CN1711279B

CN1711279B - Epstein Barr virus peptide epitopes, polyepitopes and delivery system therefor

Info

Publication number: CN1711279B
Application number: CN200380102880.3A
Authority: CN
Inventors: 拉吉夫·康纳; 贾库马尔·杜赖斯瓦米
Original assignee: Queensland Institute of Medical Research QIMR
Current assignee: QIMR Berghofer Medical Research Institute
Priority date: 2002-11-07
Filing date: 2003-11-03
Publication date: 2010-05-26
Anticipated expiration: 2023-11-03
Also published as: CN1711279A; AU2002952524A0

Abstract

Immunogenic cytotoxic T cell epitope peptides of Epstein Barr Virus LMP1 protein are provided having between nine and seventeen amino acids and minimal consensus sequences selected from QRH, AGNDG, QNW, VLYS and DSNSNE. These peptides, either individually or in polyepitope constructs, are useful in pharmaceutical compositions, vaccines and methods of treating Epstein Barr Virus associated diseasessuch as Hodgkin's Disease and/or Nasopharyngeal Carcinoma, although without limitation thereto. A preferred mode of polyepitope delivery is an adenovirus-based system.

Description

Epstein Barr virus peptide epitopes, multi-epitope and delivery system thereof

Technical field

The present invention relates to the immunogenic peptide of Epstein Barr virus.Especially, the present invention relates to proteic cytotoxic T cell epi-position derived from Epstein Barr virus LMP1.The present invention also provides the pharmaceutical composition that contains one or more Epstein Barr virocyte toxicity T cell epitopes, multi-epitope, based on the method for the vaccine delivery system of adenovirus and the treatment disease relevant with Epstein Barr virus, these diseases for example have Hokdkin disease and/or nasopharyngeal carcinoma but are not limited thereto.The present invention also provides a kind of method of the Epstein of evaluation Barr virocyte toxicity T cell epitope.

Background technology

Epstein Barr virus (EBV) is not only one of the widest human virus of distribution, some contradiction be, it also relevant (Anagnostopoulos 1996) with some tumours.This comprises various B cells and T cell non-Hodgkin's, Hokdkin disease, and some lymphocytic epithelium warty cancers, and its typical case is nasopharyngeal carcinoma (NPC).The relation of these tumours and EBV, and EBV puts down in writing at the existing document of external carcinogenic potential, and (Rickinson 1996; Khanna 2000).CD8 ⁺The T cytoactive is played an important role in control EBV infects, and this is derived from being realized to the small-molecular peptides of the infected cells on surface by MHC I type molecular presentation by identification.EBV specificity cell toxicity T lymphocyte (CTL) preparation can make external by the memory T cell of stimulation from healthy virus carrier's peripheral blood, and this carrier is contained (LCL) cell (Khanna1992 of lymph sprout cell system that is transformed by self EBV; Murray 1992).In a kind of LCL, EBV expresses six nuclear antigen (EMNAs1,2,3A, 3B, 3C and LP), and two resting form membranins (LMP1 and LMP2).Wherein, EBNA3 family (EBNA 3A, 3B, 3C) at CTL to (Khanna 1992 for immundominance in the replying of many human leucocyte antigens (HLA); Murray etc. 1992).

The tumour cell of Hokdkin disease and nasopharyngeal carcinoma all can be expressed viral protein, is known as CTL the target epi-position is provided.These two kinds of malignant tumours are expressed nuclear antigen EBNA1, BARF0, LMP1 and LMP2.EBNA1 comprises that a glycine-L-Ala repeats (GAr), and this antigen is used as cis and suppresses signal in the proteasome degraded, thereby and blocks the interior CTL epi-position endogenous of this antigen and present (Levitskaya 1995).The CTL of BARF0 epi-position presents by the differential splicing of virus transcription body to be influenced, and the result has produced the dominance protein isoforms (Kienzle 2000) that the CTL determinant has been removed.Opposite with EBNA1 and BARF0, LMP1 and LMP2 both are the strong targets of EBV specific CTL, so many attentions all concentrate on the target epi-position of differentiating in these two kinds of antigens, and (Lee 1997; Khanna 1998; Meij 2002).

LMP1 is a transmembrane protein, and its kytoplasm N-terminal and C-terminal structural domain are separated by 6 TMDs.LMP1 has been considered to one of most important resting form albumen, is used for the normal B transformation of EBV mediation, and can be in transgenic mouse uniquely inducing malignant tumor generate and hyperplasia (Kulwichit 1998).And also known LMP1 presents pleiotropy to the cell phenotype of B cell, and comprising induce (Wang 1990) of active antigen, (Henderson 1991 in the expression of apoptosis supressor; Laherty 1992), and via the NF-kB activation of TRAF signal pipeline (Hammarskjold 1992; Mosialos 1995).Previous studies show that activated receptors sample molecule on a kind of structure of acting as of LMP1, irrelevant with combining of part.The C-terminal of LMP1 conducts by C-terminal active agent zone (being meant CTAR1, amino acid/11 94-231 and CTAR2, amino acid 332-386 and CTAR3, amino acid 275-330) priming signal.CTAR1 is relevant with inducing of NF-κ B with the CTAR2 zone, and CTAR2 is main NF-kB activation agent site, and the CTAR3 structural domain is combined (Gires 1999) by report recently with Janus kinases 3.

Summary of the invention

The present inventor has adopted a kind of new analysis based on IFN-γ, carries out the sequential analysis that LMP1 specific T-cells is widely replied in many papovas carrier.This method combines with the functional cell oxicity analysis and dissolves the ability of the target cell that is infected by EBV with evaluation LMP1 specific CTL.

Put it briefly, the invention provides a kind of new and effective EBV peptide derived from LMP1, it is suitable for the immunotherapy to the tumour that comprises B cell and T cell non-Hodgkin's, Hokdkin disease and the lymphocytic epithelium warty cancer as nasopharyngeal carcinoma (NPC) so potentially.

The present invention also provides a kind of vaccine delivery system based on adenovirus, and this system is especially effective when being used to carry EBV multi-epitope structure.

Therefore, provide a kind of isolating EBV ctl peptide epi-position among the present invention, it has comprised proteic at least 9 the successive amino-acid residues of LMP1, and wherein said EBV ctl peptide is not YLQQNWWTL (SEQ ID NO:1) or YLLEMLWRL (SEQ ID NO:3).

First aspect the invention provides a kind of isolating EBV ctl peptide epi-position, and it comprises aminoacid sequence QRH (SEQ ID NO:4).

In a preferred embodiment, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)QRHSDEHHH(SEQ ID NO：9)；

(ii)GQRHSDEHH(SEQ ID NO：10)；

(iii) YYHGQRHSD (SEQ ID NO:11); With

(iv)WMYYHGQRH(SEQ ID NO：12)。

In other embodiment of first aspect, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)YYHGQRHSDEHH(SEQ ID NO：13)；

(ii) IWMYYHGQRHSD (SEQ ID NO:14); With

(iii)LIWMYYHGQRHSDEHHH(SEQ ID NO：15)

Second aspect the invention provides a kind of isolating EBV ctl peptide epi-position, and it comprises aminoacid sequence AGNDG (SEQ ID NO:5).

In a preferred embodiment of second aspect, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i) AGNDGGPPQ (SEQ ID NO:16); With

(ii)PSDSAGNDG(SEQ ID NO：17)。

In other embodiment of second aspect, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)SDSAGNDGGPPQ(SEQ ID NO：18)；

(ii) DSAGNDGGPPQL (SEQ ID NO:19); With

(iii)PHSPSDSAGNDGGPPQL(SEQ ID NO：20)。

The third aspect the invention provides a kind of isolating EBV ctl peptide epi-position, and it comprises aminoacid sequence QNW (SEQ ID NO:6), especially, does not wherein comprise sequence YLQQNWWTL (SEQ ID NO:1).

In a preferred embodiment of the third aspect, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)IALYLQQNW(SEQ ID NO：21)；

(ii)ALYLQQNWW(SEQ ID NO：22)；

(iii) QNWWTLLVD (SEQ ID NO:23); With

(iv)LYLQQNWWT(SEQ ID NO：24)。

In other embodiment of the third aspect, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)IALYLQQNWWTL(SEQ ID NO：25)；

(ii) YLQQNWWTLLVD (SEQ ID NO:26); With

(iii)LIIALYLQQNWWTLLVD(SEQ ID NO：27)。

Fourth aspect the invention provides a kind of isolating EBV ctl peptide epi-position, and it comprises aminoacid sequence VLYS (SEQ ID NO:7).

In the preferred embodiment therein, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)ALLVLYSFAL(SEQ ID NO：28)；

(ii)LLVLYSFAL(SEQ ID NO：29)；

(iii) ALLVLYSFA (SEQ ID NO:30); With

(iv)VLYSFALML(SEQ ID NO：31)。

In other embodiment of fourth aspect, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)ALLVLYSFALML(SEQ ID NO：32)；

(ii)GALLVLYSFALM(SEQ ID NO：33)；

(iii)DWTGGALLVLYS(SEQ ID NO：34)；

(iv) GGALLVLYSFAL (SEQ ID NO:35); With

(v)DWTGGALLVLYSFALML(SEQ ID NO：36)。

The 5th aspect the invention provides a kind of isolating EBV ctl peptide epi-position, and it comprises aminoacid sequence DSNSNE (SEQ ID NO:8), especially, does not wherein comprise aminoacid sequence ESDSNSNEG (SEQ ID NO:2).

In the preferred embodiment aspect the 5th, described epi-position comprises the aminoacid sequence that is selected from by the following group of forming:

(i)DSNSNEGRH(SEQ ID NO：37)；

(ii) SGHESDSNSNEG (SEQ ID NO:38); With

(iii)TDDSGHESDSNSNEGRH(SEQ ID NO：39)。

The present invention also provides a kind of varient EBV CTL epi-position.

In special embodiment, described varient contains aminoacid sequence as shown in table 4 (SEQ ID NO:40-44 and 47-50).

The 6th aspect the invention provides a kind of isolating albumen, and this albumen comprises at least one the EBV CTL epi-position according to above-mentioned each side.

Preferably, this isolating albumen is multi-epitope albumen, and it comprises the aminoacid sequence that is selected from the group of being made up of ALLVLYSFA (SEQ ID NO:30) and IALYQQNW (SEQ ID NO:21).

In a preferred embodiment, this isolating multi-epitope albumen comprises each EBV CTL epi-position as shown in table 5.

In a preferred embodiment, this isolating multi-epitope albumen comprises the aminoacid sequence shown in SEQID NO:81.

A seventh aspect of the present invention provides a kind of isolating nucleic acid, the EBV CTL epi-position or the multi-epitope of the above-mentioned either side of this nucleic acid encoding.

In a preferred embodiment, the listed multi-epitope aminoacid sequence of this isolating nucleic acid encoding such as SEQ ID NO:81.

In this embodiment, more preferably this isolating nucleic acid encoding has the listed nucleotide sequence as SEQ ID NO:82.

This aspect also provides a kind of isolating nucleic acid, the EBV CTL epi-position varient of the above-mentioned each side of this nucleic acid encoding.

In special embodiment, described isolating nucleic acid comprises a kind of nucleotide sequence as shown in table 4 (SEQ ID NO:63-65,67-69,71-76 and 78-80).

Eight aspect the invention provides a kind of expression structure, and it comprises the isolating nucleic acid of the 6th aspect of the one or more regulatory nucleotide sequences that are operably connected in the expression vector.

In a preferred embodiment, this expression structure is based on adenovirus.

In a special embodiment, described expression structure is the multi-epitope structure of most EBV CTL epi-positions in the code book invention.

The 9th aspect the invention provides a kind of host cell or organism, and it includes the expression structure of eight aspect.

The tenth aspect the invention provides a kind of pharmaceutical composition, and it comprises according to the EBV CTL epi-position of above-mentioned either side or isolating nucleic acid or its expression structure of encoding.

Preferably, this pharmaceutical composition is a kind of immunotherapy composition.

More preferably, this pharmaceutical composition is a kind of vaccine.

The tenth on the one hand, the invention provides the method for a kind of treatment disease relevant with EBV, comprises animal is used one or more EBV CTL epi-positions of the present invention or its step of expression structure of encoding.

Method in this respect comprises administration of protein and nucleic acid composition, is used for treatment of diseases relevant with EBV and/or prevention.

Although be not limited thereto, the disease relevant with EBV preferably includes various B and T cell non-Hodgkin's, Hokdkin disease, and several lymphocytic epithelium warty cancer, the wherein form of nasopharyngeal carcinoma (NPC) for being gazed at especially.

Preferably, this animal is a Mammals.

More preferably, this animal is the people.

In a preferred embodiment, the method for the tenth aspect further comprises the step of selecting one or more EBV CTL epi-positions according to the people's that will receive treatment HLA type.

The 12 aspect the invention provides a kind of method of the EBV of evaluation CTL epi-position, and aforesaid method may further comprise the steps:

(i) preparation is in a large number derived from the proteic different peptides of LMP1;

(ii) above-mentioned one or more described peptides are combined with one or more T lymphocytes available from EBV seropositivity individuality; With

(iii) measure to the lymphocytic IFN-γ of above-mentioned one or more T output in the replying of above-mentioned one or more peptides, wherein IFN-γ output is higher than reference quantity and represents that promptly described a kind of or a plurality of peptide has at least a EBV CTL epi-position.

In a preferred embodiment, the method for this aspect comprises that further step (iv) detects above-mentioned one or more T lymphocytes whether step is (ii) produced and can dissolve the target cell that one or more are infected by EBV.

The 13 aspect the invention provides a kind of antibody, and this antibody capable combines with one or more EBV CTL epi-positions according to above-mentioned each side of the present invention.

The 14 aspect, the invention provides and a kind ofly detect whether animal carries or the method for contacted EBV once, described method comprises such step: one or more T cells that will separate from above-mentioned individuality contact with one or more EBV peptides of the present invention, and above-mentioned thus one or more T cells can indicate this animal to carry EBV or once contacted EBV to the replying of at least one in one or more peptides.

Preferably, this animal is a Mammals.

More preferably, this animal is the people.

This specification unless otherwise indicated, comprises everywhere " " be meant " included " rather than " foreclose ", therefore the integral body of being stated can comprise one or more integral body that other is not stated.

Accompanying drawing and explanation of tables

The healthy contributor of table 1:EBV immunity or the HLA antigenic type (I type) of NPC sufferer are included in this specification sheets.

Table 2: by the EBV specific T-cells from LMP1 sequence list that healthy virus carrier recognized.

Table 3:T cell is to the answer frequency of MHC I type and the restricted LMP1 epi-position of MHC II type, and this epi-position is present among not agnate healthy individual and the NPC patient.

Table 4: by the isolated sequence that is present in the restricted LMP1 epi-position of HLA I type among the EBV of Caucasian, African, Chinese, New guineans and Indonesian.The nucleotide sequence of each epi-position of encoding all is expressed out with the sequence change that is present in the particular separation body.The peptide sequence varient of SEQ ID NO:30 is represented as SEQ ID NO:40.The peptide sequence varient of SEQID NO:21 is represented as SEQ ID NO:41-43.Peptide sequence varient YLLEMLWRL (SEQ ID NO:3) is represented as SEQ ID NO:44-48.Peptide sequence varient YLQQNWWTL (SEQ ID NO:1) is represented as SEQ ID NO:49 and 50.The nucleotide sequences of encoding wild type and varient epi-position is represented as SEQ ID NO:62-80.

Table 5:EBV multi-epitope peptide sequence and HLA specificity.YLLEMLWRL(SEQ IDNO：3)；YLQQNWWTL(SEQ ID NO：1)；ALLVLYSFA(SEQ IDNO：30)；IAYLQQNW(SEQ ID NO：21)；SSCSSCPLSKI(SEQ ID NO：51)；PYLFWLAAI(SEQ ID NO：52)；TYGPVFMCL(SEQ ID NO：53)；RRRWRRLTV(SEQ ID NO：54)；LLSAWILTA(SEQ ID NO：55)；LTAGFLIFL(SEQ ID NO：56)；VMSNTL LSAW(SEQ ID NO：57)；IEDPPFNSL(SEQ ID NO：58)；CLGGLLTMV(SEQ ID NO：59)。

Fig. 1: the LMP1 specific T-cells in the seropositivity individuality of one group of health is replied distribution plan in the body.A: use over lapping synt hetic peptides (10 μ g/ml) to stimulate PMBC from the seropositivity individuality of health from LMP1, and INF-γ output with as material and the described ELISPOT analytical method measurement of method part.The result is represented as per 10 ⁶The number of spot formation cell among the PBMC (spot forming cell, SFC).The chart of the T cytoactive in the B:LMP1 albumen distributes.C: in the ELISPOT analytical method by the aminoacid sequence of the peptide epitopes that the virus carrier discerned of health: peptide 9 (SEQ ID NO:36); Peptide 17 (SEQ IDNO:60); Peptide 21 (SEQ ID NO:27); Peptide 24 (SEQ ID NO:15); Peptide 25 (SEQ ID NO:61); Peptide 27 (SEQ ID NO:39); Peptide 38 (SEQ ID NO:20).

Fig. 2: by CD4 ⁺With CD8 ⁺Detect in the T cell colony excretory IFN-γ body, continue again with the stimulation of LMP1 peptide.With the anti-human CD4 described in materials and methods or CD8 immune magnetic particle with CD4 ⁺Cell or CD8 ⁺Cell is got rid of from fresh PBMCs.These cells are resuspended in growth medium, use DWTGGALLVLYSFALML peptide (SEQ ID NO:36 again; Group A) with TDDSGHESDSNSNEGRH peptide (SEQ IDNO:39; Group B) (10 μ g/ml) stimulates, and detects INF-γ secretion situation with the ELISPOT analytical method then.

Fig. 3: minimum epitope sequences is figure with the ELISPOT analytical method.Stimulate from contributor MM (group A and B) with overlapping peptide (5 μ g/ml), RE (group C), the PBMC of LL (group D), and INF-γ output is with measuring as material and the described ELISPOT analytical method of method part.The result is represented as per 10 ⁶Spot forms the number of cell (SFC) among the PBMC.

Fig. 4: minimum epitope sequences is figure with the ELISPOT analytical method.Stimulate from contributor LL (group A) with overlapping peptide (1 μ g/ml), MM (group B), the PBMC of RE (group C and D), and INF-γ output is measured in as material and the described ELISPOT analytical method of method part.The result is represented as per 10 ⁶Spot forms the number of cell (SFC) among the PBMC.

Fig. 5: A: to the HLA I type restriction analysis of IALYLQQNW specific CTL clone MM22.Some have with contributor MM, and identical HLA I type is allelic to contact with ctl clone MM22 with LCL and the PHA protoblast that allos EBV transforms from body.The PHA protoblast carries out quick in advance with the IALYLQQNW peptide epitopes.With the ratio of target be that 2: 1 effector is used in this analysis.B: the CTL that is discerned by ctl clone MM23 discerns the IALYLQQNW epi-position.To PHA protoblast sensitization, be exposed to IALYL QQNW specific CTL clone MM23 with a series of dilution peptides then.

Fig. 6: A: the ALLVLYSFA specific CTL system that derives from contributor LL is carried out HLA I type restriction analysis.Under this peptide existence or non-existent situation, some have with contributor LL, and identical HLA I type is allelic to contact with epitope specificity CTL system with allos PHA protoblast from body.With the ratio of target be that 10: 1 effector is used in this analysis.B: the CTL that is come from contributor LL is the CTL identification ALLVLY SFA epi-position of being discerned.To PHA protoblast sensitization, be exposed to ALLVLYSFA specific CTL system with a series of dilution peptides then.

Fig. 7: A: the CTL identification of varient and the restricted LMP1 epi-position of prototype HLAA2 YLLEMLWRL.The diluent sensitization of PHA protoblast and a series of each peptide is exposed to arbitrary YLLEMLWRL specific CTL clone SB7 then.B: on the T2 cell, do the MHC stability analysis with varient and the restricted LMP1 epi-position of prototype HLAA2.Originally the T2 cell uses every kind of peptide (10 μ g/ml) of 200 μ l to hatch under 26 ℃ 14-16 hour, then hatches under 37 ℃ 2-3 hour.The expression of HLAA2 on these cells analyzed with HLAA2 monoclonal antibody specific FACS.

Fig. 8: the structure iron of recombinant adenoviral vector, this vector expression coding contains the proteic synthetic DNA of multi-epitope (SEQ ID NO:79) of 13 kinds of restricted LMP1 of HLAI type and LMP2 epi-position (SEQ ID NO:79 also sees table 5).The proteic dna sequence dna of this multi-epitope of encoding is to use sequence specific primers (being expressed as LMP-A, LMP-B, LMP-C and LMP-D) and constitutes based on the technology of mutual guiding (mutual priming) with overlapping extension.BamH I restriction enzyme site of this segmental nucleic acid sequence encoding (from 5 ' end), one section Kozak sequence, a methionine(Met) initiator codon, 13 minimum LMP1 CTL epi-positions that front and back link to each other, a terminator codon, and an EcoR I restriction enzyme site at 3 ' end.The insertion fragment of LMP multi-epitope is to downcut and be cloned into the pAdTrack expression vector from the pcDNA3.1 expression vector.After amplification and the reorganization, the Ad5-LMPpoly carrier is wrapped in the transfection sexual gland virus by human embryonic kidney (HEK) 293 cells of transfection in E.coli.Gather in the crops recombinant adenovirus by freezing with thawing.

Fig. 9: by the endogenous processing of the CTL epi-position of Ad5LMP multi-epitope coding.

Figure 10: the immunogenicity of Ad5LMP multi-epitope.

The immunization of Figure 11: Ad5-LMPpoly is to expressing EL4-A2/K ^bThe LMP1 of tumour cell provides protection.Two groups of mouse, 6 every group, respectively with Ad5-LMPpoly or control group adenovirus (10 ⁸PFU/ mouse) immunity.EL4-A2/K is used in back 21 days of immunity ^b-LMP1 cell (10 ⁷An individual cell/mouse) mouse is carried out subcutaneous attack, and after attack 16 days monitoring tumour sizes.The tumour size data with on average ± standard deviation represents.

Detailed Description Of The Invention

This specification has been described many new and the unexpected EBV CTL epi-positions derived from LMP 1 albumen. New analyze to identify EBV CTL epi-position based on IFN-γ with a kind of, and analyze to assess the ability that the LMP1 specific CTL dissolves the target cell that is infected by EBV with the function cytotoxic.

Astoundingly, CTL epi-position of the present invention preferentially derived from the membrane spaning domain of CTAR1 and LMP1 albumen, does not in fact comprise CTAR2 domain and N end.

EBV peptide derived from LMP1 of the present invention is applicable to the immunotherapy of the disease that treatment is relevant with EBV potentially. Especially, the tumour relevant with EBV of considering among the present invention comprises B cell and T cell non-Hodgkin's, the lymphocytic epithelium warty cancer that Hodgkin's disease and for example nasopharyngeal carcinoma (NPC) are such.

For realizing purpose of the present invention, " separate " mean material and be removed by its nature and maybe can reach by human use. The material that separates can fully or basically be separated with its composition of following under nature, or can be handled so that it with its nature in the composition followed coexist. The material that separates can be natural, chemical synthesis or recombinant forms.

" protein " refers to a seed amino acid condensate, and this amino acid can be natural amino acid nature or non-, D-or L-type amino acid, or chemically derived amino acid known in the art.

" peptide " refers to have and is no more than 50 amino acid whose protein.

" polypeptide " refers to have the amino acid whose protein that surpasses more than 50.

" EBV CTL epi-position " refers to a kind of amino acid sequence by the EBV genome encoding, in external or body, in the situation that has MHC I type to exist, when this amino acid sequence ran at least a T cell clone type (clonotype), it can cause immune response by at least a T cell clone type (clonotype). T cell epitope is not got rid of in this definition in addition, for example helper cell or B cell epitope.

Consensus amino acid sequences shown in SEQ ID NO:4-8 is common region (listing in table 2) minimum in the EBV ctl peptide of the present invention (SEQ ID NO:2,9-25 and 27-39).

Compatibly, above-mentioned EBV ctl peptide epi-position is by at least 9 but be no more than 20 continuous amino acid and formed.

In certain embodiments, above-mentioned EBV ctl peptide epi-position is by 9, and 12 or 17 continuous amino acid form.

In a preferred embodiment, the amino acid of above-mentioned EBV ctl peptide epi-position with the following group that makes of choosing group: SEQ ID NO:2; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31 and SEQ IDNO:37.

The present invention has also considered the albumen of the separation that polypeptide for example or multi-epitope albumen are such, its comprise of the present invention one or more, or preferred a large amount of EBV CTL epi-position. For example, described epi-position can be individualism or repeat to exist, wherein also comprise tandem repetition epi-position. " interval base " amino acid also can be included between one or more these EBV CTL epi-positions in the albumen that is present in above-mentioned separation.

In one embodiment, a kind of albumen of purifying may be made up of one or more or preferred a large amount of EBV CTL epi-position of the present invention.

In another embodiment, basically can serve as reasons one or more or preferred a large amount of EBV CTL epi-position of the present invention of a kind of albumen of purifying forms.

" basically by ... form " refer to that except EBV CTL epitope sequences every kind of EBV ctl peptide epi-position has and is no more than 5, or preferably is no more than 3 amino acid.

With regard to special multi-epitope albumen, these extra amino acid residues can refer to " the interval base " amino acid.

Also find, multi-epitope albumen of the present invention can comprise one or more other EBV ctl peptides extraly, for example one or more list in the LMP epi-position of the prior art of table 2 or table 5, and/or EBV CTL epi-position YLLEMLWRL (SEQ ID NO:2) and YLQQNWWTL (SEQ ID NO:1).

In a preferred embodiment, the multi-epitope albumen of separation comprises a large amount of restricted LMP1 of MHC I type and/or LMP2 CTL epi-position.

Preferably, have at least a kind of above-mentioned CTL epi-position to have the amino acid sequence that is selected from by the following group that consists of: ALLVLYSFA (SEQ ID NO:30) and IALYQQNW (SEQ IDNO:21).

In an especially preferred embodiment, the multi-epitope of separation comprises 13 kinds of EBVCTL epi-positions, and this epi-position has respectively following amino acid: YLLEMLWRL (SEQ IDNO:3); YLQQNWWTL (SEQ ID NO:1); ALLVLYSFA (SEQ IDNO:30); IAYLQQNW (SEQ ID NO:21); SSCSSCPLSKI (SEQ ID NO:51); PYLFWLAAI (SEQ ID NO:52); TYGPVFMCL (SEQ IDNO:53); RRRWRRLTV (SEQ ID NO:54); LLSAWILTA (SEQ ID NO:55); LTAGFLIFL (SEQ ID NO:56); VMSNTLLSAW (SEQ IDNO:57); IEDPPFNSL (SEQ ID NO:58); CLGGLLTMV (SEQ IDNO:59).

More preferably, the multi-epitope albumen of this separation has the amino acid sequence shown in SEQ ID NO:81 (figure .8).

CTL epi-position in the multi-epitope among table 5 and Fig. 8 is the screened widely epi-position of MHC I type specificity that comprised. For example, estimate that these most preferred HLA specificitys have comprised about 90% Asia, Africa and Caucasia crowd.

The multi-epitope albumen (SEQ ID NO:81) that is also noted that the separation among Fig. 8 is confirmed can induce by the CD8 in the mouse of multi-epitope immunity by the application's inventor⁺CTL replys, and this is replied and can protect mouse not to be subjected to the attack of tumour.

Also considered simultaneously the variant of EBV CTL epi-position.

In general, term used herein " variant " be EBV CTL epi-position of the present invention, wherein deleted one or more amino acid or replaced by different amino acid, but do not change its immunogenicity in essence. Well known some amino acid can be changed to other amino acid with extensively similar characteristic, but does not change its immunogenicity (the conservative replacement).

Selective replace (selecting substitution) can cause the essence of function to change, and the conservative degree of selectively changing is littler, and its degree that can be stood is also relatively low. In general, may cause the maximum replacement that changes of protein properties be those wherein (a) hydrophily residue (such as Ser or Thr) replaced by hydrophobic residue (such as Ala, Leu, Ile, Phe or Val); (b) cysteine or proline are replaced by other any residue; (c) residue (such as Arg, His or Lys) that has a positively charged side chain is replaced by electronegative residue (such as Glu or Asp); Or the residue (such as Phe or Trp) that (d) has bulky side chain is had the residue (such as Ala or Ser) of little side chain or the residue (such as Gly) of unprotected side chain replaces.

The specific example of abiogenous variant EBV CTL epi-position is provided by table 4 and SEQ IDNO:44-44 and 47-50, and these variants are derived from the EBV separator in specific race zone, and will be discussed in detail following.

As seen from Table 4, the variant EBV CTL epi-position that provides and SEQ ID NO:1, SEQ ID NO:21 and SEQ ID NO:30 have an amino acid whose difference. The variant of SEQ ID NO:3 has the difference of 1,2 or 3 amino acid residue.

The present invention has also discussed EBV CTL epi-position of the present invention " derivative "; the for example chemical modification by amino acid residue; biotinylation; be combined with fluorescent dye; add Epitope tag (such as c-myc; hemagglutinin and FLAG mark); and be convenient to expression of recombinant proteins, detection and purifying (such as glutathione-S-transferase, green fluorescent protein; six histidines and maltose-binding protein, but be not limited thereto) fusion partner (fusion partners) generate.

Amino acid whose chemical modification includes but not limited to; acylation is modified; amidineization; the pyridoxylation of lysine; standard reductive alkylation; with 2; 4,6-trinitrobenzen sulfuric acid (TNBS) carries out trinitrobenzen with amino and turns usefulness into, modifies with sulfhydryl by the acid amides that the cysteine performic oxidation is become the sulfo group third ammonia ammonia carry out carboxyl and modifies; the formation of mercurial derivative; the formation of the mixture of disulphide and other mercaptan compound, with the reaction of maleimide, the carboxy methylation of iodoacetic acid or iodoacetamide; and under alkaline pH the carbamylation of cyanate, although be not limited thereto.

In this respect, those skilled in the art can be with reference to (John Wiley and Sons NY such as CURRENT PROTOCOLSIN PROTEIN SCIENCE Eds.Coligan, 1995-2000) the 15th chapter wherein has widely relevant with chemical modification method.

EBV CTL epi-position of the present invention, EBV multi-epitope and the protein that comprises this epi-position can be produced by any known method of the prior art, includes but not limited to, chemical synthesis, the proteolytic cleavage of DNA recombinant technique and LMP albumen is to produce fragments of peptides.

In one embodiment, EBV CTL epi-position can prepare by chemical synthesis, and it is synthetic to comprise solid phase and liquid phase. These methods are well known in the art, although the embodiment of chemical synthesising technology is arranged in the list of references, provide such as the 15th chapter of (John Wiley and Sons, Inc.NY USA 1995-2001) such as the 9th chapter of SYNTHETIC VACCINES Ed.Nicholson (Black hole Scientific Publications) and CURRENTPROTOCOLS IN PROTEIN SCIENCE Eds.Coligan. Aspect this, list of references also sees WO 99/02550 and WO 97/45444.

In another embodiment, restructuring EBV CTL epi-position of the present invention, or preferred, the protein that comprises EBV CTL epi-position, can adopt the standard test step to prepare easily by those skilled in the art, such as Sambrook etc., molecular cloning: laboratory manual (cold spring port publishing house, 1989), the 16th and 17 chapters particularly; CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Eds.Ausubel etc., (John Wiley and Sons, Inc.NY, USA, 1995-2001), particularly the 10th and 16 chapters; And CURRENTPROTOCOLS IN PROTEIN SCIENCE Eds.Coligan etc., (John Wiley and Sons, Inc.NY, USA, 1995-2001), particularly the 1st, 5 and 6 chapters.

Nucleic acid and expression structure

The invention provides a kind of nucleic acid of separation, its encode EBV CTL epi-position of the present invention or multi-epitope.

Code book is invented through the nucleotide sequence of the EBV CTL epi-position of selection and variant thereof as shown in table 4. Code book is invented the nucleotide sequence of other EBV CTL epi-position, can easily be derived out by published complete LMP1 nucleic acid sequence encoding, for example the Genbank number of obtaining X58140.

The present invention also provides the nucleotide sequences of coding EBV peptide variant, example (SEQ ID NO:63-65,67-69,71-76 and 78-80) as shown in table 4.

Nucleotide sequences such as the SEQ ID NO:82 and shown in Figure 8 of the separation of coding multi-epitope albumen.

Term used herein " nucleic acid " refer to strand or double-stranded mRNA, RNA, cRNA, RNAi and DNA, wherein DNA comprises cDNA and genomic DNA.

" polynucleotides " are the nucleic acid with the continuous nucleotide more than 80 or 80, and " oligonucleotides " have and be less than 80 continuous nucleotide.

" probe " can be strand or double chain oligonucleotide or polynucleotides, suitably is used for behind the mark detecting its complementary series at Northern or Southern hybridization.

" primer " is generally single stranded oligonucleotide, preferably has 15-50 continuous nucleotide, it can with its complementary nucleic acid template annealing, and by for example Taq polymerase, depend on polymerase or the Sequenase (Sequenase of RNA^TM) such archaeal dna polymerase does to extend in order to the mode that relies on template.

The present invention also comprises the nucleotides that utilizes codon sequence redundancy to modify. At one more particularly in the example, Codon usage can be modified with the expression maximization with the nucleic acid in particular organisms or the cellular type.

The present invention also comprises the purposes of modified purine (for example trophicardyl, methyl inosine and methyladenosine) and modified pyrimidine (such as thiouridine and methylcystein).

The present invention also provides expression structure, and it comprises encodes at least one, or the nucleotide sequence of preferred a large amount of EBV CTL epi-positions of the present invention.

Rightly, above-mentioned expression structure comprises the described nucleotide sequence of the one or more regulatory nucleotide sequences that are operably connected in the expression vector.

" regulatory nucleotide sequence " that be present in the expression vector can comprise enhancer, promoter, donor splicing site/receptor signal, the Kozac sequence, terminator and polyadenylation sequence, as known in the art and promote to be operably connected to the expression of the nucleotide sequence on it, or promote the protein expression that is encoded.

Here used " being operably connected " refers to that described regulatory nucleotide sequence and nucleotides sequence of the present invention show locational relatedly, in order to cause, regulates or controls it and transcribe.

Regulatory nucleotide sequence is suitable to host cell or the biology that is used for expressing usually. Be known in the art and be applicable to a large amount of various suitable expression vector of various host cells and suitable regulating and controlling sequence.

For promoter, structural promoter is (such as CMV, SV40, cowpox, HTLV1 and human elongation factor promoter) with inductivity/inhibition promoter (as tet-inhibition promoter and IPTG-metallothionein-or moulting hormone-inducible promoter) all be well known in the art, and be contained among the present invention. To recognize that also promoter can be in conjunction with the hybrid promoter that surpasses a promoter element.

Preferably, described expression structure also comprises one or more selected markers, these marks are applicable to the screening of the bacterium (such as bla, kanR and tetR) that is converted or the screening of the mammalian cell (such as hygromycin, G418 and puromycin) that is converted.

Expression structure can also comprise a fusion partner (fusion partner), is typically provided by expression vector), so recombinant protein of the present invention is expressed with described fusion partner as fusion. The main advantage of fusion partner is evaluation and/or the purifying that can assist described fusion.

The example of fusion partner was described in preamble. Typically, when dividing isolated fusion protein with affinity chromatography, fusion partner is particularly useful. When using the pure fused polypeptide of affinity chromatography, the relevant matrix that is used for chromatography is respectively antibody, albumin A-or G-, and glutathione, amylose, and nickel or cobalt are puted together resin. The form of many such all available kits of matrix obtains, and for example is applicable to (HIS₆) QIAexpress of fusion partner^TMSystem (Qiagen) and Pharmacia GST purification system.

The host cell that is suitable for expressing can be protokaryon or eukaryotic, such as Escherichia coli (for example DH5 α), yeast cells, be applied to the Sf9 cell of baculovirus expression system, mammal cell line such as lymphoblast are, mouse EL4 cell and the splenocyte that separates from the host living beings that is converted as people or mouse that is converted are although be not limited thereto.

Expression structure can import in host cell or the biology by many arbitrary technology of having known, such technology includes but not limited to: heat shock transforms, electroporation, the transfection of DEAE-glucan, microinjection, liposome connects the transfection of mediation, calcium phosphate precipitation, Protoplast fusion, microparticle bombardment, virus Transformation and similar techniques.

In a special embodiment, the invention provides a kind of expression structure that exists with the form of multi-epitope expression structure.

Preferably, described this multi-epitope structure is suitable for being used as dna vaccination.

According to this embodiment, the nucleic acid of a large amount of EBV CTL epi-positions of encoding is passable, for example for example adopt the such technology of Splice Overlap by Extension PCR to synthesize by synthetic oligonucleotides, such as Thomson etc., described in 1995, the Proc.Natl.Acad.Sci.USA 92 5845 like that.

In some special forms, expression structure of the present invention can be used for expressing and transporting viral source carrier, such as poxvirus and adenovirus.

When being used as the vaccine delivery system, the form that the expression structure of viral source can VLPs or be applied to animal as " exposed " nucleic acid structure.

In a special embodiment, comprise the vaccinia virus promoter according to the multi-epitope expression structure of this embodiment, as be present in the p7.5 promoter in the sub-carrier of plasmid. For example, Khanna etc., 1992 propose to recombinate to produce TK-recombinant vaccinia virus with marker rescue.

In a preferred embodiment, the invention provides a kind of expression structure based on adenovirus, be used for the vaccine delivery system. Structure based on adenovirus can infect large-scale mammal or human cell, comprises dormancy or proliferative cell.

This expression structure based on adenovirus can comprise structural or inductivity/inhibition promoter, such as tetracycline inductivity/inhibition system.

The preferred form of this expression structure based on adenovirus be lack at least one E1 gene derived from replication competent type A5 adenovirus not. Not replication competent type like this provides with the Aden-X system of clone technology.

Particularly preferred expression structure based on adenovirus and vaccine delivery system hereinafter are provided in detail.

Pharmaceutical composition and vaccine

The present invention also provides the pharmaceutical composition that comprises one or more EBV CTL epi-positions of the present invention, comprises variant and derivative thereof, or the expression of nucleic acid structure of the identical epi-position of encoding.

Preferred pharmaceutical composition is " immunotherapy compositions against cancer ", and providing can be to treatment and the prevention of the foregoing disease relevant with EBV.

In a special embodiment, described pharmaceutical composition is vaccine.

The form of vaccine can be the form that is rich in protein vaccine (proteinacious vaccine), it comprises one or more EBV CTL epi-positions of the present invention, comprised subunit vaccine or dna vaccination form, its a special example is multi-epitope expression structure as previously described.

The potential suitable technology relevant with dna vaccination is found in WO 96/03144 and Thomson etc., and 1998, J.Immunol.160 1717.

Can through systemic administration in animal or via the elementary transduction antigen presenting cell as dendritic cells, then will be applied in the animal body by transducer cell based on transporting of adenovirus structure.

Ranieri, 1999 and Gahn etc., when having proposed to be used for the treatment of the disease relevant with EBV by the dendritic cells of adenoviral transduction, calendar year 2001 transports example.

Rightly, pharmaceutical composition further comprises pharmaceutically acceptable carrier, diluent or excipient.

" pharmaceutically acceptable carrier, diluent or excipient " refers to carry out safely solid or the liquid filler material of systemic administration, diluent or make material of capsule etc. Special pathway when using can use various carrier well known in the art. These carriers comprise carbohydrate to be selected from, starch, cellulose and derivative thereof, the wheat tooth, gelatin, talcum, calcium sulfate, vegetable oil, artificial oil, polyalcohol, alginic acid, phosphate buffer, emulsifying agent waits a salt solution and salt, and salt is as the mineral acid salt that has comprised hydrogen chloride, bromide and sulfide, organic acid such as acetate, propionate and malonate and apirogen water (pyrogen-free water).

Describe pharmaceutically acceptable carrier for one piece, the useful list of references of diluent and excipient sees Remington ' s Pharmaceutical Sciences (Mack Publishing Co.N.J.USA, 1991).

Available any secure way provides composition of the present invention for the patient. Can use for example per os, rectum, intestines are injected outward, the hypogloeeis, the oral cavity, intravenous, in the joint, in the muscle, in the corium with subcutaneous injection, or through sucking, in the eyeball, in the peritonaeum, in the ventricles of the brain or through transdermal etc. and similar approach thereof. For example, be suitable for immunogenic composition with hypodermic injection in the muscle, be rich in using of protein vaccine and dna vaccination.

Medicine type comprises tablet, powder (dispersions), suspension, injection, solution, syrup, tablet, capsule, suppository, spray, the paster of transdermal etc. and similar type thereof. Medicine type also can comprise injection or transplant a kind of custom-designed controlled radiological unit, or other is through the graft that other effect is arranged of modified forms. The radiation of in check treatment reagent can be subjected to its coated impact, for example, has hydrophobic polymer to comprise that the acrylic acid tree refers to, wax, higher fatty acid wine alcohol, many lactic acid and multiethylene-glycol acid (polyglycolic acids), and the plain derivative of some fibre such as hydroxypropyl methylcellulose. In addition, in check radiation also can be with other polymer poly compound matrix, and liposome and/or microsphere particle affect.

Being fit to pharmaceutical composition of the present invention oral or the outer injection of intestines can present by individual; such as capsule; pouch; or tablet; per unit contains one or more medicaments of the present invention of more than one scheduled volumes, with powder or particle or with solution, and the suspension in the aqueous solution; non-aqueous solution, oil are scattered in emulsion or the emulsion of aqueous dispersion in oil in the water. This composition can be prepared with any dosage, but every kind of method all comprises such step: the carrier that will contain one or more essential components is combined with one or more above-mentioned medicaments. In general, the solid phase carrier of medicament of the present invention and liquid phase carrier or meticulous separation or both, make composition after doing on all four mixing, if need afterwards, again product is moulded the external form of wanting.

Available method with medicament design compatibility is used above-mentioned composition with medicine effective quantity. In content of the present invention, the dosage that sufferer is used is enough to produce favourable replying with it the patient behind one section appropriate time. Application dosage should be judged by the doctor, decides on the various factors of subject, and such as the age, sex, body weight and general health situation thereof etc.

In the example of a special immunotherapy compositions against cancer and vaccine, " pharmaceutically acceptable carrier, diluent or excipient " can be an adjuvant. Will understand that in the art " adjuvant " refers to comprise the composition of one or more materials, it can strengthen immunogenicity and the effectiveness of vaccine combination. The example of the unqualified property of suitable adjuvant comprises saualane and squalene (or other animal oil); Block copolymer; Washing agent as-80； A, mineral oil such as Drakeol or Marcol, vegetable oil such as peanut oil; Corynebacteria derive adjuvant such as ammonia benzyl imidazoles; Propionibacterium derive adjuvant such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigen; Tetanus toxoid; The diptehria toxoid; Interleukins such as interleukin 2 and interleukin 12; Monokine such as interleukin 1; TNF; Interferon such as gamma-interferon; Composition such as saponin(e-aluminium hydroxide or Quil-A aluminium aluminium hydroxide; Liposome;

WithAdjuvant; The bacillus tubercle cell wall extract; Synthetic glycopeptide such as muramyl dipeptide or other derivative; Avridine; The lipid A derivative; Dextran sulfate; Independent DEAE-extran or with the mixture of aluminum phosphate; Carboxyl polymethylene such as carbopol ' EMA; Acrylic copolymer emulsion such as Neocryl A640 (for example US 5047238); Emulsion such as the Montanide ISA 720 of aqueous dispersion in oil; Polio virus, cowpox or animal poxvirus albumen; Or its mixture.

For subunit vaccine, a system of this vaccine join embodiment be with

Prepare, described as WO97/45444.

Antibody

The present invention also comprises the antibody at EBV CTL epi-position of the present invention.

Antibody of the present invention can be mono-clonal or polyclonal antibody.Antibody is made, the method of purifying and use is well known, and is found in Coligan etc., CURRENTPROTOCOLS IN IMMUNOLOGY (John Wiley and Sons NY, chapter 2 1991-1994) and Harlow, E. and 0Lane, D, antibody: laboratory manual, the cold spring port, cold spring harbor laboratory, 1988, both are merged recently herein by reference.

In general, antibody of the present invention can with a polypeptide of the present invention, fragment, varient or derivative in conjunction with or put together combination.For example, antibody can comprise polyclonal antibody.Prepare this antibody, can be with polypeptide of the present invention, fragment, varient or derivative are expelled to the type of production species, and this can comprise mouse and rabbit, with the polyclonal antiserum that obtains.Those skilled in the art know the method for preparing polyclonal antibody.Operable typical method sees aforesaid Coligan etc., CURRENT PROTOCOLS IN IMMUNOLOGY, Harlow and Lane, 1988.

Be used to replace available from the polyclonal antiserum of producing species, monoclonal antibody can prepare in standard method, is carried before such method for example is specified in

And Milstein, 1975, Nature 256, described in 495, or a plurality of its nearest body such as Coligan etc. of modifying, CURRENT PROTOCOLS IN IMMUNOLOGY is described, be with one or more polypeptide, fragment, varient or derivative are inoculated into the species of production usefulness, and spleen cell or other antibody produced cell of getting its infinite multiplication prepare monoclonal antibody.

The present invention also is included in above-mentioned mono-clonal or the polyclonal antibody, comprises the Fc or the Fab fragment of antibody.Alternatively, antibody can comprise at isolating proteic single-chain antibody Fv of the present invention (scFvs).This scFv for example can prepare according to the method in following each literary composition, US No5091513, EP239400 or Winter and Milstein, 1991, Nature's 349 293

Mark is relevant with antibody of the present invention or antibody fragment.

Mark selects to comprise following group: chromogen, catalyzer, enzyme, fluorescent agent, chemical cold light molecule, lanthanide ion such as europium (Eu ³⁴), radio isotope and direct vision mark.The direct vision mark can be formed use by following material, comprises glue or nonmetal particle, colouring particles, and enzyme or substrate, the organic polymer body, emulsion granules, liposome, or other contains the vesicle that signal produces material, and analogue.

In US 4366241, the many enzymes that can be used to make marks of US 4843000 and US 4849338 set forth in detail, they integrate with the present invention by reference at this.Enzyme labelling useful among the present invention comprises alkaline phosphatase, horseradish peroxidase, luciferase, b-tilactase, glucose oxidase, N,O-Diacetylmuramidase, malate dehydrogenase (malic acid dehydrogenase) and similar substance.Protein labeling can use separately or combine with second kind of enzyme in the solution and use.

Though be not limited thereto, through embodiment, the fluorescence staining agent can be fluorescein isothiocyanate (FITC), green difficult to understandly (oregon green), fluorescein isothiocyanate (TRITL), allophycocyanin (APC) and R-phycoerythrin (RPE).

So the present invention can easily be understood and be put to practical function, the bootable technician of following non-limiting examples, wherein embodiment one proposes the explanation and the immunological characteristic description of EBV CTL epi-position of the present invention, and second embodiment proposes adenovirus mediated EBV multi-epitope mode of transport of the present invention.

Embodiment

Embodiment 1

Materials and methods

The foundation of clone with keep

With B95.8, BL74 and QIMR-WIL virus isolated strain are done exogenous virus with periphery B cell and are transformed, and set up the LCLs that EBV transforms by seropositive contributor.Remain on to these clone routines and added 2mM L-glutaminate, 100IU/ml penicillin and 100 μ g/ml Streptomycin sulphates add among the substratum RPMI1640 of 10% foetal calf serum (FCS).In addition, peptide transportation thing (TAP)-negative B x T hybrid cell line.174 * CEM.T2 (being called T2) (Salter 1986) stabilized peptide analysis.

In order to produce phytohemagglutinin (PHA) protoblast, stimulate peripheral blood lymphocytes (PBMC) (CSL Ltd with PHA, Melbourne, Australia), cultivate after three days, the growth medium and the highly purified recombinant human IL-2 (rIL-2) that contain MLA 144 supernatant liquors are added (Khanna 1992).Change rIL-2 and MLA supernatant liquor per two weeks so that the PHA protoblast is bred (no longer adding PHA), until six weeks.

Virus isolated strain

LCLs (lymphoblastoid cell lines) is set up by one group of incoherent healthy contributor, and these contributors are seropositive African, Caucasian and New guineans.The method of setting up is, under the condition that 0.1 μ g/ml ring bag rhzomorph A exists (Moss 1988), allows the idiopathic growth of periphery lymphocyte of cultivating.Ading up to 29 spontaneous LCLs (11 Caucasians, 11 New guineans, 2 Africans, and 5 Southeast Asians) is used to make each independent individual to recapture its inherent EBV isolates.In addition, by EBV carrier's nasopharyngeal carcinoma vivisection 16 kinds of inferior people's the virus isolated strains southeast that directly check order.

The PCR of EBV gene fragment and dna sequencing

The specific oligonucleotide primers that the side connects LMP1 gene different zones is selected for pcr amplification (table 1).The product of result's PCR is with QIAquick column spinner (QiagenInc.Chatsworth, CA) purifying, and the experimental procedure of abideing by the producer is with PRISMready reaction dyedeoxy terminator cycle sequencing kit (AppliedBiosystems Inc., Foster City CA) does order-checking from two ends.

Synthetic peptide

The aminoacid sequence of peptide is by the LMP1 sequence of announcing, these LMP1 sequences be former from Caucasia former template 1EBV bacterial strain B95.8.With the Merrifield solid phase method (MimotopesPty Ltd, Melbourne, Australia) synthetic 45 peptides on the automatic peptide synthesizer, length is 17 amino acid, 8 residues are overlapping, and across whole LMP1 sequence.The peptide sample size is dissolved among the 20%DMSO to 2mg/ml.

EBV seropositivity contributor

Recruit one group and carry out this research from not agnate 42 healthy virus carriers and nasopharyngeal carcinoma patient.Every contributor carries out HLA somatotype (table 2) through the dna typing method of serology and PCR-based.

INF-γ-ELISPOT analyzes

ELISPOT analyze be the PBMC that is used for assessing a large amount of seropositivity contributors that carry the LMP1 peptide can inducing T cell after stimulating in the expression (Bharadwaj2000) of INF-γ.In brief, (Millipore, Bedford USA) coat anti--IFN-γ mAb of the 10 μ g/mL in 100 μ L/ holes in advance, and (Mabtech, Stockholm's 1-D1K Sweden), spend the night in 4 ℃ with the well-mixed cellulose ester membrane flat board in 96 holes.Every plate washs 3 times with phosphate-buffered salt (PBS), and block with 5% ox tire serum at the active position of tool.PBMCs from the EBV carrier of the health of known HLA somatotype separates from whole blood with Ficoll-Hypaque (Sigma) density gradient centrifugation.PBMCs is added in the ternary hole, and every pore volume 100 μ L contain 2.5 * 10 ⁵Individual cell adds peptide then among the PBMCs to final concentration and reaches 5 μ g/Ml.Do one group of test that contains three kinds of different cell count simultaneously, three holes contain 2.5 * 10 respectively ⁵, 1.5 * 10 ⁵And 1 * 10 ⁵Individual PBMC cell.Analyze the reaction of PBMCs to the different concns peptide, peptide concentration is respectively 10 μ g/mL, 5 μ g/mL and 1 μ g/ml.In negative control, with the PBMCs single culture in not adding the growth medium of peptide.Dull and stereotyped in 37 ℃, 5% CO ₂Overnight incubation under the concentration (about 16-20 hour), wash three times with PBST (0.05% Tween-20 is in PBS) and PBS respectively again, the 1 μ g/mL that every then hole adds 100 μ l resists-IFN-γ mAb, 7-B6-1 (Mabtech, Stockholm, Sweden), and in incubated at room 3-4 hour.Respectively wash three times with PBST and PBS respectively before and after again after hatching, add 1 μ g/mL antibiosis protein chain mycin-alkaline phosphatase conjugate (Sigma) of 100 μ l again in every hole.Flat board was at room temperature hatched 2 hours.And then, put together matrix (BCIP/NBT with alkaline phosphatase respectively with PBST and PBS washing hole each three times; Sigma) carry out 30 minutes to 1 hour color reaction with 5-bromo-4-chloro-3-indyl phosphoric acid salt and nitroblue tetrazolium(NBT), just can see the indivedual IFN-γ founder cells that are the black spots point-like.Automatically calculate spot number (ImagePro) (Bharadwaj 2001) with image analysing computer software, and form cell (SFC)/10 with spot ⁶PBMCs represents.Its number of the T cell of secretion of gamma-IFN is to calculate by deduct the negative control value from the SFC counting of experimental group.

Remove CD4 ⁺ With CD8 ⁺ Cell

With anti-people CD4 ⁺Or CD8 ⁺The immune magnetic particulate is removed the CD4 among the fresh PBMCs ⁺Or CD8 ⁺Cell (Dynabeads M450-CD4 and M450-CD8), respectively (Dynal, Oslo is Norway) according to the suggestion of manufacturer.Put together the anti-CD 4 antibodies double staining by removed PBMCs is puted together anti--CD8 and PE-with FITC-, and determine the validity of removal with the flow cytometer of Coulter EPICSXL cell counter.FACScan analyzes and can determine that this experimental technique can remove＞95% CD4 ⁺Or CD8 ⁺Cell.Cell is counted again, cell is resuspended among the R10 again, and is arranged in the in triplicate ELISPOT analysis.

Polyclone CTL system and LMP1 specific CTL clone's foundation

According to the previous method (Moss1988 that announces; Khanna1998) set up polyclone CTL system and LMP1 specific CTL clone.In brief, 2ml pore volume in 24 orifice plates, use by 10 μ M peptides triggered 1 * 10 ⁶Individual from body LS 2 * 10 ⁶Individual PBMCs (replying thing is 2: 1 to stimulator ratio) 1 hour.Add the substratum that contains IL-2 (10U/ml) after 3 days and continue propagation by cell.Stimulate lymphocyte with gamma activity (8000rad) once more from body LCLs during by the 7th day.In substratum after ten days, these cells at the peptide sensitivity from the paotoblastic standard of body PHA ⁵¹Be used as the polyclone effector in the Cr radiometric analysis.

In order producing the LMP1 derived peptide there is specific peripheral blood ctl clone, in the 2ml hole of 24 orifice plates of pore volume in substratum, uses the (autoimmune cell (1 * 10 of 10ug peptide/ml) the peptide sensitivity ⁶) reactivate healthy contributor's PBMCs (2 * 10 ⁶).After 3 days, cell is inoculated on 0.35% the agarose, and keeps at the T cell growth medium that contains Г IL-2 (50IU/ml).After 3 days, the clone who grows is transferred in the 96-hole culture dish of round bottom (Life Technologies), and is incubated in the T cell culture medium that contains Г IL-2 (50-100IU/ml).

The T cell kills and wounds the T cell

This technology (Burrows, 1992) is based on cell in the CTL substratum to presenting the antigenic activity of peptide each other, if suitable peptide will cause CTL-CTL to kill and wound.Ctl clone (about 300 cells/well) is incubated in the 25 μ l T cell culture mediums that contain 10 μ M peptides.Assess cytolytic situation with inverted microscope in 37 ℃ of cultivations after 3 hours.

Cytotoxicity analysis

Quick in advance with the synthetic peptide epitopes to target cell, use then ⁵¹Cr was hatched 90 minutes.Hatch back these cells of washing in growth medium, and these cells are used as 5 hours ⁵¹Target in the Cr radiometric analysis (Moss, 1988).

The MHC stability analysis

Combining of the restricted LMP1 epi-position of the HLA A2-varient that obtains for assessment MHC and EBV isolates is with T2 cell (2 * 10 ⁵) under 26 ℃, hatched 14-16 hour with every kind of peptide (100 μ g/ml) of 200 μ l, then be to hatch under 37 ℃ 2-3 hour.(HB82 ATCC), measures the expression of HLA A2 by FACS with monoclonal antibody specific to hatch the back employing.

The result

The intravital LMP1-specific T-cells of not agnate healthy virus carrier is replied

In order fully to describe memory T cell to the replying of LMP1 in the seropositivity virus carrier body, we use the ELISPOT analytical method, and this analytical method can be sketched the contours of the LMP1-specific immune response apace in vivo and can not prolong culturing in vivo.Stimulate separation from seropositivity virus carrier's PBMC (table 2) with the overlapping LMP1 peptide of whole group, and detect the cell of producing INF-γ.Figure 1A and B representative are done the data (17 amino acid lengths, 8 residues are overlapping) that ELISPOT analyzes with whole group overlapping peptide.In 21 healthy contributors that detect, there are 77 kinds in 45 kinds of peptides had in the intensive body to reply that (the SFC scope is from 39-824/10 ⁶Individual PBMC) (Figure 1A).These interesting phenomenons of replying are that most t cell responses all directly is present in LMP1 at these and strides epi-position in membrane portions and the CTAR1 structural domain, yet do not find any reaction at CTAR2 and N-terminal (Figure 1B).Especially, almost there is 80% the zone all can be in the CTAR1 structural domain by the T cell recognition.Fig. 1 lists all peptides of understanding 17 amino acid lengths being discerned by different contributors.Can the T cell subsets of replying be arranged to these peptides in order to identify, we isolate PBMCs from the contributor of 7 potential energies identification 17mer peptide, be divided into two groups on CD4 cancellation type and CD8 cancellation type, and then detect with the ELISPOT analytical method.There are 5 kinds in these peptides to CD4 ⁺With CD8 ⁺The T cell all has activity, and this shows that these sequences have comprised MHC I type and MHC II type restricted epitope.On the other hand, peptide DWTGGA LLVLYSFALML clearly shows it to depending on CD8 ⁺The T cell activity, however mainly be by CD4 to replying of peptide TDDSGHES DSNSNEGRH ⁺T cell-mediated (Fig. 2 A and B).The activity that depends on CD4 of peptide TDDSGHESDSNSNEGRH is consistent with the previous Leen and the viewed result that works together thereof, and they figure has drawn the HLA DQ2-restricted epitope in this sequence.

The LMP1 specific T-cells is replied is figure in detail

In ELISPOT analyzes, use 17mer peptide to obtain LMP1 specific C D8 through brachymemma ⁺T cell response be figure in detail.Originally experiment draws immunogenicity zone in the 17mer peptide with 12mer plyability peptide.The representativeness of overlapping peptide of taking from 4 individual contributors is shown in Fig. 3 A-D.It may be factor of determination (referring to table 2) that 12 kinds of total 12mer peptides are considered to.Based on these 12mer peptides, the eclipsed 9mer peptide of crossing over the 12mer peptide is used to define minimum epitope sequences.From 4 different contributors' representative data shown in Fig. 4 A-D.Having that 18 kinds of 9mer peptides are considered to may be minimum epitope sequences (table 3).Wherein two kinds of minmal sequences (YLLEMLWRL and YLQQNWWTL) have been accredited as CTL epi-position (Khanna, 1998) before, and other sequence then is to be found for the first time in this research.

The characteristic of new LMP1 CTL epi-position

In order to further describe the characteristic of being analyzed determined minimum t cell epitope by ELISPOT, we prepare has specific polyclone CTL clone and clone CTL clone to these epi-positions.With the PBMCs of synthetic peptide epitopes stimulation from healthy seropositivity contributor, and in standard ⁵¹Ctl clone or polyclone clone are set up in Cr radiation experiment.Fig. 5 and Fig. 6 are a series of data of two kinds of new epi-positions (IALYLQQNW and ALLVLYSFA).Earlier with the stimulating of peptide sensitivity from body PBMC, then again with radioactivity from the continuous repetitive stimulation of body LCL to produce IALYLQQNW peptide specific ctl clone.With ⁵¹The Cr radiometric analysis is directed to has the active ctl clone of IALYLQQNW specific CTL to screening from body and allosome PHA protoblast and LCLs of peptide sensitivity.Data among Fig. 5 A clearly illustrate that the target cell of only sharing HLA B57 with CTL is discerned by IALYLQQNW specific CTL clone, and this shows that this epi-position is limited by HLA B57 allelotrope only.Further determined the meticulous specificity (Fig. 5 B) of this ctl clone from the titration of the minimum epi-position of body PHA protoblast.

Previous HIV CTL epi-position be studies show that similar HLA I type molecule can present identical peptide such as CTL epi-position (Goulder etc., 2000).Because the general hypotype of HLA B57 and HLA B58 has only the difference of several amino acid, and enjoy similar peptide, so we just probe into the possibility that IALTLQQNW also can be subjected to the allelic restriction of HLA B58 in conjunction with primitive.We also demonstrate really to the healthy seropositivity contributors' of two HLA B58 research has equal intensive to reply (table 3) to minimum IALTLQQNW epi-position.These observe demonstration, and this epi-position shows the dual HLA restriction to HLAB57 and HLAB58.With having identified the HLA-A2 restricted CTL epitope with above-mentioned similar mode.Confirm the HLA A2 restricted (Fig. 6 A) of ALLVLYSFA epi-position with peptide specific T clone.The meticulous specificity of ALLVLYSFA epitope specificity ctl clone confirms (Fig. 6 B) by the dose response analysis of minimum peptide subsequently.

Other 17-mer peptide, TDDSGHESDSNSNEGRH shows CD8 in ELISPOT analyzes ⁺T cell response has its reaction size of 3/21 healthy contributor between 131-190SFC/10 in the analysis ⁶Between the PBMC (Figure 1A).Foundation from two different contributors' polyclone CTL clone can discern with peptide bag quilt from body PHA protoblast (data unlisted come).Two contributors (MM and RE) discern minimum peptide ESDSNSNEG and DSNSNEGRH respectively, but these minimum epi-positions can't cause the generation of CTL clone.Drawn out a restricted t cell epitope of HLA DQ2-(table 3) between the 17mer sequence before it should be noted that Leen and colleague thereof.

Different ethnic groups are to the identification of minimum HLA I type-restricted LMP1 CTL epi-position

In order to determine that we filter out PBMC with the ELISPOT analytical method from one group of not agnate healthy virus carrier to the frequency of HLA I type-restricted LMP1 CTL epi-position identification.The detailed summary of analyzing is listed in table 3.This comprises 4 kinds of different restricted t cell epitopes of HLA I type restricted and a kind of HLA II type in analyzing.We observe among the contributor that 35-45% test is arranged to the restricted YLLEMLWRL of HLA-A2, YLQQNWWTL and ALLVLYSFA epi-position have strongly replys.These are replied higher consistence in the Caucasian, these epi-positions are then lower by the frequency that the South East Asia contributor discerns.The restricted IALYLQQNW epi-position of HLA-B57 is discerned by 5/7 HLA B57 positive individuals.Interesting is that the positive contributors of some HLA B58 (3/5) also discern this epi-position, and effective equally with the positive contributor of HLA B57, have confirmed further that thus this epi-position has dual HLA I type restricted.The restricted LMP1 epi-position of HLA II type TDDSGHESDSNSNEGRH can be discerned by the virus carrier of all three kinds of health.

Be derived from the restricted LMP1 CTL table of its HLA I type in the EBV isolates of different geographic regions The sequential analysis of position

In the previous research (Khanna, 1998) in the present invention and our laboratory, the epi-position in the LMP1 is all passed through the bacterial strain with reference to I type EBV, and the CTL of B95.8 reactivate confirms.Yet when treatment EBV associated malignancies, if the effective basis of CTL treatment of this epi-position, we just must at first determine the conservative degree of these epitope sequences in other EBV bacterial strain of different crowd all over the world.Therefore, we isolate many EBV from healthy virus carrier and NPC vivisection, and sequential analysis is done in the DNA zone of its coding LMP1 epi-position.The detailed summary of the The sequencing results of four kinds of LMP1 epi-positions (IALYLQQNW, ALLVLYSFA, YLLEMLWRL and YLQQNWWTL) is listed in table 4.From Caucasia, Africa and South East Asia contributor's virus isolated strain, its HLAA2 restricted (ALLVLYSFA and YLQQNWW TL) and HLA B57 and B58 restricted (IALYLQQNW) epi-position all highly keep (table 4).Have only the minority virus isolated strain to demonstrate slight epitope sequences variation.In New guineans' strain isolated, though YLQQNWW TL and IALYLQQNW epi-position are normally conservative, yet the situation that replaces is more general in ALLV LYSFA.Every single strain isolated all shows an identical change, promptly becomes L-Ala (table 4) at the 7th by Serine.

To the sequential analysis of HLA A2 restricted epitope, disclosed an interesting heritable variation form from the YLLEMLWRL in the EBV strain isolated of different ethnic groups.Caucasian's strain isolated has 5/11 pair of YLLEM LWRL epi-position to show former template B95.8 sequence, and other strain isolated of 7/11 then has some changes, influences one or more residues (table 4).In four strain isolateds, the leucine of position 2, the arginine of the methionine(Met) of position 5 and position 8 is substituted by phenylalanine respectively, Isoleucine and glycine.Other two strain isolateds in the position 2 with position 5 or have only position 5 to show replacement.The sequential analysis of inferior crowd's virus isolated strain is southeast shown that nearly all strain isolated all has identical replacement situation, comprising position 2 (L → F) and the position 5 (replacement of M → I).Strain isolated is presented at position 4 sudden change (E → D).Must importantly emphasize at this, come generally all to show identical mutant form (table 4) in the virus isolated strain of spontaneous LCLs and NPC vivisection.In South East Asia crowd's strain isolated, do not detect any wild-type sequence.Interesting is the dominant inheritance variant form (Y of the epi-position YLLEMLWRL epi-position of finding in the crowd of South East Asia FLE ILWRL), this also often appears in Caucasia crowd's the virus isolated strain.

For the genetic variant of indicator YLLEMLWRL epi-position further, the synthetic peptide of our synthetic every kind of varient sequence, and do the assessment that HLA combination and immunity are discerned with the EBV specific CTL.Data as shown in Figure 7 show that when body PHA protoblast was cloned hydrolysis by specific CTL, the synthetic peptide functioning efficiency of most of varient sequences was significantly lower than former breeding wild type YLLEMLWRL peptide in sensitization.Has only a kind of varient sequence YLLE ILWRL is considered to and wild-type epi-position same effectively (Fig. 7 A).In addition, using the varient peptide sequence (Y that is common in South East Asia crowd's strain isolated FLE IWhen LWRL) hatching the T2 cell, and the expression (Fig. 7 B) that can't save HLA A2.Other varient peptide (Y FLE ILW GL, YL ME ILWRL and YLLE ILWRL) but increased MHC expression on the T2 cell significantly, it is incorrect owing to the interaction between TXi Baoshouti and MHC-peptide complex body that this expression causes these varients to lose its antigenicity, rather than do not combine with MHC.

Embodiment 2

Materials and methods

Reorganization LMP1 multi-epitope inserts segmental structure

The dna sequence dna of coding multi-epitope aminoacid sequence is by the design of universal code.We will have 5 long oligonucleotide (primer 1-5 of 20 base pair eclipsed, length range is between 74-100 base) represented the Polyepitope DNA sequence, by overlapping extension and progressively the montage mode of asymmetric PCR (stepwise asymmetric PCR) (referring to Fig. 8) they are annealed together.In brief, multi-epitope sequence specific primers LMPA and LMPB are 5 circulations of pyritous PCR reaction (94 ℃, 1 minute) amplification with beginning, and reaction volume is that 20 μ l include prolongation enzyme mixture and PCR damping fluid.During 5 loop ends, the PCR program can be suspended at 72 ℃, above-mentioned reaction liquid 2 μ l is transferred in the 20 μ l reaction solutions of 72 ℃ of new preheatings, carries out five circulations in addition again with primer LMPC and multi-epitope sequence-specific forward primer.At the 10th circulation time, program can be suspended once more, more once reaction solution behind the 2 μ l is added in the 20 new μ l reaction solutions, carries out five circulations again with primer LMPD and multi-epitope sequence-specific forward primer.Before all oligonucleotide all are added into, this progressively PCR repeated always.In the end in step, 2 last μ l reaction solutions are done 25 circulations of amplification with multi-epitope sequence-specific forward and reverse primer.

This segmental nucleotide sequence (terminal) Bam HI restriction enzyme site of encoding successively from 5 ', the Kozak consensus sequence, methionine(Met) initiator codon, minimum LMP1 of 13 successive and LMP2 CTL epi-position (table 5), terminator codon, EcoRI restriction enzyme site (Fig. 8).The PCR fragment of total length gel-purified is cloned into the BamHI/EcoRI site of inserting the pcDNA3 expression vector, checks sudden change by order-checking.

Preparation Ad5-LMP polyepitope vaccines carrier

Use Adeno-X system (CLONTECH, Palo Alto, { Mizuguchi 1998 ﹠amp of method of attachment efficiently CA); 1999} assembles and produces the adenovirus (referring to Fig. 8) based on Ad5.The LMP1 multi-epitope inserts EcoRI and the BamH1 site that fragment is received pAd-TrackCMV.By transfection human embryos kidney (HEK) 293 cells, the Ad5 carrier package of will recombinating is gone in the infectious adenovirus then, and by freeze-thaw from transfected cell harvesting recombinant adenovirus (referring to Ad5-LMPpoly).

The foundation of clone and keeping

With the B95.8 virus isolated strain periphery B cell is carried out exogenous virus and transform, to set up the LCLs that EBV transforms from the seropositivity contributor.These clones are maintained always routinely is added with 2mM L-glutaminate, (Gibco Invi-trogen Corp among the RPMI 1640 of 100IU/ml penicillin and 100 μ g/ml Streptomycin sulphates and 10%FCS (growth medium).，Carlsbad，CA)。In addition, embryo's kidney clone 293{Graham, 1977} are maintained among the DMEM that contains 10% fetal bovine serum.

With the Ad5-LMPPoly carrier to HLA A2/K ^b The immunity of transgenic mouse

The HLA A2/K that is used for this research ^bTransgenic mouse other places was again described (Theobald, 1997).These mouse are expressed a kind of chimeric I type molecule, and this molecule is by allelic α 1 of human A*0201 and α 2 structural domains, and mouse H-2K ^bα 3 structural domains of I type molecule are formed.With 10 ⁸(plaque forming units, PFU) reorganization Ad5-LMPpoly or control group adenovirus are carried out the intraperitoneal vaccination to these mouse to plaque forming unit.3 weeks back harvest spleen cell, and test epi-position-specific T-cells is replied.Test with CTL in ELISPOT and the body and to detect these t cell responses.

Tumor challenge and multi-epitope immunity

Use two kinds of different vaccination strategies and estimate the effect of LMP polyepitope vaccines.In first group of experiment, use or Ad5-LMPpoly or control group adenovirus (10 ⁹Every mouse of PFU/) to HLA A2/K ^bMouse carries out the intraperitoneal immunity.After 3 weeks of immunity, with 10 ⁷Individual EL4-A2/K alive ^bThese mouse of-LMP1 cell are by subcutaneous attack.After the attack, monitored these mouse termly 21 days, and with tape measure tumour size.In second group of experiment, use EL4-A2/K earlier ^b-LMP1 (every mouse 10 ⁷Individual cell) tumour cell is attacked HLA A2/K ^bMouse.Attack after 10 days, when diameter of tumor size about position 0.2cm, use again or Ad5-LMPpoly or these mouse of control group adenovirus immunity.The result of treatment of LMP polyepitope vaccines is estimated in the degeneration of monitoring tumour termly.According to the criterion of animal Ethics Committee, any mouse of diameter of tumor＞1.0cm all must make its death.

The result

The endogenous processing of the CTL epi-position of recombinant adenovirus LMP multi-epitope coding and CTL identification

Whether the coded LMP epi-position (table 5) of multi-epitope (Ad5-LMPpoly) has been processed by endogenous in order to monitor, and we are had specific LMP1 or LMP2 specific CTL polyclone clone to the cellular exposure of the target that is infected by Ad5-LMPpoly to various epi-positions.These CTL clones are to come from healthy virus carrier.With the LCLs that HLA paired inoblast and Ad5-LMPpoly infect, system discerns (Fig. 9) by discrete LMP Specific CTL Cells.These results clearly illustrate, are included in HLA I type Restricted CTL multilist potential energy in the adenovirus LMP multi-epitope and are effectively processed and be the cell of passing target.

In ensuing experiment, adenovirus LMP multi-epitope is used to reply to produce secondary CTL at external repetitious stimulation PBMC, and PBMC is derived from healthy EBV seropositivity individuality.The polyclone culture that is produced is used as anti-from the paotoblastic effector of body PHA, and these protoblasts are earlier by LMP1 or LMP2 peptide epitopes sensitization.The data of Figure 10 clearly illustrate that LMP multilist potential energy arouses multiple CTL efficiently and replys, and it replys the epi-position that the expressed HLA allelotrope of each contributor is limited specificity.For example, the LMP multi-epitope can stimulate that (HLA A2-is restricted at epi-position YLQ, LMP1) and CLG (HLA A2-is restricted, LMP2) intensive t cell response, observe also that (HLA A2-is restricted at epi-position ALL, LMP1) and YLL (HLA A2-, A68-and A69-are restricted, replying LMP1).

On the contrary, the LCL that transforms with EBV stimulates the expansion of inducing very limited LMP specific T-cells, and the level of dissolution of the cell of the target of LMP peptide sensitization be lower than can be detected degree (data not shown).

The reversed growth of LMP1 expressing tumor of the immunization of Ad5-LMPpoly vaccine

Whether can provide provide protection in order to detect adenovirus LMP1 polyepitope vaccines institute inductive t cell response at the tumour cell of expressing LMP1, with Ad5-LMPpoly or control group adenovirus to two groups of HLA A2/K ^bMouse (10 every group) carries out first immunisation twice, each 14 days at interval, uses EL4-A2/K then ^b-LMP1 cell is attacked, and monitors the tumor growth situation of these mouse termly.Though two groups all have tumour to grow, the tumor growth situation is more violent in the adenovirus mice immunized of control group, and the attack of tumour is not shown any provide protection (Figure 11).Must emphasize in this, not show at EL4/A2K with the animal of control group adenovirus or Ad5-LMPpoly immunity ^bThe provide protection that cell is attacked, this expression epitope specificity immunne response is the key (data not shown) of this provide protection.

Discuss

Hokdkin disease and nasopharyngeal carcinoma have an important common trait, i.e. the antigenic expression of EBV all is subject to EBNA1, LMP1 and LMP2 usually.Wherein EBNA1 is by HLA I type trail protection; to avoid processing (Levitskaya; 1995),, can be used for developing new immunization strategy to expand antigen-specific T-cells immunity with treatment Hokdkin disease and nasopharyngeal carcinoma so have only LMP1 and LMP2 to be only the antigen of target.Its methods of treatment is mainly replied LMP is antigenic increasing the T cell, this in treatment recurrence nasopharyngeal carcinoma relevant and Hokdkin disease with EBV of great use.We adopt ELISPOT to analyze fully to describe among the papova carrier LMP1 specific T-cells and reply.In the contributor of all these tested person, the healthy Caucasian of 55-60% has detected the LMP1 specific T-cells and has replied.On the other hand, then more rare among this healthy virus carrier who replys in South East Asia and the nasopharyngeal carcinoma patient.

Research at present has an interesting phenomenon, and promptly the t cell responses at LMP1 more than 80% all is to directly act on to stride film district and CTAR1 structural domain.What is interesting is that especially different testees all can act preferentially on the CTAR1 structural domain.In the 15mer determiner by the evaluation of ELISPOT analytical method, have 3/7 to be located in the CTAR1 structural domain.On the other hand, we can't detect the T cell any peptide that is positioned at far-end C-terminal CTAR2 structural domain is replied, yet the CTAR2 structural domain all is necessary concerning the function of some LMP1 mediations.Previous research shown the sudden change in this independent zone or disappearance will completely destroy LMP1 the signal conduction and the regulate process of mediation.Though the definite reason that the T cell is not replied the CTAR2 zone is also unknown, might be that EBV can protect this important function zone to exempt from and should control to avoid being subjected to potential.Just might find the reason that the CTAR2 structural domain is played the immunity effect if know this mechanism of avoiding, so can not only block the transformation of LMP1 mediation, can also destroy the latent infection of normal cell and malignant cell.

Previous researchs and proposes, and the LMP1 sequence of whole world different geographic regions has the variability of height.The target of the potential immunotherapy when using LMP1 as the HD of treatment recurrence and NPC disease the time, this heritable variation is considered to a topmost obstruction.Therefore, determine that B95.8 deutero-LMP1 epitope sequences is very important being subjected to situation from the guarantor in the EBV strain isolated of the different geographic regions in the whole world.Research one big group is from the people's of different areas peripheral blood deutero-EBV strain isolated and NPC vivisection, and we find from the t cell epitope sequence of LMP1 high conservative normally.Here there are 3 kinds to demonstrate slight variation in 4 kinds of epi-positions that checked order.Unique sequence that great variation is arranged is HLA A2 supertype (supertype)-restricted YLLEMLWRL, and it has different sequence form in the EBV strain isolated from different areas.It is especially obvious that this shows in inferior southeast strain isolated, because the heritable variation of same form is arranged in its YLLEMLWRL epitope regions.And separating between the strain isolated of NPC and spontaneous LCLs, its heritable variation situation is identical.Though also find the common sequences varient in the YLLE MLWRL epi-position in Caucasian and PNG, these isolated epi-positions can additionally show different heritable variation at each specific geographic region.Antigenicity analysis is done in the heritable variation of YLLEMLWRL epi-position to be found, varient sequence (YFLEILWRL) is compared with wild-type sequence, its series of variation that generally sees the South East Asia crowd is not only difficult to be discerned by epitope specificity CTL, also significantly not by HLA A2 combination.On the contrary, though some other series of variation (YFLEILWGL and YLMEILWRL) is difficult for by the T cell recognition, they are to the not obviously influence of combination of HLA A2.Though there is the definite reason/mechanism of heritable variation of so high degree also not bright in this epi-position, might be to help to protect these strain isolateds to avoid the influence that the EBV specific CTL is replied from the sudden change in this epi-position in the strain isolated in NPC popular South East Asia.

In view of LMP1 has highly carcinogenic possibility, thus very impossible based on the vaccine of total length LMP1 or immunotherapy strategy be the preferential selection of treatment NPC and HD.And, studies show that originally that in the virus carrier of most of health LMP1 only causes low-level t cell response usually.Infer that this is because LMP1 is positioned at the zone of running through film, makes it be difficult for entering typical MHC I type metabolic pathway.Use the LMP1 and the LMP2 epi-position of high conservative can overcome these potential restrictions significantly as polyepitope vaccines.

Correspondingly, the present application people is verified can cause that with the proteic adenovirus carrier immunization of LMP multi-epitope of expressing the minimum LMP1 that contains a series of vicinities and LMP2 CTL epi-position multiple independently MHC-Restricted CTL replys.These epi-positions are not only carried out the processing of effective endogenous by the human cell, also can arouse LMP antigen is had specific memory-type CTL to reply in the virus carrier of health.And the adenovirus polyepitope vaccines also can cause elementary t cell response, and this shows that it has result of treatment in the tumor challenge system.

Need lay special stress in this, the vaccine based on multi-epitope that is used for HD and NPC has many places that are better than traditional vaccine, and it is based on the LMP antigen of total length.Multi-epitope albumen is especially unstable, and because their restricted 2 grades and 3 level structures, they can be decomposed in tenuigenin apace.On the other hand, the LMP antigen of total length can not be decomposed fast, and can cause that the interior multiple signal conduction of cell causes the generation of the secondary cancer of injection site.Other significant advantage comprises that polyepitope vaccines does not need other related substances with small relatively structure, just can cause long-term protectiveness CTL and reply to resist a large amount of CTL epi-positions.At last, also may be able to overcome the potential problems of the LMP1 heritable variation that extensively exists among the different geographical crowds in the whole world based on the vaccine of multi-epitope.

If can be applied to the patient of significance quantity based on the treatment of the NPC of CTL and HD, the colony of target must must be high by the allelic frequency of the HLA that exists with high frequency.In this article, except by A11, the confined LMP1 of A24 and B40, the allelotrope that the LMP2 specificity is replied makes us feeling interest especially, because these allelotrope very common (A11,56% in the southern china crowd; A24,27%; B40,28%), especially wherein NPC is an endemic illness.Therefore, we develop and a kind of new LMP multi-epitope treatment vaccine, use the adenovirus of not having a replication that contains LMP1 and LMP2 two epi-positions, and these epi-positions generally are stored in the HLA allelotrope among the different ethnic populations, A2, A11, A23, A24, B27, B40 and B57 limit.But estimate these preferential MHC I type restricted epitope useful effects of screening in the Asia more than 90%, Africa and Caucasia crowd.

The main purpose of whole patent specification is to describe the preferred embodiments of the invention, but and the present invention is not limited to arbitrary embodiment or specific feature collection.Explain the embodiment that describes to make different changes or modification in this literary composition and can not abandon the spirit and scope of the present invention.

All computer programs of mentioning in this specification sheets, method of calculation, patent and scientific literature all intactly are incorporated in this specification sheets herein by reference.

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Table 1

Contributor's code	The HLA type	Ethnic derivation
Contributor's code	The HLA type	Ethnic derivation	DM NK LC LL MM RE GC MW DY TN MG MB JD IM LM SS SE SB CS JT MS DS WS EL KG JG AS JF CV TL PR PA WE SUr CHu SUm SO AN CHa SUc	A24 A29 B44 A2 B14 B44 A1 B8 B18 A2 B7 B44 A1 A3 B44 B57 A1 A3 B50 B57 A1 A2 B7 B51 A1 A3 B8 B35 A11 A32 B35 B44 A11 A24 B54 B75 A1 A3 B7 A1 A24 B7 B58 A31 A33 B35 B58 A1 A11 B8 B51 A28 A32 B12 A2 A24 B8 B60 A2 A29 B44 B60 A2 B35 B57 A3 A23 B35 B44 A2 A32 B44 B62 A1 A24 B7 B8 A2 B44 B60 A2 A32 B27 B60 A2 A3 B7 B35 A1 A3 B7 B57 A1 B8 B57 A2 A3 B7 B35 is somatotype somatotype A2 B13 B58 A2 A4 B35 B60 A2 A11 B60 A2 A33 B44 B46 A2 A3 B13 B61 A2 A3 B7 B35 A2 B57 A1 A24 B35 B57 A3 A33 B57 B75 A11 A24 B57 B62 A11 A24 B51 B58 not	Caucasian Caucasian Caucasian Caucasian Caucasian Indian Caucasian Caucasian Caucasian Vietnamese Caucasian Indian Indian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Vietnamese Thailander/Chinese Thailander/Thailander of Thailander of Thailander of Chinese Thailander/Thailander/Chinese of Thailander of Thailander of Thailander of Chinese Thailander

Contributor's code	The HLA type	Ethnic derivation
Contributor's code	The HLA type	Ethnic derivation	CI	A2 A33 B46 B58

Table 3

Epi-position	Testee HLA type	Testee's code	Ethnic derivation	Ctl response (SFC/10 ⁶PBMC)
Epi-position	Testee HLA type	Testee's code	Ethnic derivation	Ctl response (SFC/10 ⁶PBMC)	ALLVLYSFA LALYLQQNW	A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 B57 B57 B57 B57 B57 B57 B57 B57 B58 B58 B58 B58 B58	LL GC NK SE EL TL PR PA WE DS WS SUr SB CHu SUm CI(NPC) RE SB MM SUm KG SO AN Cha MB JD TL SUc(NPC) CI(NPC)	Caucasian Caucasian Caucasian Caucasian Caucasian Vietnamese Thailander/Chinese Thailander/Thailander of Caucasian Thailander of Caucasian Caucasian Thailander of Chinese Thailander/Chinese Thailander/Chinese Indian Caucasian Caucasian Thailander/Indian Indian Vietnamese Thailander/Chinese of Thailander of Thailander of Thailander of Chinese Caucasian Thailander	192 96 88 10 80 128 0 0 0 1184 592 0 480 0 0 0 897 455 80 0 1952 48 100 0 240 955 504 0 0

TDDSGHESDSNSNEGRH

Table 4

Table 5

Epitope sequences	The epi-position code	EBV antigen	The LMP1 location	HLA is restricted	Bibliography
Epitope sequences	The epi-position code	EBV antigen	The LMP1 location	HLA is restricted	Bibliography	YLLEMLWRL	YLL	LMP1	aa125-133	HLAA2, A68 and A69	(Khanna1998)
YLQQNWWTL	YLQ	LMP1	aa159-167	HLAA2	(Khanna1998)	YLLEMLWRL	YLL	LMP1	aa125-133	HLAA2, A68 and A69	(Khanna1998)
YLQQNWWTL	YLQ	LMP1	aa159-167	HLAA2	(Khanna1998)	ALLVLYSFA	ALL	LMP1	aa51-60	HLAA2	This research
IALYLQQNW	IAL	LMP2	aa156-164	HLAB57，B58	This research	ALLVLYSFA	ALL	LMP1	aa51-60	HLAA2	This research
IALYLQQNW	IAL	LMP2	aa156-164	HLAB57，B58	This research	SSCSSCPLSKI	SSC	LMP2	aa340-350	HLAA11	(Lee1997)
PYLFWLAAI	PYL	LMP2		HLAA23	(Khanna1998)	SSCSSCPLSKI	SSC	LMP2	aa340-350	HLAA11	(Lee1997)
PYLFWLAAI	PYL	LMP2		HLAA23	(Khanna1998)	TYGPVFMCL	TYG	LMP2	aa419-427	HLAA24	(Lee1997)
RRRWRRLTV	RRR	LMP2	aa236-244	HLAB27	(Lee1997)	TYGPVFMCL	TYG	LMP2	aa419-427	HLAA24	(Lee1997)
RRRWRRLTV	RRR	LMP2	aa236-244	HLAB27	(Lee1997)	LLSAWILTA	LLS	LMP2	aa447-455	HLAA2.3	(Lee1997)
LTAGFLIFL	LTA	LMP2	aa453-461	HLA2.1	(Lee1997)	LLSAWILTA	LLS	LMP2	aa447-455	HLAA2.3	(Lee1997)
LTAGFLIFL	LTA	LMP2	aa453-461	HLA2.1	(Lee1997)	VMSNTLLSAW	VMS	LMP2	aa442-451	HLAA25	(Lee1997)
IEDPPFNSL	IED	LMP2	aa200-208	HLAB40	(Lee1997)	VMSNTLLSAW	VMS	LMP2	aa442-451	HLAA25	(Lee1997)
IEDPPFNSL	IED	LMP2	aa200-208	HLAB40	(Lee1997)	CLGGLLTMV	CLG	LMP2	aa426-434	HLAA2.1	(Lee1997)

Sequence table

<110>The Council of the Queensland Institute of Medical

Research

Khanna，Rajiv

Jaikumar，Duraiswamy

<120>Epstein Barr Virus Peptide Epitopes，Polyepitopes

and Delivery System Therefor

<130>11441US2

<140>PCT/AU2003/01451

<141>2003-11-03

<150>2003901792

<151>2003-04-15

<150>2002952524

<151>2002-11-07

<160>125

<170>PatentIn version 3.3

<210>1

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>1

Tyr Leu Gln Gln Asn Trp Trp Thr Leu

1 5

<210>2

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>2

Glu Ser Asp Ser Asn Ser Asn Glu Gly

1 5

<210>3

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>3

Tyr Leu Leu Glu Met Leu Trp Arg Leu

1 5

<210>4

<211>3

<212>PRT

<213>Epstein Barr Virus

<400>4

Gln Arg His

1

<210>5

<211>5

<212>PRT

<213>Epstein Barr Virus

<400>5

Ala Gly Asn Asp Gly

1 5

<210>6

<211>3

<212>PRT

<213>Epstein Barr Virus

<400>6

Gln Asn Trp

1

<210>7

<211>4

<212>PRT

<213>Epstein Barr Virus

<400>7

Val Leu Tyr Ser

1

<210>8

<211>6

<212>PRT

<213>Epstein Barr Virus

<400>8

Asp Ser Asn Ser Asn Glu

1 5

<210>9

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>9

Gln Arg His Ser Asp Glu His His His

1 5

<210>10

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>10

Gly Gln Arg His Ser Asp Glu His His

1 5

<210>11

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>11

Tyr Tyr His Gly Gln Arg His Ser Asp

1 5

<210>12

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>12

Trp Met Tyr Tyr His Gly Gln Arg His

1 5

<210>13

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>13

Tyr Tyr His Gly Gln Arg His Ser Asp Glu His His

1 5 10

<210>14

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>14

Ile Trp Met Tyr Tyr His Gly Gln Arg His Ser Asp

1 5 10

<210>15

<211>17

<212>PRT

<213>Epstein Barr Virus

<400>15

Leu Ile Trp Met Tyr Tyr His Gly Gln Arg His Ser Asp Glu

His His

1 5 10 15

His

<210>16

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>16

Ala Gly Asn Asp Gly Gly Pro Pro Gln

1 5

<210>17

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>17

Pro Ser Asp Ser Ala Gly Asn Asp Gly

1 5

<210>18

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>18

Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro Pro Gln

1 5 10

<210>19

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>19

Asp Ser Ala Gly Asn Asp Gly Gly Pro Pro Gln Leu

1 5 10

<210>20

<211>17

<212>PRT

<213>Epstein Barr Virus

<400>20

Pro His Ser Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro

Pro Gln

1 5 10 15

Leu

<210>21

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>21

Ile Ala Leu Tyr Leu Gln Gln Asn Trp

1 5

<210>22

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>22

Ala Leu Tyr Leu Gln Gln Asn Trp Trp

1 5

<210>23

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>23

Gln Asn Trp Trp Thr Leu Leu Val Asp

1 5

<210>24

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>24

Leu Tyr Leu Gln Gln Asn Trp Trp Thr

1 5

<210>25

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>25

Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu

1 5 10

<210>26

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>26

Tyr Leu Gln Gln Asn Trp Trp Thr Leu Leu Val Asp

1 5 10

<210>27

<211>17

<212>PRT

<213>Epstein Barr Virus

<400>27

Leu Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu

Leu Val

1 5 10 15

Asp

<210>28

<211>10

<212>PRT

<213>Epstein Barr Virus

<400>28

Ala Leu Leu Val Leu Tyr Ser Phe Ala Leu

1 5 10

<210>29

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>29

Leu Leu Val Leu Tyr Ser Phe Ala Leu

1 5

<210>30

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>30

Ala Leu Leu Val Leu Tyr Ser Phe Ala

1 5

<210>31

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>31

Val Leu Tyr Ser Phe Ala Leu Met Leu

1 5

<210>32

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>32

Ala Leu Leu Val Leu Tyr Ser Phe Ala Leu Met Leu

1 5 10

<210>33

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>33

Gly Ala Leu Leu Val Leu Tyr Ser Phe Ala Leu Met

1 5 10

<210>34

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>34

Asp Trp Thr Gly Gly Ala Leu Leu Val Leu Tyr Ser

1 5 10

<210>35

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>35

Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe Ala Leu

1 5 10

<210>36

<211>17

<212>PRT

<213>Epstein Barr Virus

<400>36

Asp Trp Thr Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe Ala

Leu Met

1 5 10 15

Leu

<210>37

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>37

Asp Ser Asn Ser Asn Glu Gly Arg His

1 5

<210>38

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>38

Ser Gly His Glu Ser Asp Ser Asn Ser Asn Glu Gly

1 5 10

<210>39

<211>17

<212>PRT

<213>Epstein Barr Virus

<400>39

Thr Asp Asp Ser Gly His Glu Ser Asp Ser Asn Ser Asn Glu

Gly Arg

1 5 10 15

His

<210>40

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>40

Ala Leu Leu Val Leu Tyr Ala Phe Ala

1 5

<210>41

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>41

Ile Ala Leu Tyr Leu His Gln Asn Trp

1 5

<210>42

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>42

Ile Ala Leu Tyr Leu Gln His Asn Trp

1 5

<210>43

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>43

Leu Ala Leu Tyr Leu Gln Gln Asn Trp

1 5

<210>44

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>44

Tyr Phe Leu Glu Ile Leu Trp Arg Leu

1 5

<210>45

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>45

Tyr Phe Leu Glu Ile Leu Trp Gly Leu

1 5

<210>46

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>46

Tyr Leu Leu Glu Ile Leu Trp Arg Leu

1 5

<210>47

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>47

Tyr Phe Leu Asp Ile Leu Trp Arg Leu

1 5

<210>48

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>48

Tyr Leu Met Glu Ile Leu Trp Arg Leu

1 5

<210>49

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>49

Tyr Leu His Gln Asn Trp Trp Thr Leu

1 5

<210>50

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>50

Tyr Leu Gln His Asn Trp Trp Thr Leu

1 5

<210>51

<211>11

<212>PRT

<213>Epstein Barr Virus

<400>51

Ser Ser Cys Ser Ser Cys Pro Leu Ser Lys Ile

1 5 10

<210>52

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>52

Pro Tyr Leu Phe Trp Leu Ala Ala Ile

1 5

<210>53

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>53

Thr Tyr Gly Pro Val Phe Met Cys Leu

1 5

<210>54

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>54

Arg Arg Arg Trp Arg Arg Leu Thr Val

1 5

<210>55

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>55

Leu Leu Ser Ala Trp Ile Leu Thr Ala

1 5

<210>56

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>56

Leu Thr Ala Gly Phe Leu Ile Phe Leu

1 5

<210>57

<211>10

<212>PRT

<213>Epstein Barr Virus

<400>57

Val Met Ser Asn Thr Leu Leu Ser Ala Trp

1 5 10

<210>58

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>58

Ile Glu Asp Pro Pro Phe Asn Ser Leu

1 5

<210>59

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>59

Cys Leu Gly Gly Leu Leu Thr Met Val

1 5

<210>60

<211>18

<212>PRT

<213>Epstein Barr Virus

<400>60

Leu Val Leu Gly Ile Trp Ile Tyr Leu Leu Glu Met Leu Trp

Arg Arg

1 5 10 15

Leu Gly

<210>61

<211>17

<212>PRT

<213>Epstein Barr Virus

<400>61

Arg His Ser Asp Glu His His His Asp Asp Ser Leu Pro His

Pro Gln

1 5 10 15

Gln

<210>62

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>62

gccctccttg tcctctattc ctttgct

27

<210>63

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>63

gcgctccttg tcctctattc ctttgct

27

<210>64

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>64

gcactcttgg tcctctattc ctttgct

27

<210>65

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>65

gcgctccttg tcctctatac ctttgct

27

<210>66

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>66

attgctctct atctacaaca aaactgg

27

<210>67

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>67

attgctctct atctacacca aaactgg

27

<210>68

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>68

cttgctctct atctacaaca aaactgg

27

<210>69

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>69

attgctctct atctacaaca caactgg

27

<210>70

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>70

tacttattgg agatgctctg gcgactt

27

<210>71

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>71

tacttcttgg agattctctg gcgactt

27

<210>72

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>72

tacttcttgg agattctctg ggggctt

27

<210>73

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>73

tacttattgg agattctctg gcgactt

27

<210>74

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>74

tacttcttgg agattctctg gcggctt

27

<210>75

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>75

tacttcttgg acattctctg gcggctt

27

<210>76

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>76

tacctaatgg agattctctg gcgactt

27

<210>77

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>77

tatctacaac aaaactggtg gactcta

27

<210>78

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>78

tatctacacc aaaactggtg gactcta

27

<210>79

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>79

tatctacaac acaactggtg gactcta

27

<210>80

<211>27

<212>DNA

<213>Epstein Barr Virus

<400>80

tacttcttgg aaattctctg gcggctt

27

<210>81

<211>122

<212>PRT

<213>Synthetic protein sequence

<400>81

Met Pro Tyr Leu Phe Trp Leu Ala Ala Ile Ser Ser Cys Ser

Ser Cys

1 5 10 15

Pro Leu Ser Lys Ile Thr Tyr Gly Pro Val Phe Met Cys Leu

Arg Arg

20 25 30

Arg Trp Arg Arg Leu Thr Val Leu Leu Ser Ala Trp Ile Leu

Thr Ala

35 40 45

Leu Thr Ala Gly Phe Leu Ile Phe Leu Cys Leu Gly Gly Leu

Leu Thr

50 55 60

Met Val Val Met Ser Asn Thr Leu Leu Ser Ala Trp Ile Glu

Asp Pro

65 70 75

80

Pro Phe Asn Ser Leu Tyr Leu Leu Glu Met Leu Trp Arg Leu

Tyr Leu

85 90 95

Gln Gln Asn Trp Trp Thr Leu Ala Leu Leu Val Leu Tyr Ser

Phe Ala

100 105 110

Leu Ile Ala Leu Tyr Leu Gln Gln Asn Trp

115 120

<210>82

<211>366

<212>DNA

<213>Synthetic nucleotide sequence

<400>82

atgccctacc tgttctggct ggccgccatc agcagctgca gcagctgccc

cctgagcaag 60

atcacctacg gccccgtgtt catgtgcctg aggaggaggt ggaggaggct

gaccgtgctg 120

ctgagcgcct ggattctgac cgccctgacc gccggcttcc tgatcttcct

gtgcctgggc 180

ggcctgctga ccatggtggt gatgagcaac accctgctga gcgcctggat

cgaggacccc 240

cccttcaaca gcctgtacct gctggagatg ctgtggaggc tgtacctgca

gcagaactgg 300

tggaccctgg ccctgctggt gctgtacagc ttcgccctga tcgccctgta

cctgcagcag 360

aactgg

366

<210>83

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>83

Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu Leu Val

1 5 10

<210>84

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>84

Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu Leu

1 5 10

<210>85

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>85

Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu

1 5 10

<210>86

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>86

Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp Thr

1 5 10

<210>87

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>87

Leu Ile Ile Ala Leu Tyr Leu Gln Gln Asn Trp Trp

1 5 10

<210>88

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>88

His Glu Ser Asp Ser Asn Ser Asn Glu Gly Arg His

1 5 10

<210>89

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>89

Gly His Glu Ser Asp Ser Asn Ser Asn Glu Gly Arg

1 5 10

<210>90

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>90

Asp Ser Gly His Glu Ser Asp Ser Asn Ser Asn Glu

1 5 10

<210>91

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>91

Asp Asp Ser Gly His Glu Ser Asp Ser Asn Ser Asn

1 5 10

<210>92

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>92

Thr Asp Asp Ser Gly His Glu Ser Asp Ser Asn Ser

1 5 10

<210>93

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>93

Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro Pro

1 5 10

<210>94

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>94

Ser Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly Pro

1 5 10

<210>95

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>95

His Ser Pro Ser Asp Ser Ala Gly Asn Asp Gly Gly

1 5 10

<210>96

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>96

Pro His Ser Pro Ser Asp Ser Ala Gly Asn Asp Gly

1 5 10

<210>97

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>97

Thr Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe Ala

1 5 10

<210>98

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>98

Trp Thr Gly Gly Ala Leu Leu Val Leu Tyr Ser Phe

1 5 10

<210>99

<211>12

<212>PRT

<213>Epstein Barr Virus

<400>99

Asp Trp Thr Gly Gly Ala Leu Leu Val Leu Tyr Ser

1 5 10

<210>100

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>100

Leu Val Leu Tyr Ser Phe Ala Leu Met

1 5

<210>101

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>101

Gly Ala Leu Leu Val Leu Tyr Ser Phe

1 5

<210>102

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>102

Gly Gly Ala Leu Leu Val Leu Tyr Ser

1 5

<210>103

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>103

Thr Gly Gly Ala Leu Leu Val Leu Tyr

1 5

<210>104

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>104

Trp Thr Gly Gly Ala Leu Leu Val Leu

1 5

<210>105

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>105

Asp Trp Thr Gly Gly Ala Leu Leu Val

1 5

<210>106

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>106

Gln Gln Asn Trp Trp Thr Leu Leu Val

1 5

<210>107

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>107

Leu Gln Gln Asn Trp Trp Thr Leu Leu

1 5

<210>108

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>108

Leu Tyr Leu Gln Gln Asn Trp Trp Thr

1 5

<210>109

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>109

Ile Ile Ala Leu Tyr Leu Gln Gln Asn

1 5

<210>110

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>110

Leu Ile Ile Ala Leu Tyr Leu Gln Gln

1 5

<210>111

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>111

Gly Asn Asp Gly Gly Pro Pro Gln Leu

1 5

<210>112

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>112

Ala Gly Asn Asp Gly Gly Pro Pro Gln

1 5

<210>113

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>113

Ser Ala Gly Asn Asp Gly Gly Pro Pro

1 5

<210>114

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>114

Asp Ser Ala Gly Asn Asp Gly Gly Pro

1 5

<210>115

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>115

Ser Asp Ser Ala Gly Asn Asp Gly Gly

1 5

<210>116

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>116

Ser Pro Ser Asp Ser Ala Gly Asn Asp

1 5

<210>117

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>117

His Ser Pro Ser Asp Ser Ala Gly Asn

1 5

<210>118

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>118

Pro His Ser Pro Ser Asp Ser Ala Gly

1 5

<210>119

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>119

Ser Asp Ser Asn Ser Asn Glu Gly Arg

1 5

<210>120

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>120

His Glu Ser Asp Ser Asn Ser Asn Glu

1 5

<210>121

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>121

Gly His Glu Ser Asp Ser Asn Ser Asn

1 5

<210>122

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>122

Ser Gly His Glu Ser Asp Ser Asn Ser

1 5

<210>123

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>123

Asp Ser Gly His Glu Ser Asp Ser Asn

1 5

<210>124

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>124

Asp Asp Ser Gly His Glu Ser Asp Ser

1 5

<210>125

<211>9

<212>PRT

<213>Epstein Barr Virus

<400>125

Thr Asp Asp Ser Gly His Glu Ser Asp

1 5

Claims

1. an isolating EBV ctl peptide epi-position is made up of the aminoacid sequence that is selected from the following group: IALYLQQNW (SEQ ID NO:21); ALYLQQNWW (SEQ IDNO:22); QNWWTLLVD (SEQ ID NO:23); LYLQQNWWT (SEQ IDNO:24); IALYLQQNWWTL (SEQ ID NO:25); LIIALYLQQNWWTLLVD (SEQ ID NO:27).

2. the varient of the isolating EBV ctl peptide epi-position of claim 1, it is made up of following aminoacid sequence:

(i) there is 1 amino acid different with SEQ ID NO:21;

(ii) there is 1 or 2 amino acid different with SEQ ID NO:22;

(iii) there are 1 or 2 amino acid different with SEQ ID NO:23;

(iv) there are 1 or 2 amino acid different with SEQ ID NO:25; Or

(v) there are 1 or 2 amino acid different with SEQ ID NO:27;

Wherein said varient can cause immunne response by at least a T cell clone type.

3. the varient of the isolating EBV ctl peptide epi-position of claim 2, it is made up of aminoacid sequence arbitrary in SEQ IDNO:49 and 50.

4. isolating protein, it comprises a large amount of adjacent EBV CTL epi-position and/or EBV CTL epi-position varients, and wherein at least a described EBV CTL epi-position and/or EBVCTL epi-position varient are according to each epi-position and/or varient among the claim 1-3.

5. the isolating protein of claim 4, it is a multi-epitope protein, comprises aminoacid sequence IALYLQQNW (SEQ ID NO:21).

6. the isolating multi-epitope protein of claim 5, it comprises 13 kinds of EBV CTL epi-positions, and these epi-positions have aminoacid sequence YLLEMLWRL (SEQ ID NO:3) respectively; YLQQNWWTL (SEQ ID NO:1); ALLVLYSFA (SEQ ID NO:30); IALYLQQNW (SEQ ID NO:21); SSCSSCPLSKI (SEQ ID NO:51); PYLFWLAAI (SEQ ID NO:52); TYGPVFMCL (SEQ ID NO:53); RRRWRRLTV (SEQ ID NO:54); LLSAWILTA (SEQ ID NO:55); LTAGFLIFL (SEQ ID NO:56); VMSNTLLSAW (SEQ ID NO:57); IEDPPFNSL (SEQ ID NO:58); CLGGLLTMV (SEQ ID NO:59).

7. the isolating multi-epitope protein of claim 6, it comprises the aminoacid sequence shown in SEQ ID NO:81.

8. isolating nucleic acid, each isolating EBVCTL epi-position or epi-position varient among its coding claim 1-3.

9. isolating nucleic acid, each isolating protein among its coding claim 4-7.

10. the isolating nucleic acid of claim 8, it is made up of the nucleotide sequence shown in SEQ ID NO:78 or 79.

11. the isolating nucleic acid of claim 9, it is made up of the nucleotide sequence shown in SEQ ID NO:80.

12. an expression structure, it comprises among the claim 8-11 on one or more regulatory nucleotide sequence that is operably connected in the expression vector each isolating nucleic acid.

13. the expression structure of claim 12, it is based on adenovirus.

14. the expression structure of claim 12, the aminoacid sequence of its coding shown in SEQ ID NO:81.

15. comprise each the isolating host cell of expression structure of claim 12-14.

16. pharmaceutical composition, it comprises at least a isolating EBV ctl peptide epi-position and/or at least a isolating EBVCTL peptide epitopes varient according to claim 2 or 3 according to claim 1, and pharmaceutically acceptable carrier, thinner or vehicle.

17. the pharmaceutical composition of claim 16, it comprises the isolating EBV CTL epi-position of being made up of aminoacid sequence IALYLQQNW (SEQ ID NO:21).

18. the pharmaceutical composition of claim 16 or 17, it comprises multi-epitope protein, and this multi-epitope protein comprises the aminoacid sequence shown in SEQ ID NO:81.

19. a pharmaceutical composition, it comprises expression structure and pharmaceutically acceptable carrier, thinner or the vehicle of claim 12.

20. the pharmaceutical composition of claim 19, it comprises the expression structure of encoding amino acid sequence IALYLQQNW (SEQ ID NO:21).

21. the pharmaceutical composition of claim 20, it comprises an expression structure, and this structured coding has the multi-epitope protein of the aminoacid sequence shown in SEQ ID NO:81.

22. the pharmaceutical composition of claim 21, it comprises the nucleotide sequence shown in SEQ ID NO:82.

23. each pharmaceutical composition among the claim 16-22, said composition are a kind of immunotherapy compositions.

24. the pharmaceutical composition of claim 23, said composition are vaccines.

25. the isolating EBV CTL epi-position varient of the isolating EBV CTL epi-position of at least a claim 1 and/or at least a claim 2 or 3 is used for the treatment of and/or prevents application in the medicine of disease relevant with EBV in the animal in preparation.

26. the application of claim 25, at least a epi-position wherein comprise aminoacid sequence IALYLQQNW (SEQ ID NO:21).

27. the application of claim 25, at least a peptide epitopes wherein are the multi-epitope protein that comprises the aminoacid sequence shown in SEQID NO:81.

28. each expression structure of claim 12-14 is used for the treatment of and/or prevents application in the medicine of disease relevant with EBV in the animal in preparation.

29. the application of claim 28, expression structure coding wherein comprises the multi-epitope protein of aminoacid sequence IALYLQQNW (SEQ ID NO:21).

30. the application of claim 29, expression structure wherein comprise the nucleotide sequence shown in SEQ ID NO:82.

31. each application among the claim 25-30, wherein relevant with EBV disease is selected from B cell and T cell non-Hodgkin's, Hokdkin disease, and lymphocytic epithelium warty cancer.

32. right is wanted 31 application, wherein relevant with EBV disease is nasopharyngeal carcinoma (NPC).

33. each application among the claim 25-32, animal wherein is a Mammals.

34. the application of claim 33, Mammals wherein is the people.

35. the application of claim 34, the one or more of wherein at least a EBV peptide epitopes are to select according to the people's that will receive treatment HLA type.

36. at least a according to claim 1 the EBV peptide epitopes or be used for detecting in preparation whether a certain animal carries or the application of the composition of the method for once contacted EpsteinBarr virus according to the varient of claim 2 or 3, described method comprises and will separate from one or more T cells of described individuality and at least a according to the EBV peptide epitopes of claim 1 or according to the contacted step of the varient of claim 2 or 3 that described one or more T cells can indicate this animal whether to carry or once contacted Epstein Barr virus to replying of at least a EBV peptide epitopes.

37. the application of claim 36, animal wherein is a Mammals.

38. the application of claim 36, animal wherein is the people.