CN100507566C

CN100507566C - Method for isolating hepatitis c virus peptides

Info

Publication number: CN100507566C
Application number: CNB03809696XA
Authority: CN
Inventors: 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
Original assignee: Intercell Austria AG
Current assignee: Valneva Austria GmbH
Priority date: 2002-09-13
Filing date: 2003-08-27
Publication date: 2009-07-01
Anticipated expiration: 2023-08-27
Also published as: CN101525375A; CN1695060A

Abstract

Described is a method for isolating Hepatitis C Virus peptides (HPs) which have a binding capacity to a MHC/HLA molecule or a complex comprising said HCV-peptide and said MHC/HLA molecule characterized by the following steps: - providing a pool of HCV-peptide, said pool containing HCV-peptides which bind to said MHC/HLA molecule and HCV-peptides which do not bind to said MHC/HLA molecule, -contacting said MHC/HLA molecule with said pool of HCV-peptides whereby a HCV-peptide which has a binding capacity to said MHC/HLA molecule binds to said MHC/HLA molecule and a complex comprising said HCV-peptide and said MHC/HLA molecule is formed, -detecting and optionally separating said complex from the HCV-peptide which do not bind to said MHC/HLA molecule and optionally isolating and characterising the HCV-peptide from said complex.

Description

The method of isolating hepatitis c virus peptides

Invention field

The present invention relates to a kind of method of the HCV-of separation peptide, particularly separate the method have in conjunction with the HCV t cell epitope of the ability of MHC/HLA molecule.

Background of invention

Immune system is a complex network between relevant cell type and the molecule, and they are evolved in order to protect multicellular organism opposing infectious microorganism.Can be divided into congenital (or nature) immunity and adaptability (or acquired) immunity of evolving early.Innate immune system identification be to pathogen total usually with basic pattern.For a limited number of molecular structures, plant the acceptor of system's coding and evolve.On the contrary, the cell-B of adaptive immune system and T lymphocyte-can discern multiple antigenic structure.These acceptors according to the cellular type of expressing them, are named as B-cell receptor (BCR, its soluble form is called antibody) and TXi Baoshouti (TCR, just cell surface combining form), produce by the body cell reorganization, show that the clone distributes.Therefore, have only small amounts of cells to have certain species specificity at first.After antigen runs into these cells, begin division (clonal expansion), generation can resist the effect colony of this antigen.After antigen was removed, the specialized cell subgroup of this antigen of specific recognition still had immunological memory.In a word, adaptive immune system is (to compare with congenital immunity) slowly, yet is special, strengthens behind a kind of appointment pathogen/antigen in repeated exposure.

The T cell has central role in adaptive immunity.Their acceptor (TCR) recognizing cells lip-deep " main histocompatibility complex " (MHC or HLA): peptide complexes.These peptides are called as t cell epitope, represent the catabolite of antigen.The T cell has two primary categories: CD8 positive cell toxicity T cell (CTL) is limited to MHC I class.The positive helper cell of CD4 (HTL) is limited to MHC II class.HTL is that the various features of adaptive immunity is necessary: so-called " professional antigen presenting cell " activation (APC), the conversion of immunoglobulin (Ig) (Ig) classification, centrum germinativum's reaction and Ig affinity maturation, the activation of CTL, immunological memory, the adjusting of immune response etc.

Peptide in the MHC molecule collecting cell is the TCR that passs the T cell at cell surface with them.MHC has two primary categories: by the I class of the positive CTL identification of CD8 with by the II class of the positive HTL identification of CD4.

MHC I quasi-molecule is made up of the α chain of the 45kDa of film grappling and the B2M (b2m) of the non-covalent 12kDa that is connected.Resolve three-dimensional structure (Stern and Wiley1994) by the X ray crystallisation and show that the α chain has a breach, it adapts to the peptide of 8-11 amino acid length in closed at both ends.The I quasi-molecule is generally expressed, and the peptide that they are presented derives from cytoplasmic protein matter.They are degraded by proteasome, and the peptide of generation is arrived in the endoplasmic reticulum (ER) by active transport.Assisting down of several chaperones, form MHC at this: peptide complexes, transporte to cells surface (Heemels 1995).Therefore, MHC I class reflects the protein group of cell in its surface, makes that the T cell can interior pathogen of recognizing cells or malignant cell.

MHC II quasi-molecule is made up of the two kinds of film anchorin matter (α chain and β chain) that are respectively 35kDa and 30kDa.They have constituted a breach together, at both ends open, can adapt to the peptide of variable-length, usually 12-25 amino acid.Although these differences are arranged, I class and II quasi-molecule have surprising structural similarity (Stern and Wiley 1994).The II quasi-molecule is only expressed on professional APC, comprises dendritic cells (DC), B cell and monocytes/macrophages.These cells are specially with picked-up of endosome approach and process antigen.After biosynthesizing, the II quasi-molecule is compound by so-called constant chain (Ii) immediately, stops the combination of peptide in the ER.When containing the II class: when the endosome of the vesicle of Ii compound (vesicles) and the catabolite that contains exogenous antigen merged, Ii is degraded, and was only compound by so-called CLIP peptide up to the breach of MHC combination.The latter exchanges (Villadangos 2000) with antigenic peptides under the help of chaperone such as HLA-DM.At last, MHC: peptide complexes is presented on the APC surface once more, and they interact with HTL in many ways.

The MHC system is polygenes and polymorphism extremely, is high complexity.Human I class α chain has three locus, be called as HLA-A ,-B and-C.Equally, three II class α chain gene seats (DRA, DQA, DPA) are arranged.As for II class β chain gene seat, situation is complicated more, because there are 4 different DR β chains (DRB1,2,3,5) to add DQB and DPB.Except the DR α chain DRA of singlet, each locus is present in a plurality of different allele in the colony (tens to hundreds of) (Klein 1986).Different allele has big different peptide binding specificity.For example, allele is named as HLA-A ^*0201 or HLA-DRB1 ^*0401 or HLA-DPA ^*0101/DPB ^*0401.

T cell epitope is identified (Vanden Eynde 1997) with several different methods.For example, T clone and clone have been used for examination cDNA expression library in the COS cell of suitably HLA molecule transfection.In addition, also utilize biochemical method.The latter comprises wash-out native ligand from the lip-deep MHC molecule of target cell, separates these peptides by several chromatographic step, utilize epi-position rebuild determination and analysis they with lymphocytic reactivity, by mass spectroscopy check order (

Deng people 1994, people such as Cox 1994).

Recently, the appearance of test of super-sensitive cytokines measurement as IFN-γ ELIspot allows to utilize the lymphocyte of direct ex vivo to screen overlapping synthetic peptide (Maecker 2001, Kern2000, Tobery 2001).Originally, people (1999 and 2000) such as Kern uses the array in overlapping 9mer peptide storehouse external the CD8+T cell epitope to be mapped.Afterwards, people such as Tobery, 2001 have improved this method, and confirm to contain nearly the storehouse of 64 kinds of 20mer peptides can be used for screening CD8+ and CD4+T cell epitope in mouse.These two kinds of methods all are based on by cell inner dyeing people 2000 such as () Kern or ELIspot and measure the generation that people such as (, 2001) Tobery measures INF-γ, and the monitoring antigentic specificity is replied.Utilize the potpourri of 15-mers, the CD4+T cell response approximates detected replying when using complete soluble protein as antigen greatly, and the CD8+T cell response is significantly higher than and utilizes soluble protein to stimulate detected negligible replying usually, and this is not astonishing.And, the CD8+T cell response of the potpourri of fifteen amino acid peptide is similar to replying that the potpourri that utilizes the 8-12 amino acid peptide of selecting the minimum epi-position of the known MHC I class of representative obtains.Most probably, in these cases, be responsible for peptide " pruning " is become optimum length (people such as Maecker, 2001) with the membrane-bound peptase of cell.

An interesting alternative approach is with the synthetic combined peptide library of special lymphocyte examination.For example, the decapeptide library that 200 kinds of potpourris arranging in the location scanning mode are formed successfully be used to identify the clonotype colony that stimulates the T cell the peptide part (people such as Wilson, J.Immunol., 1999,163:6424-6434).

Many t cell epitopes have been used, and so-called " oppositely immunization " measures (Rammensee 1999).In this case, the protein that produces possible t cell epitope is known, and the HLA binding motif is scanned its primary sequence.General synthetic tens to hundreds of candidate's peptide, and even a complete set of overlapping peptide, detects they and the combining of HLA molecule.Usually select the best combination agent further to characterize the reactivity of they and T cell.For example, by the HLA transgenic mice,, can realize this purpose by in external or body, causing the T cell.

Hepatitis C virus (HCV) is a member of flaviviridae (flaviviridiae), worldwide about 1.7 hundred million people of chronic infection.At least 6 HCV genotype and the hypotype more than 50 have been described.In the U.S., Europe and Japanese,

genotype

1,2,3 is the most common.The genotypic geographic distribution of HCV alters a great deal with the genotype difference, and 1a mainly is distributed in the U.S. and West Europe part, and 1b preponderates in southern Europe and Central Europe (Bellentani 2000).

HCV propagates by parenteral or through skin (percutan) approach, duplicates in liver cell.About 15% patient experience is with virus sweep and recover relevant acute self limiting hepatitis.The infected of about 80% becomes chronic carrier.Infect usually and continue several years asymptomatically, slow progress, yet, final HCV be cirrhosis, latter stage hepatopathy and the main cause (Liang 2000) of liver cancer.Intensity that HTL and CTL reply and quality have determined the patient to recover (spontaneously or the result of treatment) still to develop into chronic infection (Liang 2000).

The standard care of HCV comprises the use of uniting of the interferon-' alpha ' of PEGization and antiviral Ribavirin.According to the genotype difference, there is about 50% HCV patient to produce virus and replys.The low tolerance of this treatment and sizable spinoff obviously need new treatment intervention, comprise therapeutic vaccine (Cornberg 2002).Yet, also do not have at present which kind of epi-position in the detail knowledge MHC combination to cause the immune response (Ward 2002) of success.Therefore, exploitation needs the t cell response of analysis-by-synthesis at complete HCV based on the therapeutic vaccine of epi-position.

The HCV virion contains the normal chain single stranded RNA genome of a 9.5-kilobase, its about 3000 amino acid whose big single polyprotein of encoding.The latter is processed as at least 10 kinds of protein (Liang 2000) by host and HCV encoded protein hydrolysing activity.Importantly, the RNA polymerase that HCV RNA-relies on has wrong tendency, causes viral quasispecies evolution, produces immunologic escape mutation (Farci 2000).

Summary of the invention

One object of the present invention is to provide a kind of method, be used for peptide at specificity MHC molecular screening HCV, preferably carry and be fit to and specific HCV t cell epitope, it is selected from has specifying the unknown specific multiple HCV peptide of MHC molecule, prevents and resist the effective ways that HCV infects thereby provide.

Therefore, the invention provides a kind of method, be used to separate the HCV peptide that has in conjunction with the ability of MHC/HLA molecule, or a kind of compound that contains this HCV peptide and this MHC/HLA molecule, this method comprises the following steps:

-HCV peptide that can combine that provides HCV peptide storehouse, this storehouse to contain and the HCV peptide that does not combine with this MHC/HLA molecule with this MHC/HLA molecule,

-make this MHC/HLA molecule contact this HCV peptide storehouse, thus the HCV peptide that has in conjunction with the ability of this MHC/HLA molecule is combined with this MHC/HLA molecule, form the compound that contains this HCV peptide and this MHC/HLA molecule,

-detect this compound, it is separated with the HCV peptide in conjunction with this MHC/HLA molecule not,

-randomly from this compound, separate and characterize this HCV peptide.

The present invention also provides a kind of method, is used to separate the t cell epitope that has in conjunction with the ability of MHC/HLA molecule, or a kind of compound that contains this epi-position and this MHC/HLA molecule, and this method comprises the following steps:

-HCV peptide that can combine that provides HCV peptide storehouse, this storehouse to contain and the HCV peptide that does not combine with this MHC/HLA molecule with the MHC/HLA molecule,

-randomly from this compound, separate and characterize this HCV peptide,

-with the T raji cell assay Raji measure described through randomly separating this HCV peptide or the t cell activation ability of this compound and

-provide the HCV peptide that randomly separates as t cell epitope or compound with t cell activation ability.

The method according to this invention makes it possible to screen the ability that combines with specificity MHC/HLA molecule with a kind of screening system.Identification of M HC binding molecule is a kind of important tool of the characterization of molecules of pathogen, tumour etc.Therefore, the present invention can screen multiple (" storehouse ") possible HCV peptide as part immediately according to the function affinity of they and MHC molecule.Also be intended to necessary condition with the binding affinity of MHC molecule, although be not adequate condition as the HCV peptide of t cell epitope.Also screen suitable HCV t cell epitope, and measure its t cell activation ability.Therefore, provide the method for separating the HCV t cell epitope according to the present invention in conjunction with screening technique with the combination of suitable T raji cell assay Raji, wherein utilized MHC, can from possible HCV peptide storehouse, identify these t cell epitopes in conjunction with mensuration according to of the present invention.

In the prior art, always carry out these mensuration to having the specific part of known combination/MHC, different with it, the method according to this invention provides these determination methods conducts to containing the screening instruments in unknown specific part storehouse.In the prior art, these determination methods are generally carried out each single part, to detect the binding affinity of they and MHC/HLA molecule.People such as Kwok (2001) utilize the storehouse that can reach 5 kinds of over lapping synt hetic peptideses at most to produce the MHC II class tetramer; Then with the latter's specific MHC II class among the PBMC that dyes: the T cell that peptide complexes is special, this compound are to produce in the association reaction in the storehouse of 5 kinds of peptides.Yet,, do not think that this method can increase the amount of ligand in each storehouse (people 2001 such as Novak) owing to susceptibility and specific reason.About a specific problem be, if there is more than one bond in the storehouse, then produce the MHC tetramer, each tetramer has more than one bond.This will get rid of, and it is used for the T cell dyeing that epi-position is identified in the described method of prior art.Very different with it, the method according to this invention allows to identify more than one bond from the potpourri of the high complexity that contains more than one bonds.

Utilize the character in the storehouse that the present invention screens unimportant: any natural or HCV peptide that non-natural exists can be contained in this storehouse, it a) specificity in conjunction with the MHC/HLA molecule, and/or b) can be discerned by the T cell-specific.This group HCV peptide in this storehouse and MHC molecule combine character the unknown; Therefore, contain a kind of bond of the MHC of appointment molecule and at least a non-binding dose in this storehouse usually.Therefore at least 10 kinds of different HCV peptides are contained in this storehouse.In fact, obviously more different HCV peptide kinds, for example 20 kinds or more, 100 kinds or more, 1000 kinds or more, perhaps 10000 kinds or more are contained in storehouse used according to the invention.Also can (for example contain 10 in the bigger library of examination ⁶More than, 10 ⁸More than, and even 10 ¹⁰Above different HCV peptide kinds).Yet this depends primarily on the availability of these HCV peptide libraries.

The preferred part storehouse that the method according to this invention is used is selected from: the peptide storehouse, the storehouse of overlapping peptide particularly, the protein fragments storehouse, the modified peptides storehouse, storehouse by the antigen presenting cell acquisition, the form of its total lysate or fraction preferably, the fraction of wash-out on the surface of these cells or the MHC/HLA molecule particularly, comprise the cell fragment (cell that particularly contains HCV, tumour cell or tissue are particularly from liver) the storehouse, comprise the storehouse of peptide library, the storehouse of (many) peptides that produce by the recombinant DNA library, particularly derive from pathogen or tumour cell, from the storehouse of protein and/or the protein fragments of HCV, or its potpourri.

The HCV peptide in this storehouse may come from natural origin (natural and/or derive form), also may synthesize generation (for example by chemosynthesis or recombinant technique).If (many) peptides part is contained in this storehouse, preferably produce these peptides with peptide synthesizer or by recombinant technique.According to a preferred embodiment, (many) peptides storehouse can be produced by the recombinant DNA library, for example derives from HCV or contains (tumour) cell of HCV, expresses by external translation (for example ribosomal display) or by xenogenesis host (as Escherichia coli etc.).

The character of the concrete MHC molecule of selecting for this method (this term also comprises MHC sample molecule certainly) is also inessential.Therefore, these molecules can be selected from any kind in principle, and primate particularly is as people (HLA, see below), chimpanzee, other mammal, for example maquaques, rabbit, cat, dog or rodent, as mouse, rat, cavy etc., important animal on agricultural as ox, horse, sheep and fish, but provides vaccine certainly preferred people's (or " humanization ") molecule for the mankind.In order to be concrete animal, particularly agricultural goes up important animal, provides vaccine as ox, horse, sheep and fish, preferably uses the special MHC molecule of these animals.

Therefore preferred HLA molecule contains the I quasi-molecule, its derive from HLA-A ,-B or-C locus, particularly A1, A2, A3, A24, A11, A23, A29, A30, A68; B7, B8, B15, B16, B27, B35, B40, B44, B46, B51, B52, B53; Cw3, Cw4, Cw6, Cw7; The II quasi-molecule, derive from HLA-DP ,-DQ or-DR locus, particularly DR1, DR2, DR3, DR4, DR7, DR8, DR9, DR11, DR12, DR13, DR51, DR52, DR53; DP2, DP3, DP4; DQ1, DQ3, DQ5, DQ6; With unconventional MHC/HLA and MHC/HLA sample molecule, the member of their energy specific binding ligands, particularly HLA-E, HLA-G, MICA, MICB, Qa1, Qa2, T10, T18, T22, M3 and CD1 family.

According to a preferred embodiment, the method according to this invention is characterised in that this MHC/HLA molecule is selected from HLA I quasi-molecule, HLA II quasi-molecule, unconventional MHC/HLA and MHC/HLA sample molecule or its potpourri, perhaps their potpourri.

Preferably, make the optional sign step of the HCV peptide that carries out compound with the following method, method is selected from: mass spectroscopy, sequencing polypeptides, in conjunction with measuring, particularly SDS-stability is measured, and identifies part by the method for following mensuration retention factors (retention factor), and assay method comprises chromatography, HPLC particularly, or other spectral technique, particularly ultraviolet ray (UV), infrared ray (IR), nuclear magnetic resonance (NMR), circular dichroism (CD) or electron spin resonance (ESR), or its combination.

According to a preferred embodiment, method of the present invention is characterised in that it combines with a kind of cytokine secretion mensuration, preferably combine with Elispot mensuration (a kind of cell within a cell factor dyeing), FACS or ELISA (enzyme-linked immunoassay) (referring to, for example " modern immunological method ").

Preferred T raji cell assay Raji comprises described potpourri and the T mixing with cells and the incubation that separate, measure the propagation of the T cell of cytokine secretion or separation then, and/or the rise of mensuration activation mark (particularly CD69, CD38), or the downward modulation of surface indicia (particularly CD3, CD8 or TCR), and/or rise/downward modulation of the mensuration mRNA relevant with t cell activation, particularly by real-time RT-PCR measure (referring to, for example " modern immunological method ", " modern molecular biology method ").

Further preferred T raji cell assay Raji is selected from measures the TXi Baoshouti downstream composition (p56lck of TCR particularly, ITAMS and zeta chain, ZAP70, LAT, SLP-76, fyn and lyn) phosphorylation/dephosphorylized T raji cell assay Raji, measure the protein activated T cells mensuration that interior Ca++ concentration of cell or Ca++-rely on, measure the T raji cell assay Raji that immune cynapse forms, measure effector molecule (perforin particularly, granzyme or granulolysin) the T raji cell assay Raji that discharges, or the combination of these T raji cell assay Rajis (referring to, for example " modern immunological method ", " modern cell biology method ").

In order to identify the molecule determinant of the immunoprotection that resists HCV, use speciesspecific epitope's catching method, this method is used the synthetic peptides of representing

HCV genotype

1,2,3 conservative parts.Concentrate on conserved region and can guarantee the broad applicability of epi-position.And perhaps these districts can not easily be suddenlyd change by virus, thereby the danger that the immunologic escape mutation is evolved minimizes.

Utilize method of the present invention to detect new HCV epi-position.According to another aspect, the HCV t cell epitope that the present invention therefore also provides available the method according to this invention to identify, this t cell epitope preferably are selected from the peptide A120-A124, the B25-B30 that contain with good grounds table 1 or table 2, B46-B48, B84-B92, C106, C113-C114,1627,1628,1629,1604 polypeptide.These peptides are HLA-DRB1 at least ^*0101, ^*0401, ^*0404, ^*Therefore 0701 new part covers the 45-55% at least (seeing Table 2) of main colony.

Preferred polypeptide is selected from peptide 1630, C97,1547, B94-B98, the A272-A276 according to table 1 or table 2.These peptides are HLA-DRB1 at least ^*0101, ^*0401, ^*Therefore 0701 new part covers the 40-50% at least (seeing Table 2) of main colony.

Preferred polypeptide is selected from peptide B120, B122, C108, C134, the C152 according to table 1 or table 2.These peptides are HLA-DRB1 at least ^*0101, ^*0404, ^*0701 new part, therefore at least 45% (the seeing Table 2) that covers main colony.

Preferred polypeptide is selected from the

peptide

1606,1607,1577,1578 according to table 1 or table 2.These peptides are HLA-DRB1 at least ^*0401, ^*0404, ^*Therefore 0701 new part covers at least 45% (table 2) of main colony.

Preferred polypeptide is selected from peptide B50-52,1623, the C130 according to table 1 or table 2.These peptides are HLA-DRB1 at least ^*0101, ^*0401, ^*0404 new part, therefore at least 40% (the seeing Table 2) that covers main colony.

Preferred polypeptide is selected from peptide 1603, the C96 according to table 1 or table 2.These peptides are HLA-DRB1 at least ^*0101, ^*0701 new part, therefore at least 40% (the seeing Table 2) that covers main colony.

Preferred polypeptide is selected from the peptide C191 according to table 1 or table 2, is HLA-DRB1 at least ^*0401, ^*0701 new part, therefore at least 40% (the seeing Table 2) that covers main colony.

Preferred polypeptide is selected from peptide A216-A224, A242-A244, the C92-C93 according to table 1 or table 2.These peptides are HLA-DRB1 at least ^*0101, ^*0401 new part, therefore at least 35% (referring to table 2) that covers main colony.

Preferred polypeptide is selected from the peptide A174 according to table 1 or table 2, be at least HLA-DRB1*0404, ^*Therefore 0701 new part covers the 25-30% at least (referring to table 2) of main colony.

Preferred polypeptide is selected from peptide B32-B38, B100-B102, the C135 according to table 1 or table 2.These peptides are HLA-DRBl at least ^*0101, ^*Therefore 0404 new part covers the 20-25% at least (referring to table 2) of main colony.

Preferred polypeptide is selected from the peptide C162 according to table 1 or table 2, is HLA-DRB1 at least ^*0401, ^*Therefore 0404 new part covers the 20-25% at least (referring to table 2) of main colony.

Preferred polypeptide is selected from the

peptide

1618,1622,1624,1546,1556 according to table 1 or table 2.These peptides are HLA-DRB1 at least ^*0701 new part, therefore at least 25% (referring to table 2) that covers main colony.

Preferred polypeptide is selected from peptide A114, B58, B112-B118, B18-B22, C112, C116, C122, C127, C144, C159-C160, the C174,1558,1581 according to table 1 or table 2.These peptides are HLA-DRBl at least ^*0101 new part, therefore at least 20% (referring to table 2) that covers main colony.

Preferred polypeptide is selected from peptide C95, is a kind of new part of HLA-DRB1*0401 at least, therefore at least 20% (referring to table 2) that covers main colony.

Preferred polypeptide is selected from peptide C129, C157-C158, A254-A258,1605, C109, the C161 according to table 1 or table 2.These peptides contain HLA-DRB1 at least ^*0404 new part, therefore at least 5% (referring to table 2) that covers main colony.

Preferred polypeptide is selected from the

peptide

1547,1555,1558,1559,1560,1563,1592,1604,1605,1616,1621,1623,1625,1627,1630,1649,1650,1651,1652,1654,1655,1656 according to table 1 or table 2, and these peptides are at HLA-DRB1 ^*Therefore show immunogenicity (seeing example II) in 0401 transgenic mice, represent or contain the HLA II class t cell epitope that is confirmed, its at least with HLA-DRB1 ^*0401 in conjunction with (referring to table 3).

Preferred polypeptide is selected from the

peptide

1545,1552,1555,1558,1559,1560,1577,1592,1604,1605,1615,1617,1621,1627,1631,1632,1641,1647,1650,1651,1652,1653,1654,1655 according to table 1 or table 2, these peptides show immunogenicity (seeing example II) in the HLA-A*0201 transgenic mice, therefore represent or contain the HLA I class t cell epitope that is confirmed, its at least with HLA-A ^*0201 in conjunction with (seeing Table 3).

Demonstration is the HLA-B with t cell activation ability ^*The preferred polypeptide of 0702 epi-position is selected from

polypeptide

1506,1526,1547,1552,1553,1555,1558,1562,1563,1565,1577,1578,1580,1587,1592,1604,1605,1621,1623,1624,1627,1628,1647,1650,1651, contain sequence LPRRGPRL (1506 is contained) 1843 and contain sequence SPGALVVGVI (contained in 1587) 1838 as minimum HLA-B*0702 epi-position.

Peptide 1526,1565,1631 is at the HLA-DRB1 that contains known II class epi-position ^*Also show immunogenicity in 0401 transgenic mice.Peptide 1526,1553,1565,1587,1623,1630 is at the HLA-A that contains known A2 epi-position ^*Also show immunogenicity in 0201 transgenic mice.

Preferred polypeptide is selected from table 3,5 listed peptide and black matrix peptides in the table 7 (" focus ").

Above-mentioned preferred polypeptide also comprises all fragments that contain the epi-position minmal sequence, promptly combines required 8-or 9-mer with the MHC/HLA molecule.

Preferably, also contain 1-30 at N end, C end or at N end and C end two ends according to epi-position of the present invention or peptide, preferably 2-10,2-6 naturally occurring amino acid residue particularly.For the present invention, term " naturally occurring " amino acid residue relates to the amino acid residue that naturally occurring protein exists at the specific site with respect to epi-position or peptide.For example, number the HLA-A2 epi-position (table 1) of 1565 amino acid contained sequence HMWNFISGI for containing peptide, the naturally occurring amino acid residue that is positioned at the N end is K; Naturally occurring three amino acid residues that are positioned at the C end are QYL.Therefore, " non-natural exists " amino acid residue is and amino acid sequence different any amino acid residue with respect to the specific site place of this epi-position or peptide.

According to a preferred embodiment of the invention, this epi-position or peptide are particularly held at N, C holds or also contain the amino acid that non-natural exists at N end and C end two ends, preferably 1-1000, preferred 2-100 is individual, particularly 2-20 the amino acid residue that non-natural exists.In this concrete preferred embodiment, the combination of non-natural and naturally occurring amino acid residue also is possible.This epi-position also can contain the amino acid (promptly being different from 20 kinds of " classics " amino acid whose amino acid residues, as the S-S combination of D-amino acid or Cys) of modification, as other amino acid residue, or replaces naturally occurring amino acid residue.

Obviously, also comprise epi-position or the peptide that is produced by these epi-positions or peptide by the amino acid exchange according to epi-position of the present invention or peptide, they improve, keep or significantly do not hinder at least the t cell activation ability of this epi-position.Therefore, these epi-positions or peptide also comprise and do not contain the original series that derives from specific HCV strain, but trigger the epi-position or the peptide of t cell response identical or that preferably strengthen.These epi-positions are called as " irregular " (heteroclitic).They comprise can trigger the T cell identical with original epi-position, preferably have in the stronger body or any epi-position of external t cell activation ability.The present invention also comprises the homology epi-position separately from other HCV strain.

Irregular epi-position can be by reasonably design acquisition, consider that promptly each residue is to the effect in conjunction with MHC/HLA, as described in people 1999 such as people such as Ramensee 1999 or Sturniolo, in conjunction with may with the systems exchange of the interactional residue of TCR, and use the sequence that obtains at the T cell detection of original epi-position.For the those skilled in the art that do not have more experiences, this design is possible.

Another kind of possibility comprises the T cell screening peptide library of using at original epi-position.A kind of preferable methods is the location scanning of synthetic peptide library.For example, people 1999 such as people 1996 such as Blake and Hemmer and list of references have herein been described these methods in detail.

Alternative as the epi-position of the amino acid sequence representative in homology HCV source or irregular epi-position also can be used the material of these epi-positions of simulation, for example " plan peptide " or " retro-inverso-peptide ".

Being with the material preparation that can improve its ability that stimulates the T cell on the other hand or modifying of the epi-position of design improvement.Comprise t helper cell epi-position, lipid or liposome or preferred the modification, as described in WO01/78767.

The another kind of method that improves the T cytositimulation ability of epi-position is to prepare with the immunostimulation material, and for example cell factor or chemotactic factor (CF) are as proleulzin ,-7 ,-12 ,-18, I class and II interferoid (IFN), particularly IFN-γ, GM-CSF, TNF-α, flt3-part etc.

According on the other hand, the present invention relates to the purposes in the HLA restriction vaccine of preparation treatment or prevention of hepatitis C (HCV) infection according to a kind of HCV epi-position of the present invention or HCV peptide.

The present invention also comprises the purposes of epi-position according to the present invention in the vaccine of preparation treatment or prevention of hepatitis C (HCV) infection.

Therefore, the present invention also comprises the vaccine that a kind of treatment or prevention of hepatitis C (HCV) infect, and it comprises according to epi-position of the present invention.

And, the HLA specificity vaccine that treatment or prevention of hepatitis C (HCV) infect, it comprises according to epi-position of the present invention or peptide, also is one aspect of the present invention.

Preferably, this vaccine also contains immunomodifier, preferably is selected from range of polycationic substances, polycation polypeptide particularly, and immunomodulatory nucleic acid particularly contains the deoxyinosine and/or the BrdU of oligodeoxynucleotide or its potpourri.

Preferably, this vaccine also contains polycationic polymer, preferably polycation peptide, particularly poly arginine, polylysine or antimicrobial peptide.

Polycationic compounds used according to the invention can be any polycationic compounds that shows according to the feature of WO 97/30721.Preferred polycationic compounds is selected from basic polypeptide, organic polycation, alkaline polyaminoacid or its potpourri.

The chain length of these polyaminoacid should be at least 4 amino acid residues.Particularly preferably be the material that contains the peptide combination, as polylysine, poly arginine and contain more than 8, particularly 20 above amino acid residues and contain more than 20%, the particularly polypeptide of 50% above basic amino acid, or its potpourri.Other preferred polycation and pharmaceutical composition thereof are described in WO 97/30721 (for example polyethyleneimine) and WO 99/38528.Preferably, these polypeptide contain 20-500 amino acid residue, a particularly 30-200 residue.

These polycationic compounds can produce or the reorganization generation by chemistry, perhaps can come from natural origin.

Kation (many) peptide also can be a polycation antibacterium microbial polypeptide.These (many) peptides can be protokaryon or animal or plant source, perhaps can produce or the reorganization generation by chemistry.These peptides also may belong to the sozin class.These host defense peptides or sozin also are a kind of preferred forms according to polycationic polymer of the present invention.A kind of compound, it can be used as end-product and activates (perhaps downward modulation) adaptive immune system, preferably by APC (comprising dendritic cells) mediation, usually as polycationic polymer.

The present invention is particularly preferred to be that (A 1416/2000 for the cathelicidin antimicrobial peptide or derivatives thereof of deriving as range of polycationic substances; be incorporated herein by reference); particularly derive from the antimicrobial peptide of mammal cathelicidin; preferably derive from people, ox or mouse; or Neurologically-active compounds, as (people) growth hormone (as described in WO01/24822).

The polycationic compounds that comes from natural origin comprises HIV-REV or the HIV-TAT (cationic peptide of deriving, feeler (antennapedia) peptide, other derivant of chitosan or chitin) or by biological chemistry or recombinant production other peptide by these peptides or protein derived.Other preferred polycationic compounds has cathelin or material, particularly mouse relevant with cathelin or that it is derived, ox or people cathelins, and/or cathelicidin.Cathelin material relevant or that derive contains all or part of cathelin sequence, contains 15-20 amino acid residue at least.Derive and to comprise that natural amino acid replaced or be modified to the amino acid that does not belong to 20 kinds of standard amino acids.And, also can in these cathelin molecules, introduce other cationic residues.These cathelin molecules preferably make up with antigen according to the present invention/vaccine combination.Yet under the situation of not adding other adjuvant, these cathelin molecules are also effective surprisingly as a kind of adjuvant of antigen.Therefore, using or do not using under the situation of other immune activation thing, using these cathelin molecules in bacterin preparation is possible as effective adjuvant.

The preferred range of polycationic substances of used according to the invention another is synthetic peptide, and it contains at least 2 KLK-motifs, is separated by the joint of a 3-7 hydrophobic amino acid, particularly L (WO02/32451 is incorporated herein by reference).

Immunological regulation used according to the invention (or immunogenicity) nucleic acid can be synthesize, prokaryotes or eucaryote source.For prokaryotes sources, according to systematic evolution tree, DNA should derive from low kind of growing (for example insect etc.).In a preferred embodiment of the invention, immunogenicity oligodeoxynucleotide (ODN) is a kind of dna molecular of synthetic generation or the potpourri of these molecules.Also comprise derivant or the modification of ODN, analog (the thiophosphate residue replaces phosphate) as the thiophosphate replacement, as U.S. Patent number US 5,723,335 and US 5,663,153 is described, and preferably stablize immunostimulatory compositions but do not change other derivant and the modification of its immune property.The DNA motif that a kind of preferred sequence motifs is one 6 base, it contains (non-methylated) CpG dinucleotide, and flank is two 5 ' purine and two 3 ' pyrimidines (5 '-Pur-Pur-C-G-Pyr-Pyr-3 ').More common in high vertebrate DNA according to CpG motif contained among the ODN of the present invention ratio in microorganism, on methylation patterns, show difference.Surprisingly, stimulating the sequence of mouse APC is not very effective for people's cell.The preferred palindrome used according to the invention or non-palindrome ODN are for example open in Australian patent application A1973/2000, A 805/2001, EP 0 468 520 A2, WO 96/02555, WO 98/16247, WO 98/18810, WO 98/37919, WO 98/40100, WO 98/52581, WO 98/52962, WO 99/51259 and WO 99/56755, all are incorporated herein by reference.Except stimulating immune system, some ODN some immune response that also neutralizes.The present invention also comprises these sequences, for example is used for the treatment of autoimmunity disease.ODNs/DNAs can produce or the reorganization generation by chemistry, perhaps comes from natural origin.Preferred natural origin is an insect.

In addition, based on the nucleic acid (for example WO01/93903 is described) of inosine and cytidine or contain deoxyinosine and/or the picodna of BrdU residue (WO 01/93905 and PCT/EP02/0544 describe, and are incorporated herein by reference) also can be preferably as immunostimulatory nucleic acids of the present invention.

Certainly, also can use the potpourri of different immunogenic nucleic acids according to the present invention.

Preferably, this vaccine also contains a kind of pharmaceutically acceptable carrier.

According to another preferred embodiment, this vaccine contains to be selected from epi-position or the peptide that following form provides: peptide, peptide analogues, protein, naked DNA, RNA, viral vectors, virus-like particle, reorganization/embedded virus, with proteins/peptides/RNA pulse or with the recombinant bacteria or the dendritic cells of the DNA transfection that contains this epi-position or peptide.

According on the other hand, the present invention relates to T cell, T cell clone or T cell colony (goods), their specific recognition are according to any HCV epi-position of the present invention or peptide, particularly aforesaid HCV epi-position.A kind of preferable use of these T cells is their in-vitro multiplications and for example by the therapeutical uses of adoptive transfer to the patient.Therefore, the present invention also provides T cell, T cell clone or T cell colony (goods) to be used for the treatment of purposes in HCV patient's the composition in preparation.

According to these T cells of the present invention (clone or be), particularly can discern above-mentioned HCV peptide, also can be used for identifying irregular epi-position, irregular epi-position is different from the epi-position of initial evaluation, but cause identical T cell.

Individuality is used with effective amount according to these cells of the present invention, composition or vaccine.

According on the other hand, the present invention also relates to have the peptide of general formula QRKTKRNTN, QRKTKRNT, or 1615,1616,1617, the 9mer peptide with general formula SAKSKFGYG, SAKSKYGYG or SARSKYGYG that particularly derives from back three kinds of peptides is as HLA-B ^*The purposes of 08 epi-position particularly is used for being HLA-B ^*08 specificity vaccine prepares pharmaceutical preparation; Peptide with general formula R KTKRNTNR is as HLA-B ^*The purposes of 2705 epi-positions particularly is used for being HLA-B ^*2705 specificity vaccines prepare pharmaceutical preparation; Peptide with general formula ARLIVFPDL is as HLA-B ^*2705 and HLA-B ^*The purposes of 2709 specificity vaccines.And, also relate to the focus epi-position and contain purposes in the vaccine of DNA composition of synthetic peptide, recombinant protein and/or this epi-position in preparation, this epi-position is selected from peptide 1835,84EX, 87EX, 89EX, 1426,1650,1836,1846,1651,1800,1799, C114,1827, C112, C114EX, 1827EX, 1798,1604,1829,1579,1624,1848,1547, A1A7, A122EX, A122,1825, A241, B8B38, C70EX, C92, C97, C106 and the C134 according to table 7.

Particularly, two or more epi-position focuses can make up, and this needs or do not need joint sequence.Preferred joint sequence for example is made up of 3-5 glycocoll or alanine or lysine residue.This can realize by peptide is synthetic, yet the combination of focus can produce quite long polypeptide.

In this case, the DNA of these constructs of clones coding and expression and the corresponding recombinant protein of purifying are alternativess.These recombinant proteins can be used as antigen, and (IC31, pR...) combination can cause the t cell response of all epi-positions of carrying at them with suitable adjuvant.Simultaneously, these artificial polypeptide lack the activity (enzyme, toxicity, immunosupress ...) that natural HCV antigen may have.

Carry t cell epitope or its combination that other several method is arranged.Comprise: recombinant viral vector, as vaccinia virus, canary pox virus, adenovirus; The RNA carrier of self-replicating; Use the plasmid of encoding hot spots or " naked DNA " inoculation of its combination; Recombinant bacteria (for example salmonella); With the dendritic cells of synthetic peptide pulse, or recombinant protein, or RNA or use the DNA transfection, their equal encode T cell epi-position focus or its combinations.

The present invention will illustrate in greater detail by the following example and accompanying drawing, but be not limited to these.

Description of drawings

Fig. 1 shows 40 kinds of peptide mixers, and wherein each all contains the 15-23mer peptide that can reach 20 kinds of HCV sources.

Fig. 2 shows use peptide storehouse and empty DRB1 ^*The epi-position prize law of 0401 molecule.

Fig. 3 shows use peptide storehouse and empty DRB1 ^*The epi-position prize law of 0404 molecule.

Fig. 4 shows various peptides and DRB1 ^*0401 combination.

Fig. 5 shows various peptides and DRB1 ^*0404 combination.

Fig. 6 shows various peptides and DRB1 ^*0101 combination.

Fig. 7 shows and DRB1 ^*The peptide of 0701 combination.

Fig. 8 shows mouse IFN-γ ELIspot, wherein uses the HLA-DRB1 from inoculation Ipep1604+IC31 ^*The splenocyte of 0401tg mouse or the CD8+ of separation or CD4+ cell.

Fig. 9 shows mouse IFN-γ ELIspot, wherein uses the HLA-DRB1 from inoculation Ipep1604+IC31 ^*The splenocyte of 0201tg mouse or the CD8+ of separation or CD4+ cell.

Figure 10 shows mouse IFN-γ ELIspot, wherein uses the HLA-DRB1 from inoculation Ipep1604+IC31 ^*The splenocyte of 0702tg mouse or the CD8+ of separation or CD4+ cell.

Embodiment

The embodiment general introduction

Present embodiment shows the performance of the present invention for special pathogen hepatitis C virus (HCV).

First uses the method according to this invention, and this method is the basis with using of " empty HLA molecule ".The peptide ligand mixture incubation in these molecules and possible HCV source screens according to the specificity binding events.Use the possibility of the potpourri of high complexity to allow from hundreds of and even thousands of kinds of possible parts, to identify several bonds very apace.Use HLA-DRB1 ^*0101 ,-DRB1 ^*0401 ,-DRB1 ^*0404 ,-DRB1 ^*0701 molecule and overlapping 15-23mer storehouse have proved this point.Importantly, the allelic analysis of the multiple different HLA of this use can be identified the part that mixes that can combine with more than one HLA allele.The t cell epitope that mixes is based on the special valuable ingredients of the vaccine of epi-position.Compare with being limited to the allelic epi-position of a kind of HLA, they can treat more part of crowd.

The peptide of I quasi-molecule and suitable length (being 8-11mer) can use identical method.The part storehouse may be the overlapping peptide that synthesizes.Another possibility is enzyme or the described antigen of non-enzymic digestion.The latter realizes by basic hydrolysis, produces all possible catabolite, has been successfully used to identify t cell epitope (Gavin 1993).Enzymic digestion can be carried out by enough proteinase.A reasonable method is to use and native antigen processing approach proteins associated enzyme, uses proteasome (Heemels1995) as the epi-position of I class restriction, perhaps the epi-position using-system proteinase (Villadangos 2000) of II class restriction.The part storehouse also may be made up of naturally occurring part, and for example the cracking or the wash-out of the cell of epi-position obtain these parts by carrying separately.In this, be important to note that and also can use non-peptide part, as glycolipid.As everyone knows, may encode or (for example CD1 family) non-classical I quasi-molecule of belonging to outside the MHC can be presented multiple non-peptide part (Kronenberg 1999) to lymphocyte by MHC (for example HLA-G, HLA-E, MICA, MICB).Use the non-classical I quasi-molecule of reorganization " sky " allow by as described here similarly method carry out the evaluation of association reaction and bond.

Rapid identification can with part that the HLA molecule combines after, the method that the method according to this invention also provides direct sign to reply at the specific T-cells of these bonds.A possibility is directly to use the HLA that separates in so-called " synthetic T raji cell assay Raji ": ligand complex.The latter comprises HLA: ligand complex stimulates the antigentic specificity of T cell again, and second kind of signal that common stimulation is provided, as the activation of activation antibody to CD28.This mensuration can be carried out by enough ELIspot readout devices.

The another one possibility is immune HLA transgenic mice, with the immunogenicity of proof by the part of epi-position catching method evaluation as described in example II.

Materials and methods

Peptide

In order to identify the conserved region between the HCV genotype 1,2,3, the about 90 kinds of full genomes that can openly obtain from Genebank of parallelism.Find that guard 43% HCV code area altogether at least 80% clinical separation strain.If always find two different amino acid (for example arginine or lysine) in certain site, these two kinds of variants all will be analyzed.Identified and be longer than 8 amino acid whose 148 conserved region altogether.Conserved region is covered by the peptide of～500 kinds 15 amino acid residues (15mer), have in 15 amino acid of every kind of peptide 14 previous overlapping with it.Length is that 8-14 amino acid whose conserved region covered by other 80 (non-overlapped) 15mers.Use Syro II synthesizer (Multisyntech, Witten, Germany) with the parallel synthetic 15mers of standard F-moc chemical method (one time 288).Per the 4th 15mer is by the mass spectroscopy inspection.The 15mer that need not be further purified is used in experiment.In addition, also use ABI 433A synthesizer (Applied Biosystems, Weiterstadt, Germany) utilize standard F-moc chemical method to synthesize the peptide of 63 kinds of 16-xx aa, use the C18 post (from the ODS ACU of YMC or 218TP, Vydac) by RP-HPLC purifying (Biocut 700E, Applied Biosystems, Langen, Germany).Use Reflex III mass spectrometer (Bruker, Bremen, Germany) to characterize purity and identity by MALDI-TOF.These peptides are dissolved among the 100%DMSO, and concentration is～10mg/ml (～5mM).Peptide storehouse (each 20 kinds of peptides) stoste prepares in 100%DMSO, the final concentration of every kind of peptide be 0.5mg/ml (～0.25mM).All peptides that the present invention uses are all listed in table 1.Peptide YAR (YARFQSQTTLKQKT), HA (PKYVKQNTLKLAT), P1 (GYKVLVLNPSVAAT), P2 (HMWNFISGIQYLAGLSTLPGNPA), P3 (KFPGGGQIVGVYLLPRRRGPRL), P4 (DLMGYIPAV) and CLIP (KLPKPPKPVSKMRMATPLLMQALPM) are being used as control peptide in measuring.

Epi-position catch with peptide in conjunction with mensuration

Solubility HLA II class DRA1 ^*0101/DRB1 ^*0101/Ii, DRA1 ^*0101/DRB1 ^*0401/Ii, DRA1 ^*0101/DRB1 ^*0404/Ii and DRA1 ^*0101/DRB1 ^*The 0701/Ii molecule is expressed in the SC-2 cell, and as people such as Aichinger, 1997 described purifying.In the peptide association reaction, solubility DRB1 ^*0101, DRB1 ^*0401, DRB1 ^*0404 molecule uses with the concentration of～0.5uM, if propose differently, every kind of peptide all adds (5 μ M) with 10 times of molar excess.The concentration of DMSO is no more than 4% in the association reaction.In the presence of protease inhibitor cocktail (Roche) and 0.1% octyl group-β-D-glucopyranoside (Sigma), in PBS damping fluid (pH 7.4), react following 48 hours of room temperature.Utilize SDS stability to measure the combination that people such as (, 1987) Gorga estimates peptide: trimerization HLA II class α: β: peptide complexes tolerance SDS, appearances～60kDa is with in SDS-PAGE Western engram analysis.In conjunction with peptide can't be stable each HLA II class α and the β chain respectively conduct～35kDa and～migration of the band of 25kDa.In brief, the HLA-peptide complexes is at room temperature handled with 1%SDS, analyzes in about 2.5 hours by at room temperature carrying out the SDS-PAGE electrophoresis with 20mA.By electroblotting protein transduction is moved on on the pvdf membrane, with anti-α chain TAL.1B5 or/and β chain MEM136 antibody staining.In order to detect Western trace signal, use ECL solution (Amersham).For DRB ^*0101 molecule is estimated strong combination in contrast with HA and P1 peptide, combination in estimating with the P2 peptide, and YAR is as negative control.For DRB1 ^*0401, the strongest is YAR and HA peptide in conjunction with contrast, and P1 and P2 respectively as in, weak bond.For DRB1 ^*0404 molecule uses P1 and the strong combination of P2 peptide evaluation, uses combination in the control of YAR peptide, and the HA peptide is as negative control.To DRB1 ^*0701 binding affinity detects people such as (, 1992) Reay by the peptide competition experiments.In brief, the high-affinity of biotinylation CLIP peptide has been used to monitor HLA in conjunction with (with reference to peptide): the formation of peptide complexes.When its affinity is higher, if perhaps affinity equals the affinity of CLIP, when suppress 50% in conjunction with the time, in association reaction, add with the detection of peptides of volumetric molar concentrations such as CLIP peptide and can compete CLIP.For the peptide than low-affinity, they should excessively add than reference peptide, and competition occupies HLA in conjunction with ditch.Suppress (IC with reference to peptide (biotinylated CLIP) in conjunction with 50% ₅₀) concentration value of required competition peptide can be used for estimating the peptide binding affinity.In addition, relatively exist or do not have the amount that combines with the HLA molecule under the situation of compete peptide with reference to peptide, can measure the purpose peptide in conjunction with activity.In this peptide competition assay, the condition of peptide combination is similar to the above.DRB1 ^*0701 molecule uses with the concentration of～0.5 μ M, and adding final concentration in all samples is the biotinylation CLIP of 2 μ M.The competition peptide adds with three different concentration: 2nM, 20 μ M and 200 μ M.Association reaction carries out in PBS damping fluid (pH 7.4), 37 ℃ 18 hours.With solubility DRB1 ^*The amount of the biotinylation CLIP of 0701 molecule combination is measured by ELISA.In brief, by with 50 μ lPBS in 10 μ g/ml dilutions be incubated overnight for 4 ℃, with mouse anti DR antibody L243 bag by MaxiSorp 96 orifice plates (Nunc, Denmark).By with the T-PBS that contains 3% BSA 37 ℃ of following incubations 2 hours, the non-specific binding in blocking-up and hole, association reaction at room temperature " was caught " 2 hours then.After fully washing, detect the peptide complexes of HLA combination with alkaline phosphatase-streptavidin (Dako) and Sigma 104 phosphatase substrates.Read the optical density of plate device (VICTOR) monitoring 405nm with microwell plate.Not biotinylated CLIP, P1 and the P2 peptide positive control of the strong combination that judges.Peptide P3 and P4 are respectively as weak combination and uncombined contrast.

The immunity of HLA transgenic mice

The immunogenicity of the synthetic peptide in HCV source is following at HLA-DRB1 ^*0401-and HLA-A ^*Detect in the 0201-transgenic mice: each group of every group of 3 mouse (female, 8 ages in week) to flank injection down (every mouse altogether 100 μ g peptides+30 μ g oligonucleotides CpI (Purimex,

, Germany)).After inoculating a week, take out spleen,, measure the specific cell (mouse ELISpot mensuration) that produces IFN-γ with peptide and the stripped splenocyte that activates of irrelevant negative control peptide that is used for inoculating.

The mouse boosting cell ELIspot that discharges for unicellular IFN-γ measures

ELISpot plate (MAHA S4510, Millipore, Germany) is with PBS rinsing (200 μ l/ hole), with anti-mouse IFN-γ mAb (clone R46A2; 100 μ l/ holes, 5 μ g/ml are dissolved in 0.1M NaHCO ₃, pH 9.2-9.5) and the bag quilt, and under 4 ℃, be incubated overnight.Wash plate 4 times with PBS/0.1% Tween 20, at room temperature with PBS/1% BSA (200 μ l/ hole) incubation 2 hours, the blocking-up non-specific binding.Preparation is from the splenocyte of the mouse of inoculation, with 1 * 10 ⁶-3 * 10 ⁵Cells/well inoculation is in the presence of immunizing antigen (peptide), control peptide or independent nutrient culture media, at 37 ℃/5%CO ₂Under be incubated overnight.Wash plate subsequently 4 times, with the anti-mouse IFN-of biotinylation γ mAb (clone AN18.17.24,100 μ l/ holes, 2 μ g/ml are in PBS/1% BSA) incubation, 37 ℃ 2 hours.After the washing, add streptavidin-peroxidase (Roche Diagnostics, Vienna, Austria) (1/5000,100 μ l/ hole among the PBS), culture plate is incubation 2 hours more at room temperature.Subsequently, (100 μ l/ holes, the potpourri of 10ml 100mM Tris pH 7.5 wherein add 200 μ l40mg/ml DAB stock solutions, contain 50 μ l80mg/ml NiCl to add substrate in the plate of washing ₂Stoste and 5 μ l, 30% H ₂O ₂).After 20-30 minute with washing the plate cessation reaction from the beginning.Dry culture plate is read plate device (BIOREADER 2000, BioSys, Karben, Germany) evaluation with ELISpot.

The IFN-γ ELIspot of end user PBMC

Collect to carry out serology HLA somatotype from HCV RNA-negative treatment responder or spontaneous recuperator's PBMC.Whole blood collection is arrived in the ACD Vacutainer test tube (Becton DickinsonEurope, Erembodegem, Germany).Utilize Leuco-sep test tube (Greiner, Frickenhausen, Germany) at Lymphoprep (Nycomed Pharma AS, Oslo, Norway) go up and separate PBMC, with PBS (Invitrogen Life Technologies (GIBCOBRL in the past), Carlsbad, CA, USA) washing is 3 times, with 2 * 10 ⁷The concentration of/ml is resuspended in the freezing nutrient culture media, this nutrient culture media is by 1640, the 9 parts of hyclone (FCS of 4 parts of RPMI that added 1mM Sodium Pyruvate, 2mM L-glutaminate, 0.1mM nonessential amino acid, 50 μ M 2 mercapto ethanols (all from Invitrogen LifeTechnologies); From PAA, Linz, Austria) and 1 part of DMSO (SIGMA, Deisenhofen, Germany) composition.(New York stores down at-80 ℃ in USA) and spends the night PBMC, transfers in the liquid nitrogen then for Nalgene Nunc International, Rochester at 1 ℃ of refrigerated container.ELIspot measures and to carry out substantially as previously mentioned people such as () Lalvani.In brief, Multi Screen 96 hole filter MAIP S4510 (Millipore, Bedford is MA) with 10 μ g/ml (0.75 μ g/ hole) anti-people IFN-γ monoclonal antibody (Mab) B140 (Bender Med Systems, Vienna, Austria) bag is spent the night under 4 ℃.Wash plate 2 times with PBS (Invitrogen Life Technologies), (RPMI 1640 with the ELIspot nutrient culture media, 1mM Sodium Pyruvate, 2mM L-glutaminate, 0.1mM nonessential amino acid, 50 μ M2-mercaptoethanols (all from Invitrogen Life Technologies) and 10% people AB type serum (PAA have been added, Linz, Austria)) sealing.Frozen PBMC melts in 37 ℃ of water-baths fast, with the washing of ELISPOT nutrient culture media once, be incubated overnight (37 ℃, 5%CO ₂).Next day, with 200,000PBMC/ hole inoculating cell was cultivated 20 hours altogether with various peptides (10 μ g/ml) or peptide storehouse (final concentration of every kind of peptide is 5 μ g/ml).Take out cell and use lavation buffer solution (PBS; 0.1% Tween 20, from SIGMA) washing 6 times after, (0.015 μ g/ hole) the biotinylated anti-people IFN-γ MAb B308-BT2 (Bender Med Systems) that adds 100 μ l 1:10000 dilution 37 ℃ of following incubations 2 hours, perhaps spends the night under 4 ℃.After the washing, add streptavidin-alkaline phosphatase (DAKO, Glostrup, Denmark) of 1.2 μ g/ml, 37 ℃ following 1 hour.Adding 100 μ l/ hole BCIP/NBT alkaline phosphatase substrates (SIGMA) measures.

Human PBMC's external initiation (priming)

In the presence of IL-2 and IL-7, with antigen (peptide or peptide mixer) repetitive stimulation human PBMC.This causes the few clonal expansion of selectivity of T cells with antigenic specificity.Can pass through IFN-γ ELIspot evaluation of measuring to for example replying of each epi-position.The new PBMC that melts cultivates (2-4 * 10 in 6 orifice plates ⁶/ mL living cells), nutrient culture media is RPMI-1640 (GibcoBRL), 1% nonessential amino acid (GibcoBRL, cat11140-035), 1% penicillin (10,000U/ml)-streptomysin (10,000 μ g/ml) (GibcoBRL cat#15140-122), 1%L-glutamine (GibcoBRL), 0.1% beta-mercaptoethanol (GibcoBRL), 1% Sodium Pyruvate (GibcoBRL), adds 10% people AB type serum (PAA, Linz, Austria).In every hole, add peptide (each 10 μ M).Adding final concentration is the rhIL-7 (Strathmann Biotech) of 10ng/mL.Added 20-30U/mL rhIL-2 (Strathmann Biotech) at the 4th day.From culture plate, took out all cells at the 10th day, with nutrient culture media (the same) washing once, counting.For the external initiation of next one circulation, living cells is cultivated from body PBMC and aforesaid peptide, rh-IL-2 altogether with (1.2 gray(Gy)s/minute, 20 minutes) as the radiation gamma of feeder cells (with 100,000 inoculations of every hole).ELIspot carries out as mentioned above, difference be to use 200,000 responsive cells (take turns external initiation and stimulate in advance for 2) and 60,000 irradiations from the body responsive cell.

Example I. by measuring the peptide storehouse of arranging, to the Rapid identification that mixes the HLA binding peptide from HCV with matrix form

In order to cover the conserved region in the HCV polyprotein matter, synthesized the peptide (table 1) more than 640 kinds.For Rapid identification HLA part and novel t cell epitope, prepare 40 peptide storehouses, 20 kinds of single peptides are all contained in each storehouse.These storehouses are configured to every kind of peptide and are present in 2 storehouses (matrix form).This allows to identify the reactive peptide (Fig. 1 HCV peptide matrix) of the point of crossing that is positioned at the row and column potpourri.

Table 1. derives from the synthetic peptide of HCV conserved region

Catch for epi-position, each peptide storehouse and solubility reorganization HLA II quasi-molecule incubation are by the combination of SDS stability evaluation of measuring specificity.Use HLA molecule DRB1 ^*0401, DRB1 ^*0404 and DRB1 ^*0101 result shows in Fig. 2 and Fig. 3 respectively: find 28 peptide storehouses and DRB1 ^*0401 molecule combination: from 1,2,6,7,8,9,10,11,12,13,14,16,17,18,19, No. 20 of " OK " storehouse with from 23,25,26,27,29,30,31,34,36,38,39 and No. 40 (Fig. 2) in " row " storehouse.In 40 peptide storehouses detecting, 35 and DRB1 are arranged ^*0404 molecule be combined into the positive (Fig. 3), and all peptide storehouses all show and DRB1 ^*The combination of 0101 molecule is active.By finding the intersection in reactive storehouse in the array, determine possible bond, and reexamine binding affinity separately.

All peptides that confirm are respectively summed up in table 2.With DRB1 ^*0401 be combined among Fig. 4 shows: 54 kinds of peptides are accredited as the part of this HLA type.Usually there are several overlapping 15mers to combine in the delegation, allow to identify its core land with HLA.Only there is the peptide (seeing Table 1) of the different representative variant of one or two amino acid to combine with two kinds of solubility HLA II quasi-molecules usually.This " duplicating " is considered to represent same epi-position.Therefore, identified can with DRB1 ^*31 kinds of parts of 0401 combination comprise 11 kinds of previously known II class epi-positions.Yet, in the latter, have only two kinds of (A202-A206 and B60-B68) known DR4 of being limited to (seeing Table 2).20 kinds of parts are candidates of new epi-position.For DRB1 ^*0404, determined 64 kinds of bonds of 28 kinds of possible epi-positions of called after, wherein 4 kinds belong to known epi-position (Fig. 5, table 2).For DRB1 ^*0101, identified 83 kinds of peptides (Fig. 6, table 2) of representing 44 kinds of possible epi-positions.Wherein, there are 7 kinds to describe in the past, but have different HLA restrictions.

Also utilize the peptide competition assay to detect all peptides and the DRB1 that confirm respectively that can combine one of at least with above-mentioned 3 kinds of HLA types ^*The affinity of 0701 molecule (Fig. 7, table 2).50 kinds of parts have been identified at this.Wherein there are 7 kinds corresponding to known II class epi-position, but have only a kind of DRB1 of being described to ^*0701 epi-position (A202-A206).

The peptide in the HCV source that table 2. combines with solubility HLA II quasi-molecule.The storehouse (see figure 1) that the reaches 20 kinds of peptides HLA II quasi-molecule different with 4 kinds that use is arranged with matrix form derives from the peptide of about 400 kinds of 15-mer of HCV conserved region to 23-mer by the analysis of epi-position prize law.Confirm the specificity combination of various peptides.

^{* *}Strong combination

^*Middle combination

^*Weak combination

Not combination of nb

The peptide number table of black matrix is shown in the immunogenic HLA-part that has confirmation in the HLA transgenic mice.

The black matrix peptide sequence is represented the core land according to prediction algorithm deduction as described herein.

¹⁾At DRB1 ^*Has immunogenicity in 0401 transgenic mice

As HLA-DRB1 ^*0701 is described, utilizes the peptide competition assay to check that some highly mix and/or have computerized algorithm (SYFPEITHI, TEPITOPE) peptide of Yu Ce affinity and solubility HLA-DRB1 ^*The combination of 1101 molecules.Use several known DR11 epi-positions in contrast, confirm external in conjunction with HLA-DRB1 ^*1101 molecules.At the new HLA-DRB1 that identifies ^*In 1101 bonds, be numbered A120, A122, A141, C114, C134,1426,1628,1629 peptide has high-affinity, be numbered C106, C135,1578,1547,5 kinds of peptides of 1604 have middle affinity, 4 kinds of peptides that are numbered B46, B48, B86, B96 are weak affinity part.

In a word, exist at least and HLA-DRB1 ^*0101, ^*0401, ^*0404, ^*0701 He ^*8 kinds of new parts of 1101 combinations (table 2: peptide numbering A120, A122, A141,1604,1547,1628,1629 and table 6: peptide numbering 1426); At least with HLA-DRB1 ^*0101, ^*0401, ^*0404 He ^*10 kinds of parts of 0701 combination (table 2: peptide numbering A120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114,1627,1628,1629,1604); At least with HLA-DRB1 ^*0101, ^*0401 He ^*5 kinds of new parts of 0701 combination are at least with HLA-DRB1 ^*0101, ^*0404 He ^*5 kinds of new parts of 0701 combination are at least with HLA-DRB1 ^*0401, ^*0404 He ^*4 kinds of new parts of 0701 combination are at least with HLA-DRB1 ^*0101, ^*0401 He ^*3 kinds of new parts of 0404 combination are at least with HLA-DRB1 ^*0101 He ^*2 kinds of new parts of 0701 combination, at least with HLA-DRB1*0401 and ^*1 kind of new part of 0701 combination is at least with HLA-DRB1 ^*0101, ^*3 kinds of new parts of 0401 combination are at least with HLA-DRB1 ^*0404 He ^*1 kind of new part of 0701 combination is at least with HLA-DRB1 ^*0101 He ^*4 kinds of new parts of 0404 combination are at least with HLA-DRB1 ^*5 kinds of new parts of 0701 combination are at least with HLA-DRB1 ^*13 kinds of new parts of 0101 combination are at least with HLA-DRB1 ^*1 kind of new part of 0401 combination and at least with HLA-DRB1 ^*6 kinds of new parts of 0404 combination.

And, confirmed 12 kinds of known HLA II class epi-positions, under several situations, proved and allelic combination of not reporting as yet (table 2, last group).

The physical bond of clear and definite and HLA II class, directly confirmed for the immunogenicity of specifying part, for example peptide is numbered A120-A124, B46-B48,1627,1604,1630,1547,1623, B112-118,1558, all combine, at HLA-DRB1 with one or more HLA II class allele ^*Also show immunogenicity (referring to example II) in 0401 transgenic mice.

In order to determine the best epi-position in the longer polypeptide, mouse can inoculate the longer polypeptide that mixes the candidate list bit sequence.Can measure generation by stimulate mice spleen cell or lymph-node cell and IFN-γ ELIspot again with overlapping 15-mers then at the specific C D4+T cell response of the natural process and the epi-position of presenting.The final confirmation of the new HLA-part of identifying can realize by using from these peptides of T cell detection of people.Ideally, comprise treatment responder or infect spontaneous recuperator.The immunogenicity of peptide in the HLA transgenic mice in example II .HCV-source

The immunogenicity of synthetic peptide (from conserved region) in the HLA transgenic mice in research HCV source: in inoculation experiments, induce peptide specific to produce the IFN-gamma cells for 36 kinds in 68 kinds of peptides finding to detect.Sum up as table 3, some peptides are at DR4-and/or A ^*In 0201 transgenic mice or immunogenic (+, in 1,000,000 splenocytes less than 100 peptide specific cells) or or even immunogenic by force (++, in 1,000,000 splenocytes, surpass 100 peptide specific cells).

Table 3:

Peptide 1526,1565,1631 is at HLA-DRB1 ^*Also show immunogenicity in 0401 transgenic mice, contain known II class epi-position.Peptide numbering 1526,1553,1565,1587,1623,1630 is at HLA-A ^*Also show immunogenicity in 0201 transgenic mice, contain known A2 epi-position.

In order further to characterize new epi-position provided herein, can define these epi-positions and can be limited by the accurate HLA of the minimum epi-position in the sequence of T cell recognition.Can carry out (" modern immunological method ", John Wiley ﹠amp by well known to a person skilled in the art several different methods; Sons, Inc.).

At first, can utilize the program that can openly obtain to predict t cell epitope according to the motif of combination.Comprise, for example: http://bimas.dcrt.nih.gov/molbio/hla_bind/ (people 1994 such as Parker), http: // 134.2.96.22l/scripts/MHCServer.dll/home.htm (people 1999 such as Rammensee), http://mypage.ihost.com/usinet.hamme76/ (people 1999 such as Sturniolo).A kind of prediction algorithm in back provides identifies the possibility that mixes t helper cell epi-position (peptide that can combine with several HLAII quasi-molecules).These predictions can be by the combine confirmation of detection of peptides with HLA everywhere.

Distinguish that fast a kind of method of replying the restriction of I class or II class to a kind of peptide is to repeat ELIspot mensuration with pure CD4+ or CD8+T cytological effect colony.For example, this subgroup that can utilize the magnetic cell sorting to separate separately realizes.The artificial antigen that also can detect pure CD8+T cell in ELIspot measures and only express a kind of purpose HLA molecule is delivery cell.An example is the positive T2 cell (174CEM.T2, people such as Nijman, 1993) of HLA-A*0201.In addition, also can in the presence of the monoclonal antibody of specific inhibition CD4+ or CD8+T cell subsets, use full PBMCs to carry out ELIspot and measure.HLA restriction accurately can use some allele specific oligonucleotide blocking-up monoclonal antibody to measure with similar method.For example, add the HLA-A24 monoclonal antibody specific and can block replying at the HLA-A24 restricted epitope.

Can be in order to define by the minimum epi-position in the peptide sequence of T cell recognition, people can use from the splenocyte of the transgenic mice of immunity or from people's identification separately the T cell detection of epi-position overlapping with peptide brachymemma (for example 8-, 9-, 10-mers).

EXAMPLE III. the HLA restriction of the immunogenic peptide in the HCV source of in transgenic mice, studying

Several groups of (the HLA-A of every group of 5 mouse ^*0201-, HLA-DRB1 ^*0401-and HLA-B ^*0702 transgenic mice, male, age in 8-14 week) to metapedes lift every mouse 100 μ g peptide+IC31 of injection (each foot pad 50 μ g) down.(PCT/EP01/12041, WO 02/32451A1 and PCT/EP01/06433, WO 01/93905A1; IC31 is the combination of WO 01/93905 and the disclosed immunizing agent of WO02/32451).

Inoculate after 6 days, the single cell suspension of the spleen that preparation merges, use MSCA separating kit (Miltenyi in addition, Germany) from splenocyte suspension, separate pure fraction, for A2 and B7tg mouse is that (for the B7 mouse is the CD8+ fraction to the CD8+ fraction, it contains 97% CD8 and 1.5% cd4 cell, for the A2tg mouse, contain 83% CD8 and 8% cd4 cell), for the DR4tg mouse is CD4+ fraction (for the DR4tg mouse is the CD4+ fraction, contains 98% cd4 cell and 0.2% cd8 cell).All cells (not being isolated cells, positive and corresponding negative fraction) exsomatizes with relevant peptide (for example Ipep1604) to stimulate again, uses irrelevant peptide as negative control (known HLA-DRB1 ^*The epi-position Ipep 1505 in 0401CMV source, HLA-B ^*The epi-position Ipep 1787 in 0702HIV source, or HLA-A ^*The epi-position lpep1124 in 0201 tyrosinase source), in measuring, ELISpot detects the cell that produces INF-γ.

The example that Fig. 8-10 shows is that Ipep1604 (WCCSMSYTWTGALITPC is with immunizing agent IC31 combination) can induce a large amount of specificitys to produce the T cell of INF-γ in all 3 transgenosis I classes and the strain of II class mouse.This not only shows with whole cells in spleen source, and for A2 and B7 CD8+ cell, shows for the enriched fraction of DR4 tg mouse with the CD4+ cell.Similarly, see more weak replying with Ipep1605 (WCCSMSYSWTGALITPC), Ipep1605 is a series of variation representing threonine with serine.

Therefore, Ipep1604 contains for HLA-A ^*0201 and HLA-B ^*0702 I class epi-position and for HLA-DRB1 ^*The II class epi-position of 0401 molecule.

Shown in table 2 and table 6, Ipep 1604 in the mode of a specific admixture in conjunction with the II quasi-molecule.Therefore, it contains at least for HLA-DRB1 ^*0101, DRB1 ^*0404, DRB1 ^*0701 and DRB1 ^*Other epi-position of 1101.

The similar methods analyst of some other peptide: Ipeps1605,1623,1547,1558,1559,1560,1565,1592,1650,1654 and 1655 confirms to contain people HLA-DRB1 ^*0401 epi-position.In addition, for the great majority in these epi-positions, in conjunction with the HLA-DRB1 that is not limited to shown in table 2 and table 6 ^*0401.

Ipeps 1565,1605 and 1650 confirms to contain people HLAA ^*0201 epi-position.

Ipeps 1506,1587 confirms to contain people HLA-B ^*0702 epi-position.

The Ipep 1843 that contains sequence LPRRGPRI shows it is HLA-B contained in 1506 ^*0702 minimum epi-position:

Figure 10 shows mouse IFN-γ ELlspot, wherein uses the HLA-A from inoculation Ipep1506+IC31 or Ipep1835+IC31 ^*The splenocyte of 0702 transgenic mice or the CD8+ of separation or CD4+ cell.

Figure 10 A) and B) after being presented at single inoculation Ipep1506+IC31 or Ipep1835+IC31, after stimulating with overlapping 15mers, 15mers A30 and A37 (seeing Table 1) react again.The common sequences of these 15mers is LPRRGPRL (lpep 1843, see Table 4).

Figure 10 C) these discoveries have been confirmed: behind single inoculation Ipep1506+IC31 or Ipep1835+IC31, can detect at the tangible interferon-of Ipep1843 and induce.In both cases, utilize Ipep1790 as the negative control that stimulates again, Ipep 1790 is the HLA-B in a kind of HIV NEF-source ^*0702 epi-position (sequence RPMTYKAAL).

The Ipep 1838 (seeing Table 4) that contains sequence SPGALWGVI shows it is a kind of HLA-B contained in 1587 ^*0702 minimum epi-position:

Adopt a kind of diverse ways for Ipep1587: the HLA-B that checks the Ipep1587 sequence ^*0702 binding motif, synthetic a pair of small peptide.

Use solubility HLA-B at the state of conflict peptide in conjunction with in measuring ^*0702 and the detecting with reference to peptide LPCVLWPVL of FITC-mark, the latter is the known HLA-B of a kind of EBV of deriving from ^*0702 epi-position (people such as Stuber, 1995).When using 48 hours with 80 times of molar excess under 37 ℃, peptide Ipep1838 shows～30% competition.Therefore may present minimum HLA-B contained among the Ipep 1587 ^*0702 epi-position.

EXAMPLE IV: in the IFN-γ ELIspot that uses the human PBMC who treats responder or spontaneous recovery patient from HCV, have the evaluation and the confirmation of reactive novel HCV peptide

Use from more than 50 people to the PBMC of the individuality (but being all patients of HCV antibody positive HCV-RNA feminine gender) of interferon/effective individuality of Ribavirin standard care or spontaneous removing HCV, the peptide mixer (Fig. 1) of 40 kinds of matrix forms of screening in IFN-γ ELlspot, these potpourris contain the synthetic peptide (table 1) that derives from the HCV conserved region.Be considered to contain the relevant T cell colony of being responsible for removing HCV from these individual PBMC.Therefore, utilize these PBMC to find or the peptide that confirms may be represented the structural determinant of immunoprotection/removing HCV.According to this first submatrix results of screening, in order in the IFN-γ ELIspot of use, to detect a large amount of peptide of individual choice again from the PBMC of selected donor.In addition, also synthesized several new peptide that mixes from the sequence or the shortage Key residues (as halfcystine) of overlapping reactive peptide.These are summarized in the table 4.

Table 4: other peptide that derives from the HCV conserved region

The peptide numbering	Peptide sequence (monamino acid coding)
The peptide numbering	Peptide sequence (monamino acid coding)	1006	MWNFISGIQYLAGLSTLPGN
1334	HMWNFISGI	1006	MWNFISGIQYLAGLSTLPGN
1334	HMWNFISGI	1425	NFISGIQYLAGLSTLPGNPA
1426	HMWNFISGIQYLAGLSTLPGNPA	1425	NFISGIQYLAGLSTLPGNPA
1426	HMWNFISGIQYLAGLSTLPGNPA	1798	IGLGKVLVDILAGYGAGVAGALVAFK
1799	AAWYELTPAETTVRLR	1798	IGLGKVLVDILAGYGAGVAGALVAFK
1799	AAWYELTPAETTVRLR	1800	DYPYRLWHYPCTVNYTIFKI
1836	DYPYRLWHYPCTVNFTIFKI	1800	DYPYRLWHYPCTVNYTIFKI
1836	DYPYRLWHYPCTVNFTIFKI	1801	AYSQQTRGLL
1827	TAYSQQTRGLLG	1801	AYSQQTRGLL
1827	TAYSQQTRGLLG	1829	SMSYTWTGALITP
1838	SPGALVVGVI	1829	SMSYTWTGALITP
1838	SPGALVVGVI	1843	LPRRGPRL

Use the results are summarized in the table 5 of programmed screening of each peptide.Patient altogether～20% (G05, G18, H02, H03, H04, H10, H12, H19, H32, H38) shows tangible IFN-γ T-cell response to one or more peptides.In some cases, observed ELIspots quantity obviously raises, but does not have statistics to be higher than background significantly.In these cases, PBMC (donor H03, H10, H33, H38) peptide stimulated in vitro (2 take turns external initiation, referring to " materials and methods ") is separately replied to strengthen peptide specific.Confirmed several peptides like this, the result also is summarized in the table 5.

Peptide A3-A7 represents the overlapping 15mers of sequence coverage TNPKPQRKTKRNTNRRPQD.Since they all with from the PBMC of donor H03 reaction, the minmal sequence of this epi-position is positioned at sequence PQRKTKRNTNR.Prediction algorithm shows that QRKTKRNTN and QRKTKRNT represent HLA-B ^*08 part, and RKTKRNTNR most probable and HLA-B ^*2705 combinations.

Peptide C64-C70 represents the overlapping 15mers of sequence coverage KGGRKPARLIVFPDLGVRVCE.C64 and C70 respectively with from the PBMC of donor H32 and H38 reaction.Therefore the minmal sequence of this epi-position is positioned at sequence A RLIVFPDL.Prediction algorithm shows that ARLIVFPDL is HLA-HLA-B ^*2705 and HLA-B ^*A kind of part of 2709.

The summary of the HCV peptide of table 5. pair responding property of PBMC

Digitized representation is according to T cell/10 of the secretion peptide specific IFN-γ of ELIspot result's calculating ⁶PBMC (duplicate mensuration); Value〉3 times of 8 (〉 background) to be considered to statistics significant.Donor H32 and H33 are the patients of spontaneous recovery.

The peptide that EXAMPLE V .HCV derives combines with HLA II quasi-molecule

Except the listed peptide of table 1, also synthesized several new peptides, wherein mixed sequence from overlapping reactive peptide, perhaps do not contain Key residues such as halfcystine (table 4).Again detect the affinity of they and II class solubility HLA molecule, result's compare with the result who uses original peptide to obtain (table 6).

The combining of peptide that the HCV that table 6. is selected derives and 15-mer homologue thereof and solubility HLA II quasi-molecule (" +++" strong affinity, affinity in " ++ ", affinity a little less than "+", "-" no affinity, " nd " do not do; Line out below the core binding motif).

For peptide 1798, compare with short homologue (B84-B96), DRB1 ^*0101 and DRB1 ^*It may be owing to have the long sequence (26 amino acid) of the secondary structure that stops combination that the affinity of 0401 molecule is eliminated.Can expect that in vivo, after the proteolysis cutting, peptide 1798 will produce two shorter II class epi-positions.The halfcystine of in peptide 1827 and 1829, removing (C) residue (being respectively the derivant of peptide C114 and 1604) for DRB1 ^*The combination of 0401 molecule is seemingly vital, still not necessarily changes the affinity with other DR hypotype that detects.

The evaluation and the sign of example VI .HCV-epi-position focus

At this, t cell epitope focus (hereinafter referred to as " focus ") is defined as containing at least the short peptide sequence of an above t cell epitope.For example, after two or more epi-positions may be positioned at each other nearerly (being defined as less than 5-10 amino acid) than near-earth, perhaps be located immediately at each other after, perhaps partly and even overlapping fully.Focus may only contain I class or II class epi-position, or the two combination.Epi-position in the focus may have different HLA restrictions.

Because the high complexity in brief introduction, mentioned and optionally I class and II class antigen process approach, t cell epitope can't easily be predicted in a kind of peptide sequence.Although be extensive use of, the computerized algorithm that is used for the t cell epitope prediction has high false negative rate and false positive rate.

Therefore, because each t cell epitope is not obvious in a kind of peptide sequence, the situation of focus is all the more so.The experimental technique of several fundamental differences combined according to the invention carries out t cell epitope to be identified, comprises that epi-position is caught, HLA transgenic animals and person monocytic cell's stimulated in vitro.All these three kinds of methods can both systematically be used to cover the overlapping peptide of purpose antigen, can comprehensively identify epi-position (relate to the CMV/ epi-position and catch patent).This analysis-by-synthesis is not limited to a kind of concrete HLA allele, but all possible directed epi-position of disperseing in colony, this analysiss afterwards the epi-position focus may be tangible.In a kind of antigen, have only the minority sequence to show the focus feature.Therefore, the evaluation of focus is a surprising incident always.

T-cell epitope focus has important advantage: focus can activate, and can be by an experimenter's different T cell clone identifications.Focus (when containing the epi-position of different HLA restriction) can with the T cell interaction from the unmatched Different Individual of HLA.

Based on the vaccine of epi-position up to now at the general HLA allele of selecting, for example HIA-A2 that in only about half of Caucasian, expresses.Because other gene frequency is lower, the vaccine based on epi-position with extensive world crowd coverage must contain multiple different epi-position.A kind of chemical individual of vaccine (for example peptide) quantity is produced, is prepared the constraint and the product stability that cause and limited.

Focus makes these vaccines based on epi-position have world crowd coverage widely because their peptides by limited quantity provide may be a large amount of epi-position.

T cell epitope focus in the table 7:HCV conserved region

Focus (comprising some variation) shows that with black matrix contained epi-position shows with normal font in the focus.Numbering and sequence and the HLA I class and the II class coverage of peptide have been listed.Data Source is meant embodiment and the Biao in this instructions, perhaps list of references.

Example VII A: HCV epi-position focus Ipep 1426 contains HLA-A at least ^*0201 and the II class t cell epitope of several specific admixtures

The fundamental purpose of this experiment is that the immunogenicity of " focus " Ipep 1426 is compared with various epi-positions, and Ipep 1426 contains at least one HLA-A ^*0201 epi-position (Ipep 1334) and 2 II class epi-positions (Ipeps 1006 and 1425) that mix.For this reason, stimulate at external use 1426 or 1334,1006,1425 potpourri from the peripheral blood lymphocytes (PBMC) of the healthy of several HLA somatotypes.Carrying out three-wheel stimulates, and produces few clone T clone.Reply all 4 kinds of peptides by interferon-(IFN-γ) ELIspot assay then.

The interior contained various epi-positions of peptide 1426 and sequence inducing T cell are similarly replied.Particularly, at HLA-A ^*The CD8 positive T cell of the epi-position 1334 of 0201 restriction successfully produces.

Table 8: the IFN-γ secretion of the few clone of inducing peptide T clone.These clones derive from the healthy individual of two HLA somatotypes, produce by take turns external initiation with 3 of peptide 1426 or peptide 1006+1425+1334 potpourri.Estimate reactivity in these clones of CD4 and CD8 positive T cell (" +++" is extremely strong, and " ++ " is strong, and "+" is remarkable, and "-" be secretion of gamma-IFN not) by IFN-γ ELIspot.

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Sequence table

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<400>26

<210>27

<211>18

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1627

<222>(1)..(18)

<223>

<400>27

<210>28

<211>20

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1628

<222>(1)..(20)

<223>

<400>28

<210>29

<211>18

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1629

<222>(1)..(18)

<223>

<400>29

<210>30

<211>18

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1604

<222>(1)..(18)

<223>

<400>30

<210>31

<211>17

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1630

<222>(1)..(17)

<223>

<400>31

<210>32

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C97

<222>(1)..(15)

<223>

<400>32

<210>33

<211>21

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1547

<222>(1)..(21)

<223>

<400>33

<210>34

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B94

<222>(1)..(15)

<223>

<400>34

<210>35

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B95

<222>(1)..(15)

<223>

<400>35

<210>36

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B96

<222>(1)..(15)

<223>

<400>36

<210>37

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B97

<222>(1)..(15)

<223>

<400>37

<210>38

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B98

<222>(1)..(15)

<223>

<400>38

<210>39

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A272

<222>(1)..(15)

<223>

<400>39

<210>40

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A273

<222>(1)..(15)

<223>

<400>40

<210>41

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A274

<222>(1)..(15)

<223>

<400>41

<210>42

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A275

<222>(1)..(15)

<223>

<400>42

<210>43

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A276

<222>(1)..(15)

<223>

<400>43

<210>44

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B120

<222>(1)..(15)

<223>

<400>44

<210>45

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B122

<222>(1)..(15)

<223>

<400>45

<210>46

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C108

<222>(1)..(15)

<223>

<400>46

<210>47

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C134

<222>(1)..(15)

<223>

<400>47

<210>48

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C152

<222>(1)..(15)

<223>

<400>48

<210>49

<211>17

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1606

<222>(1)..(17)

<223>

<400>49

<210>50

<211>17

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1607

<222>(1)..(17)

<223>

<400>50

<210>51

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1577

<222>(1)..(16)

<223>

<400>51

<210>52

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1578

<222>(1)..(16)

<223>

<400>52

<210>53

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B50

<222>(1)..(15)

<223>

<400>53

<210>54

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B51

<222>(1)..(15)

<223>

<400>54

<210>55

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B52

<222>(1)..(15)

<223>

<400>55

<210>56

<211>21

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1623

<222>(1)..(21)

<223>

<400>56

<210>57

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C130

<222>(1)..(15)

<223>

<400>57

<210>58

<211>18

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1603

<222>(1)..(18)

<223>

<400>58

<210>59

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C96

<222>(1)..(15)

<223>

<400>59

<210>60

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C191

<222>(1)..(15)

<223>

<400>60

<210>61

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A216

<222>(1)..(15)

<223>

<400>61

<210>62

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A217

<222>(1)..(15)

<223>

<400>62

<210>63

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A218

<222>(1)..(15)

<223>

<400>63

<210>64

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A219

<222>(1)..(15)

<223>

<400>64

<210>65

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A220

<222>(1)..(15)

<223>

<400>65

<210>66

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A221

<222>(1)..(15)

<223>

<400>66

<210>67

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A222

<222>(1)..(15)

<223>

<400>67

<210>68

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A223

<222>(1)..(15)

<223>

<400>68

<210>69

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A224

<222>(1)..(15)

<223>

<400>69

<210>70

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A242

<222>(1)..(15)

<223>

<400>70

<210>71

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A243

<222>(1)..(15)

<223>

<400>71

<210>72

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A244

<222>(1)..(15)

<223>

<400>72

<210>73

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C92

<222>(1)..(15)

<223>

<400>73

<210>74

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C93

<222>(1)..(15)

<223>

<400>74

<210>75

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A174

<222>(1)..(15)

<223>

<400>75

<210>76

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B32

<222>(1)..(15)

<223>

<400>76

<210>77

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B33

<222>(1)..(15)

<223>

<400>77

<210>78

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B34

<222>(1)..(15)

<223>

<400>78

<210>79

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B35

<222>(1)..(15)

<223>

<400>79

<210>80

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B36

<222>(1)..(15)

<223>

<400>80

<210>81

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B37

<222>(1)..(15)

<223>

<400>81

<210>82

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B38

<222>(1)..(15)

<223>

<400>82

<210>83

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B100

<222>(1)..(15)

<223>

<400>83

<210>84

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B101

<222>(1)..(15)

<223>

<400>84

<210>85

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B102

<222>(1)..(15)

<223>

<400>85

<210>86

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C135

<222>(1)..(15)

<223>

<400>86

<210>87

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C162

<222>(1)..(15)

<223>

<400>87

<210>88

<211>17

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1618

<222>(1)..(17)

<223>

<400>88

<210>89

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1622

<222>(1)..(16)

<223>

<400>89

<210>90

<211>19

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1624

<222>(1)..(19)

<223>

<400>90

<210>91

<211>20

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1546

<222>(1)..(20)

<223>

<400>91

<210>92

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1556

<222>(1)..(16)

<223>

<400>92

<210>93

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A114

<222>(1)..(15)

<223>

<400>93

<210>94

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B58

<222>(1)..(15)

<223>

<400>94

<210>95

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B112

<222>(1)..(15)

<223>

<400>95

<210>96

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B113

<222>(1)..(15)

<223>

<400>96

<210>97

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B114

<222>(1)..(15)

<223>

<400>97

<210>98

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B115

<222>(1)..(15)

<223>

<400>98

<210>99

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B116

<222>(1)..(15)

<223>

<400>99

<210>100

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B117

<222>(1)..(15)

<223>

<400>100

<210>101

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B118

<222>(1)..(15)

<223>

<400>101

<210>102

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B18

<222>(1)..(15)

<223>

<400>102

<210>103

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B19

<222>(1)..(15)

<223>

<400>103

<210>104

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B20

<222>(1)..(15)

<223>

<400>104

<210>105

<11>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B21

<222>(1)..(15)

<223>

<400>105

<210>106

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B22

<222>(1)..(15)

<223>

<400>106

<210>107

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C112

<222>(1)..(15)

<223>

<400>107

<210>108

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C116

<222>(1)..(15)

<223>

<400>108

<210>109

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C122

<222>(1)..(15)

<223>

<400>109

<210>110

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C127

<222>(1)..(15)

<223>

<400>110

<210>111

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C144

<222>(1)..(15)

<223>

<400>111

<210>112

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C159

<222>(1)..(15)

<223>

<400>112

<210>113

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C160

<222>(1)..(15)

<223>

<400>113

<210>114

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C174

<222>(1)..(15)

<223>

<400>114

<210>115

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1558

<222>(1)..(15)

<223>

<400>115

<210>116

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1581

<222>(1)..(16)

<223>

<400>116

<210>117

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C95

<222>(1)..(15)

<223>

<400>117

<210>118

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C129

<222>(1)..(15)

<223>

<400>118

<210>119

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C157

<222>(1)..(15)

<223>

<400>119

<210>120

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C158

<222>(1)..(15)

<223>

<400>120

<210>121

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A254

<222>(1)..(15)

<223>

<400>121

<210>122

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A255

<222>(1)..(15)

<223>

<400>122

<210>123

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A256

<222>(1)..(15)

<223>

<400>123

<210>124

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A257

<222>(1)..(15)

<223>

<400>124

<210>125

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A258

<222>(1)..(15)

<223>

<400>125

<210>126

<211>18

<212>PRT

＜213〉hepatitis C virus

<220>

<21>1605

<222>(1)..(18)

<223>

<400>126

<210>127

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C109

<222>(1)..(15)

<223>

<400>127

<210>128

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C161

<222>(1)..(15)

<223>

<400>128

<210>129

<211>37

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1555

<222>(1)..(37)

<223>

<400>129

<210>130

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1559

<222>(1)..(15)

<223>

<400>130

<210>131

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1560

<222>(1)..(15)

<223>

<400>131

<210>132

<211>34

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1563

<222>(1)..(34)

<223>

<400>132

<210>133

<211>34

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1592

<222>(1)..(34)

<223>

<400>133

<210>134

<211>19

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1616

<222>(1)..(19)

<223>

<400>134

<210>135

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1621

<222>(1)..(16)

<223>

<400>135

<210>136

<211>19

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1625

<222>(1)..(19)

<223>

<400>136

<210>137

<211>36

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1649

<222>(1)..(36)

<223>

<400>137

<210>138

<211>32

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1650

<222>(1)..(32)

<223>

<400>138

<210>139

<211>32

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1651

<222>(1)..(32)

<223>

<400>139

<211>28

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1652

<222>(1)..(28)

<223>

<400>140

<210>141

<211>23

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1654

<222>(1)..(23)

<223>

<400>141

<210>142

<211>23

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1655

<222>(1)..(23)

<223>

<400>142

<210>143

<211>23

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1656

<222>(1)..(23)

<223>

<400>143

<210>144

<211>20

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1545

<222>(1)..(20)

<223>

<400>144

<210>145

<211>41

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1552

<222>(1)..(41)

<223>

<400>145

<210>146

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1557

<222>(1)..(15)

<223>

<400>146

<210>147

<211>19

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1615

<222>(1)..(19)

<223>

<400>147

<210>148

<211>19

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1617

<222>(1)..(19)

<223>

<400>148

<210>149

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1631

<222>(1)..(16)

<223>

<400>149

<210>150

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1632

<222>(1)..(16)

<223>

<400>150

<210>151

<211>30

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1641

<222>(1)..(30)

<223>

<400>151

<210>152

<211>37

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1647

<222>(1)..(37)

<223>

<400>152

<210>153

<211>28

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1653

<222>(1)..(28)

<223>

<400>153

<210>154

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A141

<222>(1)..(15)

<223>

<400>154

<210>155

<211>23

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1426

<222>(1)..(23)

<223>

<400>155

<210>156

<211>67

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1506

<222>(1)..(67)

<223>

<400>156

<210>157

<211>56

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1526

<222>(1)..(56)

<223>

<400>157

<210>158

<211>37

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1553

<222>(1)..(37)

<223>

<400>158

<210>159

<211>34

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1562

<222>(1)..(34)

<223>

<400>159

<210>160

<211>34

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1565

<222>(1)..(34)

<223>

<400>160

<210>161

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1580

<222>(1)..(16)

<223>

<400>161

<210>162

<211>56

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1587

<222>(1)..(56)

<223>

<400>162

<210>163

<211>8

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1843

<222>(1)..(8)

<223>

<400>163

<210>164

<211>10

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1838

<222>(1)..(10)

<223>

<400>164

<210>165

<211>9

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1

<222>(1)..(9)

<223>

<400>165

<210>166

<211>8

<212>PRT

＜213〉hepatitis C virus

<220>

<221>2

<222>(1)..(8)

<223>

<400>166

<210>167

<211>9

<212>PRT

＜213〉hepatitis C virus

<220>

<221>3

<222>(1)..(9)

<223>

<400>167

<210>168

<211>9

<212>PRT

＜213〉hepatitis C virus

<220>

<221>4

<222>(1)..(9)

<223>

<400>168

<210>169

<211>9

<212>PRT

＜213〉hepatitis C virus

<220>

<221>5

<222>(1)..(9)

<223>

<400>169

<210>170

<211>9

<212>PRT

＜213〉hepatitis C virus

<220>

<221>6

<222>(1)..(9)

<223>

<400>170

<210>171

<211>29

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1835

<222>(1)..(29)

<223>

<400>171

<210>172

<211>19

<212>PRT

＜213〉hepatitis C virus

<220>

<221>84EX

<222>(1)..(19)

<223>

<400>172

<210>173

<211>13

<212>PRT

＜213〉hepatitis C virus

<220>

<221>87EX

<222>(1)..(13)

<223>

<400>173

<210>174

<211>25

<212>PRT

＜213〉hepatitis C virus

<220>

<221>89EX

<222>(1)..(25)

<223>

<400>174

<210>175

<211>20

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1836

<222>(1)..(20)

<223>

<400>175

<210>176

<211>20

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1846

<222>(1)..(20)

<223>

<400>176

<210>177

<211>20

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1800

<222>(1)..(20)

<223>

<400>177

<210>178

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1799

<222>(1)..(16)

<223>

<400>178

<210>179

<211>12

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1827

<222>(1)..(12)

<223>

<400>179

<210>180

<211>25

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C114EX

<222>(1)..(25)

<223>

<400>180

<210>181

<211>23

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1827EX

<222>(1)..(23)

<223>

<400>181

<210>182

<211>26

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1798

<222>(1)..(26)

<223>

<400>182

<210>183

<211>13

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1829

<222>(1)..(13)

<223>

<400>183

<210>184

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1579

<222>(1)..(16)

<223>

<400>184

<210>185

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1848

<222>(1)..(16)

<223>

<400>185

<210>186

<211>17

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A1A7

<222>(1)..(17)

<223>

<400>186

<210>187

<211>21

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A122X

<222>(1)..(21)

<223>

<400>187

<210>188

<211>16

<212>PRT

＜213〉hepatitis C virus

<220>

<221>1825

<222>(1)..(16)

<223>

<400>188

<210>189

<211>15

<212>PRT

＜213〉hepatitis C virus

<220>

<221>A241

<222>(1)..(15)

<223>

<400>189

<210>190

<211>18

<212>PRT

＜213〉hepatitis C virus

<220>

<221>B8B38

<222>(1)..(18)

<223>

<400>190

<210>191

<211>20

<212>PRT

＜213〉hepatitis C virus

<220>

<221>C70EX

<222>(1)..(20)

<223>

<400>191

Claims

1. separate the hepatitis c virus peptide that has in conjunction with the ability of MHC/HLA molecule, or contain the method for the compound of hepatitis c virus peptide and this MHC/HLA molecule, the method is characterized in that the following step:

-provide a hepatitis c virus peptide storehouse of containing at least 10 kinds of different hepatitis c virus peptides, this storehouse to contain hepatitis c virus peptide that can combine and the hepatitis c virus peptide that does not combine with this MHC/HLA molecule with this MHC/HLA molecule,

-make this MHC/HLA molecule contact this hepatitis c virus peptide storehouse, thus the hepatitis c virus peptide that has in conjunction with the ability of this MHC/HLA molecule is combined with this MHC/HLA molecule, form the compound that contains this hepatitis c virus peptide and this MHC/HLA molecule,

-detect this compound, randomly make its with do not separate in conjunction with the hepatitis c virus peptide of this MHC/HLA molecule and

-randomly from this compound, separate and characterize this hepatitis c virus peptide.

2. separate the hepatitis C virus t cell epitope that has in conjunction with the ability of MHC/HLA molecule, or contain the method for the compound of this epi-position and this MHC/HLA molecule, the method is characterized in that the following step:

-provide a hepatitis c virus peptide storehouse of containing at least 10 kinds of different hepatitis c virus peptides, this storehouse to contain hepatitis c virus peptide that can combine and the hepatitis c virus peptide that does not combine with this MHC/HLA molecule with a kind of MHC/HLA molecule,

-detect this compound, it is separated with the hepatitis c virus peptide in conjunction with this MHC/HLA molecule not,

-randomly from this compound, separate and the sign hepatitis c virus peptide,

-in the T raji cell assay Raji, measure the described hepatitis c virus peptide that randomly separates or this compound the t cell activation ability and

-provide the hepatitis c virus peptide that randomly separates as hepatitis C virus t cell epitope or compound with t cell activation ability.

3. according to the method for claim 1 or 2, it is characterized in that this hepatitis c virus peptide set is selected from: the peptide storehouse; The protein fragments storehouse; The modified peptides storehouse; By the storehouse that antigen presenting cell obtains, it is the form of total lysate or fraction; The storehouse that comprises cell fragment; The storehouse that comprises peptide library; By the peptide of recombinant DNA library generation or the storehouse of polypeptide; Storehouse from the protein and/or the protein fragments of hepatitis C virus; Or its potpourri.

4. according to the method for claim 3, wherein said peptide storehouse is the storehouse of overlapping peptide.

5. according to the method for claim 3, the wherein said storehouse that is obtained by antigen presenting cell is the fraction of wash-out on the surface of these cells or the MHC/HLA molecule.

6. according to the method for claim 3, the storehouse that wherein comprises cell fragment is the storehouse of the cell, tumour cell or the tissue that comprise HCV.

7. according to the method for claim 6, wherein said cell, tumour cell or tissue are from liver.

8. according to the method for claim 3, the wherein said peptide storehouse that is produced by the recombinant DNA library derives from pathogen or tumour cell.

9. according to each method among the claim 1-3, it is characterized in that described MHC/HLA molecule is selected from: HLA I quasi-molecule, HLA II quasi-molecule, non-traditional MHC/HLA and MHC/HLA-sample molecule or its potpourri, or their potpourri.

10. according to each method among the claim 1-3, it is characterized in that utilizing being selected from the hepatitis c virus peptide that following method characterizes this compound: mass spectroscopy; Sequencing polypeptides; In conjunction with measuring; Identify hepatitis c virus peptide by measuring retention factors, method is a chromatography, or ultraviolet ray, infrared ray, nuclear magnetic resonance, circular dichroism or electron spin resonance, or its combination.

11. according to the method for claim 10, wherein said is that SDS-stability is measured in conjunction with measuring.

12. according to the method for claim 10, wherein said chromatography is HPLC.

13., it is characterized in that it and a kind of cytokine secretion measure combination according to each method among the claim 1-3.

14., it is characterized in that it and Elispot mensuration, the dyeing of the cell within a cell factor, FACS or ELISA make up according to the method for claim 13.

15., it is characterized in that described T raji cell assay Raji comprises this compound and the T mixing with cells and the incubation that separate, measures the cytokine secretion or the propagation of the T cell of described separation subsequently according to each method among the claim 1-3.

16., it is characterized in that described T raji cell assay Raji comprises the rise of measuring activation tagging, or the downward modulation of surface indicia according to each method among the claim 1-3.

17. according to the method for claim 16, wherein said activation tagging is CD69, CD38, surface indicia is CD3, CD8 or TCR.

19., it is characterized in that described T raji cell assay Raji comprises by real-time RT-PCR to measure accent/downward modulation on the mRNA that participates in t cell activation according to each method among the claim 1-3.

20., it is characterized in that described T raji cell assay Raji is selected from: measure phosphorylation/dephosphorylized T raji cell assay Raji that the TXi Baoshouti downstream becomes branchs, measure the interior Ca of cell according to each method among the claim 1-3 ⁺⁺Concentration or Ca ⁺⁺The protein activated T cells of-dependence is measured, and measures the T raji cell assay Raji that immune cynapse forms, the T raji cell assay Raji that the measuring effect molecule discharges, or the combination of these T raji cell assay Rajis.

21. according to the method for claim 20, wherein said TXi Baoshouti downstream composition is selected from ITAMS and zeta chain, ZAP70, LAT, SLP-76, fyn and the lyn of p56lck, TCR.

22. according to the method for claim 20, wherein said effector molecule is perforin, granzyme or granulysin (granulolysin).