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Method for isolating hepatitis c virus peptides

A hepatitis C virus, molecular technology, applied in the field of HCV T cell epitopes, can solve the problem of not knowing the immune response in detail

Inactive Publication Date: 2009-07-01
INTERCELL AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no detailed understanding of which epitopes in the MHC combination lead to a successful immune response (Ward 2002)

Method used

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  • Method for isolating hepatitis c virus peptides
  • Method for isolating hepatitis c virus peptides
  • Method for isolating hepatitis c virus peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0127] Example I. Rapid identification of promiscuous HLA-binding peptides from HCV by assaying peptide libraries arranged in a matrix

[0128] To cover conserved regions within the HCV polyprotein, more than 640 peptides were synthesized (Table 1). For the rapid identification of HLA ligands and novel T cell epitopes, 40 peptide libraries were prepared, each containing 20 unique peptides. These libraries were constructed with each peptide present in 2 libraries (matrix format). This allowed the identification of reactive peptides located at the intersection of row and column mixtures (Figure 1 HCV peptide matrix).

[0129] Table 1. Synthetic peptides derived from HCV conserved regions

[0130]

[0131]

[0132]

[0133] For epitope capture, each peptide library was incubated with soluble recombinant HLA class II molecules and specific binding was assessed by SDS stability assay. Using the HLA molecule DRB1 ＊ 0401, DRB1 ＊ 0404 and DRB1 ＊ The results for 0101 are...

Embodiment II

[0151] To determine the best epitope within a longer polypeptide, mice can be vaccinated with the longer polypeptide incorporating the candidate epitope sequence. Generation of specific CD4+ T cell responses against naturally processed and presented epitopes can then be assayed by restimulation of murine splenocytes or lymph node cells with overlapping 15-mers and IFN-γ ELIspot. Final confirmation of newly identified HLA-ligands can be achieved by testing these peptides with T cells from humans. Ideally, include responders to treatment or those who recover spontaneously from infection. Example II. Immunogenicity of HCV-derived peptides in HLA transgenic mice

[0152] Immunogenicity of HCV-derived synthetic peptides (from conserved regions) was studied in HLA transgenic mice: 36 out of 68 peptides examined were found to induce peptide-specific IFN-γ producing cells in vaccination experiments. As summarized in Table 3, some peptides in DR4- and / or A ＊ 0201 transgenic mice wer...

Embodiment III

[0161] Example III. HLA restriction of HCV-derived immunogenic peptides studied in transgenic mice

[0162] Several groups of 5 mice per group (HLA-A ＊ 0201-, HLA-DRB1 ＊ 0401- and HLA-B ＊ 0702 transgenic mice, male, 8-14 weeks old) were subcutaneously injected with 100 μg of peptide+IC31 per mouse (50 μg per footpad) into the hind footpad. (PCT / EP01 / 12041, WO 02 / 32451A1 and PCT / EP01 / 06433, WO 01 / 93905A1; IC31 is a combination of immunological agents disclosed in WO 01 / 93905 and WO02 / 32451).

[0163] Six days after inoculation, single-cell suspensions of pooled spleens were prepared, and pure fractions, CD8+ fractions for A2 and B7tg mice (for B7 CD8+ fraction for mice containing 97% CD8 and 1.5% CD4 cells, 83% CD8 and 8% CD4 cells for A2tg mice), CD4+ fraction for DR4tg mice (for DR4tg small Mouse is the CD4+ fraction, containing 98% CD4 cells and 0.2% CD8 cells). All cells (not isolated cells, positive and corresponding negative fractions) were restimulated ex vivo with ...

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PUM

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Abstract

A method is described for isolating a hepatitis C virus peptide (HP) having the ability to bind to an MHC / HLA molecule, or a complex comprising the HCV peptide and the MHC / HLA molecule, which method is characterized by the following Steps: - providing a library of HCV peptides, the library containing HCV peptides that bind the MHC / HLA molecule and HCV peptides that do not bind the MHC / HLA molecule, - contacting the MHC / HLA molecule with the library of HCV peptides, whereby HCV peptides having the ability to bind to the MHC / HLA molecules are combined with the MHC / HLA molecules to form a complex containing the HCV peptides and the MHC / HLA molecules, detecting the complex, optionally making it unbound HCV peptide isolation of the MHC / HLA molecule, optionally isolating and characterizing the HCV peptide from the complex.

Description

field of invention [0001] The present invention relates to a method for isolating HCV-peptides, in particular HCV T-cell epitopes having the ability to bind MHC / HLA molecules. Background of the invention [0002] The immune system is a complex network of related cell types and molecules that have evolved to protect multicellular organisms against infectious microorganisms. It can be divided into innate (or natural) immunity and adaptive (or acquired) immunity that evolved earlier. Innate immune system recognition is a commonly shared and fundamental paradigm for pathogens. For a limited number of molecular structures, germline-encoded receptors have evolved. In contrast, the cells of the adaptive immune system - B and T lymphocytes - are able to recognize a wide variety of antigenic structures. These receptors, named B-cell receptors (BCR, whose soluble form is called an antibody) and T-cell receptors (TCR, just the cell-surface-bound form) according to the cell type that...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/576G01N33/50G01N33/569C07K7/04C07K14/18A61K39/29A61K38/04A61K38/16
Inventor M·比施勒A·哈贝尔C·克雷德F·麦特内尔O·奥塔瓦O·维特维斯卡W·扎内尔S·津克H·克拉普斯
Owner INTERCELL AG
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