the genome of m . tuberculosis [ cole et al . ( 1998 ) nature 393 : 537 - 544 ] contains open reading frames ( orfs ) that have been deleted from the avirulent bcg strain . the polypeptides encoded by these orfs are designated herein “ m . tuberculosis bcg negative ” polypeptides (“ mtbn ”) and the orfs are designated “ mtbn .” the invention is based on the discovery that a mtbn polypeptide ( mtbn4 ) elicited a skin response in animals infected with m . tuberculosis , but not in animals sensitized to either bcg or m . avium , a non - m . tuberculosis - complex strain of mycobacteria ( see example 1 below ). these findings indicate that mtbn ( e . g ., mtbn1 - mtbn8 ) can be used in diagnostic tests that discriminate infection of a subject by m . tuberculosis from exposure to both mycobacteria other than the m . tuberculosis - complex and bcg . the m . tuberculosis - complex includes m . tuberculosis , m . bovis , m . microti , and m . africanum . thus they can be used to discriminate subjects exposed to m . tuberculosis , and thus potentially having or being in danger of having tuberculosis , from subjects that have been vaccinated with bcg , the most widely used tuberculosis vaccine . diagnostic assays that are capable of such discrimination represent a major advance that will greatly reduce wasted effort and consequent costs resulting from further diagnostic tests and / or therapeutic procedures in subjects that have given positive results in less discriminatory diagnostic tests . furthermore , the results in example 1 show that mtbn4 , as expressed by whole viable m . tuberculosis organisms , is capable of inducing a strong immune response in subjects infected with the organisms and thus has the potential to be a vaccine . the mtbn polypeptides of the invention include , for example , polypeptides encoded within the rd1 , rd2 , and rd3 regions of the m . tuberculosis genome [ mahairas et al . ( 1996 ) j . bacteriol . 178 : 1274 - 1282 ]. of particular interest are polypeptides encoded by orfs within the rd1 region of the m . tuberculosis genome . however , the invention is not restricted to the rd1 , rd2 , and rd3 region encoded polypeptides and includes any polypeptides encoded by orfs contained in the genome of one or more members of the m . tuberculosis genome and not contained in the genome of bcg . the amino acid sequences of mtbn1 - mtbn8 are shown in fig1 a and 1b and the nucleotide sequences of mtbn1 - mtbn8 are shown in fig2 a - 2e . the invention encompasses : ( a ) isolated dna molecules containing mtbn sequences ( e . g ., mtbn1 - mtbn8 ) encoding mtbn polypeptides ( e . g ., mtbn1 - mtbn8 ) and isolated portions of such dna molecules that encode polypeptide segments having antigenic and immunogenic properties ( i . e ., functional segments ); ( b ) the mtbn polypeptides themselves ( e . g ., mtbn1 - mtbn8 ) and functional segments of them ; ( c ) antibodies ( including antigen binding fragments , e . g ., f ( ab ′) 2 , fab , fv , and single chain fv fragments of such antibodies ) that bind to the mtbn polypeptides ( e . g ., mtbn1 - mtbn8 ) and functional segments ; ( d ) nucleic acid molecules ( e . g ., vectors ) containing and capable of expressing one or more of the mtbn ( e . g ., mtbn1 - mtbn8 ) sequences and portions of dna molecules ; ( e ) cells ( e . g ., bacterial , yeast , insect , or mammalian cells ) transformed by such vectors ; ( f ) compositions containing vectors encoding one or more m . tuberculosis polypeptides ( or functional segments ) including both the mtbn ( e . g ., mtbn1 - mtbn8 ) polypeptides ( or functional segments thereof ) and previously described m . tuberculosis polypeptides such as esat - 6 , 14 kda antigen , mpt63 , 19 kda antigen , mpt64 , mpt51 , mtc28 , 38 kda antigen , 45 / 47 kda antigen , mpb70 , ag85 complex , mpt53 , and katg ( see also u . s . pat . no . 6 , 087 , 163 ); ( g ) compositions containing one or more m . tuberculosis polypeptides ( or functional segments ), including both the polypeptides of the invention and previously described m tuberculosis polypeptides such as those described above ; ( h ) compositions containing one or more of the antibodies described in ( c ); ( i ) methods of diagnosis involving either ( 1 ) administration ( e . g ., intradermal injection ) of any of the above polypeptide compositions to a subject suspected of having or being susceptible to m tuberculosis infection , ( 2 ) in vitro testing of lymphocytes ( b - lymphocytes , cd4 t lymphocytes , and cd8 t lymphocytes ) from such a subject for responsiveness ( e . g ., by measuring cell proliferation , antibody production , cytokine production , or ctl activity ) to any of the above polypeptide compositions , ( 3 ) testing of a bodily fluid ( e . g ., blood , saliva , plasma , serum , urine , or semen or a lavage such as a bronchoalveolar lavage , a vaginal lavage , or lower gastrointestinal lavage ) for antibodies to the mtbn polypeptides ( e . g ., mtbn1 - mtbn8 ) or functional segments thereof , or the above - described polypeptide compositions ; ( 4 ) testing of a bodily fluid ( e . g ., as above ) for the presence of m . tuberculosis , mtbn ( e . g ., mtbn1 - mtbn8 ) polypeptides or functional segments thereof , or the above - described polypeptide compositions in assays using the antibodies described in ( c ); and ( 5 ) testing of a tissue ( e . g ., lung or bronchial tissue ) or a body fluid ( e . g ., as above ) for the presence of nucleic acid molecules ( e . g ., dna or rna ) encoding mtbn polypeptides ( e . g ., mtbn1 - mtbn8 ) ( or portions of such a nucleic acid molecules ) using nucleic acid probes or primers having nucleotide sequences of the nucleic molecules , portions of the nucleic molecules , or the complements of such molecules ; and ( j ) methods of vaccination involving administration to a subject of the compositions of either ( f ), ( g ), ( h ) or a combination of any two or even all 3 compositions . with respect to diagnosis , purified mtbn proteins , functional segments of such proteins , or mixtures of proteins and / or the functional fragments have the above - described advantages of discriminating infection by m . tuberculosis from either infection by other bacteria , and in particular , non - pathogenic mycobacteria , or from exposure ( by , for example , vaccination ) to bcg . furthermore , compositions containing the proteins , functional segments of the proteins , or mixtures of the proteins and / or the functional segments allows for improved quality control since “ batch - to - batch ” variability is greatly reduced in comparison to complex mixtures such as purified protein derivative ( ppd ) of tuberculin . the use of the above - described polypeptide and nucleic acid reagents for vaccination also provides for highly specific and effective immunization . since the virulent m . tuberculosis polypeptides encoded by genes absent from avirulent bcg are likely to be mediators of virulence , immunity directed to them can be especially potent in terms of protective capacity . where vaccination is performed with nucleic acids both in vivo and ex vivo methods can be used . in vivo methods involve administration of the nucleic acids themselves to the subject and ex vivo methods involve obtaining cells ( e . g ., bone marrow cells or fibroblasts ) from the subject , transducing the cells with the nucleic acids , preferably selecting or enriching for successfully transduced cells , and administering the transduced cells to the subject . alternatively , the cells that are transduced and administered to the subject can be derived from another subject . methods of vaccination and diagnosis are described in greater detail in u . s . pat . no . 6 , 087 , 163 , the disclosure of which is incorporated herein by reference in its entirety . the following example is meant to illustrate , not limit the invention . mtbn4 elicits a specific skin reaction in guinea pigs infected with m . tuberculosis four groups of outbred female guinea pigs ( 18 per group ) were used to test the usefulness of the mtbn4 polypeptide as a m . tuberculosis - specific diagnostic reagent . the four groups were treated as follows . group 1 animals were infected by aerosol with approximately 100 m . tuberculosis strain h37rv cells . group 2 animals were sensitized intradermally with 106 live m . bovis bcg japanese cells . group 3 animals were sensitized intradermally with 106 live m . avium cells . seven weeks after infection or sensitization , the animals were injected intradermally with 1 μg of ppd ( 6 animals from each group ), 2 μg of purified recombinant mpt64 ( 6 animals from each group ), or 2 μg of mtbn4 ( 6 animals from each group ). the diameter of the resulting erythema was measured 24 hours later . data are expressed as mean diameter of erythema ( in mm ) and standard deviations are indicated ( fig3 ). no erythema was detected in the group 4 animals with any test substance and thus no data are shown for this group . on the other hand , group 1 animals ( solid bars ) showed a significant response with all three test substances . group 2 animals ( open bars ) showed a significant response to ppd and mpt64 but not mtbn4 . group 3 animals showed a significant response to ppd only ( hatched bars ). thus , ppd which contains antigenic / immunogenic molecules common to the m . tuberculosis - complex as well as other mycobacterial strains , gave the least discriminatory results in that it induced responses in animals infected with or sensitized to mycobacteria of the m . tuberculosis - complex ( m . tuberculosis and bcg ) as well as another non - pathogenic mycobacterium ( m . avium ). while mpt64 , which is encoded and expressed by both m . tuberculosis and bcg , did not elicit a response in animals infected with m . avium , it did elicit responses in both the m . tuberculosis infected and the bcg sensitized animals . finally , mtbn4 elicited a response in only the m . tuberculosis animals . thus it induced the most specific response and , most importantly , allowed for discrimination between animals infected with m . tuberculosis and those sensitized to bcg . although the invention has been described with reference to the presently preferred embodiment , it should be understood that various modifications can be made without departing from the spirit of the invention . accordingly , the invention is limited only by the following claims .
the present invention is directed to reagents useful for generating immune responses to mycobacterium tuberculosis and for diagnosing infection and disease in a subject that has been exposed to m . tuberculosis .