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modified hepatitis C virus protein

A kind of technology of hepatitis C virus and composition, applied in the direction of virus, virus peptide, immunoglobulin, etc.

Active Publication Date: 2016-09-28
THE MACFARLANE BURNET INST FOR MEDICAL RES & PUBLIC HEALTH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently available treatments are limited to administration of ribavirin and pegylated interferon, which exhibit limited efficacy of 40-80% and cause severe side effects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0283] Example 1: Cysteine ​​disulfide bond substitution mutations alone in the context of full-length E2 glycoprotein co-expression with E1

[0284] As proposed by Krey et al., 2010 (supra), the disulfide bond assignments for the domain structure of E2 are shown in Figure 1 and Table 2. In the context of E1E2 derived from the genotype 1a isolate H77c in HCVpp, the effect of replacing individual cysteines with alanines on E2 folding and function was assessed. Substitution of Cys to Ala abolished the ability of HCVpp to infect Huh7 hepatoma cells, suggesting that the nine disulfide bonds of E2 are critical for cell entry ability (Fig. 2A). Western blot analysis of transfected cell lysates with MAb H52 (for E2) and MAb A4 (for E1 ) showed that E1 and E2 were expressed at wild-type levels (Fig. 2B). However, the use of the conformation-dependent E2-specific MAb, H53, in immunoprecipitation of biosynthetically-tagged HCVpp indicated that the mutation had caused defects in the gly...

Embodiment 2

[0288] Example 2: Simultaneous mutagenesis of cysteine ​​pairs involved in disulfide bond formation

[0289] Cys-to-Ala scanning of the HIV envelope glycoprotein gp120 / gp41 complex reveals that cysteine ​​mutations alone do not favor functional protein folding, while simultaneous Ala substitutions are in 2 out of 10 disulfide bonds Both fold and function are rescued (van Anken et al., Mol Biol Cell 19:4298-309, 2008). To reduce the tendency of proteins to mispair due to the presence of unpaired cysteines, Ala-substitutions of each disulfide bond pair were performed simultaneously. For the double Cys-to-Ala mutant, intracellular expression and polyprotein processing of E1 and E2 were confirmed by Western blot ( Figure 3A ), however, lacked HCVpp entry activity (data not shown). Incorporation of H53-reactive E2 into HCVpp was observed for C452A / C459A (domain II), C581A / C585A and C652A / C677A (domain III), which did not form heterodimers with E1 ( Figure 3A ). In these 3 cas...

Embodiment 3

[0290] Embodiment 3: in the truncated E2 glycoprotein (E2 661 Single cysteine ​​and pairwise disulfide bond substitution mutations in -his)

[0291] Next, in the receptor binding domain of E2 (residues 384-661, E2 661 -His) to assess the effect of the Cys-to-Ala mutation, E2 folds independently of E1, retains the 3-domain structure described by Krey et al., 2010 (supra), and retains CD81 and SRB1 binding function. All mutants were secreted from transfected 293T cells at wild-type levels, as shown by immunoprecipitation of metabolically tagged proteins with anti-His antibody via the C-terminal 6-His tag ( Figure 4A , upper tile). E2 661 All but one of the MAb H53-reactivity profiles of the -His mutants largely mirrored those observed for the corresponding E1E2 mutants ( Figure 4A , 2nd and 3rd tiles, see Fig. 2B). Thus, Cys residues essential for H53 reactivity include C494, C508 (DII), C552, C564 (DI), C607 and C644 (DIII), while C452, C459, C486, C503 (DII), C569, C581...

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Abstract

The present invention provides compositions comprising hepatitis C virus (HCV) envelope 2 (E2) polypeptides, including receptor-binding variants, and various uses thereof, wherein the polypeptides are modified to comprise: (i) A cysteine ​​mutated or interrupted at 2, 3 or 4 cysteines from C452, C486, C569, and C597; and, wherein, relative to an HCV E2 polypeptide without cysteine ​​modification, the polypeptide passes Intermolecular disulfide bonds form substantially few multimers and CD81 binding is substantially retained. The present invention provides methods for preparing a composition comprising at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70% monomeric HCV E2 polypeptide, so The method comprises expressing a polypeptide in a host cell and isolating the expressed product, wherein the polypeptide is an HCV E2 polypeptide including receptor binding variants, and wherein the polypeptide is modified to comprise: (i) a protein selected from the group consisting of C452 Cysteines mutated or interrupted at 2, 3 or 4 cysteines of C486, C569 and C597.

Description

technical field [0001] The present invention relates to modified hepatitis C virus (HCV) E2 proteins and methods of making and using the same. Background technique [0002] The specific published literature cited by the author in this application document is collected at the end of this specification. [0003] Reference to prior art in this specification should not be considered as an acknowledgment or in any way to suggest that said prior art forms part of the common general knowledge in any country. [0004] Hepatitis C virus (HCV) is a major public health problem, with more than 123 million estimated chronic infections worldwide. There is no vaccine or post-exposure prophylaxis available. Currently available treatments are limited to the administration of ribavirin and pegylated interferon, which exhibit limited efficacy of 40-80% and cause severe side effects. HCV is the only member of the genus Hepacivirus of the family Flaviviridae, and is divided into 6 major genot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/18A61K39/29
CPCC07K16/109C07K2317/76C12N7/00C12N2770/24222C12N2770/24261C12N2770/24262C07K14/005A61K39/00A61K39/12C07K14/1833G01N33/56983
Inventor 海蒂·卓默尔潘提利斯·庞博瑞斯凯萨琳·麦卡弗里
Owner THE MACFARLANE BURNET INST FOR MEDICAL RES & PUBLIC HEALTH LTD
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