Mycobacterial mutants inducing IL-12
View Patent ↗Provided are mycobacteria deleted in at least a portion of a region 3 ESAT-6-like gene cluster. Also provided are mycobacteria comprising a mutation in an roc-1 gene. Additionally, vaccines comprising these mycobacteria are provided. Further provided are methods of making a recombinant mycobacterium , methods of inducing an immune response in a mammal, methods of inhibiting IL-12 production in a mammal, and methods of stimulating IL-12 production in a mammal. Vaccine adjuvants are also provided, as are methods of inducing immunity to a target antigen in a mammal.
1. A recombinant mycobacterium comprising a deletion of a gene of a region 3 ESAT-6-like gene cluster, wherein the mycobacterium is not an M. tuberculosis.
2. The mycobacterium of claim 1 , wherein the region 3 ESAT-6-like gene cluster is deleted.
3. The mycobacterium of claim 1 , wherein the deletion comprises at least genes analogous to Ms0615-Ms0626 as identified in the GenBank M. smegmatis genome sequence nc — 008596.
4. The mycobacterium of claim 1 , wherein the mycobacterium is M. smegmatis, M. bovis, M. avium, M. phlei, M. fortuitum, M. lufu, M. paratuberculosis, M. habana, M. scrofulacium, M. intracellulare , or M. kansasii.
5. The mycobacterium of claim 1 , wherein the mycobacterium is an M. smegmatis.
6. The mycobacterium of claim 1 , genetically complemented with Region 3 from an M. tuberculosis.
7. The mycobacterium of claim 6 , wherein the mycobacterium is genetically complemented with genes comprising RV0283-RV0291 from M. tuberculosis.
8. The mycobacterium of claim 1 , further comprising a recombinant gene operably linked to a promoter that directs expression of the gene when the mycobacterium infects a mammalian cell.
9. The mycobacterium of claim 8 , wherein the gene encodes an antigen of a mammalian pathogen.
10. A method of inducing an immune response in a mammal, the method comprising inoculating the mammal with the mycobacterium of 54.
11. A method of inducing an immune response in a mammal, the method comprising inoculating the mammal with the mycobacterium of 60.
12. A method of making the recombinant mycobacterium of claim 1 , the method comprising deleting a gene of a region 3 ESAT-6-like gene cluster, wherein the mycobacterium is not an M. tuberculosis.
13. A method of making the recombinant mycobacterium of claim 6 , the method comprising deleting a gene of a region 3 ESAT-6-like gene cluster in the mycobacterium , wherein the mycobacterium is not an M. tuberculosis , and genetically complementing the mycobacterium with an M. tuberculosis region 3 ESAT-6-like gene cluster.
14. A recombinant mycobacterium comprising a mutation in a roc-1 gene, wherein the mutation increases the ability of the mycobacterium to induce IL-12 and/or TNF-α production in a mammalian macrophage infected by the mycobacterium , and wherein the mycobacterium is not a virulent Mycobacterium tuberculosis.
15. The mycobacterium of claim 14 , wherein the mutation is a deletion.
16. The mycobacterium of claim 15 wherein the deletion is a deletion of the entire roc-1 gene (Δroc-1).
17. The mycobacterium of claim 14 , wherein the mycobacterium is M. smegmatis, M. bovis, M. avium, M. phlei, M. fortuitum, M. lufu, M. paratuberculosis, M. habana, M. scrofulacium, M. intracellulare , an attenuated or avirulent M. tuberculosis or M. kansasii.
18. The mycobacterium of claim 17 , wherein the mycobacterium is M. bovis BCG.
19. The mycobacterium of claim 17 , wherein the mycobacterium is an M. tuberculosis H37ra.
20. The mycobacterium of claim 17 , wherein the mycobacterium is an M. tuberculosis further comprising a deletion in a second gene, wherein the second gene by itself attenuates the virulence of the M. tuberculosis.
21. A method of making the recombinant mycobacterium of claim 14 , the method comprising genetically creating a mutation in a roc-1 gene, wherein the mutation increases the ability of the mycobacterium to induce IL-12 and/or TNF-α production in a mammalian macrophage infected by the mycobacterium , and wherein the mycobacterium is not a virulent Mycobacterium tuberculosis.
22. A method of inhibiting IL-12 production in a mammal, the method comprising treating the mammal with (i) purified trehalose 6′-6′ dimycolates from a virulent Mycobacterium tuberculosis , or (ii) a purified keto mycolate esterified to trehalose, or (iii) a purified methoxy mycolate esterified to trehalose, or (iv) a purified keto mycolate esterified to trehalose and a purified methoxy mycolate esterified to trehalose.
23. A method of stimulating IL-12 production in a mammal, the method comprising treating the mammal with (i) purified trehalose 6′-6′ dimycolates from Mycobacterium tuberculosis Δroc-1, or (ii) purified α-mycolate, or (iii) purified epoxy mycolate esterified to trehalose.
24. A vaccine or vaccine adjuvant comprising (i) purified trehalose 6′-6′ dimycolates from Mycobacterium tuberculosis Δroc-1, (ii) purified α-mycolate, or (iii) purified epoxy mycolate esterified to trehalose.
25. The method of claim 10 , wherein the mammal is a mouse.