Hepatitis C virus asialoglycoproteins
AssigneeChiron Corporation
InventorsRobert O. Ralston, Frank Marcus, Kent B. Thudium, Barbara A. Gervase, John A. Hall, Kim M. Berger, Qui-Lim Choo, Michael Houghton, George Kuo
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Abstract
Two Hepatitis C Virus envelope proteins (E1 and E2) are expressed without sialylation. Recombinant expression of these proteins in lower eukaryotes, or in mammalian cells in which terminal glycosylation is blocked, results in recombinant proteins which are more similar to native HCV glycoproteins. When isolated by GNA lectin affinity, the E1 and E2 proteins aggregate into virus-like particles.
Claims25
A hepatitis C virus (HCV) glycoprotein composition comprising a pharmaceutically acceptable excipient and a purified HCV E1/E2 glycoprotein aggregate, wherein said glycoproteins of said aggregate have mannose-terminated glycosylation, wherein less than about 10% of the total N-linked carbohydrate on said glycoproteins is sialic acid, and further wherein said aggregate comprises a glycoprotein expressed from the E1 region of HCV and a glycoprotein expressed from the E2 region of HCV, said aggregate produced by the method comprising the steps of:
- contacting a composition containing HCV glycoproteins with a mannose-binding protein specific for mannose-terminated glycoproteins;
- and
- isolating the portion of the composition which binds to said mannose-binding protein.
A composition comprising a pharmaceutically acceptable excipient and a purified hepatitis C virus (HCV) glycoprotein having mannose-terminated glycosylation, wherein less than about 10% of the total N-linked carbohydrate on said HCV glycoprotein is sialie acid, wherein said HCV glycoprotein is selected from the group consisting of a glycoprotein expressed from the E1 region of HCV and a glycoprotein expressed from the E2 region of HCV, produced by the method comprising the steps of:
- growing a mammalian host cell transformed with a structural gene encoding said HCV glycoprotein in a suitable culture medium, wherein said structural gene is linked to a sequence encoding a secretion leader that directs the glycoprotein to the endoplasmic reticulum;
- causing expression of said structural gene and secretion leader sequence under conditions inhibiting sialylation, wherein said conditions inhibiting sialylation comprise inhibiting transport of glycoproteins from the endoplasmic reticulum to the golgi;
- and
- isolating said HCV glycoprotein from said cell culture by contacting said HCV glycoprotein with a mannose-binding protein specific for mannose-terminated glycoproteins, and isolating the protein which binds to said mannose-binding protein.
An isolated hepatitis C virus (HCV) glycoprotein having mannose-terminated glycosylation, wherein less than about 10% of the total N-linked carbohydrate on said HCV glycoprotein is sialic acid, wherein said HCV glycoprotein is selected from the group consisting of a glycoprotein expressed from the E1 region of HCV, a glycoprotein expressed fiom the E2 region of HCV, and an E1/E2 aggregate glycoprotein, wherein said glycoprotein is produced by the method comprising the steps of:
- growing a mammalian host cell transformed with a structural gene encoding said glycoprotein in a suitable culture medium, wherein said structural gene is linked to a sequence encoding a secretion leader that directs the glycoprotein to the endoplasmic reticulum;
- causing expression of said structural gene and secretion leader sequence under conditions inhibiting sialylation, wherein said conditions inhibiting sialylation comprise inhibiting transport of glycoproteins from the endoplasmic reticulum to the golgi;
- and
- isolating said HCV glycoprotein from said cell culture by contacting said HCV glycoprotein with a mannose-binding protein specific for mannose-terminated glycoproteins.
An isolated hepatitis C virus (HCV) E1/E2 glycoprotein aggregate having mannose-terminated glycosylation, wherein less than about 10% of the total N-linked carbohydrate on said HCV glycoprotein is sialic acid, and further wherein said HCV glycoprotein aggregate comprises a glycoprotein expressed from the E1 region of HCV and a glycoprotein expressed from the E2 region of HCV, wherein said glycoprotein aggregate is produced by the method comprising the steps of:
- growing a host cell transformed with a structural gene encoding said glycoprotein in a suitable culture medium;
- causing expression of said structural gene under conditions inhibiting sialylation;
- and
- isolating said HCV glycoprotein aggregate from said cell culture by contacting said HCV glycoprotein with a mannose-binding protein specific for mannose-terminated glycoproteins.
Dependencies are inferred from the claim text.