Polynucleotide functionally coding for the LHP protein from Mycobacterium tuberculosis, its biologically active derivative fragments, as well as methods using the same

Abstract

The present invention is directed to a polynucleotide carrying an open reading frame coding for an antigenic polypeptide from Mycobacterium tuberculosis , named lhp, which is placed under the control of its own regulation signals which are functional in mycobacteria, specially in mycobacteria belonging to the Mycobacterium tuberculosis complex and also in fast growing mycobacteria such as Mycobacterium smegmatis . The invention is also directed to the polypeptide LHP encoded by lhp and most preferably to suitable antigenic portions of LHP as well as to oligomeric polypeptides containing more than one unit of LHP or an antigenic portion of LHP. The invention concerns also immunogenic and vaccine compositions containing a polypeptide or an oligomeric polypeptide such as defined above, as well as antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. In another embodiment, the present invention is directed to a polynucleotide carrying the natural regulation signals of lhp which is useful in order to express heterologous proteins in mycobacteria. Finally, the present invention is directed to oligonucleotides comprising at least 12 consecutive nucleotides from the regulation sequence of lhp which are useful as reagents for detecting the presence of Mycobacterium tuberculosis in a biological sample.

What is claimed is:  
     
         1 . A purified polynucleotide wherein said polynucleotide is chosen from the group consisting of: 
 a) a polynucleotide comprising the following nucleotide sequence of SEQ ID NO 1: CTGCAGCAGGTGACGTCGTTGTTCAGCCAGGTGGGCGGCACCGGCGGCGGC AACCCAGCCGACGAGGAAGCCGCGCAGATG GGCCTGCTCGGCACCAGTCCGCTGTCGAACCATCCGCTGGCTGGTGGATCA GGCCCCAGCGCGGGCGCGGGCCTGCTGCG CGCGGAGTCGCTACCTGGCGCAGGTGGGTCGTTGACCCGCACGCCGCTGAT GTCTCAGCTGATCGAAAAGCCGGTTGCCC CCTCGGTGATGCCGGCGGCTGTTGCCGGATCGTCGGTGACGGGTGGCGCCG CTCCGGTGGGTCCGGGAGCGATGGGCCAG GGTTCGCAATCCGGCGGCTCCACCAGCCCGGGTCTGGTCGCGCCGGCACCG CTCGCGCAGGAGCGTGAAGAAGACGACGA GGACGACTGGGACGAAGAGGACGACTGGTGAGCTCCCGTAATGACAACAG ACTTCCCGGCCACCCGGGCCGGAAGACTTG CCAACATTTTGGCGAGGAAGGTAAAGAGAGAAAGTAGTCCAGCATGGCAG AGATGAAGACCGATGCCGCTACCCTCGGGC AGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCG ACCAGGTGGAGTCGACGGCAGGTTCGTTG CAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGGT GCGCTTCCAAGAAGCAGCCAATAAGCAGAA GCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATA CTCGAGGGCCGACGAGGAGCAGCAGCAGG CGCTGTCCTCGCAAATGGGCTTCTGACCCGCTAATACGAAAAGAAACGGAG CAAAAACATGACAGAGCAGCAGTGGAATT TCGCGGGTATCGAGGCCGCGGCAAGCGCAATCCAGGGAAATGTCACGTCCA TTCATTCCCTCCTTGACGAGGGGAAGCAG TCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTAC CAGGGTGTCCAGCAAAAATGGGACGCCAC GGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGCGCGGACGATCAGCG AAGCCGGTCAGGCAATGGCTTCGACCGAAG GCAACGTCACTGGGATGTTCGCATAGGGCAACGCCGAGTTCGCGTAGAATA GCGAAACACGGGATCGGGCGAGTTCGACC TTCCGTCGGTCTCGCCCTTTCTCGTGTTTATACGTTTGAGCGCACTCTGAGA GGTTGTCATGGCGGCCGACTACGA    b) a polynucleotide comprising the following nucleotide sequence of SEQ ID NO 2, starting at its 5′ end with the nucleotide in position 1 of SEQ ID NO 1 and ending at its 3′ end with the nucleotide in position 524 of SEQ ID NO 1, or a biologically active polynucleotide derivative of SEQ ID NO 2:CTGCAGCAGGTGACGTCGTTGTTCAGCCAGGTGGGCGGCACCGGCGGCGGC AACCCAGCCGACGAGGAAGCCGCGCAGATG GGCCTGCTCGGCACCAGTCCGCTGTCGAACCATCCGCTGGCTGGTGGATCA GGCCCCAGCGCGGGCGCGGGCCTGCTGCG CGCGGAGTCGCTACCTGGCGCAGGTGGGTCGTTGACCCGCACGCCGCTGAT GTCTCAGCTGATCGAAAAGCCGGTTGCCC CCTCGGTGATGCCGGCGGCTGTTGCCGGATCGTCGGTGACGGGTGGCGCCG CTCCGGTGGGTCCGGGAGCGATGGGCCAG GGTTCGCAATCCGGCGGCTCCACCAGCCCGGGTCTGGTCGCGCCGGCACCG CTCGCGCAGGAGCGTGAAGAAGACGACGA GGACGACTGGGACGAAGAGGACGACTGGTGAGCTCCCGTAATGACAACAG ACTTCCCGGCCACCCGGGCCGGAAGACTTG CCAACATTTTGGCGAGGAAGGTAAAGAGAGAAAGTAGTCCAGC    c) a polynucleotide comprising the following nucleotide sequence of SEQ ID NO 3, starting at its 5′ end with the nucleotide in position 1 of SEQ ID NO 1 and ending at its 3′ end with the nucleotide in position 481 of SEQ ID NO 1, or a biologically active polynucleotide derivative of SEQ ID NO 3: CTGCAGCAGGTGACGTCGTTGTTCAGCCAGGTGGGCGGCACCGGCGGCGGC AACCCAGCCGACGAGGAAGCCGCGCAGATG GGCCTGCTCGGCACCAGTCCGCTGTCGAACCATCCGCTGGCTGGTGGATCA GGCCCCAGCGCGGGCGCGGGCCTGCTGCG CGCGGAGTCGCTACCTGGCGCAGGTGGGTCGTTGACCCGCACGCCGCTGAT GTCTCAGCTGATCGAAAAGCCGGTTGCCC CCTCGGTGATGCCGGCGGCTGTTGCCGGATCGTCGGTGACGGGTGGCGCCG CTCCGGTGGGTCCGGGAGCGATGGGCCAG GGTTCGCAATCCGGCGGCTCCACCAGCCCGGGTCTGGTCGCGCCGGCACCG CTCGCGCAGGAGCGTGAAGAAGACGACGA GGACGACTGGGACGAAGAGGACGACTGGTGAGCTCCCGTAATGACAACAG ACTTCCCGGCCACCCGGGCCGGAAGACTTG    d) a polynucleotide comprising the following nucleotide sequence of SEQ ID NO 4, starting at its 5′ end with the nucleotide in position 525 of SEQ ID NO 1 and ending at its 3′ end with the nucleotide in position 826 of SEQ ID NO 1 coding for the LHP polypeptide: ATGGCAGAGATGAAGACCGATGCCGCTACCCTCGGGC AGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCG ACCAGGTGGAGTCGACGGCAGGTTCGTTG CAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGGT GCGCTTCCAAGAAGCAGCCAATAAGCAGAA GCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATA CTCGAGGGCCGACGAGGAGCAGCAGCAGG CGCTGTCCTCGCAAATGGGCTTCTG    e) a polynucleotide comprising at least 12 consecutive nucleotides of a polynucleotide chosen among the group consisting of SEQ ID NO2, SEQ ID NO 3 or SEQ ID NO 4;    f) A polynucleotide having a sequence fully complimentary to a polynucleotide chosen among the group consisting of SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4;    g) A polynucleotide hybridizing under stringent hybridization conditions with polynucleotide chosen among the group consisting of SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4.    
     
     
         2 . A polynucleotide according to  claim 1  wherein said polynucleotide codes for an antigenic protein from  Mycobacterium tuberculosis  comprising the following amino acid sequence of SEQ ID NO 4:MAEMKTDAATLGQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQ AAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF  
     
     
         3 . A polynucleotide according to  claim 1  which is labeled with a marker compound.  
     
     
         4 . A purified polynucleotide comprising: 
 a) a polynucleotide of sequence SEQ ID NO 2 or a biologically active polynucleotide derivative of SEQ ID NO 2; and    b) a polynucleotide coding for a polypeptide.    
     
     
         5 . A purified polynucleotide comprising: 
 a) a polynucleotide of sequence SEQ ID NO 3 or a biologically active polynucleotide derivative of SEQ ID NO 2; and    b) a polynucleotide coding for a polypeptide.    
     
     
         6 . A recombinant vector containing a polynucleotide according to any one of  claims 1  to  5 .  
     
     
         7 . The recombinant vector according to  claim 6 , which is plasmid pIPX61 that has been deposited at the CNCM on May 14, 1996 under the Accession Number I-1705.  
     
     
         8 . The recombinant vector according to  claim 6 , which is plasmid pIPX30 that has been deposited at the CNCM on Feb. 13, 1997 under the Accession Number I-1845.  
     
     
         9 . A recombinant cell host containing a purified polynucleotide according to any one of  claims 1  to  5  or a recombinant vector according to any one of  claims 6  to  8 .  
     
     
         10 . The recombinant cell host according to  claim 9  which is a mycobacterium cell host belonging to the  Mycobacterium tuberculosis  complex.  
     
     
         11 . The recombinant cell host according to  claim 10  which is  Mycobacterium tuberculosis.    
     
     
         12 . The recombinant cell host according to  claim 10  which is  Mycobacterium bovis -BCG.  
     
     
         13 . The recombinant cell host according to  claim 9  which is the  E. coli  strain deposited at the CNCM on May 14, 1996 under the Accession Number I-1705.  
     
     
         14 . The recombinant cell host according to  claim 9  which is the  E. coli  strain deposited at the CNCM on Feb. 13, 1997 under the Accession Number I-1845.  
     
     
         15 . A recombinant cell host containing a polynucleotide of SEQ ID NO 2 or a recombinant vector carrying SEQ ID NO 2 which is  Mycobacterium smegmatis.    
     
     
         16 . A purified polypeptide expressed by a recombinant cell host according to any one of  claims 9  to  13  and  15 .  
     
     
         17 . A purified polypeptide of  claim 16  which was chosen from the group of polypeptides consisting in: 
 a) a polypeptide which comprises the following amino acid sequence of SEQ ID NO 5:MAEMKTDAATLGQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQ AAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF;  
 b) a polypeptide comprising: 
 i) amino acid in position 1 to amino acid in position 48 of SEQ ID NO 5; or  
 ii) amino acid in position 60 to amino acid in position 100 of SEQ ID NO 5;  
 
 c) a polypeptide comprising at least one antigenic portion of a polypeptide a) or b).  
 
     
     
         18 . An oligomeric polypeptide comprising at least two units of a polypeptide according to  claim 17 .  
     
     
         19 . The oligomeric polypeptide of  claim 18  comprising up to 10 units of a polypeptide according to  claim 17 .  
     
     
         20 . A purified polypeptide comprising at least one antigenic portion of a polypeptide according to  claim 17 .  
     
     
         21 . The purified polypeptide according to  claim 18  wherein the antigenic portion of the polypeptide of sequence SEQ ID NO 4 is chosen among the group consisting in the following antigenic portions: 
 a) SEQ ID NO 6: NH2-MAEMKTDAATLGQEAGNFERISGDLKTQIDQVESTAGS LQGQWRGAAGT-COOH;  
 b) SEQ ID NO 7: NH2-QEAANKQKQELDEISTNIRQAGVQYSRADEEQQQ ALSSQMGF-COOH;  
 c) SEQ ID NO 8: NH2-QEAGNFERISGDLKTQIDQV-COOH;  
 d) SEQ ID NO 9: NH2-GDLKTQIDQVESTAGS-COOH;  
 e) SEQ ID NO 10: NH2-GSLQGQWRGAAGTAAA-COOH;  
 f) SEQ ID NO 11: NH2-QEAANKQKQELDEIST-COOH;  
 g) SEQ ID NO 12: NH2-STNIRQAGVQYSRADEEQQQALSSQMGF-COOH;  
 h) SEQ ID NO 13: NH2-RADEEQQQALSSQMGF-COOH.  
 
     
     
         22 . The purified polypeptide of  claim 20  comprising from 2 to 10 antigenic portions of the polypeptide of SEQ ID NO 4.  
     
     
         23 . A purified polypeptide or an oligomeric polypeptide according to any one of  claim 16  which is in the form of a MAP construct.  
     
     
         24 . A purified polypeptide or an oligomeric polypeptide according to  claim 16  which comprises an additional T-epitope.  
     
     
         25 . A monoclonal or a polyclonal antibody directed specifically against a purified polypeptide or an oligomeric polypeptide according to any one of  claims 16  to  24 .  
     
     
         26 . An immunogenic composition comprising a purified polypeptide or an oligomeric polypeptide according to any one of  claims 16  to  24 .  
     
     
         27 . A vaccine composition comprising a purified polypeptide or an oligomeric polypeptide according to any one of  claims 16  to  24 .  
     
     
         28 . The vaccine composition according to  claim 27  wherein said vaccine composition comprises additionally an antigenic protein from  Mycobacterium tuberculosis  or an antigenic portion of an antigenic protein from  Mycobacterium tuberculosis.    
     
     
         29 . The vaccine composition according to  claim 28  wherein said vaccine composition comprises additionally the ESAT-6 antigenic protein or an antigenic portion of the ESAT-6 protein.  
     
     
         30 . A diagnostic method for detecting the presence of a  Mycobacterium tuberculosis  bacterium in a biological sample, said diagnostic method comprising the steps of: 
 a) bringing into contact the biological sample expected to contain a given pathogenic microorganism with a purified monoclonal or polyclonal antibody according to  claim 25;     b) detecting the antigen-antibody complexes formed.    
     
     
         31 . A diagnostic method for detecting the presence of a  Mycobacterium tuberculosis  bacterium in the serum of an infected patient, said diagnostic method comprising the steps of: 
 a) bringing into contact the serum sample expected to contain a given pathogenic microorganism with a purified polypeptide or an oligomeric polypeptide according to any one of  claims 16  to  24 ;    b) detecting the antigen-antibody complexes formed.    
     
     
         32 . A diagnostic kit for the in vitro diagnosis of an infection by  Mycobacterium tuberculosis,  comprising the following elements: 
 a) a purified preparation of a monoclonal or a polyclonal antibody according to  claim 25;     b) suitable reagents allowing the detection of the antigen/antibody complexes formed, these reagents preferably carrying a label compound, or being recognized themselves by a labeled reagent;    c) optionally a reference biological sample containing the  Mycobacterium tuberculosis  antigen recognized by the purified monoclonal or polyclonal antibody (positive control);    d) optionally, a reference biological sample that does not contain the  Mycobacterium tuberculosis  antigen recognized by the purified monoclonal or polyclonal antibody (negative control).    
     
     
         33 . A diagnostic kit for the in vitro diagnosis of an infection by  Mycobacterium tuberculosis,  comprising the following elements: 
 a) a purified preparation of a purified polypeptide or an oligomeric polypeptide according to any one of  claims 16  to  24 ;    b) suitable reagents allowing the detection of the antigen/antibody complexes formed, these reagents preferably carrying a label compound, or being recognized themselves by a labeled reagent;    c) optionally, a reference biological sample containing a polyclonal or monoclonal antibody recognizing the purified polypeptide or the oligomeric polypeptide of step a) (positive control);    d) optionally, a reference biological sample that does not contain a polyclonal or monoclonal antibody recognizing the purified polypeptide or the oligomeric polypeptide of step a) (negative control).    
     
     
         34 . A method for detecting  Mycobacterium tuberculosis  is a biological sample comprising the steps of: 
 a) bringing into contact a purified polynucleotide according to any one of  claims 1  to  3  with a biological sample;    b) detecting the hybrid nucleic acid molecule formed between said purified polynucleotide and the nucleic acid molecules contained within the biological sample.    
     
     
         35 . The method of  claim 34 , wherein before step a), the nucleic acid molecules of the biological sample have been made available to a hybridization reaction.  
     
     
         36 . A method for detecting a bacterium belonging to the  Mycobacterium tuberculosis  complex or to  Mycobacterium bovis  in a biological sample comprising the steps of: 
 a) bringing into contact a purified polynucleotide according to any one of  claims 1  to  3  that has been immobilized onto a substrate with a biological sample.    b) bringing into contact the hybrid nucleic acid molecule formed between said purified polynucleotide and the nucleic acid contained in the biological sample with a labeled polynucleotide according to any one of  claims 1  to  3 , provided that said polynucleotide and polynucleotide of step a) have non-overlapping sequences.    
     
     
         37 . The method of  claim 36 , wherein, before step a), the nucleic acid molecules of the biological sample have been made available to a hybridization reaction.  
     
     
         38 . The method of any one of claims  36  or  37 , wherein, before step b), the nucleic acid molecules that are not hybridized with the immobilized purified polynucleotide are removed.  
     
     
         39 . A method for detecting a bacterium belonging to the  Mycobacterium tuberculosis  complex in a biological sample comprising the steps of: 
 a) bringing into contact the nucleic acid molecules contained in the biological sample with a pair of purified polynucleotides according to any one of  claims 1  to  3 ;    b) amplifying said nucleic acid molecules;    c) detecting the nucleic acid fragments that have been amplified, for example by gel electrophoresis or with a labeled polynucleotide according to any one of  claims 1  to  3 .    
     
     
         40 . The method of  claim 39 , wherein before step a), the nucleic acid molecules of the biological sample have been made available to a hybridization reaction.  
     
     
         41 . A kit for detecting a bacterium belonging to the  Mycobacterium tuberculosis  complex or to  Mycobacterium bovis  in a biological sample comprising: 
 a) a purified polynucleotide according to any one of  claims 1  to  3 ;    b) reagents necessary to perform a nucleic acid hybridization reaction.    
     
     
         42 . A kit for detecting a bacterium belonging to the  Mycobacterium tuberculosis  complex or to  Mycobacterium bovis  in a biological sample comprising: 
 a) a purified polynucleotide according to any one of  claims 1  to  3  that is immobilized onto a substrate;    b) reagents necessary to perform a nucleic acid hybridization reaction;    c) a purified polynucleotide according to any one of  claims 1  to  3  which is radioactively or non-radioactively labeled, provided that said polynucleotide and the polynucleotide of step a) have non-overlapping sequences.    
     
     
         43 . A kit for detecting a bacterium belonging to the  Mycobacterium tuberculosis  complex or to  Mycobacterium bovis  in a biological sample comprising: 
 a) a pair of purified oligonucleotides according to any one of  claims 1  to  3 ;    b) reagents necessary to perform a nucleic acid amplification reaction;    c) optionally, a purified polynucleotide according to any one of claims useful as a probe.    
     
     
         44 . A recombinant vector according to  claim 6 , which is plasmid pIPX26 that has been deposited at the CNCM on May 14, 1996 under the Accession Number I-1706.  
     
     
         45 . A recombinant vector according to  claim 6 , which is plasmid pPX1 that has been deposited at the CNCM on May 14, 1996 under the Accession Number I-1707.  
     
     
         46 . A recombinant cell host according to  claim 9 , which is the  E. coli  strain that has been deposited at the CNCM on May 14, 1996 under the Accession Number I-1706.  
     
     
         47 . A recombinant cell host according to  claim 9 , which is the  E. coli  strain that has been deposited at the CNCM on May 14, 1996 under the Accession Number I-1707.  
     
     
         48 . The vaccine composition according to  claim 27  comprising a recombinant cell host containing a polynucleotide encoding a polypeptide according to  claim 1  or a recombinant vector containing said polynucleotide.  
     
     
         49 . The vaccine composition according to  claim 48 , wherein said polynucleotide or said vector encodes both the lhp or the ESAT-6 antigenic polypeptides or antigenic portion thereof.  
     
     
         50 . The vaccine composition according to  claim 48  comprising a recombinant cell host expressing lhp and a recombinant cell host expressing ESAT-6.  
     
     
         51 . The vaccine composition according to any one of  claims 48  to  50 , wherein the recombinant cell host is an eukaryotic cell host.  
     
     
         52 . The vaccine composition according to any one of  claims 48  to  50 , wherein the recombinant cell host is a prokaryotic cell host.  
     
     
         53 . The vaccine composition according to  claim 52 , wherein the recombinant cell host is chosen from the group of bacteria consisting in: 
 a) an attenuated bacterium belonging to the tuberculosis-complex;    b)  E. coli;      c) a bacterium belonging to the Salmonella genus;    d) a bacterium belonging to the Pseudomonas genus.    
     
     
         54 . A polynucleotide useful as a primer or a probe according to  claim 1  which is chosen from the group consisting of: 
 a) SEQ ID NO 14: 5′-CTGCAGCAGGTGACGTCGTTG-3′ 
 b) SEQ ID NO 15: 5′-CCGGGTGGCCGGGAAGTCTGTGT-3′ 
 c) SEQ ID NO 16: 5′-ACTACTTTCTCTTTCTACCTTCC-3′ 
 
     
     
         55 . A pair of oligonucleotide primers useful as a primer or a probe, which pair is chosen from the group consisting of: 
 a) SEQ ID NO 14 and SEQ ID NO 15;    b) SEQ ID NO 14 and SEQ ID NO 16.