Attenuated recombinant mycobacteria useful as immunogens or as vaccine components

Abstract

This invention provides recombinant mycobacterium strains of pathogenic origin that have been attenuated by the inactivation of a gene coding for a metabolic protein, specifically a gene coding for a protein necessary for the biosynthesis of a purine or a pyrimidine base, and more precisely, the purC gene that codes for an enzyme of the metabolic pathway of purine biosynthesis. The recombinant mycobacterium of this invention have a reduced capacity to propagate in a mammalian host, but remain viable in the host for a period of time sufficient to induce an protective immune response against the natural pathogenic mycobacterium counterpart.

We claim:  
     
         1 . A recombinant mycobacterium strain of pathogenic origin capable of replicating in a macrophage of a host, said strain containing in its chromosome or on a plasmid a counterpart to a gene in the wild type mycobacterium coding for a protein necessary for the biosynthesis of a purine or a pyrimidine base, wherein said counterpart gene in the recombinant mycobacterium has been inactivated by at least one point mutation or by addition, deletion, or substitution of one or more base pairs.  
     
     
         2 . The recombinant mycobacterium strain according to    claim 1   , wherein said mycobacterium strain belongs to the  Mycobacterium tuberculosis  complex.  
     
     
         3 . The recombinant mycobacterium strain according to    claim 1   , wherein said mycobacterium strain belongs to the  Mycobacterium avium  species.  
     
     
         4 . The recombinant mycobacterium strain according to    claim 1   , wherein said mycobacterium strain belongs to the  Mycobacterium intracellulare  species.  
     
     
         5 . The recombinant mycobacterium strain according to    claim 1   , wherein the gene in the wild type mycobacterium codes for a protein necessary for the biosynthesis of at least one of the following bases chosen among the group consisting of guanine or adenine.  
     
     
         6 . The recombinant mycobacterium strain according to    claim 1   , wherein the gene in the wild type mycobacterium codes for a protein necessary for the biosynthesis of at least one of the following bases chosen among the group consisting of cytosine, uracil, or thymine.  
     
     
         7 . The recombinant mycobacterium strain according to    claim 5   , wherein the gene in the wild type mycobacterium is the purC or the purL gene.  
     
     
         8 . The recombinant mycobacterium strain MYC 1551 (C.N.C.M. No. I-1871).  
     
     
         9 . The recombinant mycobacterium strain MYC 1552 (C.N.C.M. No. I-1872).  
     
     
         10 . A recombinant vector containing SacB gene coding for a levane saccharase enzyme and a nucleotide sequence consisting of a gene coding for a protein necessary for the biosynthesis of a purine or a pyrimidine base that has been inactivated by at least one point mutation or by addition, deletion, or substitution of one or more base pairs.  
     
     
         11 . The recombinant vector according to    claim 10   , wherein said modified gene is a gene coding for a base chosen among the group consisting of guanine or adenine.  
     
     
         12 . The recombinant vector according to    claim 10   , wherein said modified gene is a gene coding for a base chosen among the group consisting of cytosine, uracil, or thymine.  
     
     
         13 . The recombinant vector according to    claim 12   , wherein said modified gene is the purC or the purL gene.  
     
     
         14 . The recombinant vector according to    claim 10   , wherein the gene coding for a protein necessary for the biosynthesis of a purine or a pyrimidine base is interrupted by the insertion of an exogenous polynucleotide.  
     
     
         15 . The recombinant vector according to    claim 14   , wherein the exogenous polynucleotide is selectable marker gene.  
     
     
         16 . The recombinant vector according to    claim 15   , wherein the selectable marker gene is a gene coding for resistance to an antibiotic.  
     
     
         17 . The recombinant vector according to    claim 16   , wherein the antibiotic resistance gene is a gentamycin resistance coding gene or a kanamycin resistance coding gene.  
     
     
         18 . The recombinant vector according to    claim 14   , wherein the exogenous polynucleotide comprises a gene coding for an antigenic protein heterologous with respect to the mycobacterium strain to be transfected.  
     
     
         19 . The recombinant vector according to any one of claims  13 ,  15 ,  16 , or  17  which is contained in the recombinant mycobacterium strain MYC 1551 (C.N.C.M. No. I-1871).  
     
     
         20 . The recombinant vector according to any one of claims  13 ,  15 ,  16 , or  17  which is contained in the recombinant mycobacterium strain MYC 1552 (C.N.C.M. No. I-i 872).  
     
     
         21 . A purified polynucleotide comprising the SacB gene placed in the neighborhood of a gene coding for a protein necessary for the biosynthesis of a purine or a pyrimidine base that has been inactivated by at least one point mutations or by addition or deletion of one or more base pairs.  
     
     
         22 . A method for producing a recombinant mycobacterium strain according to    claim 1    comprising the steps of: 
 a) providing a vector according to    claim 10   ;  
 b) transforming the mycobacterium strain with said vector;  
 c) selecting the clones of the transformed mycobacteria for the replacement of the nucleotide sequence of interest by propagating said transformed clones in a culture medium supplemented with sucrose; and  
 d) isolating the recombinant strain.  
 
     
     
         23 . The method according to    claim 22    wherein the vector contains a marker gene, and wherein step c) is preceded by a first selection step of transformed clones by propagating said clones in a culture medium supplemented with a selection molecule.  
     
     
         24 . An immunogenic composition comprising a recombinant mycobacterium strain according to    claim 1   .  
     
     
         25 . A vaccine composition containing a recombinant mycobacterium according to    claim 1    in combination with a pharmaceutically compatible excipient.  
     
     
         26 . A polynucleotide containing all or part of the genome of a mycobacterium strain which is devoid of a wild gene encoding a protein involved in the biosynthesis of a purine or a pyrimidine base.  
     
     
         27 . A recombinant mycobacterium according to    claim 1    which has been additionally transformed with a recombinant vector carrying a polynucleotide encoding an antigenic peptide or protein which contains at least one antigenic epitope.  
     
     
         28 . An immunotherapeutic composition comprising a recombinant mycobacterium strain according to    claim 1   .  
     
     
         29 . The immunotherapeutic composition according to    claim 28   , wherein the recombinant mycobacterium strain is alive.  
     
     
         30 . The immunotherapeutic composition according to    claim 28   , wherein the recombinant mycobacterium strain is not alive.  
     
     
         31 . A pharmaceutical composition containing a recombinant mycobacterium strain according to    claim 1    together with a pharmaceutically acceptable carrier.  
     
     
         32 . A method of treating or preventing pathogenic mycobacterial infection comprising the step of administering a composition according to any one of claims  24 ,  25 ,  28  or  31  to a patient in need thereof.