Claims
- 1. A non-naturally occurring Mycobacterium tuberculosis comprising a deletion of an RD1 region or a region controlling production of a vitamin, wherein the M. tuberculosis exhibits attenuated virulence in a mammal when compared to the M. tuberculosis without the deletion.
- 2. The M. tuberculosis of claim 1, wherein the M. tuberculosis is capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks.
- 3. The M. tuberculosis of claim 1 or 2, wherein the M. tuberculosis is capable of protecting the mammal from challenge by a virulent M. tuberculosis.
- 4. The M. tuberculosis of claim 3, wherein the mammal is a human.
- 5. The M. tuberculosis of claim 4, wherein the human is a human child.
- 6. The M. tuberculosis of any one of claims 1-5, wherein the mammal is immunocompetent.
- 7. The M. tuberculosis of any one of claims 1-5, wherein the mammal is immunocompromised.
- 8. The M. tuberculosis of any one of claims 1-7, wherein the M tuberculosis is an H37Rv strain.
- 9. The M. tuberculosis of any one of claims 1-7, wherein the M. tuberculosis is a CDC1551 strain.
- 10. The M. tuberculosis of any one of claims 1-9, wherein the deletion is of the RD1 region.
- 11. The M. tuberculosis of claim 10, wherein the deleted RD1 region has at least 95% homology to SEQ ID NO:1.
- 12. The M. tuberculosis of claim 10, wherein the deleted RD1 region comprises SEQ ID NO:1.
- 13. The M. tuberculosis of any one of claims 10-12, further comprising a second deletion, wherein a virulent M. tuberculosis having the second deletion exhibits attenuated virulence.
- 14. The M. tuberculosis of claim 13, wherein the second deletion causes the M. tuberculosis to be auxotrophic.
- 15. The M. tuberculosis of claim 14, wherein the second deletion is a region controlling production of a vitamin.
- 16. The M. tuberculosis of claim 15, wherein the vitamin is pantothenic acid or nicotinamide adenine dinucleotide (NAD).
- 17. The M. tuberculosis of claim 15, 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 14, 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 selected from the group consisting of proline, tryptophan, leucine or lysine.
- 21. The M. tuberculosis of claim 19, wherein the amino acid is lysine.
- 22. The M. tuberculosis of claim 20, wherein the second deletion is a ΔlysA deletion.
- 23. The M. tuberculosis of claim 22, wherein the ΔlysA deletion has at least 95% homology to SEQ ID NO:4.
- 24. The M. tuberculosis of claim 22, wherein the ΔlysA deletion comprises SEQ ID NO:4.
- 25. The M. tuberculosis of any one of claims 1-9, wherein the deletion is of a region controlling production of a vitamin.
- 26. The M. tuberculosis of claim 25, wherein the vitamin is pantothenic acid or NAD.
- 27. The M. tuberculosis of claim 25, wherein the vitamin is pantothenic acid.
- 28. The M. tuberculosis of claim 27, wherein the deletion is a ΔpanCD deletion.
- 29. The M. tuberculosis of claim 28, wherein the ΔpanCD deletion has at least 95% homology to SEQ ID NO:2.
- 30. The M. tuberculosis of claim 28, wherein the ΔpanCD deletion comprises SEQ ID NO:2.
- 31. The M. tuberculosis of claim any one of claims 27-30, further comprising a second deletion, wherein a virulent M. tuberculosis having the second deletion exhibits attenuated virulence.
- 32. The M. tuberculosis of claim 31, wherein the second deletion is in a region controlling production of a second vitamin or an amino acid.
- 33. The M. tuberculosis of claim 32, wherein the second deletion is in a region controlling production of an amino acid.
- 34. The M. tuberculosis of claim 33, wherein the amino acid is selected from the group consisting of proline, tryptophan, leucine or lysine.
- 35. The M. tuberculosis of claim 34, wherein the second deletion is a ΔlysA deletion.
- 36. The M. tuberculosis of claim 35, wherein the ΔlysA deletion has at least 95% homology to SEQ ID NO:4.
- 37. The M. tuberculosis of claim 35, wherein the ΔlysA deletion comprises SEQ ID NO:4.
- 38. The M tuberculosis of any one of claims 1-30, wherein the deletion is made by serial passage of a virulent M. tuberculosis and selection for a mutation having the deletion.
- 39. The M tuberculosis of any one of claims 1-30, wherein the deletion is made by genetic engineering.
- 40. The M. tuberculosis of claim 39, wherein the deletion is made by specialized transduction.
- 41. The M. tuberculosis of claim 39, wherein the deletion is made by sequential two-step recombination.
- 42. The M. tuberculosis of claim 41, wherein the sequential two-step recombination uses a sacB selective marker.
- 43. The M. tuberculosis of any one of claims 1-42, further comprising a foreign DNA stably integrated into genomic DNA of the M. tuberculosis.
- 44. The M. tuberculosis of claim 43, 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.
- 45. The M. tuberculosis of claim 44, wherein the foreign DNA encodes at least one protein antigen selected from the group consisting of antigens from Mycobacterium leprae, Mycobacterium tuberculosis, malaria sporozoites, malaria merozoites, diphtheria toxoid, tetanus toxoids, Leishmania spp., Salmonella spp., Mycobacterium africanum, Mycobacterium intracellulare, Mycobacterium avium, Treponema spp., Pertussis, Herpes virus, Measles virus, Mumps virus, Shigella spp., Neisseria spp., Borrelia spp., rabies, polio virus, Human immunodeficiency virus, snake venom, insect venom, and Vibrio cholera; steroid enzymes; interleukins 1 through 7; tumor necrosis factor α and β; interferon α, β, and γ; and reporter molecules luciferase, β-galactosidase, β-glucuronidase and catechol dehydrogenase.
- 46. A mycobacterium in the Mycobacterium tuberculosis complex, genetically engineered to comprise a deletion of an RD1 region or a region controlling production of a vitamin.
- 47. The mycobacterium of claim 46, wherein the mycobacterium exhibits attenuated virulence in a mammal when compared to the mycobacterium without the deletion.
- 48. The mycobacterium of claim 46 or 47, wherein the mycobacterium is a Mycobacterium bovis.
- 49. The mycobacterium of claim 46 or 47, wherein the mycobacterium is a Mycobacterium tuberculosis.
- 50. The mycobacterium of any one of claims 46-49, further comprising a second deletion, wherein a virulent mycobacterium in the M. tuberculosis complex having the second deletion exhibits attenuated virulence.
- 51. The mycobacterium of claim 50, wherein the second deletion is a deletion of a region controlling production of a vitamin or an amino acid.
- 52. A non-naturally occurring mycobacterium in the M. tuberculosis complex comprising a deletion of a region controlling production of a vitamin, wherein the mycobacterium is capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks.
- 53. The mycobacterium of claim 52, wherein the mycobacterium is an M. tuberculosis.
- 54. The mycobacterium of claim 52, wherein the mycobacterium is an M. bovis.
- 55. The mycobacterium of claim 52-54, wherein the mycobacterium is an M. bovis BCG.
- 56. The mycobacterium of any one of claims 52-57, wherein the mycobacterium exhibits attenuated virulence in a mammal when compared to the mycobacterium without the deletion.
- 57. The mycobacterium of any one of claims 52-56, wherein the mycobacterium is capable of protecting a mammal from challenge by a virulent mycobacterium in the M. tuberculosis complex.
- 58. The mycobacterium of any one of claims 52-57, wherein the vitamin is pantothenic acid.
- 59. The mycobacterium of claim 58, wherein the deletion is a ΔpanCD deletion.
- 60. The mycobacterium of claim 59, wherein the ΔpanCD deletion has at least 95% homology to SEQ ID NO:2.
- 61. The mycobacterium of claim 59, wherein the ΔpanCD deletion comprises SEQ ID NO:2.
- 62. The mycobacterium of any one of claims 52-61, further comprising a second deletion, wherein a virulent mycobacterium in the M. tuberculosis complex having the second deletion exhibits attenuated virulence.
- 63. The mycobacterium of claim 62, wherein the second deletion is of an RD1 region.
- 64. The mycobacterium of claim 63, wherein the deleted RD1 region has at least 95% homology to SEQ ID NO:1.
- 65. The mycobacterium of claim 63, wherein the deleted RD1 region comprises SEQ ID NO:1.
- 66. The mycobacterium of claim 62, wherein the second deletion is of a region controlling production of an amino acid.
- 67. The mycobacterium of claim 66, wherein the amino acid is selected from the group consisting of proline, tryptophan, leucine or lysine.
- 68. The mycobacterium of claim 66, wherein the second deletion is a ΔlysA deletion.
- 69. The mycobacterium of claim 68, wherein the ΔlysA deletion has at least 95% homology to SEQ ID NO:4.
- 70. The mycobacterium of claim 68, wherein the ΔlysA deletion comprises SEQ ID NO:4.
- 71. The mycobacterium of any one of claims 52-70, further comprising a foreign DNA stably integrated into genomic DNA of the mycobacterium.
- 72. The mycobacterium of claim 71, 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.
- 73. The mycobacterium of claim 72, wherein the foreign DNA encodes at least one protein antigen selected from the group consisting of antigens from Mycobacterium leprae, Mycobacterium tuberculosis, malaria sporozoites, malaria merozoites, diphtheria toxoid, tetanus toxoids, Leishmania spp., Salmonella spp., Mycobacterium africanum, Mycobacterium intracellulare, Mycobacterium avium, Treponema spp., Pertussis, Herpes virus, Measles virus, Mumps virus, Shigella spp., Neisseria spp., Borrelia spp., rabies, polio virus, Human immunodeficiency virus, snake venom, insect venom, and Vibrio cholera; steroid enzymes; interleukins 1 through 7; tumor necrosis factor α and β; interferon α, β, and γ; and reporter molecules luciferase, β-galactosidase, β-glucuronidase and catechol dehydrogenase.
- 74. A non-naturally occurring mycobacterium in the Mycobacterium tuberculosis complex comprising a deletion of a region controlling production of lysine, wherein the mycobacterium is capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks.
- 75. The mycobacterium of claim 74, wherein the mycobacterium is an M. tuberculosis.
- 76. The mycobacterium of claim 74, wherein the mycobacterium is an M. bovis.
- 77. The mycobacterium of claim 74, wherein the mycobacterium is an M. bovis BCG.
- 78. The mycobacterium of any one of claims 74-77, wherein the mycobacterium exhibits attenuated virulence in a mammal when compared to the mycobacterium without the deletion.
- 79. The mycobacterium of any one of claims 74-78, wherein the mycobacterium is capable of protecting a mammal from challenge by a virulent mycobacterium in the M. tuberculosis complex.
- 80. The mycobacterium of any one of claims 74-79, wherein the deletion is a ΔlysA deletion.
- 81. The mycobacterium of claim 80, wherein the ΔlysA deletion has at least 95% homology to SEQ ID NO:4.
- 82. The mycobacterium of claim 80, wherein the ΔlysA deletion comprises SEQ ID NO:4.
- 83. The mycobacterium of any one of claims 74-82, further comprising a second deletion, wherein a virulent mycobacterium in the M. tuberculosis complex having the second deletion exhibits attenuated virulence.
- 84. A mycobacterium in the M. tuberculosis complex, genetically engineered to comprise a two deletions, wherein a virulent mycobacterium in the M. tuberculosis complex having either deletion exhibits attenuated virulence.
- 85. The mycobacterium of claim 84, wherein the mycobacterium is a Mycobacterium tuberculosis.
- 86. The mycobacterium of claim 84 or 85, wherein the mycobacterium is capable of sustaining an infection in an immunocompetent mouse for at least 20 weeks.
- 87. The mycobacterium of any one of claims 84-86, wherein the mycobacterium is capable of protecting the mammal from challenge by a virulent mycobacterium.
- 88. The mycobacterium of claim 87, wherein the mammal is a human.
- 89. The mycobacterium of claim 88, wherein the human is a human child.
- 90. The mycobacterium of any one of claims 87-89, wherein the mammal is immunocompetent.
- 91. The mycobacterium of any one of claims 87-89, wherein the mammal is immunocompromised.
- 92. The mycobacterium of any one of claims 84-91, wherein at least one of the two deletions is a deletion of an RD1 region, a region controlling production of a vitamin, or a region controlling the production of an amino acid.
- 93. The mycobacterium of claim 92, wherein the deletion is of an RD1 region.
- 94. The mycobacterium of claim 93, wherein the deleted RD1 region has at least 95% homology to SEQ ID NO:1.
- 95. The mycobacterium of claim 93, wherein the deleted RD1 region comprises SEQ ID NO:1.
- 96. The mycobacterium of any one of claims 84-91, wherein the deletion is of a region controlling production of a vitamin.
- 97. The mycobacterium of claim 96, wherein the vitamin is pantothenic acid or NAD.
- 98. The mycobacterium of claim 96, wherein the vitamin is pantothenic acid.
- 99. The mycobacterium of claim 96, wherein the deletion is a ΔpanCD deletion.
- 100. The mycobacterium of claim 99, wherein the ΔpanCD deletion has at least 95% homology to SEQ ID NO:2.
- 101. The mycobacterium of claim 99, wherein the ΔpanCD deletion comprises SEQ ID NO:2.
- 102. The mycobacterium of any one of claims 84-91, wherein the deletion is of a region controlling production of an amino acid.
- 103. The mycobacterium of claim 102, wherein the amino acid is selected from the group consisting of proline, tryptophan, leucine or lysine.
- 104. The mycobacterium of claim 102, wherein the second deletion is a ΔlysA deletion.
- 105. The mycobacterium of claim 104, wherein the ΔlysA deletion has at least 95% homology to SEQ ID NO:4.
- 106. The mycobacterium of claim 104, wherein the ΔlysA deletion comprises SEQ ID NO:4.
- 107. The mycobacterium of claim 92, wherein one deletion is of an RD1 region and the other deletion is of a region that controls production of a vitamin.
- 108. The mycobacterium of claim 107, wherein the deletion of the RD1 region comprises SEQ ID NO:1 and the deletion of a region that controls production of a vitamin comprises SEQ ID NO:2.
- 109. The mycobacterium of claim 84, wherein one deletion is of an RD1 region and the other deletion is of a region that controls production of an amino acid.
- 110. The mycobacterium of claim 109, wherein the deletion of the RD1 region comprises SEQ ID NO:1 and the deletion of a region that controls production of an amino acid comprises SEQ ID NO:4.
- 111. The mycobacterium of claim 84, wherein one deletion is of a region that controls production of a vitamin and the other deletion is of a region that controls production of an amino acid.
- 112. The mycobacterium of claim 111, wherein the deletion of a region that controls production of a vitamin comprises SEQ ID NO:2 and the deletion of a region that controls production of an amino acid comprises SEQ ID NO:4.
- 113. A tuberculosis vaccine comprising the M. tuberculosis of any one of claims 1-45 in a pharmaceutically acceptable excipient, wherein the is vaccine is capable of protecting a mammal from challenge by a virulent M. tuberculosis.
- 114. The vaccine of claim 113, wherein the mammal is a human.
- 115. The vaccine of claim 114, wherein the human is a human child.
- 116. The vaccine of any one of claims 113-115, wherein the mammal is immunocompromised.
- 117. A tuberculosis vaccine comprising the mycobacterium of any one of claims 46-112 in a pharmaceutically acceptable excipient, wherein the vaccine is capable of protecting the mammal from challenge by a virulent mycobacterium.
- 118. The vaccine of claim 117, wherein the mycobacterium is Mycobacterium bovis and the mammal is a cow.
- 119. The vaccine of claim 117, wherein the mycobacterium is Mycobacterium tuberculosis and the mammal is a human.
- 120. A method of protecting a mammal from a virulent Mycobacterium tuberculosis, the method comprising treating the mammal with the vaccine of any one of claims 113-116.
- 121. The method of claim 120, wherein the vaccine is administered subcutaneously or intradermally.
- 122. A method of protecting a mammal from a virulent mycobacterium in the Mycobacterium tuberculosis complex, the method comprising treating the mammal with the vaccine of any one of claims 117-119.
- 123. The method of claim 122, wherein the vaccine is administered subcutaneously or intradermally.
- 124. A method of preparing a tuberculosis vaccine, the method comprising deleting an RD1 region or a region controlling production of a vitamin or an amino acid from an Mycobacterium tuberculosis to produce the M. tuberculosis of any one of claims 1-45, wherein the is vaccine is capable of protecting the mammal from challenge by a virulent M. tuberculosis.
- 125. A method of preparing a tuberculosis vaccine, the method comprising genetically engineering a mycobacterium in the Mycobacterium tuberculosis complex to delete an RD1 region or a region controlling production of a vitamin from the mycobacterium to produce the mycobacterium of any one of claims 46-112, wherein the is vaccine is capable of protecting the mammal from challenge by a virulent mycobacterium in the M. tuberculosis complex.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 60/358,152, filed Feb. 19, 2002. That application is incorporated by reference herewith in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided by the terms of AI26170 awarded by National Institutes of Health.
Provisional Applications (1)
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Number |
Date |
Country |
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60358152 |
Feb 2002 |
US |