Claims
- 1. An isolated hepatitis C virus (HCV) asialoglycoprotein selected from the group consisting of E1 and E2.
- 2. The asialoglycoprotein of claim 1, wherein said asialoglycoprotein is E1.
- 3. The asialoglycoprotein of claim 1, wherein said asialoglycoprotein is E2.
- 4. The asialoglycoprotein of claim 2, wherein said E1 asialoglycoprotein is recombinant E1.
- 5. The asialoglycoprotein of claim 2, wherein said E1 asialoglycoprotein is recombinant E2.
- 6. A method for producing hepatitis C virus (HCV) asialoglycoproteins suitable for use in a vaccine or immunoassay, which method comprises:
growing a lower eukaryote transformed with a structural gene encoding an HCV asialoglycoprotein selected from the group consisting of E1 and E2 in a suitable culture medium; causing expression of said structural gene; and recovering said HCV asialoglycoprotein from said cell culture.
- 7. The method of claim 6, wherein said lower eukaryote is yeast.
- 8. The method of claim 7, wherein said yeast is Saccharomyces.
- 9. The method of claim 7, wherein said yeast is phenotypically pmrl.
- 10. The method of claim 6, wherein said HCV asialoglycoprotein structural gene further comprises a polynucleotide encoding a secretion leader functional in said lower eukaryote.
- 11. The method of claim 10, wherein said secretion leader comprises the α-factor secretion leader.
- 12. A method for producing hepatitis C virus (HCV) asialoglycoproteins suitable for use in a vaccine or immunoassay, which method comprises:
growing a mammalian host cell transformed with a structural gene encoding an HCV asialoglycoprotein selected from the group consisting of E1 and E2 in a suitable culture medium; causing expression of said structural gene under conditions inhibiting sialylation; and recovering said HCV asialoglycoprotein from said cell culture.
- 13. The method of claim 12, wherein said condition inhibiting sialylation comprises expression of E1 or E2 at a rate sufficient to inhibit transport of glycoproteins from the endoplasmic reticulum to the golgi.
- 14. The method of claim 12, wherein said conditions inhibiting sialylation comprise:
presence of a sufficient amount of a calcium modulator to cause release of proteins within the host cell's endoplasmic reticulum.
- 15. The method of claim 14, wherein said calcium modulator is thapsigargin.
- 16. A method for purifying hepatitis C virus (HCV) asialoglycoproteins, which method comprises:
contacting a composition containing HCV asialoglycoproteins with a mannose-binding protein; and isolating the portion of the composition which binds to said mannose-binding protein.
- 17. The method of claim 16, wherein said mannose-binding protein is a lectin selected from the group consisting of ConA and GNA.
- 18. The method of claim 16, wherein said mannose-binding protein is immobilized on a support.
- 19. The method of claim 18, wherein:
said contacting comprises incubation of said composition containing HCV asialoglycoproteins in a column comprising a mannose-binding lectin immobilized on a support, for a period of at least one hour; and said isolating comprises eluting said HCV asialoglycoproteins with mannose.
- 20. An assay kit for detecting the presence of hepatitis C virus (HCV) asialoglycoproteins, said kit comprising:
solid support; a mannose-binding protein; and an antibody specific for said HCV asialoglycoprotein; wherein one of said antibody and said mannose-binding protein is bound to said solid support.
- 21. The assay kit of claim 20, wherein said mannose-binding protein is GNA.
- 22. The assay kit of claim 20, wherein said antibody is bound to said support and said mannose-binding protein is bound to a detectable label.
- 23. The assay kit of claim 20, wherein said mannose-binding protein is bound to said support and said antibody is bound to a detectable label.
- 24. In a method for determining exposure to or infection by hepatitis C virus (HCV), the method wherein any HCV within a sample of body fluid is concentrated by contact with a mannose-binding protein prior to assay.
- 25. The method of claim 24, wherein said mannose-binding protein is GNA.
- 26. A cell transformed with a vector for recombinant expression of a hepatitis C virus (HCV) asialoglycoprotein, wherein said vector comprises a structural gene encoding a glycosylation signal, an HCV asialoglycoprotein, a regulatory sequence operable in said host cell and capable of regulating expression of said HCV asialoglycoprotein, and a selectable marker; wherein said cell does not sialylate glycoproteins.
- 27. The cell of claim 26, wherein said cell is a glycosylation-defective yeast strain.
- 28. The cell of claim 26, wherein said vector comprises a vaccinia virus vector.
- 29. A method for reducing or eliminating the presence of hepatitis C virus (HCV) in plasma, serum, or other biological liquids which method comprises:
contacting said biological liquid with a mannose-binding protein specific for mannose-terminated glycoproteins; and separating said biological liquid from said mannose-binding protein.
- 30. The method of claim 29 wherein said mannose-binding protein is GNA.
- 31. A method of inducing an immune response in an animal, which method comprises:
providing a vaccine composition comprising an effective amount of a hepatitis C virus (HCV) asialoglycoprotein in a pharmaceutically acceptable vehicle; administering said vaccine composition to said animal.
- 32. The method of claim 31, wherein said HCV asialoglycoprotein is E1.
- 33. The method of claim 31, wherein said HCV asialoglycoprotein is E2
- 34. The method of claim 31, wherein said HCV asialoglycoprotein is a purified E1/E2 aggregate.
- 35. The method of claim 31, wherein said animal is a primate.
- 36. A hepatitis C virus (HCV) asialoglycoprotein composition, comprising:
purified HCV E1 /E2 asialoglycoprotein aggregate.
- 37. The composition of claim 36, wherein said HCV E1/E2 asialoglycoprotein aggregate is at least 40% pure.
- 38. The composition of claim 37, wherein said HCV E1/E2 asialoglycoprotein aggregate is at least 50% pure.
- 39. The composition of claim 38, wherein said HCV E1/E2 asialoglycoprotein aggregate is at least 60% pure.
- 40. The composition of claim 36, wherein said HCV E1 1/E2 asialoglycoprotein aggregate is substantially free of other HCV proteins.
- 41. The composition of claim 36, wherein said aggregate has a molecular weight of about 107 kD.
- 42. The composition of claim 36, wherein said aggregate has a molecular weight of about 800 kD.
- 43. The composition of claim 36, wherein said aggregate forms a particle having a diameter of about 40 nm.
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of copending U.S. Ser. No. 07/758,880, filed Sep. 13, 1991, which is a continuation-in-part of U.S. Ser. No. 07/611,419, filed Nov. 8, 1990, now abandoned, the discosures of which are incorporated herein by reference.
Continuations (1)
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Number |
Date |
Country |
Parent |
08249843 |
May 1994 |
US |
Child |
09929782 |
Aug 2001 |
US |
Continuation in Parts (2)
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Number |
Date |
Country |
Parent |
07758880 |
Sep 1991 |
US |
Child |
08249843 |
May 1994 |
US |
Parent |
07611419 |
Nov 1990 |
US |
Child |
07758880 |
Sep 1991 |
US |