Non-naturally occurring mycobacteria in the Mycobacterium tuberculosis complex are provided. These mycobacteria have a deletion of an RD1 region or a region controlling production of a vitamin, and exhibit attenuated virulence in a mammal when compared to the mycobacteria without the deletion. Also provided are non-naturally occurring mycobacteria that have a deletion of a region controlling production of lysine, and mycobacteria comprising two attenuating deletions. Vaccines comprising these mycobacteria are also provided, as are methods of protecting mammals from virulent mycobacteria using the vaccines. Also provided are methods of preparing these vaccines which include the step of deleting an RD1 region or a region controlling production of a vitamin from a mycobacterium in the M. tuberculosis complex.
1. A mycobacterium in the Mycobacterium tuberculosis complex, genetically engineered to be auxotrophic for a vitamin.
2. The mycobacterium of claim 1 , wherein the mycobacterium is a Mycobacterium bovis.
3. The mycobacterium of claim 1 , wherein the mycobacterium is a Mycobacterium tuberculosis.
4. The mycobacterium of claim 3 , wherein the M. tuberculosis exhibits attenuated virulence in a mammal when compared to the M. tuberculosis without the deletion.
5. The mycobacterium of claim 3 , further comprising a foreign DNA stably integrated into genomic DNA of the M. tuberculosis.
6. The mycobacterium of claim 1 , wherein the vitamin is pantothenic acid.
7. The mycobacterium of claim 6 , wherein the deletion is a ΔpanCD deletion.
8. The mycobacterium of claim 1 , 2 , 3 , 4 , 6 or 7 , further comprising a deletion controlling production of an amino acid.
9. The mycobacterium of claim 8 , wherein the amino acid is lysine.
10. A non-naturally occurring Mycobacterium tuberculosis comprising a deletion of the entire RD1 region, wherein the M. tuberculosis with the RD1 deletion exhibits attenuated virulence in a mammal when compared to virulent M. tuberculosis without the deletion.
11. The M. tuberculosis of claim 10 , which is genetically engineered.
12. The M. tuberculosis of claim 10 , wherein the mammal is immunocompromised.
13. The M. tuberculosis of claim 10 , wherein the RD1 region has at least 95% homology to SEQ ID NO:1.
14. The M. tuberculosis of claim 10 , further comprising a second deletion.
15. The M. tuberculosis of claim 14 , wherein the second deletion causes the M. tuberculosis to be auxotrophic.
16. The M. tuberculosis of claim 15 , wherein the second deletion is a region controlling production of a vitamin.
17. The M. tuberculosis of claim 16 , wherein the vitamin is pantothenic acid.
18. The M. tuberculosis of claim 17 , wherein the second deletion is a ΔpanCD deletion.
19. The M. tuberculosis of claim 15 , wherein the second deletion is in a region controlling production of an amino acid.
20. The M. tuberculosis of claim 19 , wherein the amino acid is lysine.
21. The M. tuberculosis of claim 19 , wherein the second deletion is a ΔlysA deletion.
22. The M. tuberculosis of claim 10 , further comprising a foreign DNA stably integrated into genomic DNA of the M. tuberculosis.
23. The M. tuberculosis of claim 22 , wherein the foreign DNA encodes at least one protein or polypeptide selected from the group consisting of an antigen, an enzyme, a lymphokine, an immunopotentiator, and a reporter molecule.