Mycobacterial genes down-regulated during latency

Abstract

A method is provided for identifying mycobacterial genes the expression of which is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving and that support exponential growth of said mycobacteria. The described method optionally provides for identifying mycobacterial genes that are simultaneously down-regulated under low DOT conditions. The down-regulated genes of the present invention form the basis of nucleic acid vaccines, or provide targets to allow preparation of attenuated mycobacteria for vaccines against mycobacterial infections. Similarly, peptides encoded by said down-regulated genes are employed in vaccines. In a further embodiment, the identified genes/peptides provide the means for identifying the presence of a mycobacterial infection in a clinical sample by nucleic acid probe or antibody detection.

1 - 3 . (canceled) 
     
     
         4 . A method of identifying a mycobacterial gene the expression of which is down-regulated during microbial latency, said method comprising:
 culturing a first  mycobacterium  under culture conditions that are nutrient-starving and that do not support exponential growth of the first  mycobacterium;      culturing a second  mycobacterium  under culture conditions that are not nutrient-starving and that support exponential growth of the second  mycobacterium;      obtaining first and second mRNA populations from said first and second mycobacteria, respectively wherein said first mRNA population is obtained from the first  mycobacterium  that has been harvested during stationary phase and wherein the second mRNA is obtained from the second  mycobacterium  that has been harvested during exponential phase growth;   preparing first and second cDNA populations from said first and second mRNA populations, respectively, during which cDNA preparation a detectable label is introduced into the cDNA molecules of the first and second cDNA populations;   isolating corresponding first and second cDNA molecules from the first and second cDNA populations, respectively;   comparing relative amounts of label or corresponding signal emitted from the label present in the isolated first and second cDNA molecules;   identifying a greater amount of label or signal provided by the isolated second cDNA molecule than that provided by the isolated first cDNA molecule; and   identifying the first cDNA and the corresponding mycobacterial gene that is down-regulated during culture of a  mycobacterium  under nutrient-starving conditions.   
     
     
         5 . A method according to  claim 4 , wherein the corresponding first and second cDNA molecules are isolated from the first and second cDNA populations, respectively, by hybridisation thereof to an array plate containing immobilised amplified DNA sequences which have been generated from mycobacterial genomic DNA, said immobilised sequences being representative of each known gene of the mycobacterial genome, and each representative sequence having been immobilised at an identified location on the plate. 
     
     
         6 . A method according to  claim 4 , wherein the first  mycobacterium  is cultured under culture conditions defined by a dissolved oxygen tension of less than 10% air saturation when measured at 37° C., and wherein the first  mycobacterium  is harvested under said culture conditions. 
     
     
         7 . A method according to  claim 4 , wherein a relative down-regulation is identified by a relative 3-fold decrease in the amount of label or signal provided by the isolated first cDNA molecule over that provided by the isolated second cDNA molecule. 
     
     
         8 - 11 . (canceled) 
     
     
         12 . An attenuated  mycobacterium  in which a gene has been modified thereby rendering the  mycobacterium  reduced in ability to enter a latent state, wherein the gene is a gene the expression of which is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving culture conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving and that support exponential growth of said mycobacterial culture;
 wherein said gene has a wild-type coding sequence selected from the group consisting of SEQ ID NOs: 4 and 34.   
     
     
         13 . (canceled) 
     
     
         14 . An attenuated microbial carrier according to  claim 31 , wherein the attenuated microbial carrier is attenuated  salmonella , attenuated vaccinia virus, attenuated fowlpox virus, or attenuated  M. bovis.    
     
     
         15 . (canceled) 
     
     
         16 . A DNA plasmid according to  claim 32 , wherein the promoter is selected from the group consisting of CMV and SV40 promoters, and/or
 the polyadenylation signal is selected from the group consisting of SV40 and bovine growth hormone polyadenylation signals.   
     
     
         17 . (canceled) 
     
     
         18 . An RNA vector comprising the RNA sequence of  claim 35  and an integration site for a chromosome of a host cell. 
     
     
         19 - 28 . (canceled) 
     
     
         29 . An isolated  M. tuberculosis  DNA sequence, wherein said DNA sequence is selected from the group consisting of:
 (i) a DNA sequence selected from:
 (a) SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46, or a derivative of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46; 
 (b) a fragment of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46, wherein said fragment has at least 15 nucleotides, or a derivative of said fragment; or 
 (c) a variant of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46, wherein the full-length nucleotide sequence of said variant has at least 70% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46; 
   wherein the peptide encoded by said derivative, fragment or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46;   (ii) a DNA sequence selected from:
 (a) SEQ ID NO: 8 or 18, or a derivative of SEQ ID NO: 8 or 18; or 
 (b) a variant of SEQ ID NO: 8 or 18, wherein the full-length nucleotide sequence of said variant has at least 70% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 8 or 18; or a derivative of said variant; 
   wherein the peptide encoded by said derivative or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 8 or 18;   (iii) a DNA sequence selected from:
 (a) SEQ ID NO: 4, or a derivative of SEQ ID NO: 4; or 
 (b) a variant of SEQ ID NO: 4, wherein the full-length nucleotide sequence of said variant has at least 80% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 4; or a derivative of said variant; 
   wherein the peptide encoded by said derivative or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 4;   (iv) a DNA sequence selected from:
 (a) SEQ ID NO: 24 or 40, or a derivative of SEQ ID NO: 24 or 40; 
 (b) a fragment of SEQ ID NO: 24 or 40, wherein said fragment has at least 15 nucleotides; or a derivative of said fragment; 
 (c) a variant of SEQ ID NO: 24 or 40, wherein the full-length nucleotide sequence of said variant has at least 90% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 24 or 40; or a derivative of said variant; 
   wherein the peptide encoded by said derivative, fragment or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 24 or 40;   and   (v) a DNA sequence selected from:
 (a) SEQ ID NO: 19; or 
 (b) a derivative of SEQ ID NO: 19; 
   wherein the peptide encoded by said derivative has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 19;   
       wherein said  M. tuberculosis  DNA sequence is the coding sequence of an  M. tuberculosis  gene, the expression of which is down-regulated during a stationary phase culture of  M. tuberculosis  under nutrient-starving culture conditions, when compared with an exponential phase culture of  M. tuberculosis  under culture conditions that are not nutrient starving and that support exponential growth of said  M. tuberculosis.    
     
     
         30 . The isolated  M. tuberculosis  DNA sequence of  claim 29 , wherein said DNA sequence is selected from:
 (i) a DNA sequence selected from:
 (a) SEQ ID NO: 34, or a derivative of SEQ ID NO: 34; 
 (b) a fragment of SEQ ID NO: 34, wherein said fragment has at least 15 nucleotides, or a derivative of said fragment; or 
 (c) a variant of SEQ ID NO: 34, wherein the full-length nucleotide sequence of said variant has at least 70% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 34; 
   wherein the peptide encoded by said derivative, fragment or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 34;   and   (ii) a DNA sequence selected from:
 (a) SEQ ID NO: 4, or a derivative of SEQ ID NO: 4; or 
 (b) a variant of SEQ ID NO: 4, wherein the full-length nucleotide sequence of said variant has at least 80% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 4; or a derivative of said variant; 
   wherein the peptide encoded by said derivative or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 4.   
     
     
         31 . An attenuated microbial carrier, comprising an  M. tuberculosis  gene having a DNA sequence according to  claim 29 . 
     
     
         32 . A DNA plasmid comprising a promoter, a polyadenylation signal, and a DNA sequence according to  claim 29 , wherein the promoter and polyadenylation signal are operably linked to the DNA sequence. 
     
     
         33 . A DNA plasmid according to  claim 32 , wherein the promoter is a CMV promoter and the polyadenylation signal is a bovine growth hormone polyadenylation signal. 
     
     
         34 . A DNA plasmid according to  claim 33 , further comprising a selectable marker, wherein said selectable marker encodes a protein that confers resistance to an antibiotic. 
     
     
         35 . An isolated RNA sequence that is encoded by an isolated  M. tuberculosis  DNA sequence, wherein said DNA sequence is selected from the group consisting of:
 (i) a DNA sequence selected from:
 (a) SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46, or a derivative of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46; 
 (b) a fragment of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46, wherein said fragment has at least 15 nucleotides, or a derivative of said fragment; or 
 (c) a variant of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46, wherein the full-length nucleotide sequence of said variant has at least 70% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46; 
   wherein the peptide encoded by said derivative, fragment or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 2, 6, 12, 16, 22, 26, 28, 32, 34, 36, 38, 42, 44 or 46;   (ii) a DNA sequence selected from:
 (a) SEQ ID NO: 8 or 18, or a derivative of SEQ ID NO: 8 or 18; or 
 (b) a variant of SEQ ID NO: 8 or 18, wherein the full-length nucleotide sequence of said variant has at least 70% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 8 or 18; or a derivative of said variant; 
   wherein the peptide encoded by said derivative or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 8 or 18;   (iii) a DNA sequence selected from:
 (a) SEQ ID NO: 4, or a derivative of SEQ ID NO: 4; or 
 (b) a variant of SEQ ID NO: 4, wherein the full-length nucleotide sequence of said variant has at least 80% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 4; or a derivative of said variant; 
   wherein the peptide encoded by said derivative or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 4;   (iv) a DNA sequence selected from:
 (a) SEQ ID NO: 24 or 40, or a derivative of SEQ ID NO: 24 or 40; 
 (b) a fragment of SEQ ID NO: 24 or 40, wherein said fragment has at least 15 nucleotides; or a derivative of said fragment; 
 (c) a variant of SEQ ID NO: 24 or 40, wherein the full-length nucleotide sequence of said variant has at least 90% nucleotide sequence identity with the full-length nucleotide sequence of SEQ ID NO: 24 or 40; or a derivative of said variant; 
   wherein the peptide encoded by said derivative, fragment or variant has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 24 or 40;   and   (v) a DNA sequence selected from:
 (a) SEQ ID NO: 19; or 
 (b) a derivative of SEQ ID NO: 19; 
   wherein the peptide encoded by said derivative has a common antigenic cross-reactivity to the peptide encoded by SEQ ID NO: 19;   
       wherein said  M. tuberculosis  DNA sequence is the coding sequence of an  M. tuberculosis  gene, the expression of which is down-regulated during a stationary phase culture of  M. tuberculosis  under nutrient-starving culture conditions, when compared with an exponential phase culture of  M. tuberculosis  under culture conditions that are not nutrient starving and that support exponential growth of said  M. tuberculosis.    
     
     
         36 . A viral vector comprising a DNA sequence according to  claim 29 . 
     
     
         37 . A viral vector comprising an RNA sequence according to  claim 35 . 
     
     
         38 . A therapeutic agent for combating mycobacterial infections, comprising an  M. tuberculosis  DNA sequence according to  claim 29 . 
     
     
         39 . A diagnostic kit for identifying a mycobacterial infection, comprising a polynucleotide probe comprising at least 8 nucleotides wherein said probe binds to a DNA sequence according to  claim 29 . 
     
     
         40 . A method of diagnosing a mycobacterial infection, comprising the steps of:
 (a) incubating a biological sample with a polynucleotide probe comprising at least 8 nucleotides wherein said probe binds to a DNA sequence according to  claim 29 ; and   (b) detecting hybridization between said probe and said DNA sequence.   
     
     
         41 . A viral vector according to  claim 37 , wherein said viral vector is a retroviral vector.