Claims for Patent: 6,120,776

Summary for Patent: 6,120,776

Title:	Diagnostic skin test for tuberculosis
Abstract:	Diagnostic methods capable of discriminating between cell mediated immunologic responses due to on the one hand active tuberculosis caused by bacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) and on the other hand vaccination with an immunogenic agent conferring immunity to tuberculosis. A diagnostic kit is also provided, comprising a polypeptide (e.g. MPT64) capable of eliciting a delayed type hypersensitivity reaction (Dth) in animals with active tuberculosis, but not in animals vaccinated against TB with an immunogenic agent (e.g. M. bovis BCG strain: Danish 1331). Also provided are polypeptide fragments comprising a T-cell epitope of MPT64 as well as nucleic acid fragments encoding these polypeptide fragments.
Inventor(s):	Hasl.o slashed.v; Kaare (S.o slashed.borg, DK), Andersen; .ANG.se Bengaard (Br.o slashed.nsh.o slashed.j, DK), Oettinger; Thomas (Hellerup, DK)
Assignee:	Statens Seruminstitut (Copenhagen, DK)
Application Number:	08/569,221
Patent Claims:	1. A kit comprising: (A) a BCG vaccine against tuberculosis, and (B) at least one diagnostic test comprising a pharmaceutical composition comprising a polypeptide, wherein: (i) the polypeptide has a length of at least 12 amino acid residues, and the polypeptide has a sequence comprising at least 12 amino acid residues from a polypeptide of a mycobacterium of the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis), and (ii) lymphoid cells previously primed with virulent mycobacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) are capable of reacting with the polypeptide and (iii) lymphoid cells previously primed with a vaccine against tuberculosis are not capable of reacting with the polypeptide, and (iv) a positive response is indicative of having or having had tuberculosis, and (v) a negative response is indicative of not having or not having had tuberculosis. 2. A kit according to claim 1, wherein the pharmaceutical composition comprises a homopolymer of the polypeptide. 3. A kit according to claim 2, wherein the homopolymer is produced by introduction of at least one cyteine residue in the N-terminal region of the polypeptide. 4. A kit according to claim 1, wherein the pharmaceutical composition comprises a heteropolymer of the polypeptide. 5. A kit according to claim 4, wherein the heteropolymer comprises the polypeptide coupled to a carrier or vehicle. 6. A kit according to claim 4, wherein the heteropolymer comprises the polypeptide coupled to another mycobacterial protein. 7. A kit according to claim 1, wherein the polypeptide has been post-translationally modified. 8. A kit according to claim 1, wherein the positive response is a positive skin response which appears 1-4 days after injection. 9. A kit according to claim 1, wherein the positive response is a positive skin response between 0.5 cm and 4.0 cm in diameter. 10. A kit acording to claim 1, wherein the polypeptide has an amino acid sequence comprising the amino acid sequence SEQ ID NO: 2. 11. A kit according to claim 1, wherein the polypeptide is encoded by a DNA molecule having a nucleotide sequence comprising the nucleotide sequence SEQ. ID NO: 1. 12. A kit according to claim 1, wherein the polypeptide has been modified in order to abolish or delete sensitizing epitopes. 13. A kit according to claim 1, wherein the polypeptide has been modified by denaturing procedures. 14. A kit according to claim 1, wherein the polypeptide comprises MPT64. 15. A kit according to claim 1, wherein the pharmaceutical composition comprises at least two different polypeptides. 16. A ket according to claim 15 comprising a first polypeptide comprising MPT64 and a second polypeptide comprising MPT59. 17. A kit according to claim 14, wherein the pharmaceutical composition comprises 0.05 to 20 .mu.g of the polypeptide. 18. A kit comprising: (A) a BCG vaccine against tuberculosis, wherein said BCG vaccine does not contain MPT64 or MPT59 or a fragment thereof having a length of at least 12 amino acids from MPT64 or MPT59, and (B) at least one diagnostic test comprising a pharmaceutical composition comprising a polypeptide, wherein: (i) the polypeptide comprises MPT64, or MPT59, or MPT64 and MPT59, or a fragment of MPT64 or MPT59, or a fragment of MPT64 and MPT59; said fragment having a length of at least 12 amino acid residues from MPT64 and/or MPT59, and (ii) lymphoid cells previously primed with virulent mycobacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) are capable of reacting with the polypeptide and (iii) lymphoid cells previously primed with a vaccine against tuberculosis are not capable of reacting with the polypeptide, and (iv) a positive response is indicative of having or having had tuberculosis, and (v) a negative response is indicative of not having or not having had tuberculosis. 19. A method of diagnosing tuberculosis caused by Mycobacterium tuberculosi, Mycobacterium africanum or Mycobacterium bovis in an individual susceptible thereto, comprising: exposing immune cells of the individual to a pharmaceutical composition comprising a polypeptide: having a length of at least 12 amino acid residues, wherein the polypeptide has a sequence comprising at least 12 amino acid resiudes from a polypeptide of a mycobacterium of the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis), and with which lymphoid cells previously primed with virulent mycobacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) are capable of reacting and with which lymphoid cells previously primed with a BCG vaccine against tuberculosis are not capable of reacting, wherein a positive response is indicative of the individual having or having had tuberculosis, and a negative response is indicative of the individual not having or not having had tuberculosis. 20. A method of diagnosing tuberculosis caused by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis in an individual susceptible thereto, comprising: exposing immune cells of the individual to a pharmaceutical composition comprising a polypeptide: having a length of at least 12 amino acid residues, wherein the polypeptide has a sequence comprising at least 12 amino acid residues from a polypeptide of a mycobacterium of the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis), and with which lymphoid cells previously primed with virulent mycobacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) are capable of reacting and with which lymphoid cells previously primed with a BCG vaccine against tuberculosis are not capable of reacting, wherein a positive response is indicative of the individual having or having had tuberculosis, and a negative response is indicative of the individual not having or not having had tuberculosis, and the polypeptide is as in the dit of claim 2-7, 10,11-13, 14-16, 17, or 18. 21. A method of diagnosing tuberculosis caused by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis in an individual susceptible thereto, comprising: exposing immune cells of the individual to a pharmaceutical composition comprising a polypeptide comprising MPT64, or MPT59, or MPT64 and MPT59, or a fragment of MPT64 or MPT59 or a fragment of MPT64 and MPT59; said fragment having a length of at least 12 amino acid residues from MPT64 and/or MPT59, and with which lymphoid cells previously primed with virulent mycobacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) are capable of reacting and with which lymphoid cells previously primed with a BCG vaccine against tuberculosis are not capable of reacting, wherein the BCG vaccine does not contain MPT64 or MPT59 or a fragment thereof having a length of at least 12 amino acids from MPT64 or MPT59, a positive response is indicative of the individual having or having had tuberculosis, and a negative response is indicative of the individual not having or not having had tuberculosis. 22. The method of claim 19 wherein the pharmaceutical composition comprises a homopolymer and/or a heteropolymer of the polypeptide. 23. The method of claim 19 wherein the polypeptide comprises: MPT64; or MPT59; or a polypeptide encoded by a nucleic acid molecule comprising MPT64-4, MPT64-5, MPT64-6, MPT64-7, MPT64-8; or a polypeptide comprising amino acids 186-215 of SEQ ID NO:2; or a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2; or a polypeptide comprising a polypeptide encoded by the sequence set forth in SEQ ID NO:1. 24. An isolated polypeptide consisting of an amino acid sequence as set forth in SEQ ID NO:2. 25. The isolated polypeptide according to claim 24, which has been modified in order to abolish or delete sensitizing epitopes. 26. The isolated polypeptide according to claim 24 containing at least one T-cell epitope. 27. An isolated polypeptide consisting of the amino acid sequence 186-215 in SEQ ID NO:2. 28. The kit of claim 1 wherein the polypeptide comprises a polypeptide encoded by a nucleic acid molecule comprising MPT64-4, MPT64-5, MPT64-6, MPT64-7, MPT64-8; or the polypeptide comprises amino acids 186-215 of SEQ ID NO:2. 29. A method of producing an isolated polypeptide as defined in claim 24, the method comprising sequentially linking component amino acids to form the polypeptide. 30. A kit according to claim 2, wherein the homopolymer comprises at least two copies of the peptide. 31. A kit according to claim 7, wherein the polypeptide has been post-translationally acylated, post-translationally glycosylated, or post translationally acylated and glycosylated. 32. A kit according to claim 8, wherein the positive skin response appears 2-3 days after injection. 33. A kit according to claim 9, wherein the positive skin response is between 1.0 cm and 3.0 cm in diameter. 34. A kit according to claim 18, wherein the polypeptide comprises MPT64 or MPT59. 35. A kit according to claim 13, wherein the denaturing procedures are selected from the group consisting of autoclaving, and treatment with formaldelyde or glutaraldehyde. 36. A kit according to claim 17 wherein the pharmaceutical composition comprises 0.5 to 2.0 .mu.g of the polypeptide. 37. A kit according to claim 17 wherein the pharmaceutical composition comprises 0.75 to 1.5 .mu.g of the polypeptide. 38. The method of claim 29 of producing an isolated polypeptide comprising solid or liquid phase peptide synthesis. 39. A kit comprising: (A) a BCG vaccine against tuberculosis comprising an amount of a BCG vaccine against tuberculosis, and (B) at least one diagnostic test comprising a pharmaceutical composition comprising a polypeptide, wherein (i) the polypeptide comprises a polypeptide encoded by a DNA molecule having a nucleotide sequence comprising the nucleotide sequence SEQ ID NO: 1 and (ii) lymphoid cells previously primed with virulent mycobacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) are capable of reacting with the polypeptide and (iii) lymphoid cells previously primed with the vaccine are not capable of reacting with the polypeptide, and (iv) intradermal injection of the polypeptide at a location of injection elicits a skin reaction having a diameter of at least 0.5 cm in an individual previously sensitized with virulent mycobacteria belonging to the tuberculosis complex and, (v) a positive response is indicative of the person having or having had tuberculosis, and (vi) a negative response is indicative of the person not having or not having had tuberculosis. 40. The kit of claim 1 wherein the diagnostic test is a diagnostic skin test for intradermal injection, the positive response is a positive response at the location of injection, and the negative response is a negative response at the location of injection. 41. The kit of claim 18 wherein the BCG vaccine is BCG strain: Danish 1331. 42. The method of claim 19 wherein exposing immune cells is by intradermal injection, the positive response is a positive skin response at the location of injection, and the negative response is a negative skin response at the location of injection. 43. The method of claim 21 wherein the BCG vaccine is BCG strain: Danish 1331. 44. The method of claim 31 wherein the polypeptide is from a mycobacterium belonging to the tuberculosis complex. 45. The method according to claim 20, wherein the BCG vaccine is from BCG strain: Danish 1331. 46. The method according to claim 20, wherein the polypeptide comprises a polypeptide encoded by a nucleic acid molecule comprising MPT64-4, MPT64-5, MPT64-6, MPT64-7, MPT64-8; or the polypeptide comprises amino acids 186-215 of SEQ ID NO:2. 47. The kit of claim 39 wherein the diagnostic test is a diagnostic skin test for intradermal injection, the positive response is a positive response at the location of injection, and the negative response is a negative response at the location of injection. 48. The method of claim 20 wherein exposing immune cells is by intradermal injection, the positive response is a positive skin response at the location of injection, and the negarive response is a negative skin response at the location of injection. 49. The kit of claim 1 wherein the polypeptide comprises a polypeptide from a mycobacterium of the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis). 50. The method according to claim 19 wherein the polypeptide comprises a polypeptide from a mycobacterium of the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis).

Details for Patent 6,120,776

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Teknika Llc	N/A	bcg vaccine	For Injection	103050	06/21/1989		2013-07-02