Methods and reagents to detect and characterize norwalk and related viruses

• US 20030129588A1
• Filed: 12/09/2002
• Published: 07/10/2003
• Est. Priority Date: 11/08/1989
• Status: Active Grant

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Jiang, Xi, Estes, Mary K., Graham, David Y.
• Granted Patent: US 6,942,865 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2770/16022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

Double-stranded cDNA was synthesized from nucleic acid extracted from Norwalk virus purified from stool specimens of volunteers. One clone was isolated from a cDNA library constructed in a pUC-13 vector after amplification of the cDNA. The specificity of this cDNA (pUCNV-953) was shown by hybridization assays. The cDNA reacted with post (but not pre-) infection stool samples from Norwalk volunteers and with highly purified Norwalk virus, but not with other common enteric viruses such as hepatitis A virus and rotavirus. Finally, the probe detected virus in the same fractions of CsCl gradients in which viral antigen was detected using a specific Norwalk virus radioimmunoassay, and particles were detected by immune electron microscopy. Single-stranded RNA probes derived from the DNA clone after subcloning into an in vitro transcription vector were also used to show that the Norwalk virus contains a ssRNA genome of about 8 kb in size. The original clone was also used to detect additional cDNAs which represent at least 7 kb of nucleic acid of the Norwalk genome. The availability of a Norwalk-specific cDNA and the first partial genome sequence information allow rapid cloning of the entire genome and of establishment of sensitive diagnostic assays. Such assays can be based on detection of Norwalk virus nucleic acid or Norwalk viral antigen using polyclonal or monoclonal antibodies to proteins expressed from the cDNA or to synthetic peptides made based on the knowledge of the genome sequence. Assays using proteins deduced from the Norwlk virus genome and produced in expression systmes can measure antibody responses. Vaccines made by recombinant DNA technology are now feasible.

Claims

1. A protein deduced from nucleotides including nucleotides 1 through 7753 of the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in an expression system.

2. The protein of claim 1, wherein said expression system is a prokaryotic expression system or a eukaryotic expression system.

3. A protein deduced from nucleotides 146 through 5359 of the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in an expression system.

4. The protein of claim 3, wherein said expression system is a prokaryotic expression system or eukaryotic expression system.

5. A protein deduced from nucleotides 5337 through 7573 of the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in an expression system.

6. The protein of claim 5, wherein said expression system is a prokaryotic expression system or eukaryotic expression system.

7. A protein deduced from nucleotides 5346 through 6935 of the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in an expression system.

8. The protein of claim 7, wherein said expression system is a prokaryotic expression system or eukaryotic expression system.

9. A protein deduced from nucleotides 6938 through 7573 of the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in an expression system.

10. The protein of claim 9, wherein said expression system is a prokaryotic expression system or eukaryotic expression system.

11. A method of making a probe to detect Norwalk or related viruses, comprising the steps of:

synthesizing one or more short or long nucleotides from the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof; and

producing said synthesized nucleotide by chemical methods or in an expression system.

12. The probe produced by the method of claim 11.

13. A method of making a probe to detect Norwalk or related viruses, comprising the steps of:

synthesizing one or more short or long nucleotides from the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof; and

producing said synthesized nucleotide by chemical methods or in an expression system.

14. The probe produced by the method of claim 13.

15. The method of claim 13, wherein said subgenomic region includes said Norwalk genome'"'"'s first open reading frame.

16. The probe produced by the method of claim 15.

17. The method of claim 13, wherein said subgenomic region includes nucleotides 146 through 5359.

18. The probe produced by the method of claim 17.

19. The method of claim 13, wherein said nucleotides code for a picornavirus 2C-like protein, a 3C-like protease, an RNA-dependent RNA polymerase or any combination thereof.

20. The probe produced by the method of claim 19.

21. The method of claim 13, wherein said nucleotide codes for a capsid protein.

22. The probe produced by the method of claim 21.

23. The method of claim 13, wherein said subgenomic region includes nucleotides 5337 through 7573.

24. The probe produced by the method of claim 23.

25. The method of claim 13, wherein said subgenomic region includes nucleotides 5346 through 6935.

26. The probe produced by the method of claim 25.

27. The method of claim 13, wherein said subgenomic region includes nucleotides 6938 through 7573.

28. The probe produced by the method of claim 27.

29. A kit for detecting an immune response to Norwalk virus, comprising:

a container including a protein deduced from the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in an expression system.

30. The kit of claim 29, wherein said protein is selected from the group consisting of the protein deduced from nucleotides 1 through 7753, the protein deduced from nucleotides 146 through 5359, the protein deduced from nucleotides 5337 through 7573, the protein deduced from nucleotides 5346 through 6935, the protein deduced from nucleotides 6938 through 7573 and any combination thereof.

31. The kit of claim 29, wherein said expression system is a prokaryotic expression system or a eukaryotic expression system.

32. A method of detecting an immune response to Norwalk virus, comprising the steps of:

collecting a serum sample from an individual suspected of having been exposed to Norwalk virus;

selecting a protein deduced from the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof;

expressing said selected protein in a prokaryotic expression system or eukaryotic expression system;

adding said expressed protein to said serum in a diagnostic assay under conditions allowing the expressed protein and the serum to react; and

measuring the amount of reaction of said serum and said expressed protein.

33. The method of claim 32, wherein said diagnostic assay is selected from the group consisting of enzyme-linked immunosorbent assays, radioimmunoassays and immunoblots.

34. The method of claim 32, wherein said selected protein is a capsid protein.

35. The method of claim 32, wherein said selected protein has the intrinsic property of being able to form particle(s).

36. The method of claim 32, wherein said selected protein is selected from the group consisting of the protein deduced from nucleotides 1 through 7753, the protein deduced from nucleotides 146 through 5359, the protein deduced from nucleotides 5337 through 7573, the protein deduced from nucleotides 5346 through 6935, the protein deduced from nucleotides 6938 through 7573 and any combination thereof.

37. A diagnostic assay to detect an immune response to Norwalk virus, comprising:

selecting a protein encoded in Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in a prokaryotic expression system or eukaryotic expression system;

using said protein as an antigen;

adding post-infection serum from a Norwalk infected individual under conditions allowing said serum to react with said antigen; and

measuring the amount of reaction of said serum and said antigen.

38. The method of claim 37, wherein said protein is a capsid protein.

39. The method of claim 37, wherein said protein has the intrinsic property of being able to form particle(s).

40. The method of claim 37, selected from the group consisting of the protein deduced from nucleotides 1 through 7753, the protein deduced from nucleotides 146 through 5359, the protein deduced from nucleotides 5337 through 7573, the protein deduced from nucleotides 5346 through 6935, the protein deduced from nucleotides 6938 through 7573 and any combination thereof.

41. A method of producing antibodies to Norwalk and related viruses, comprising:

immunizing animals with a protein deduced from the Norwalk virus genome shown in Table 1 or fragments or derivatives thereof and produced in a prokaryotic expression system or a eukaryotic expression system.

42. The method of claim 41, wherein said protein is selected from the group consisting of the protein deduced from nucleotides 1 through 7753, the protein deduced from nucleotides 146 through 5359, the protein deduced from nucleotides 5337 through 7573, the protein deduced from nucleotides 5346 through 6935, the protein deduced from nucleotides 6938 through 7573 and any combination thereof.

43. A vaccine for Norwalk virus, comprising:

a Norwalk virus antigen produced in an expression system using the cDNA sequence of Norwalk virus shown in Table 1 or fragments or derivatives thereof.

44. The vaccine of claim 43, wherein said antigen is produced using nucleotides 146 through 5359 of the Norwalk virus genome shown in Table 1 or a derivative thereof.

45. The vaccine of claim 43, wherein said antigen is produced using nucleotides 5337 through 7573 of the Norwalk virus genome shown in Table 1 or a derivative thereof.

46. The vaccine of claim 43, wherein said antigen is produced using nucleotides 5346 through 6935 of the Norwalk virus genome shown in Table 1 or a derivative thereof.

47. The vaccine of claim 43, wherein said antigen is produced using nucleotides 6938 through 7573 of the Norwalk virus genome shown in Table 1 or a derivative thereof.

48. The vaccine of claim 43, wherein said antigen has the intrinsic property of being able to form particle(s).

49. A method of immunizing an individual against Norwalk virus, comprising the step of:

orally or parenterally administering an immunologically effective dose(s) of the vaccine of claim 43.

50. A method of immunizing an individual against Norwalk virus, comprising the steps of:

orally and parenterally administering an immunologically effective dose of the vaccine of claim 43.

51. A vaccine for non-Norwalk virus agents, comprising:

an antigen recombinantly synthesized from a fragment or derivative of the cDNA sequence of Norwalk virus that encodes a capsid protein and one or more heterologous pieces of cDNA from the non-Norwalk virus agent that encode antigenic epitopes.

52. A method of immunizing individuals against Norwalk virus, comprising the step of:

orally or parenterally administering an immunologically effective dose(s) of the vaccine of claim 51.

53. A method of immunizing individual against Norwalk virus, comprising the steps of:

orally and parenterally administering an immunologically effective dose of the vaccine of claim 51.

54. A vaccine for non-Norwalk virus agents, comprising:

an antigen formed by mixing a capsid protein carrier recombinantly synthesized from a fragment or derivative of the Norwalk virus genome shown in Table 1 and one or more heterologous protein antigens or synthetic peptides containing heterologous epitopes.

55. A method of immunizing individuals against non-Norwalk virus agents, comprising the step of:

orally or parenterally administering an immunologically effective dose(s) of the vaccine of claim 54.

56. A method of immunizing individual against non-Norwalk virus agents, comprising the steps of:

orally and parenterally administering an immunologically effective dose of the vaccine of claim 54.

57. A vaccine for non-Norwalk virus agents, comprising:

an antigen formed by covalently bonding a capsid protein carrier recombinantly synthesized from a fragment or derivative of the Norwalk virus genome shown in Table 1 and one or more heterologous protein antigens or synthetic peptides containing heterologous epitopes.

58. A method of immunizing individuals against non-Norwalk virus agents, comprising the step of:

orally or parenterally administering an immunologically effective dose(s) of the vaccine of 57.

59. A method of immunizing individual against non-Norwalk virus agents, comprising the steps of:

orally and parenterally administering an immunologically effective dose of the vaccine of 57.