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Antibodies to hepatitis C virus asialoglycoproteins

a technology of asialoglycoprotein and hepatitis c virus, which is applied in the field of recombinant protein expression and virology, can solve the problems of not being suitable for the production of glycoproteins for use as antigens, and achieve the effect of facilitating secretion or releas

Inactive Publication Date: 2005-04-28
CHIRON CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prokaryotes such as E. coli do not glycosylate proteins, and are generally not suitable for production of glycoproteins for use as antigens because glycosylation is often important for full antigenicity, solubility, and stability of the protein.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning and Expression

[0055] (A) Vectors were constructed from plasmids containing the 5′ portion of the HCV genome, as described in EP 318,216 and EP 388,232. Cassette HCV(S / B) contains a StuI-BglII DNA fragment encoding the 5′ end of the polyprotein from Met1 up to Leu906, beginning at nucleotide −63 relative to Met1. This includes the core protein (C), the E1 protein (also sometimes referred to as S), the E2 protein (also referred to as NS1), and a 5′ portion of the NS2a region. Upon expression of the construct, the individual C, E1 and E2 proteins are produced by proteolytic processing.

[0056] Cassette HCV(A / B) contains a ApaLI-BglII DNA fragment encoding the 5′ end of the polyprotein from Met1 up to Leu906, beginning at nucleotide-6 relative to Met1. This includes the core protein (C), the E1 protein (also sometimes referred to as S), the E2 protein (also referred to as NS1), and a 5′ portion of the NS2a region. Upon expression of the construct, the individual C, E1 and E2 pro...

example 2

Comparison of in vitro and in vivo Products

[0065] (A) E1 and E2 were expressed both in vitro and in vivo and 35S-Met labeled using the vectors described in Example 1 above. BSC-40 and HeLa cells were infected with the rVV vectors for in vivo expression. Both the medium and the cell lysates were examined for recombinant proteins. The products were immunoprecipitated using human HCV immune serum, while in vitro proteins were analyzed directly. The resulting proteins were analyzed by SDS-PAGE.

[0066] The reticulocyte expression system (pGEM3Z with HCV(S / B) or HCV(A / B)) produced C, E1 and E2 proteins having molecular weights of approximately 18 kD, 35 kD, and 72 kD, respectively. Lysates from BSC-40 and HeLa cells transfected with rVV containing HCV(S / B), HCV(A / B) or C-E1(S / B) exhibited the same proteins. Because the reticulocyte system does not provide efficient golgi processing and therefore does not provide sialic acid, the fact that both in vitro and in vivo products exhibited iden...

example 3

Purification using Lectin

[0070] (A) HeLa S3 cells were inoculated with purified high-titer vv / SC59-HCV virus stock at a multiplicity of infection of 5 pfu / cell, and the mixture stirred at 37° C. for 30 minutes. The infected cells were then transferred to a spinner flask containing 8 liters spinner medium and incubated for 3 days at 37° C. The cells were collected again by centrifugation and resuspended in hypotonic buffer (20 mM HEPES, 10 mM NaCl, 1 mM MgCl2, 120 ml) on ice. The cells were then homogenized by Dounce Homogenizer (50 strokes), and the nuclei pelleted by centrifugation (5 minutes, 1600 rpm, 4° C., JA-20 rotor). The pellets were pooled, resuspended in 48 ml hypotonic buffer, rehomogenized, recentrifuged, pooled again, and frozen at −80° C.

[0071] The frozen supernatants were then thawed, and the microsomal membrane fraction of the post-nuclear Iysate isolated by centrifuging for 20 minutes in a JA-20 rotor at 13,500 rpm at 4° C. The supernatant was removed by aspiratio...

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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.

Description

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.DESCRIPTION [0002] 1. Technical Field [0003] This invention relates to the general fields of recombinant protein expression and virology. More particularly, the invention relates to glycoproteins useful for diagnosis, treatment, and prophylaxis of Hepatitis C virus (HCV) infection, and methods for producing such glycoproteins. [0004] 2. Background of the Invention [0005] Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family of diseases) that is believed to be virally induced, and is distinguishable from other forms of virus-associated liver disease, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) or Epstein-Ba...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/16A61K38/00A61K38/10A61K39/00A61K39/29G01N33/576A61P1/16A61P31/12C07K14/18C12N1/19C12N5/10C12N15/09C12N15/51C12P21/02C12P21/04
CPCA61K39/00C07K14/005C12N2770/24222Y10S977/915Y10S977/802Y10S977/918Y10S530/82Y10S977/804Y10S977/803Y10S530/826A61P1/16A61P31/12
Inventor RALSTON, ROBERT O.MARCUS, FRANKTHUDIUM, KENT B.GERVASE, BARBARA A.HALL, JOHN A.BERGER, KIM M.CHOO, QUI-LIMHOUGHTON, MICHAELKUO, GEORGE
Owner CHIRON CORP
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