Unmarked deletion mutants of mycobacteria and methods of using same

Abstract

Disclosed is a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, where an “unmarked mutation” is a mutated nucleotide sequence introduced into a mycobacterium where the introduced mutated nucleotide sequence does not contain a selectable marker, such as a gene conferring antibiotic resistance to the recombinant mycobacterium incorporating the mutated nucleotide sequence. Also disclosed is a method for preparing a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, as well as a vaccine comprising a recombinant slow-growing mycobacterium having at least one mycobacterial gene containing an unmarked mutation dispersed in a physiologically acceptable carrier. Further disclosed is a method of treating or preventing tuberculosis in a subject comprising administering the vaccine of the present invention in an amount effective to treat or prevent tuberculosis in the subject.

What is claimed is:  
     
         1 . A recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation.  
     
     
         2 . The recombinant slow-growing mycobacterium of  claim 1  wherein the mutant mycobacterial gene comprises a deletion, addition, substitution or point mutation.  
     
     
         3 . The recombinant slow-growing mycobacterium of  claim 2  wherein the mycobacterial gene is a gene that encodes an enzyme essential in the biosynthetic pathway of a nutrient, structural component or an amino acid.  
     
     
         4 . The recombinant slow-growing mycobacterium of  claim 3  wherein the mycobacterium is auxotrophic for lysine.  
     
     
         5 . The recombinant slow-growing mycobacterium of  claim 4  wherein the mycobacterial gene is lysA.  
     
     
         6 . The recombinant slow-growing mycobacterium of  claim 5  which is selected from the group of  M. bovis  BCG,  M. tuberculosis,  and  M. leprae.    
     
     
         7 . A method for preparing the recombinant slow-growing mycobacterium of  claim 1 , comprising: 
 (a) introducing a vector into a slow-growing mycobacterium, said vector comprising a selectable marker, a counterselectable marker, and an unmarked mutant mycobacterial gene;    (b) selecting for primary recombinants incorporating the selectable marker;    (c) culturing the primary recombinants incorporating the selectable marker;    (d) selecting for secondary recombinants that have lost the counterselectable marker; and    (f) isolating the secondary recombinants comprising the desired unmarked mutant mycobacterial gene.    
     
     
         8 . The method of  claim 7 , wherein the vector is a suicide plasmid.  
     
     
         9 . The method of  claim 8 , wherein the selectable marker confers antibiotic resistance and the counterselectable marker is one of rpsL, pyrF, and sacB.  
     
     
         10 . The method of  claim 9 , wherein the counterselectable marker is sacB.  
     
     
         11 . The method of  claim 7 , wherein the recombinant slow-growing mycobacterium is auxotrophic for lysine.  
     
     
         12 . The method of  claim 11 , wherein the mycobacterial gene is lysA.  
     
     
         13 . A vaccine that comprises (i) a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation and (ii) a physiologically acceptable carrier.  
     
     
         14 . The vaccine of  claim 13 , wherein the unmarked mutation is a deletion, addition, substitution or point mutation.  
     
     
         15 . The vaccine of  claim 14 , wherein the slow-growing mycobacterium is auxotrophic for lysine.  
     
     
         16 . The vaccine of  claim 15 , wherein the gene is lysA.  
     
     
         17 . The vaccine of  claim 16 , wherein the slow-growing mycobacterium is selected from  M. bovis BCG, M. tuberculosis,  and  M. leprae.    
     
     
         18 . A method of treating or preventing tuberculosis in a subject comprising administering the vaccine of  claim 17  in an amount effective to treat or prevent tuberculosis in the subject.