to transcribe and transform the input coding sequence. Host cells or “01” infected or transformed cell lines with recombinant gene-modifying transfer factors can be used for a variety of purposes 0 m

Yo _ ~ This abe includes, but is not limited to; Upon mass production of an integron protein, well-known methods can be used by those with experience in the art to construct gene-modifying transfer factors containing integrin coding sequences and transcriptional/translational control signals. These methods include ex vivo recombinant DNA methods; synthetic methods and Jab body/congenital recombination (see, for example; the methods described in Sambrook et al. 0 19434; Molecular Cloning A Laboratory Manual, “All Cold Harbor Lab; New York » 5 Ausubel et al.; 1949 Current Ceremonies in Molecular Biology; Greene Publishing Fellows and Wiley Intersciences » New York 0 RNA capable of encoding multiple 0 (Afiy) can also be synthesized Chemically Polypeptide may also be chemically synthesized Among the vector systems with the ability of the host's genetic modification to effect genetic modification of the coding sequence of a fusion protein These systems include, but are not limited to: Jie microorganisms Bacteria (Sia) Escherichia coli (B Subtilis “7 coli” transformed by a DNA recombinant bacteriophage, plasmid DNA, or cosmid DNA gene-editing capacity vectors containing a coding sequence or yeast (eg Saccharomycdes “(Pichia) transformed by capacity vectors genetic modification of brewer's yeast containing coding sequences; Insect cell systems infected with recombinant virus modifier vectors (eg “baculovirus” 0) containing a coding sequence; plant cell systems infected with recombinant virus modifier vectors (eg baculovirus)

( TMV «tobacco mosaic virus ; Camv ,Canliflower mosaic virus transformed with recombinant or transformed by recombinant plasmid expression vectors containing coding sequences or cell systems (Ti plasmid Sie ) plasmid expression vectors

CHO « COS cells (Mia) containing a coding sequence; or mammalian cell systems ) 333 «293 » BHK «©» 1051/76010+ system utilized JU depending on the host system any number of suitable transcription and transformation elements; including basic and inductive stimuli; In the carrier of the ability to modify genetics. For example; upon cloning in bacterial systems; it is possible to use; Ptrp-lac promoter Mixed promoter (2, plac, ptrp, ptac Phage PL Inducible triggers such as etc.; when cloning in cytomegalovirus promoter Vo systems is possible The use of stimuli such as a polyhedral enhancer + 1096© cell systems insect LA; when cloning in plant cell systems; it is possible to use stimuli derived baculovirous heat from a genome (group of genetic factors) of plant cells (eg; shock stimuli Free or RUBISCO small subunit » shock promoters or from chlorophyll binding protein alpha/beta Ye chlorophyll binding protein CaMV 355 RNA promoter Mia plant viruses Plant viruses When cloned in mammalian cell systems, it is possible to use coat protein promoter TMV (metallothionein promoter, for example Adil) promoters derived from the genome of adenovirus cells late, for example, the promoter of late adenovirus (mammalian viruses or mammalian viruses when generating lines) vaccinia virus 7.5K promoter cowpox virus 0 promoter 0

1717 Cells that contain multiple copies of the antigen coding sequence can use vectors. With suitable testable markers, EBV and BPV, 5740, with a base of Mycobacterium tuberculosis. For antigens, to perform genetic modification of Ayal antigens, systems are preferred

Bacillus-Calmette-guerrin Jie for administration in vivo + it is possible to engineer species of bacteria for genetic modification. Integration polypeptide of the invention on its cell surface. It is recommended to choose a number of © e.g. 0. Bacterial gene editing vectors Depending on the use for the purpose of transgenic products when large quantities of fusion protein are produced for pharmaceutical formulation ¢ it may be desirable for vectors that direct gene modification with elevated levels of purified fusion protein products Easily . These vectors include; for example, and not as a limitation ; genetic modification vector 7; EMBO J “YAY) et al. Ruther (pUR 278 E. coli for Escherichia coli 0) in which it is possible to associate the coding sequence in the vector in frame with coding region 1867 such that (YY

PIN Hybrid protein ¢ vectors and the like are produced. It is also possible to use 00727 to perform genetic modification of foreign polypeptides, generally prepared. Glutathione S-transferase (GST) with fusion proteins as fusion proteins These fusion proteins are soluble and can be easily purified from cell lysates by 12 followed by dilution in the presence of free glutathione-agarose “adsorption” pellets. Adsorption or thrombin cleavage sites for PGEX vector design include ais. free glutathione so that it is possible to liberate a G-woven integrase polypeptide of the subject of the invention from the factor Xa protease GST + protein purification cleavage. Aqent-1/5 / © Y.

- 18 Once a recombinant protein has been genetically modified; Where it can be identified by the calibrations of the product; Including labeling of physical or functional properties based on chemical and functional properties and immunoassays Radioactive gel electrophoresis of the product followed by analysis by hemoelectrophoresis etc. Radioimmunoassay bioassays and radioimmunoassays It can be isolated and purified by «encoded protein» once the encoded protein is identified © as a class chromatography Standard methods including exchange chromatography high performance liquid chromatography sizing column, affinity and volumetric column chromatography jon ion exchange differential solubility ¢ centrifugation centrifugation ¢ chromatography other standard technique; The effective conditions will, in part, depend on factors such as net charge, etc., and will be obvious to those with hydrophilicity + hydrophobic skill in this field. Functional properties may be assessed using any appropriate calibration such as “T cell oroliferation”. Stimulation of proliferation Mall, 11.4, and 11-7 are preferred. With regard to practice, the invention, 112 Jie cytokine, stimulated the production of 05 other proteins. of (hie) at least J Av fusion protein each fusion protein at least. For in vivo administration 7 900 Preferably purified proteins. 7 46 Live; preferably proteins with greater purity than -uses of the fusion protein coding sequence

Ya_- The fusion protein coding sequence of Jal, encoding a protein product, can be used for use as an immunogen to induce and/or enhance immune responses to M. tuberculosis. add to that ; It is also possible to bind the coding sequence to another molecular coding sequence, a cytokine Je or an adjuvant. These © polynucleotides can be used in vivo as a DNA vaccine (US Patent Nos. 75, 1147/17H and 315 LA). In this embodiment of invention; A polynucleotide modifies the genetically encoded protein in a receptor for direct induction of an immune response + polynucleotide may be injected. to an uninoculated study subject to initiate an immune response to the encoder producer; or given to an infected or vaccinated study subject to enhance secondary immune responses in a preferred embodiment; A therapeutic composition includes the coding sequence of a fusion protein or parts of the aie that is part of the gene modification vector. In particular ¢ this polynucleotide contains a functionally inactivated motif in the coding region ¢ and said motif is inducible or constitutive; And ; my choice ; Kind of tissue. in another model; The polynucleotide contains a free coding sequence with 0 regions that trigger homologous recombination at a desired site in the genome; Thus providing an intrachromosomal expression modification of the 'Koller and Smithies' coding sequence Proc.

Natl.

Acad 4 « USA 01144 817 0-/1 737 : AY ; Nature EYA-EYo: YiY . The administration of nucleic acid may be subject to study either directly; In that case, the subject is exposed to DNA or Jala's nucleic acid-carrying vector, or indirectly; in that case; Cells are first transformed by Yay ¢

l DNA in test tube 1:70; Then implanted in the subject of the study. That these two entrances mentioned are known; respectively, as in vivo or ex vivo gene transfer in a qualitative model; gives DNA directly in vivo; Where it is genetically modified production © fusion protein encoded product. It can be done by any of the many methods known in the field; se; by its construction as part of an appropriate DNA genetic modification vector and delivery so that it becomes intracellular “Sia” by infection events with an attenuated retroviral or other incomplete or weakened viral vector (see US Patent No. 49480747) or by direct injection of exposed DNA or by using microparticle bombardment, for example; 0 Dupont « Biolistic » gene gun or coating with lipids 0 cell-surface receptors or Transfecting factors or encapsulation in liposomes or microparticles microcapsules (US Pat. Nos. 8407704, 04677717, 811364, 87E287) or by binding to a linkage to a peptide that is known to be alas ¢ enter the nucleus by binding to a ligand of a cellular endocytosis mediator administering it in linkage to a ligand subject to receptor-mediated endocytosis (see and Wu Sie 70 and 17 Journal of Biological Chemistry; 767: 477-4474) that can be used to target cell types that make specific transcripts of receptors. target cell types specifically expressing the receptors 00 “etc. In another embodiment 0 it is possible to form a nucleic acid-ligand complex can be formed in which the complex includes

1- fusogenic viral peptide to disrupt endosomes; thus allowing the DNA to avoid autolysosomal decay. In another additional appearance; It is possible to target DNA in vivo for cell-specific internalization and genetic modification by targeting a specific receptor (see eg 0 Publication 007 or VUE TIA [AY © Apr 447 178/17 YY dated 1 Dec; 447 A or 10713/47 of 77 Nov 17; or ?1/YEYAA of vy July 1997 or 47 2 70771 of 04 Oct 14). alternatively; It is possible to introduce cellular DNA or to incorporate it into the DNA of a host cell for genetic modification by means of Koller and homologous recombination Proc.

Natl.

Aced.

Sci + 7 + Smithies; USA 07 : 4373-4477 Zijlstra « V+ et al 2 anc VAS 77 : 74-475 ). in a qualitative form; It is possible to use a viral vector such as a retroviral vector (see 7017 “Meth Enzymol 41+ coals Miller: 41-041) Retroviral vectors are modified to eliminate retroviral sequences that are not necessary for packaging a viral genome. packaging of the viral GENOME and its integration into the DNA of a host cell. Ve is transcribed into a fusion coding sequence; which facilitates the administration of DNA to a recipient. Further details of retroviral vectors can be found in 20 et al.; 11944; Biological Therapy 7-711 4 Biotherapy; which describes the use of a retroviral vector to administer gene 14081 to hematopoietic stem cells to make stem cells more resistant to chemotherapy. There are other references that explain the use of retroviral vectors in the genetic treatment of Ye. He, Clowes et al. 4; Journal of Clinical Investigations; 47 : 181-144, Kiem et al. 84 ¢. Salmons and Gunzberg « VEVY = 1£1Y : AY Blood « Ya)

vy _ 15 ; human gene therapy Human Gene Therapy ¢: 711-1 or 0 +, Grossman and Wilson + Current Opinions in the World of Genes and Evolution”; ? : 114-111 2 Adenoviruses are other viral vectors that can be used in gene therapy ¢ Adenoviruses are particularly attractive vectors Specifically to pass on 6 genes to respiratory epithelia. Usually, adenoviruses infect the respiratory epithelia. Cua respiratory epithelia may cause mild illness. Other targets of adenovirus-based administration systems are liver, central nervous system, endothelial cells 1101, and muscle. Adeno-associated virus (AAV) 0 has been proposed for use in in vivo (Walsh) gene transfer (1947). Social.

Exp.

Biol.

Med Am 011 K 5-1 Another entry involves converting a structure into cells in tissue culture by these methods on electroporation iim i.e. lipofection or 0 mediated with calcium phosphate mediated 0 ; Or my viral infection. Normally the conversion method includes convert and convertible name to cells. Then the cells are put under selection to isolate those cells that pick up and genetically modify the transgene. After that, the cells that are subject to the test are given - in this model, the nucleic acid is inserted into a cell before giving it in vivo (ov).

“+ input by the method known in the field”; included; For example but not limited to ; transfection i.e. electroportion or microinjection or infection with a viral or bacteriophage vector containing nucleic acid sequences; Cell fusion + chromosome-mediated gene transfer © ¢ microcell-mediated gene transfer ¢ fusion 1 + etc. There are great technical methods known in this field to introduce foreign genes into cells (see Loeffler and Behr 0 Sie « Cohen » TYA=294 : 27 meth Enzymol] 147 others « Meth.Enzymol » 0337 ,: Y4 1 pharmaceutical.

Ther « Y4A0 + Cline ¢T££—-TYA : YVV | 140000 ) and may be used with the present invention. The polynucleotides of the invention may also be used in the diagnosis of tuberculosis in order to detect specific polynucleotide sequences of Mycobacterium tuberculosis in a patient. It is possible to complete this disclosure; For example ¢ by isolating polynucleotides from a biological sample die then obtained from a patient suspected of being infected with the bacteria e when isolating polynucleotides from a biological sample » A oligonucleotide tagged with the invention and which is complementary to one or more polynucleotides will be allowed to hybridize to polynucleotides in a biological sample using nucleic acid hybridization techniques known to those skilled in this art. For example, 0 is possible to cross

1 — - mentioned in solution or with a hybridization partner on an adjuvant hardness y/o - therapeutic and prophylactic uses of the fusion protein. © Partially extruded or purified fusion proteins or portions thereof may be formulated as a vaccine therapeutic composition. A therapeutic combination such as this Julse may include adjuvants to enhance response. add to that ; These proteins may also be suspended in an oil emulsion to cause a slower release of the proteins in vivo upon injection 0 . The best proportions of each ingredient in the formulation may be determined by technical methods well known to those skilled in the art. Any variety of adjuvant agents may be used in vaccines of the present invention for the purpose of enhancing the immune response. Most cofactors contain a substance designed to protect the antigen from rapid catabolism; aluminum hydroxide fie or mineral oil and a non-specific Ve stimulus for the immune response; such as grease 7; Bortadella pertussis or Mycobacterium tuberculosis. Commercially available catalysts include; For example ; Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories (Merck 10 65 Merck Adjuvant &Co.;LLC;Rahway; State of New Jersey). Other suitable adjuvants include alum 01; Biodegradable microspheres ¢

— Yo —

Ling ) monophosphoryl lipid A, quil A, SBASIc 2 and others; 01337; the vaccine

,. AL(OH)3, SBAS7, (Y01V=101Y) pages : 11 Vaccine The present invention; it is preferable that the adjuvant induces an immune response Vaccines in vaccines containing appropriate adjuvant regimens, eg; Chemical conjugation of a Th lipid including the faces of 3-de-O-acylated monophosphoryl lipid A preferably ? (3D-MLP); in particular the combination saponin derivative and the monophosphoryl lipid saponin derivative of M and saponins 0521 as disclosed in the international patent 3D-MLP chemical from or genetically reactive composition of Less, as 0521 is suppressed by cholesterol as detected

0 reported in patent dal 797349/97© . Previous experiments demonstrated a clear synergistic effect of the chemical conjugates 30-01 and 0821 in inducing both humoral and type 1111-specific cellular immune responses. An active adjuvant formulation comprising 0521, 3D-MLP and tocopherol in an oil emulsion is described. Oil emulsion is water soluble in International Patent 20 [IVT] and is a preferred formulation.

10 Formulations containing the antigen of the present invention may be administered to the test subject known by itself or in the form of a pharmaceutical or therapeutic composition Pharmaceutical compositions containing proteins may be made by conventional processes of conventional mixing HLA dissolving, granulating, dragee-making, crushing, levigating, emulsifying, forming capsules, or

00 Forming by confinement or by drying in the case of freezing. Pharmaceutical formulations may be formulated in the traditional manner

0 #1 using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate processing of polypeptides into pharmaceutically usable formulations. The exact formulation depends on the method of administration chosen. © 0 for topical administration; Proteins may be formulated as solutions 5011005; gels; ointments – creams; suspensions » etc.; It is well known in this field. Somatic formulations include those formulations designed for parenteral administration; for example by subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection; In addition to those formulations designed for transdermal, transmucosal, oral, or pulmonary administration. for injection; Proteins in “aqueous solutions dsl” preferably in physiologically compatible buffer solutions Jie Hanks' solution or Ringer's solution or physiological saline buffer may contain 0 The solution is based on formalizing agents such as suspending agents ¢ chemical stabilizing agents and/or dispersing agents. alternatively Proteins may be in powdered form for combination with a suitable carrier; For example, sterile water without pyrogen before use. For transmucosal administration; Combustion agents are used to penetrate the barrier in the formulation. Generally, these penetration factors are known in this field.

017 _ for oral administration; Synthesis can be easily facilitated by aggregating the proteins with pharmaceutically acceptable carriers that are well known in the art. Such carriers help proteins to be formulated into tablets; pills ¢ dragees .; capsules liquids gels types of syrups types of slurries; © suspensions and the like; For oral ingestion by the person being treated. For hard oral formulations quot; For example ; Powders, capsules, and tablets » Sawa gat excipients include suitable SDA fillers such as sugars » such as mannitol sucrose 5 lactose and sorbitol ¢ Cellulose preparations such as starch Maize ; wheat starch; Las rice ¢ Wis potato « methyl » gum tragacanth 6 gelatin sodium carboxymethylcellulose « cellulose » hydroxypropylmethyl « cellulose 0 and/or polyvinylpyrrolidone (PVP) » granulating agents and binding agents agents. if desired; crumbling agents may be added; cross-linked polyvinylpyrrolidone Jie or agar or alginic acid or salt thereof sodium alginate Jie if desired; Solid dosing forms may be coated with sugar 0 or 1 may be enteric-coated using standard techniques + for liquid preparations such as: e.g.; For example ; Suspensions, elixirs, and solutions include carriers, excipients, or appropriate diluents. Glycols diluents include water; oils; Other alcohols. additionally; Flavorings, preservatives 001, coloring agents and the like may be added to oral administration; The proteins may take tablet form; lozenges; Etc . Crafted in the traditional 0 Yard

YA - - for administration by inhalation; The proteins for use according to the present invention are conveniently handled in the form of an aerosol spray from sl pressurized packs in the form of a nebulizer; With the use of a suitable propellant “on trichlorofluoromethane” dichlorodifluoromethane for © Ju

dichlorotetrafluorocthane©; J Carbon dioxide or other suitable gas. In the case of pressurized aerosol, the dosing unit may be determined by providing a valve to take a measured amount. Cartridges and capsules may be formulated from, for example; 70 For use in an inhaler or insufflator containing a powdered mixture of proteins and a suitable powder base such as lactose or starch.

Jia vaginal or rectal The proteins may also be formulated into formulations for rectal use 0 ; For example ; Conventional suppository bases containing retention enemas or injections suppositories or cocoa butter such as conventional suppository bases. Other glycerides Proteins may also be formulated as a buffer preparation 0 in addition to previously described formulations

05 . Such long-acting formulations may be administered by implantation (eg subcutaneously or intramuscularly (or by intramuscular injection, etc.; eg JU); proteins may be formulated using polymeric materials. or non-hydrophobic (such as emulsion in an acceptable oil) or jon exchange resins;

,. insignificant For example ; As a slightly soluble salt Ye

alternatively said; Other pharmacy handling systems may be used. Liposomes and emulsions are well-known examples of circulating vectors that may use dimethylsulfoxide (Jie ime) organic solvents, despite the cost of significant toxicity. Capsids of fusion proteins may also be formed into microspheres (US patent numbers United 8407604; © Ameh 43422 Ehma Hm 087) In addition, proteins may be handled using a supported release system “Jia” semi-permeable template materials of solid polymers containing the therapeutic or vaccinating agent Numerous enhanced release agents have been formulated and are well known to those skilled in the art. Supported release capsules, depending on their chemical nature, may release proteins for a period of a few weeks up to Ve over 100 days. Chemical and biological stability of the reactor Additional strategies for protein stabilization may be used Determining an effective amount of a fusion protein to induce an immunogenic response in a subject is within the range of capabilities of those skilled in the art; particularly with regard to the discovery described in detail provided in the present context. An effective dose can be initially estimated in in-test tube titrations. For example ; A dose can be formulated in animal models to achieve the induction of an immune response by techniques that are well known in the field. A person of ordinary skill in this field can bring administration to humans as close to efficacy as possible based on data from animal experiments. The measuring quantity of 0 doses and the time interval may be set independently. For example ; when used as a vaccine

A blood vaccine ¢ may administer the polypeptides and/or polynucleotides of the present invention in approximately 1 to 1 doses for a period of 6 weeks. preferably motherless | a.

Uae doses ‘ at intervals of about 7 months; Vaccines may be given stimulant periodically after that. Alternative protocols may be appropriate for individual patients. An adequate dose is about the amount of polypeptides or DNA when given as described above; It is capable of inducing an immune response in an incubated patient sufficient to protect the patient from infection with Mycobacterium tuberculosis for at least 1-7 years. In general jad is the amount of polypeptides present in one deja (or produced at a site by DNA in a single dose) with a range from about 1 pg to about 0 001 mg per kg from the family; Typically from about 0 1 picgram to about 1 milligram and preferably from about 0 picgram to about 1 μg . The appropriate dose range will vary according to the size of the patient; Accents will typically range from about 00 mil to about © mil.

Diagnostic uses of the fusion protein 1 The fusion polypeptides of the present invention are useful in the diagnosis of tuberculosis infection in vitro and in vivo. The ability of the polypeptide of the present invention to induce AS Stimulation of cell proliferation or cytokine production can be calibrated by methods disclosed in

Section fo 7 + the same source as the previous 0.

—-? 1

In another aspect, the present invention provides methods for the use of one or more fusion polypeptides for the diagnosis of tuberculosis, using an in vivo skin test. AS USED IN THE CURRENT CONTEXT + A skin test is a direct-on-protein titration in which a delayed-type (Jie) hypersensitivity reaction (DTH©) is measured Swelling, reddening, or dermatitis Subsequent to the transdermal injection of one or more polypeptides as described above This injection may be made using any suitable device 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 should be measured after 4 hours

Less than injection and more preferably about EA to VY 1 hour after injection.

0 Reaction 10111 is a cell mediated immune response; that are significant in patients who have previously been exposed to an antigen test (eg, the immunogenic portion of the polypeptide Asad used; or a variant thereof). Response may be measured visually; using a ruler. in general; be a response that exceeds the diameter of about DR; poison ; preferably more than about 1 cm in diameter; positive response; indicative of infection with tuberculosis, which may

0 active disease appears or does not appear as an active disease 0 of the invention 0 polypeptides Preferably multiple fusion peptides are formulated; As multi-containing drugstore formulations, the current skin fest; For use in skin testing Typical formulations contain . physiologically acceptable carrier Lis) gush and acceptable carrier agin

such as these on one or more of the polypeptides mentioned above in a quantity range from about 1,000 micrograms to about 100 micrograms; preferably from about 10 micrograms to about

1 _ 0 microgram has a volume of 6.1 mL. preferred; The carrier used in such pharmaceutical formulations shall be saline solution with appropriate preservatives Tween lf 5 phenol Jia 4 . in another guise; The present invention provides methods for using polypeptides for the indicative diagnosis of tuberculosis in this manifestation; Methods for the detection of Mycobacterium tuberculosis infection in a biological sample using multiple fusion peptides alone or in the form of a chemical conjugate 0 as used in the present context are provided; A "biological sample" is any antibody-containing die obtained from a patient. preferably pic Yo Whole blood sample; Sputum products ¢ serum ¢ plasma » saliva » cerebrospinal fluid or urine. More preferably the sample is a sample of blood, serum, or plasma obtained from a patient or a blood source. A polypeptide is used in titration; as described below; Determines the presence or absence of antibodies to polypeptide(s) in a sample with respect to a predefined cut-off value of 0. The presence of such antibodies indicates sensitivity to Mycobacterium antigens that are indicative of tuberculosis in models using more than one fusion polypeptide; The polypeptides used are preferably complementary (ie, one component polypeptide will be intended to detect infection in samples where infection with another component jal 0 (polypeptide 0) would not be detected

_m) Generally, in order to evaluate serum samples obtained from a series of patients known to have infection, polypeptides complemented using each polypeptide may be independently distinguished for M. tuberculosis infection. After determining which of the test samples is positive (as described below©) using each of the polypeptides, chemical conjugates may be formulated of two or more fusion polypeptides that are capable of detecting infection in most or all of the tested samples. Such complementary polypeptides Approximately 7-11 01 of sera from individuals with tuberculosis infection are negative for antibodies to any single protein Therefore complementary polypeptides may be used in the form of a chemical conjugate in order to improve the sensitivity of the Improve 0 diagnostic test sensitivity of a diagnostic test There are a variety of assay formats known to those of ordinary skill in the art to use one or more polypeptides in order to detect antibodies in a sample. Antibodies: then 0 Laboratory Manual, Cold Spring Harbor Laboratory , which has been incorporated into the present context 0 by reference to 0 in a preferred embodiment Including titration using a polypeptide immobilized on a rigid support to bind to and to remove antibody from the sample . The bound antibody may then be detected using a detection reactor that contains a reporter group. Suitable detection reactors include antibodies that bind to a polypeptide complex and free polypeptide labeled with a reporter group Yo (eg in a semi-competitive titration).

- cf. . which antibody substitutes for it; It is possible to use competitive calibration; In it, an antibody that binds to a polypeptide with a receptor group is labeled and allowed to bind to a stopped antigen after incubating the antigen with the sample. That the extension for which parts of the sample inhibit the binding of the labeled antibody to the polypeptide ail is an indication of the binding of the labeled antibody to the polypeptide ail .immobilized polypeptide © solid material solid support The solid support can be the opposite. For example; ALGE may be known to those of usual skill in the art to which nitrocellulose is attached as a test vessel to a micrometer, solid support, or other suitable membrane. alternatively; The stent may be a sphere or a disk; Such as glass or polystyrene, Jie plastic with its juice, or latex, or tree milk, fiberglass, fiberglass 0. The support can also be. polyvinylchloride or polyvinyl chloride polystyrene

Jie « fiber optic sensor lea or magnetic particle. 0784741 in US Patent No. Mia » those means that have been disclosed by using a variety of purification methods “solid support polypeptides can be attached” known to those skilled in this field. In the context of the present invention The term "covalent 0" denotes And covalent bonding, adsorption, such as adsorption 0, to both non-covalent pools, which may be a direct bonding between the antigen and the functional groups (attachment to the support), or it may be a bonding in any way from an antigen. and functional groups on a vessel in a microtitration plate or adsorption preferably by adsorption cross-linking agent on a membrane. In these cases, it is possible to achieve a polypeptide contact adsorption in Yo solution

to _ — oD regulator; With solid support for a suitable amount of time. The contact time with +temperature varies, but typically »temperature ranges from about 1 hour to 1 day. Generally + after contacting a container with a micro titer plate contacting a well of a plastic (jal microtiter plate) for example polystyrene or (polyvinylchloride) with an amount of polypeptide © ranging from about 0 ng to about 1 microgram, preferably about 100 nanograms, sufficient to bind an appropriate amount of 0 antigen. Generally, covalent attachment of a polypeptide to a rigid support can be achieved by first reacting the support with a bifunctional reactor which will in turn react With both the support and a functional group “(Jie) a hydroxyl or amino group on the polypeptide. Si; it is possible to bind the polypeptide to supports with a suitable polymer coating 10011216 2 using benzoquinone or by condensation of the aldehyde group on the support with a hydrogen 5 amine active on the polypeptide (see eg Immunotechnology catalog and handbook, 1991, at A12-A13 6 In certain embodiments the titration is a titration of an enzyme-bound immunosorbent (ELISA) This Vo Yl assay can be performed by means of a fusion peptide-polypeptide antigen contact that can be fixed to a rigid support; Commonly on a precision calibrated board wall; with the sample; This allows the antibody to the polypeptide inside the sample to bind to the polypeptide stabilizer. Then the unbound sample is removed from the stabilized polypeptide and a detection reactor capable of binding to a complex complex of the stabilized polypeptide antibody is added. After that it is determined how much of the detection reactor remains bound

fn _ - on the rigid support 10 using an appropriate method Jeo lial specific reagent 61601101 . more qualitatively; Once the polypeptide has been fixed to the stent as described above; Typically, the remaining protein binding sites are confined to the support. From © any suitable limiting factor known to those of usual skill in the field may be used; Jia bovine serum albumin or (01 Tween trademark) ( Sigma Chemical Co., (St.

Louis, MO. Then the incubation of the immobilized polypeptide with the sample; It allows the antibody to bind to an antigen. suitable dilution is possible; such as phosphate-buffered saline (PBS) before incubation. Generally ; The contact time (Ye) masked is the period of time that is sufficient to detect the presence of an antibody within a sample infected with Mycobacterium tuberculosis. It is preferable that the contact time be sufficient to achieve a binding level of at least 790 of what is achieved when equilibrium between the bound and unbound antibody. Those of you who are skilled will know that the time necessary to achieve equilibrium can be easily determined by calibrating the level of correlation that occurs over a period of time. at room temperature; It is necessary for VO to have an incubation time of about 0? minute. Afterwards the unbound sample may be removed by flushing the scleral support with a mild buffer solution; Such as containing 01 Tween #7 1 (trademark) It is then possible to add a detection reactor to the solid support. A suitable detection reactor for any compound bound to a stabilized polypeptide antibody complex can be detected by any one of a variety of means known to those in the field. Preferably contain detection factor

— a binding factor (Sie protein A, lectin of G protein, or free antigen) coupled to a reporter group. Preferred reporter groups include enzymes (such as horseradish 0) peroxidases, substrates, cofactors, inhibitors, dyes, radionuclides, fluorescent groups, biotin fluorescent groups, colloidal particles such as gold and colloidal selenium pe Colloidal gold and selenium It is possible to conjugate the binding agent to a reporter group using standard methods known to those of ordinary skill in this field. , San Francisco, CA, and Pierce, Rockford.

Illya. 0 After that, the detection reactor is incubated with an immobilized polypeptide-antibody complex for a period of time sufficient to detect the bound antibody. Generally it is possible to determine an adequate amount of time from the manufacturer's instructions or by calibrating the level of bonding that occurs over a period of time. The unrelated detection reactor is then removed and the detection reactor jag pall is detected using the reporter group. The method used to detect the reporter group depends on the nature of the reporter group 0 . For radioactive groups; Scintigraphy or autoradiography methods are generally appropriate. Microspectroscopy methods can be used to detect dye and photoactive groups or fluorescent groups Biotin may be detected using avidin conjugated to a different reporter group (commonly a radioactive group, a fluorescent group, or an enzyme) A general approach may be to detect 0 enzyme messenger groups by adding a substrate (generally for a specified period of time).

th - - (a specific period of time.), followed by spectral or other analysis of the reaction products 0 in order to determine the presence or absence of M. tuberculosis antibodies in the sample; generally the signal is compared detected from the reporter group that remains attached to the solid support oo with reference to Bla a predetermined cut-off value.In one of the preferred embodiments the cut-off value is an average rate signal obtained when the immobilized antigen was incubated with Samples from an uninfected patient In general, a sample generating a signal with three cin titres above the pre-determined cut-off value is considered positive for tuberculosis In a preferred alternative model, the cut-off value is determined using the Receiver Operator Curve according to the Sackett 0 et al + 1¢ on Clinical Epidemiology Lite Brown and Partners pages 011 and 0117 In summary, in this form, may be specified The cut-off value of a spot for pairs of true positive rates (ie sensitivity and false positive rates of quality 0) that each correspond to the potential extraction value of a diagnostic test result. The cut-off value on the spot closest to the upper left ve corner (that is, the value that includes the largest area) is the most accurate cut-off value; A sample generating a signal that is higher than the cut-off value determined in this way may be considered positive 0 alternatively; The cutoff value may change to the left along the spot; To minimize the false positive rate Se to minimize the false negative rate . Generally ¢ a signaling sample that is higher than the cut-off value determined in this way is considered positive for tuberculosis. Yay ¢ |

-ee; Calibration takes place in a rapid flow interstitial or slide test format; Whereas, Ala is done in a flow-form in the interstitial flow test. nitrocellulose Jie + immobilized antigen on a membrane; The antibodies inside the sample bind to the through test as the sample passes through the membrane. Then, a detection reactor is attached to the polypeptide and polypeptide antibody composition, whereby the solution containing the detection reactor flows through the membrane. The correlated detection reactor may then be detected as described above. in slide test format; One end of the membrane to which the polypeptide is attached is immersed in the solution containing the sample. leave. The sample aligns with the membrane through the area containing the detection reactor and into the area of the polypeptide immobilizer The concentration of the detection reactor in the polypeptide typically indicates the presence of antibodies to Mycobacterium tuberculosis in the sample » The concentration of the detection reactor at that location is typically; Like a line. 0 is visually readable. The absence of such a model indicates a negative result. Generally ; The amount of membrane-mounted polypeptide is tested to be a visually distinguishable sample when the biosample contains; The ELISA was also done on a level of antibody that will be sufficient to generate a positive signal in * clarified above. preferred; The amount of membrane-bound polypeptides is estimated at approx. nanograms 80 micrograms; More preferably from about 1 ng to about 0 serum one drop (of Mie serum) typically these tests can be performed with a very small amount of 0.0 or a patient's blood. The description of the invention is then given examples As a way of explanation, these examples are not specific to its scope for Mycobacterium tuberculosis. It retains the generation of antigens wall generators of fusion proteins, example: fusion proteins 4 Immunostaining for independent parts 0

4E1 Materials and methods 1 / 7 Construction of fusion proteins: The coding sequences of the Mtb antigens are modified by PCR in order to facilitate their fusion and later genetic modification of the fusion protein DNA is amplified using buffer solution 717 in a volume of 01 μl 01X and ? μl. 01 mmol dNTPs, ¥ μl of all 01 μmol PCR primers, 2 μl AY water, 10 μl Plu (Stratagene, La Jolla, CA) DNA ads, and 1 μl of DNA at a concentration of either Vie ng/μl (for antigen 3 151828) or 0 5 ng/μl (for TH38-1 antigens 0 kDa YA). for algal antibody 161883 ; Copying takes place at 54 °C for Y min. followed by +6 cycles at 17 °C for 11 sec, YY °C for 1 min and finally 0 at YY °C for ; minutes . for TA antigen kDa; Copying takes place at 97 degrees die for 1 min ¢ followed by 0 5 cycles of Augie AT for 0 5 sec, TA °C for 10 sec and VY °C for ? minutes and finally by VY °C VO for ; minutes . for antigen 1038-1; Replication takes place at 94 °C for a period of ? min followed by 01 cycles at 7°C for 115 seconds, TA°C for 11 seconds, 17°C sad 1.2 minutes; 0 5 cycles at 1°C for 11 seconds; TE is Celsius for 0 seconds, VY is Celsius for V0, and VY is Celsius for ; minutes . Following digestion Restriction endonuclease Restriction to produce cohesive blunt Yo ends; A specific polynucleotide is attached to each fusion polypeptide

1e - in the genetic modification plasmid ligated into an expression plasmid Each resulting plasmid contained a . Coding sequences of independent antigens for each fusion polypeptide. The gene editing vectors were PET-12b and 01771101. The three coding sequences of antigens 2, P7011, and Ra35 are ligated to encode a single fusion protein © (sequence identification number: 1 and ?) (Figs. 1 or 7b). The three coding sequences of Erdl4 antigens, 077 and 1411, are also ligated to encode the OL fusion protein (sequence ID numbers: 7 and ?; ) (Fig. FY). The three coding sequences of antigens 1051883, 3860 and . TH38-1 encoding one fusion protein (sequence ID: © and 1 ) (Figs. ?a-?d). Two of the 0 coding sequences ThHg and 1038-1 are ligated The four coding sequences of TbRa3 antigens 5 38120, 38-1 156, and DPEP are ligated to encode a fusion protein. protein (sequence identification numbers: 11 and 14) (Figs. A and 7B).The four coding sequences of antigens DPV, 1411, MSL and MTCC2 are ligated to encode a fusion protein. aly protein Ve (sequence identification numbers: 117 and 18) (Figs. 4a and 4b) The three coding sequences of antigens 9, PDV and MTI are ligated to encode a protein One fusion protein (Sequence ID numbers: 4 and 01) (for forms °o a-e-j). The five coding sequences of antigens antigens 14 1:0, DPV 5 1411, MSL, and 2 encoding a fusion protein (sequence identification numbers: 11 and 11) are ligated (Figs. 1a and 11b). The four coding sequences of the s DPV 5 Erdl4 antigens 1111 and MSL are ligated to encode a single fusion protein (sequence identification numbers: 5 and “1” (Figs. 01a and

_ 1e 0 b). The three coding sequences of sErd 14 antigens 7 and 7 are ligated to encode a single fusion protein (sequence identification numbers: 71 and YY (Figs. 11 and 11b). The two coding sequences are ligated of antigens 10118 and encoding a single fusion protein (sequence identification numbers: 17 and JOYE Figs 11 or ©11b). Sequence identification numbers (Yi, ve: (Figs. PAY and ?Ab). Recombinant proteins were expressed in Escherichia coli 7. Genetic modification is carried out using a residue year of histidine from a protein Amino-terminal portion 0 using a plasmid vector PET-176 (PET) and a polymerase gene modification system (WI « Novagen, Madison) T7RNA expression system) and an Escherichia coli strain (Novagen) was used BL21 ( DE3 ) PlysE £. coli for high level gene modification High level 0 expression Recombinant (His-Tag) fusion proteins are purified from 0 soluble supernatant or insoluble inclusion framework A volume of 00 #ml from cultures operated by 1716 agitator by 1000 chromatography using: The one step QIAexpress Ni-NTA Agarose Matrix (QIAGEN, Chatsworth, CA) in the presence of .8M urea in short; © 1 mL of a saturated culture overnight of 13121 containing an 11:1 composition is added to 0 mL of YT 25 medium containing 50 µg/mL of YE ampicillin | 0 µg/mL of chloramphenicol and grow at TY°C with 0 shaking

or _ — bacterial cultures are induced with ¥ mmol of 1716 at an optical density level of 074 of JY + and grow for ? Additional hours (00- 0.014 to 0.014). Cells were collected from Ja vv from the batch cultures by centrifugation and resuspension in 0 ml of binding buffer 1 mol of sodium phosphate © at pH + and 0 mmol from 1115-1101; At a concentration (A pH) containing ¥ mmol of PMSF and 01 μg / ml of Leupeptin in addition to a full tablet of (Boehringer Mannheim) protease inhibitor per Yo ml. Escherichia coli is analyzed E.coli by freeze thawing followed by brief sonication briefly then spinning at 11 krpm for 0 min to make the inclusion bodies into pellet form. Three times in CHAPS 17 at 10 mmol of 15-116 (pH) A pH indicates that this step significantly reduces contamination of 98. The inclusion structure is finally dissolved in 0 mL of the containing buffer buffer on A mol of urea or A mol of urea was added directly to the soluble supernatant The fusion proteins recombinant with His—Tag residue were a batch bound to Vo agarose resin 1113178 (© ml from 0 For each 550 mL of inductions by shaking at room temperature for one hour and passing the compound through a column. The flow is passed twice through the same column and the column is washed three times with 0 mL each time of buffer wash solution. 1) wash buffer 1 mole of sodium phosphate and 0 mmol of “Tris-Hel” pH 1.7 containing also 1 mole of urea. 0 bound proteins were eluted with 0 mL of immidazole at Vou mM in due buffer and *mL of pooled fractions. An assembly of segments that each contains a fusion protein

© Recombinant, then dialyzeed with 01 mmol of Tris Hol (at a hydrogen concentration (A pH) bound once to the NI-NTA bound once and rinsed eluted and dialyzed at 0 mmol of -18 1101) at a pH concentration (VA) the recombinant protein yield varied from TY 0 mg per liter of the induced bacterial culture with greater purity than aT AA titration of the 0 recombinant proteins in terms of endotoxin contamination using (BioWhittaker) Limulus titration and showed Proteins it contains less than 0 10E.UImg / 1 “- proliferation assay 1-0611 The purified fusion polypeptides are tested for their ability to induce proliferation stimulation of cell 1 in Peripheral blood mononuclear cell (PBMC) Ye .PBMCs are cultured from donors known to be positive for PPD skin testing and in which T cells have been shown to proliferate in response to PPD and soluble proteins of crude Mycobacterium tuberculosis in RPMI 1640 supplied with pooled human serum 01 and 7 Ov µg/mL of “gentamicin” purified polypeptides were added in similar volume at concentrations of 0.0 to 0 µg/mL. After six days of cultivation in 476 round-bottomed plates with a volume of 100 μl VO, 00 μl of medium was removed from each vessel for determination of TFN-y levels; As described in the context below in Section 7. Then, the plates were exposed to 1 μC/per vessel of tritiated thymidine for an additional VA hour; Collection, determination and adsorption of tritium using a gas scintillation counter. It is taken into account that the tiny parts resulting from proliferation in both of them multiply three times greater than the proliferation (Ye) observed in cells cultured in the media alone positively.

: Huh 1 / 1 /? Interferon-y titration In sterile conditions the spleens are removed from each of the mice and a suspension of monocytes is prepared in 1 followed by analysis of red blood WDA. 100 μL of cells (“01” x X cells) per vessel are placed in a 97-well flat-bottom titration plate. © cultures are induced with recombinant proteins for YE hr and the supernatant titrated from Cus having 0 interferon-y levels of the supernatant are analyzed by a SANDWich Elisa “using antibody pairs and procedures available from PharMingen Standard curves are produced using cytokine 0 recombinant mice z colors are encapsulated a) The so-called Corning process (using 0 μl/pot (1 μg/mL; in a coating solution of 0 gram 11 mossy bicarbonate 0 pH 5.7) of mAb masking cytokine (anti-interferon-y mouse mice extracted from rats (PharMingen) » Class No. (18181D); incubation for two hours at room temperature. The contents of the plate and mat are shaken after that. Using 0..5-0185 1 Tween and (05 BSA ZV) μL/bowl) overnight at 1°C and washing for six times at Tween 7 1.1-858. Standards (interferon-y) are added. (mice and supernatant samples I diluted in BSA 7 +.) “Tween/..0—PBS for 2 hours at room temperature. Plates were washed as above and followed by incubation for 2 hours at room temperature with 100 μl/μl of Ab biotin rat 7 mouse IFN-Y (class no. PharMingen/18112 D) at dilution rate in + µg/mL in Tween / + 0-PBS and + 7 BSA after washing; Plates are incubated with 0 µL/well of streptavidin-HRP (Zymed) at a dilution rate of 1:

oY = — You in 0-5 / d71 f).+ BSA ZL at room temperature for 1 hour. The panels are washed one last time. And the use of 001 microns / container of the substrate: TMB substrate (tetramethylbenzidine, Kirkegaard and Perry, Gaithersburg, MD) - '3.3.5,5) for display, and the reaction stops after the appearance of color 0 with the help of aS pas 11. : 50;56 μl/reservoir Absorbance (OD) was determined at the £04 nm level using 2070 nm as the reference wavelength and Essanmann concentration_ values using the standard curve. fusion proteins induce immune responses The three coding sequences of the Mtb antigens are inserted into the gene-editing vector 0 to produce a fusion protein The indicated antigens (lea) 18812 and 10119 are produced and 12835 as one recombinant fusion protein (Figures 1a and 1b). Both 17014 and DPV and 1411 are produced as a second production protein (YJ). In an affinity way the fusion proteins are purified for use in in vitro and test tube calibrations. Ye fusion protein z is being tested for its ability to induce T cell responses from six of the 277 test subjects when cell proliferation is measured 7 » Both fusion proteins showed a similar pattern of reactivity and as is the case for their extracted parts (Fig. 14 -14 f). A similar result is obtained when IFNy lil (Fig. Sie. (soo) is measured. Subject D160 responded to p1511 and MTI antigens independently.

© a Test subject 0160 also responded to fusion proteins that contained all three antigens (Figs. 1b ey). In contrast, cell 7 of 12160 was not observed to respond to other antigens individually. Other test subject Other 0201 did not interact with antigens against Erdl4 and 0517 or

MTT © individually; It was also not responsive to a fusion protein containing these antigens. It should be noted that when the 7 cell response to the independent fragments of the two fusion proteins was not particularly strong; The fusion proteins are subject to responses that are equal to or higher than those induced by independent antigens in most cases.

0 The fusion protein Triglyceride 12-16119-5M28 is also being tested as an immunogen in vivo. In these experiments, the fusion protein is injected into the mouse pad for immunization purposes. Each of the three OS groups received the protein in a different adjuvant adjuvant Bale formulation: Ling (SBAS2 + SBASIC et al.; 1197; fax11:517-17) and 7(1)011e. After subcutaneous immunization twice at an interval

Vo three weeks; Animals are sacrificed after one week. The lymph nodes of these animals are collected for use as effector cells in assays of cell proliferation 1 and cytokine production.

Regardless of the adjuvant used in the immunization; Proliferative responses are induced

Strong T cell proliferation against p1511; When used as a standalone antigen (Fig

11 0 0 a). Weaker responses are induced against 2235 and 8812 (Figs. 11b and 11c). when

_ oA as an immunogen; A response of 2812 ~TbH9-Ra35 fusion protein is used. Similar to those of independent antibody responses, SBAS2 and SBASIC produced adjuvant 'cytokine' when production was measured though; resulted from union. (VA) and 11.4 (VY-form is similar (strong interferon-y form and lower level interferon-y aluminum hydroxide and SBAS7 chemical responses among the three © in relation to the human antibody response Antigens for all IL-4 antigens to produce that fusion protein elicited both F15-119 responses in vivo; a figure is shown of the three adjuvants UT and H180 when induced Use with antigen-specific IgGl In addition, mice 5781/6 are immunized with a chemical conjugate of two constructs of the modification or Ral2-ThH9-Ra35(Mth32A) gene each containing coding sequences0 Immunized animals with DNA vaccines in the form of Erd14-DPV-MTI (Mth39A) vaccines showed significant protection against tuberculosis upon aerosol immunoassay following live bacteria. and coproducer tested in a long-term prophylaxis model of a boar.In these mono- or mixed 039 recombinant fusion protein studies, boar grafts are immunized with the 050 adjuvant fusion protein in formulations containing the Mtb39A adjuvant. and Mth32A are mixture proteins

SBAS2 or SBASICc showed that Indian pigs immunized with fusion protein 71a-0 (Figure shown) had the best protection against infection with tuberculosis at a subsequent immunoassay; Compared to animals in a mixture in the same adjuvant formulation, the antigens provided by the immunized Ol antigens. _ Fusion proteins in formulation 53852 largest protection ever in animals 0

_ 5e - Thus, it is possible to use fusion proteins from various mycobacterium tuberculosis antigens as more effective immunogens in vaccine formulations than mixtures of independent components. [x J "- bi-fusion protein induced immune responses | bi-fusion protein containing antigens TbH-9 and Tb38-1 is produced. Without detailed sequencing by recombinant methods The ability of the fusion protein 10381 9 to induce 1-cell proliferation and interferon-y fusion is investigated The PBMC is being used from three donors: one of the donors was shown accepted that it responded to Ve antigen 16119 but not to antigen Tb38-1 (donor 111) ' al showed a response to antigen 7038-1 but did not show a response to antigen 15119 (donor 184) showed another follower Response to both antigens (Ye donor) The result of these studies explains the functional activity of both antigens in the fusion protein (Figs. 17, 1apo' 17, sary '71, or 1b). Tetra-reactive sera of tuberculosis patients, fusion protein z Loi) containing antigen TbRa3, 3810, 1538-1 and DPEP are produced by recombinant methods + the effectiveness of tetra-fusion protein is tested = this as 2-7 sera from infected M. tuberculosis proteins

11 AA by 8/2115 . The results of these studies (Table 1) explain that all four antigens act independently in the fusion protein. Someone skilled in this field will appreciate that it is possible to change the independent antigens in each fusion protein and it is expected that The comparative effectiveness provides that for each of the antigen-determining factors © that remain functionally available In addition, it is possible to use truncated forms of proteins containing active antigen-determining factors in the construction of fusion proteins that the present invention is not limited to the scope of representative models whose purpose is to illustrate individual aspects of the invention » Any clone, nucleotide or amino acid sequences which are functionally the same are within the scope of the invention. Described herein in context is evident to those skilled in this field from the foregoing description and accompanying drawings.The purpose of these modifications lies within the scope of the attached claims.It is understood that all assumed base pair sizes of nucleotides are approximate and are used for descriptions + ay Vo Integrate all publications mentioned herein into context by referring to them in full. Table 1 Fusion Protein Efficiency 0-2 Patient with tuberculosis The usual serology above © 1158 1. Al-Hilla | #10 | Status | YA ID | TbRa | Tb38-1 | DPEP 40 | 4 | 0045 Serum 3 Kilo 0 Daltons Kd + | - | Ja - | Mag Ak | + | aq | 28 SR IT

—- 111 _ Bla] TB [eso a + [evs | + | + | + | + | - [ 8931100 TB : A [eee + | + The + Te To sow] TB [NAS A + [VR + | + da | + | - + -a + ha | 04 TB [YA] + [Yeve + | +ee | - 9004] TB [ea] + | C | - | grandfather | +107004] TB [Ave | + a | + | find - | + a 92004] TB ATA | + [vse + Tow | Aj Te | aaa | 9704 TB [Neve Grandpa + Laqa | + | - + 118004] TB safe + | vere + ga + d | - | - 2304l 18 (Vee | + [veva | + | + | + vv + | - vv + | + lq + | 18 7504l - 274004 TB vA Ls [vv + | - ga | - | + 220004 18 [Ver] + met Tw | - ga - da 2820041 18 : ta | + [ave + [ow oe 28004) TB for da + a | + [oe| - 308004] TB [v.v.Al + [ves hal + | d | D JED 404 L [ TB haw + To ee 317004] 18 Nov) x PAY Aga ee 312004 18 JhNes + Teer + To ee aa | 380004 TB JNA] - Teeny |e oe oe astooal TB | A - Ahl ny Fo ee | MG 478004 | TB OME - Tea A + Toe es 410004 | TB set + vy [we | - | - Aja Glan A + A .| 18 04 God for | + | oe Te 421004] TB} v | + [veer 0+ | - Te | - | +d 28004 TB [ev - qa a fo | - | d - | - | +4647| pl | Se] - | khi [oo aa aa 44688 ah aa - aa ae aa aa aa

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Claims (1)

And 0 protection elements 1-1 polynucleotide includes the first nucleotide sequence "that hybridises hybridized under medium intensity conditions to Adige second nucleotide sequence aah ov which It is complementary to a third nucleotide sequence 8 encoding the amino acid sequence with identity number 7 where © the first nucleotide sequence mentioned encoding polypeptide 1 is able to catalyze An immune response to each of: TbRal2 , 10119 and TbRa35 7 in the assay of cell proliferation 1 or the production of 0 interferon gamma 1 ? - a Wh polynucleotide of protective element 1 that includes a sequence first nucleotide sequence Y whose hybridises were hybridized under highly stringent conditions to a “0” second polynucleotide sequence that is complementary to a third nucleotide sequence; The third nucleotide sequence encodes the amino acid sequence 1, which is the sequence with identity number: dua oY The mentioned first nucleotide sequence 1 that encodes a polypeptide is able to stimulate a response Immunogenic response 1 to each of: and TbRa35 A 10119 , 1515212 in calibration of cell proliferation 7 or the production of interferon gamma 94 - 1 +- a polypeptide comprising an amino acid sequence encoded by _ a polynucleotide according to any of the 1 or 7 claims. 1 +- a pharmaceutical composition comprising an lh polypeptide For claim 3. 1 #- a pharmaceutical composition comprising a polynucleotide according to any of "protection 1 or 7. 1 +- expression vector containing a polynucleotide according to For any "element of protection 1 or ?. 1 7- An expression vector according to the protection element + which is a JU viral vector Y . 18- Vaccine composition comprising a polypeptide according to claimant ¥ and an adjuvant. 1 4- Vaccine composition comprising a polynucleotide according to any protective element 1 or ¥ and an adjuvant. ada-01-1 isolated host cell expressing a polypeptide of recombinant gene of claim 7. 1-1 method for producing a polypeptide of claim ¥; Include the expression ¥ recombinant gene of a polynucleotide according to any of the claim 1 Xr