TB diagnostic based on antigens from M. tuberculosis

Abstract

The present invention is based on the identification and characterization of a number of novel M. tuberculosis derived proteins and protein fragments. The invention is directed to the polypeptides and immunologically active fragments thereof, the genes encoding them, immunological compositions such as diagnostic reagents containing the polypeptides.

1 . A pharmaceutical composition comprising a combination of two or more substantially pure polypeptides, of which one or more comprises at least one amino acid sequence selected from the group consisting of: 
 a) Rv3354 (SEQ ID NO. 148);    b) an immunogenic portion of the sequence in (a); and    c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic.    
     
     
         2 . A composition according to  claim 1  comprising at least one fusion polypeptides, which comprises one or more amino acid sequences selected from the group consisting of 
 a) Rv3354 (SEQ ID NO. 148);    b) an immunogenic portion of any one of the sequences in (a); and/or    c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic;    and at least one fusion partner.    
     
     
         3 . A composition according to  claim 2 , wherein each fusion partner comprises a polypeptide fragment selected from the group consisting of: 
 (a) a polypeptide fragment derived from a virulent mycobacterium, such as ESAT-6, MPB64, MPT64, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, Ag85A, Ag85B, Ag85C, 19 kDa lipoprotein, MPT32, MPB59 and alpha-crystallin;    (b) a polypeptide selected from the group consisting of Rv0652, Rv2462c, Rv1984c, Rv2185c, Rv1636, Rv3451, Rv3872, Rv3354 and Rv2623 and an amino acid sequence analogue having at least 70% sequence identity to any one said sequences and at the same time being immunogenic;    (c) at least one immunogenic portion of any of such polypeptides in (a) or (b).    
     
     
         4 . An immunogenic composition comprising a composition according to  claim 1 .  
     
     
         5 . Use of a composition according to  claim 1  for the preparation of a pharmaceutical composition, e.g. for diagnosis of tuberculosis caused by virulent mycobacteria, e.g. by  Mycobacterium tuberculoisis, Mycobacterium africanum  or  Mycobacterium bovis.    
     
     
         6 . A diagnostic tool comprising a combination of two or more substantially pure polypeptides, of which at least one comprises at least one amino acid sequence selected from the group sonsisting of: 
 (a) Rv3354 (SEQ ID NO. 148);    (b) an immunogenic portion of the sequence in (a); and    (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic    
     
     
         7 . A substantially pure polypeptide, which comprises an amino acid sequence selected from the group consisting of: 
 (a) Rv3354 (SEQ ID NO. 148);    (b) an immunogenic portion of the sequence in (a); and    (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic    
     
     
         8 . Nucleic acid fragments in isolated form which 
 (a) comprises one or more nucleic acid sequences which encodes a polypeptide as defined in  claim 7 , or comprises a nucleic acid sequence complementary thereto; or    (b) has a length of at least 10 nucleotides and hybridizes readily under stringent hybridization conditions with a nucleotide sequence selected from Rv0652, Rv2462c, Rv1983c, Rv2185c, Rv1636, Rv3451, Rv3872, Rv3354 and Rv2623 nucleotide sequences or a sequence complementary thereto, or with a nucleotide sequence selected from a sequence in (a).    
     
     
         9 . A nucleic acid fragment according to  claim 8 , which is a DNA fragment.  
     
     
         10 . Use of a nucleic acid fragment according to  claim 8  for the preparation of a composition for the diagnosis of tuberculosis caused by virulent mycobacteria, e.g. by  Mycobacterium tuberculoisis, Mycobacterium africanum  or  Mycobacterium bovis.    
     
     
         11 . A replicable expression vector, which comprises a nucleic acid fragment according to  claim 8 .  
     
     
         12 . A transformed cell harbouring at least one vector according to  claim 11 .  
     
     
         13 . A method for producing a polypeptide according to  claim 7 , comprising 
 (a) inserting a nucleic acid fragment according to  claim 8  into a vector which is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell, culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide, and recovering the polypeptide from the host cell or culture medium;    (b) isolating the polypeptide from a whole  mycobacterium , e.g.  Mycobacterium tuberculosis, Mycobacterium africanum  or  Mycobacterium bovis , from culture filtrate or from lysates or fractions thereof; or    (c) synthesizing the polypeptide e.g. by solid or liquid phase peptide synthesis.    
     
     
         14 . A method of diagnosing tuberculosis caused by virulent mycobacteria, e.g. by  Mycobacterium tuberculoisis, Mycobacterium africanum  or  Mycobacterium bovis , in an animal, including a human being, comprising intradermally injecting, in the animal, a composition according to  claim 1 , a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis.  
     
     
         15 . A monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide according to  claim 7  in an immuno assay, or a specific binding fragment of said antibody for use as a diagnostic reagent.  
     
     
         16 . A method for diagnosing previous or ongoing infection with a virulent mycobacterium, said method comprising 
 (a) contacting a subject sample, e.g. a blood sample, with a composition according to  claim 1  or a diagnostic tool according to  claim 6 ,    (b) detecting binding of an antibody, said binding being an indication that said subject is infected by  Mycobacterium tuberculoisis  or is susceptible to  Mycobacterium tuberculoisis  infection.    
     
     
         17 . A serodiagnostic composition comprising a combination of two or more substantially pure polypeptides, of which at least one or more comprises at least one amino acid sequence selected from 
 (a) Rv3354 (SEQ ID NO. 148);    (b) an immunogenic portion of the sequence in (a); and    (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic.    
     
     
         18 . A substantially pure polypeptide according to  claim 7 , which comprises at least one amino acid sequence selected from the group consisting of: 
 (a) Rv3354 (SEQ ID NO. 148);    (b) an immunogenic portion of the sequence in (a); and    (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic;    and at least one fusion partner.    
     
     
         19 . A substantially pure polypeptide according to  claim 18 , wherein each fusion partner comprises a polypeptide fragment selected from the group consisting of: 
 (a) a polypeptide fragment derived from a virulent  mycobacterium , such as ESAT-6, MPB64, MPT64, TB10.4, CFP10, RD1-ORF5, RD1-ORF2,    Rv1036, Ag85A, Ag85B, Ag85C, 19 kDa lipoprotein, MPT32, MPB59 and alpha-crystallin;    (b) a polypeptide selected from the group consisting of Rv0652, Rv2462c, Rv1984c, Rv2185c, Rv1636, Rv3451, Rv3872, Rv3354 and Rv2623 and an amino acid sequence analogue having at least 70% sequence identity to any one said sequences and at the same time being immunogenic;    (c) at least one immunogenic portion of any of such polypeptides in (a) or (b).    
     
     
         20 . A composition comprising a combination of two or more substantially pure polypeptides, of which one or more comprises one or more amino acid sequence selected from the group consisting of: 
 a) Rv3354 (SEQ ID NO. 148);    b) an immunogenic portion of the sequence in (a); and    c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic;    and of which one or more comprises one or more amino acid sequences selected from the group consisting of:    d) Rv0652, Rv2462c, Rv1984c, Rv2185c, Rv1636, Rv3451, Rv3872, Rv3354 and Rv2623;    e) an immunogenic portion of any one of the sequences in (d); and    f) an amino acid analogue having at least 70% sequence identity to any one of the sequences in (d) or (e) and at the same time being immunogenic.