CN1711025A - Optimized polyepitope constructs and uses thereof - Google Patents

Abstract

The present invention relates to the field of biology. More particularly, it relates to polyepitope nucleic acid and peptide vaccines and methods of designing such vaccines to provide enhanced immunogenicity.

Description

Multi-epitope constructs of optimizing and application thereof are about the statement of the research of federation's support

In carrying out process of the present invention, the part work of finishing has obtained the financial support of U.S. government.U.S. government has some right of the present invention.

Technical field

The present invention relates to field of biology.More specifically, it relates to the multi-epitope nucleic acid vaccine and designs the immunogenic method that this vaccine provides enhancing.

Background technology

Developing the technology that relates to multi-epitope (" mini gene ", for example, " epigene ") vaccine.Several independently research is verified, can induce the immune response of while at multi-epitope.For example, can induce and detect reaction to a large amount of T cell-specifics.Under natural situation, Doolan etc. (Immunity, Vol.7 (1): 97-112 (1997)) use the PBMC from single donor to detect simultaneously the nearly memory T cell reaction of 17 different P.falciparum epi-positions.Ctl response when similarly, people such as Bertoni (J Clin Invest, Vol.100 (3): 503-13 (1997)) have detected the epi-position that 12 in the single donor different HBV-are originated.About the immunity of multi-epitope nucleic acid vaccine, reported several examples, wherein induced multiple t cell responses.For example, reported that wherein all epi-positions all are immunogenic and/or antigenic by about 10 mini gene vaccines that I class MHC epi-position is formed.More specifically, verified by 9 EBV (Thomson etc., Proc Natl Acad SciUSA, Vol.92 (13): 5845-9 (1995)), 7 HIV (Woodberry etc., J Virol, Vol.73 (7): 5320-5 (1999)), 10 mouse (Thomson etc., J Immunol, Vol.160 (4): 1717-23 (1998)) and the mini gene vaccine formed of (Mateo etc., J Immunol, Vol.163 (7): 4058-63 (the 1999)) epi-position in 10 tumours source be activated.Also verified, the Polyepitope DNA plasmid of the epi-position that is derived from HBV and HIV of 9 different HLA-A2.1-and the A11-restriction of encoding can be induced the CTL (Ishioka etc., J.Immunol, Vol.162 (7): 3915-25 (1999)) of anti-all epi-positions.

Therefore, can design the mini gene vaccine that contains a plurality of I class MHC and II class (being CTL and HTL) epi-position, and can present and discern all epi-positions.As if but the immunogenicity of multi-epitope constructs is subjected to the strong influence of many parameters, many also unclear before this in them.For example, the immunogenicity of the identical epi-position of expressing in the environment of different vaccine constructs (or antigenicity) can have the variation of several magnitude.Therefore, need discrimination method to optimize the polyepitope vaccines construct.Such optimization is important inducing aspect effective immune response and the final clinical efficacy.Therefore, polyepitope vaccines, particularly mini gene (minigene) vaccine that the invention provides the antigenicity of optimizing the polyepitope vaccines contain a large amount of epi-positions and immunogenic method and optimize according to these methods.

Following paragraph has been summed up some simply influences the primary variables of the mini gene immunogenicity relevant with II class MHC molecule with the I class, epi-position processing and presenting on antigen presenting cell (APC) potentially.

Immunodominance

In thousands of kinds of possible peptides, be to can be incorporated into the peptide form that I class MHC antigen is discerned by the T cell thereby small part is only arranged by compound exotic disease substance coding.This phenomenon is the exploitation of potential impact polyepitope vaccines significantly, is known as immunodominance (Yewdell etc., Annu RevImmunol, 17:51-88 (1999)).Several main variables help immunodominance.Here, we qualitatively and the variable description that will influence the generation of appropriate peptide quantitatively be the result of processing in the cell.

Connect epi-position

The connection epi-position is defined as the epi-position that constitutes side by side owing to two other epi-positions.This new epi-position is by forming from the C-end parts of first epi-position with from the N-end parts of second epi-position.The formation that connects epi-position is to be 1 potential problems of the epi-position design multi-epitope mini gene vaccine of I class and the restriction of II class, and its reason is as follows.At first, when exploitation mini gene that is made up of people's epi-position or that contain people's epi-position, typically test its immunogenicity in HLA transgenic experiments animal, the generation of mouse epi-position can cause undesirable immunodominance effect.Secondly, the generation meeting of new undesirable epi-position of people HLA I class or II quasi-molecule causes new T cell-specific in the vaccine recipient, and its infection cell or tumour of target that is not used as the t cell responses of inducing is expressed.These reactions are incoherent and invalid, by producing undesirable immunodominance effect, itself in addition can be counterproductive.

The existence that connects epi-position has been documented in many different experiment situations.People such as Gefter have at first confirmed the effect in system, and wherein arranged side by side and conllinear ground has synthesized the epi-positions (Perkins etc., J Immunol, Vol.146 (7): 2137-44 (1991)) of two different II class restrictions.This effect is so remarkable, and these new connection epi-positions are the immune system recognition of " silence " epi-position (wang etc., Cell Immunol, Vol.143 (2): 284-97 (1992)) fully.Also in the mankind, observe and point to the helper cell that connects epi-position, it is the result who carries out immunization with synthetic lipopeptid, the HTL epi-position that described lipopeptid is originated by the immunodominant CTL epi-position and the general tetanus toxoid in the HBV-source of HLA-A2-restriction is formed (Livingston etc., J.Immunol, Vol.159 (3): 1383-92 (1997)).Therefore, the generation of connection epi-position is the significant consideration when the design multi-epitope constructs.

The invention provides this problem and the method that prevents or minimize the generation that is connected epi-position of overcoming.

Flanking region (flanking region)

The epi-position of I class restriction generates (Yewdell etc., Annu Rev Immunol, 17:51-88 (1999)) by complex process.After relating to the potential repairing of the limited proteolysis of endo protease and circumscribed protease, be the displacement of striding endoplasmic reticulum (ER) film of the transportation thing relevant with antigen processing (TAP) molecule.Participating in the main cytosol protease compound of antigenic peptide generation and their precursor is proteasome (Niedermann etc., Immunity, Vol.2 (3): 289-99 (1995)), although the ER of also verified CTL precursor repairs (Paz etc., Immunity Vol.11 (2): 241-51 (1999)).Whether argued for a long time, being close to the C of epi-position and the residue of N end can influence the efficient that epi-position generates.

The output of finished epi-position and availability have become the immunogenic primary variables of decision, thereby whole mini gene renderd a service produce significantly main influence, because the amount of immunoreactive magnitude epi-position that directly combine with MHC and that show for the T cell recognition is proportional.Several researchs have confirmed true like this really.For example, observed basically induce (Wherry etc., J Immunol, Vol.163 (7): 3735-45 (1999)) with the CTL of the proportional virus-specific of epi-position density.And, used the reorganization mini gene of the best epi-position of the preprocessing of encoding to come the induction ratio epi-position of the level of observed higher level expression (Anton etc., J.Immunol, Vol.158 (6): 2535-42 (1997)) naturally in the full-length proteins.Generally, confirmed that mini gene causes (priming) than causing more effective (Restifo etc., J Immunol, Vol.154 (9): 4414-22 (1995) with complete antigen; Ishioka etc., J Immunol, Vol.162 (7): 3915-25 (1999)), although have been noted that some exception (Iwasaki etc., Vaccine, Vol.17 (15-16): 2081-8 (1999)).

Research in the past thinks that the residue in the epi-position (Hahn etc., J Exp Med, Vol.176 (5): 1335-41 (1992)) is mainly regulated immunogenicity.But other research has obtained similar conclusion, mainly is based on to be implanted in epi-position in the uncorrelated gene or in homologous genes at diverse location (Chimini etc., J Exp Med, Vol.169 (1): 297-302 (1989); Hahn etc., J Exp Med, Vol.174 (3): 733-6 (1991)).But, other experiment (DelVal etc., Cell, Vol.66 (6): 1145-53 (1991); Hahn etc., J Exp Med, Vol.176 (5): 1335-41 (1992)) show that directly the residue adjacent with the CTL epi-position can directly influence identification (Couillin etc., J Exp Med, Vol.180 (3): 1129-34 (1994); Bergmann etc., J Virol.Vol.68 (8): 5306-10 (1994)).In the mini gene vaccine, caused new arguement.People such as Shastri (Shastri etc., J Immunol, Vol.155 (9): 4339-46 (1995)) find, can the appreciable impact t cell responses by changing the distolateral wing residue of N-, and then can the suppressor T cell reaction but add the distolateral wing residue of single C-.Observed the strongest inhibition when using isoleucine, leucine, cysteine and proline as the distolateral wing residue of C-.On the contrary, Gileadi (Gileadi etc., Eur J Immunol, Vol.29 (7): 2213-22 (1999)) has reported the profound influence that the residue with the N end that is positioned at mouse influenza virus epi-position changes.People such as Bergmann find, aromatics, alkalescence can support effective epi-position to discern with alanine residue, G and P residue then are (Bergmann etc., J Immunol, Vol.157 (8): the 3242-9 (1996)) of strong inhibition.On the contrary, Lippolis (Lippolis etc., J Virol, Vol.69 (5): 3134-46 (1995)) thinks that replacing the flank residue can not influence identification.But, only tested quite conservative replacement, it can not influence the specificity of proteasome.

Roughly the specificity with proteasome is relevant with natural epi-position specificity generally to seem the specificity of these effects.For example, the specificity of proteasome partly is (Niedermann etc., Immunity, Vol.2 (3): the 289-99 (1995)) of trypsin-like, shears behind basic amino acid.However, hydrophobic effective shearing with carboxyl side acidic residues still is possible.Corresponding to these specificitys is people's such as Sherman research, R to the H sudden change of the position of the C-end back that it is found in the p53 epi-position can influence the albumen processing (Theobald etc. of proteasome mediation, J Exp Med, Vol.188 (6): 1017-28 (1998)).Several other research (Hanke etc., J Gen Virol, Vol.79 (Pt1): 83-90 (1998); Thomson etc., Proc Natl Acad Sci USA, Vol.92 (13): 5845-9 (1995)) show, can use minimum epi-position to make up mini gene, as if do not need these flanking sequences, although admit to use flanking region that the effectiveness of further optimization can be provided yet.

In a word, for HLA I class epi-position, it be unclear that flanking region to the processing of CTL epi-position and the influence of presenting.The mini gene vaccine is not carried out the network analysis of flanking region regulating and controlling effect as yet.Therefore, need to use the mini gene vaccine that to encode by the epi-position of people I class restriction to analyze generally.The invention provides such analysis, and the polyepitope vaccines construct to immunogenicity and antigenicity optimization correspondingly is provided, and the method that designs this construct.

The formation of HLA II class peptide complexes also is the result of a series of comprehensive incidents, and they are different with the processing of HLA I class.This processing approach relates to removing with the CLIP of the degraded of invariant chain (mvariant chain) combining (Ii), its transportation to particular compartment, Ii to CLIP and HLA-DM catalysis (sees (Blum etc., Crit Rev Immunol, Vol.17 (5-6): 411-7 (1997); Arndt etc., Immunol Res, Vol.16 (3): 261-72 (1997)).And various general cathepsins and concrete cathepsin S and L also have potential key effect (Nakagawa etc., Immunity, Vol.10 (2): 207-17 (1999)) in the Ii degraded.But, generation about functional epi-position, as if this process be low optionally (Chapman H.A. to a certain extent, Curr Opin Immunol, Vol.10 (1): 93-102 (1998)), the peptide of many sizes can both be attached to II class MHC (Hunt etc., Science, Vol.256 (5065): 1817-20 (1992)).As if great majority or whole possible peptide can both generate (Moudgil etc., J Immunol, Vol.159 (6): 2574-9 (1997); With Thomson etc., J Virol, Vol.72 (3): 2246-52 (1998)).Therefore, compare with the problem of flanking region, connecting being created in the specific embodiments of epi-position is more serious problem.

Summary of the invention

The invention provides the multi-epitope nucleic acid construct of can encode a plurality of CTL and/or HTL epi-position, the polypeptide construct that contains a plurality of CTL and/or HTL epi-position (preferably by described nucleic acid construct coding), contain the such nucleic acid construct and/or the cell of polypeptide construct, the composition that contains such nucleic acid construct and/or polypeptide construct and/or such cell, use such nucleic acid construct and/or polypeptide construct and/or composition and/or the immunoreactive method of cytositimulation (for example methods of treatment).

In some embodiment, the invention provides and contain following key element or by its polynucleotides of forming:

(a) multi-epitope constructs (for example, mini gene), it contains coding hepatitis type B virus (HBV) cytotoxic T lymphocyte (CTL) epi-position pol 562, pol 745, and env 332, and pol 530, pol 388, and env 249, and env 359, pol 640, and env 335, and env 183, env 313, core (core) 117, core 19, core 18, core 419, pol 392, pol 531, pol 415, and pol 47, and pol 455, core 141, pol 429, and env 236, and pol 166, pol 538, core 101, the nucleic acid of pol 354 and core 137 (being that each HBV CTL epi-position is made up of the correlated series in the table 7), wherein this nucleic acid is connected to each other in identical reading frame directly or indirectly;

(b) multi-epitope constructs in (a), it also contains the nucleic acid of coding HBV CTL epi-position pol 665 (i.e. 665 epi-positions of pol in the table 7), and this nucleic acid is connected to the CTL epi-position nucleic acid of (a) directly or indirectly in identical reading frame;

(c) multi-epitope constructs, it contains coding hepatitis type B virus (HBV) cytotoxic T lymphocyte (CTL) epi-position pol 149, core 18, pol 562, pol 538, and pol 455, env183, core 141, pol 665, and env 335, env 313, and pol 354, and pol 629, core 19, pol 150, and pol 47, pol 388, the nucleic acid of pol 531 and pol 642, and wherein this nucleic acid is connected to each other in identical reading frame directly or indirectly;

(d) (a) or (b) or multi-epitope constructs (c), it also contains one or more nucleic acid at interval, and this interval nucleic acid is connected to CTL epi-position nucleic acid directly or indirectly in identical reading frame;

(e) multi-epitope constructs (d), wherein these one or more at interval nucleic acid be positioned between the CTL epi-position nucleic acid of (a), between (a) and the CTL epi-position nucleic acid (b), (a) and (b) and (a), between the CTL epi-position nucleic acid of (c), between the CTL epi-position nucleic acid of (C);

(f) (d) or multi-epitope constructs (e), wherein this interval nucleic acid coding length is the amino acid sequence of 1～8 residue;

(g) (d) multi-epitope constructs of each in (f), wherein 2 or different (the being inequality) amino acid sequence of a plurality of intervals nucleic acid coding;

(h) (d) multi-epitope constructs of each in (g), wherein 2 or a plurality of intervals nucleic acid coding and other different amino acid sequence of amino acid sequence of nucleic acid coding at interval;

(i) (d) multi-epitope constructs of each in (h), wherein 2 or the identical amino acid sequence of a plurality of intervals nucleic acid coding;

(j) (d) multi-epitope constructs of each in (i), wherein one or more at interval nucleic acid codings contain 3 continuous alanine (Ala) residues or by its amino acid sequence of forming;

(k) (a) multi-epitope constructs of each in (j), it also contains the nucleic acid of one or more codings HTL epi-positions, and this nucleic acid is connected to CTL epi-position nucleic acid and/or nucleic acid at interval directly or indirectly in identical reading frame;

(l) multi-epitope constructs (k), wherein this HTL epi-position is PADRE ^_Epi-position;

(m) multi-epitope constructs (k), wherein this HTL epi-position is a HBV HTL epi-position;

(n) multi-epitope constructs (m), wherein this HBV HTL epi-position is selected from pol 774, pol694, pol 145, core 50, and pol 385, pol 523, and env 339, and pol 501, pol420, pol 412, and env 180, core 120, pol 96, and pol 618, pol 767 and pol 664 (being that each HBV HTL epi-position is made up of the correlated series in the table 11);

(o) (k) multi-epitope constructs of each in (n), it also contains one or more interval nucleic acid between CTL epi-position and HTL epi-position or between the HTL epi-position;

(p) (a) multi-epitope constructs of each in (o), it also contains one or more I class MHC and/or II class MHC target nucleic acid;

(q) multi-epitope constructs (p), wherein this target nucleic acid coding is selected from following target sequence: Ig κ burst, tissue plasminogen activator's burst, the insulin signaling sequence, the endoplasmic reticulum burst, LAMP-1 lysosome target sequence, LAMP-2 lysosome target sequence, HLA-DM lysosome target sequence, the HLA-DM-relating sequence of HLA-DO (HLA-DM-association sequence), Ig-α cytoplasm domain, Ig-β cytoplasm domain, Ii albumen, influenza stromatin, the HBV surface antigen, HBV cAg and yeast Ty albumen;

(r) (a) multi-epitope constructs of each in (q), it was optimized CTL and/or the processing of HTL epi-position;

(s) (a) multi-epitope constructs of each in (r), wherein this CTL nucleic acid minimizes so that CTL and/or HTL connect the quantity of epi-position through screening;

(t) (k) multi-epitope constructs of each in (s), wherein this HTL nucleic acid minimizes so that CTL and/or HTL connect the quantity of epi-position through screening;

(u) (a) multi-epitope constructs of each in (t), it contains the nucleic acid of one or more one or more flanking amino acid residues of encoding;

(the v) multi-epitope constructs of (u), wherein these one or more flanking amino acid residues are selected from: be positioned at the K of the C+1 position of CTL epi-position nucleic acid, R, N, Q, G, A, S, C and T;

(w) (a) to (multi-epitope constructs of each v), wherein this HBV CTL nucleic acid connects with the order shown in Figure 27 A;

(x) (n) multi-epitope constructs of each in (w), wherein this HBV HTL nucleic acid connects with the order shown in Figure 28 A;

(y) (c) to (multi-epitope constructs of each v) or (x), wherein this HBV CTL nucleic acid connects with order shown in Figure 34;

(z) (a) multi-epitope constructs of each in (x), its coding contain the amino acid sequence shown in Figure 24 B or the table 13,14,18 or 19 or by its peptide of forming;

(aa) multi-epitope constructs (z), it contains and is selected from following nucleotide sequence: the nucleotide of the nucleotide sequence in the table 13+1 is to 1248, the nucleotide of the nucleotide sequence in the table 14+1 is to 1032, nucleotide sequence among Figure 24 C, the nucleotide of the nucleotide sequence in the table 18+1 to 2292 and table 19 in nucleotide+1 of nucleotide sequence to 2232;

(bb) (c) to (multi-epitope constructs of each v) or (x) or (y) or (z), its coding contain the amino acid sequence shown in table 23 or 24 or by its peptide of forming;

(cc) multi-epitope constructs (bb), it contains and is selected from following nucleotide sequence: the nucleotide of the nucleotide sequence in the table 23+1 is to 618, or the nucleotide of the nucleotide sequence in the table 24+1 is to 657;

(dd) (a) multi-epitope constructs and one or more regulating and controlling sequences of each in (cc);

(ee) (a) multi-epitope constructs and one or more IRES of each in (dd);

(ff) (a) multi-epitope constructs and one or more promotors of each in (ee);

(gg) (a) multi-epitope constructs and one or more CMV promotors of each in (ff);

(hh) (a) multi-epitope constructs and 2 or a plurality of CMV promotor of each in (gg);

The (ii) multi-epitope constructs of each in (a) to (hh), and carrier;

(jj) multi-epitope constructs (ii), wherein this carrier is an expression vector;

(kk) (a) multi-epitope constructs of each in (jj), it has Figure 29 A (i), (ii) or the structure of the multi-epitope constructs (iii).

In some embodiment, (a) has Figure 29 A (i), (ii) or the carrier structure (iii) to the polynucleotides of (kk).

In some embodiment, the invention provides the polynucleotides that contain 2 multi-epitope constructs, the 1st contain above each HBV multi-epitope constructs in (a) to (kk), the 2nd contains HBV HTL epi-position, those in (n) for example, wherein the 1st is not directly to be connected with the 2nd multi-epitope constructs, and/or is not to be connected in the identical frame.Each the 1st and the 2nd multi-epitope constructs all are operably connected to regulating and controlling sequence, for example promotor or IRES.The polynucleotides that contain the 1st and the 2nd multi-epitope constructs can comprise, for example, and at least 1 promotor and at least 1 IRES, 1 promotor and 1 IRES, 2 promotors, or 2 or a plurality of promotor and/or IRES.Promotor can be CMV promotor other promotor described herein or known in the art.In preferred embodiments, 2 multi-epitope constructs have Figure 29 A (i) or (ii) shown in structure.The 2nd multi-epitope constructs can be encoded and be contained the amino acid sequence shown in Figure 24 C or the table 14 or by its peptide of forming.The nucleotide sequence of nucleotide+1 that the 2nd multi-epitope constructs can contain the nucleotide sequence that is selected from Figure 24 C and the nucleotide sequence in the table 14 to 1032.

In other embodiment, the invention provides peptide by above-mentioned polynucleotide encoding, for example, contain following element or by its peptide of forming:

(a) multi-epitope constructs (for example, mini gene), it contains hepatitis type B virus (HBV) cytotoxic T lymphocyte (CTL) epi-position pol 562, and pol 745, env 332, and pol 530, and pol 388, env 249, and env 359, and pol 640, env 335, and env 183, and env 313, core 117, core 19, core 18, core 419, pol 392, and pol 531, pol 415, and pol 47, and pol 455, core 141, pol 429, and env 236, pol 166, and pol 538, core 101, pol 354 and core 137 (be the CTL epi-position of Figure 27 A, form) by the sequence in the table 7, described epi-position is connected to each other directly or indirectly;

(b) multi-epitope constructs in (a), it also contains HBV CTL epi-position pol 665, and described epi-position is connected to the CTL epi-position of (a) directly or indirectly;

(c) multi-epitope constructs, it contains hepatitis type B virus (HBV) cytotoxic T lymphocyte (CTL) epi-position pol 149, core 18, pol 562, pol 538, and pol 455, and env 183, core 141, pol 665, and env 335, env 313, and pol 354, and pol 629, core 19, pol 150, and pol 47, pol 388, pol 531 and pol 642, and described epi-position is connected to each other directly or indirectly;

(d) (a) or (b) or multi-epitope constructs (c), it also contains one or more septs, and described sept is connected to the CTL epi-position directly or indirectly;

(e) multi-epitope constructs (d), wherein these one or more septs be positioned between the CTL epi-position of (a), between (a) and the CTL epi-position (b), (a) and (b) and (a), between the CTL epi-position of (c), between the CTL epi-position of (c);

(f) (d) or multi-epitope constructs (e), wherein the length of this sept is 1～8 amino acid residue;

(g) (d) multi-epitope constructs of each in (f), wherein 2 or a plurality of sept contain different (being inequality) amino acid sequences, or are made up of different amino acid sequences;

(h) (d) multi-epitope constructs of each in (g), wherein 2 or a plurality of sept contain the amino acid sequence different with the amino acid sequence of other sept, or are made up of described amino acid sequence;

(i) (d) multi-epitope constructs of each in (h), wherein 2 or a plurality of sept contain identical amino acid sequence, or are made up of described amino acid sequence;

(j) (d) multi-epitope constructs of each in (i), wherein one or more septs contain 3 continuous alanine (Ala) residue, or are made up of it;

(k) (a) multi-epitope constructs of each in (j), it also contains one or more HTL epi-positions, and described epi-position is connected to CTL epi-position and/or sept directly or indirectly;

(l) multi-epitope constructs (k), wherein these one or more HTL epi-positions are PADRE ^_Epi-position;

(m) multi-epitope constructs (k), wherein these one or more HTL epi-positions are HBV HTL epi-positions;

(n) multi-epitope constructs (m), wherein these one or more HTL epi-positions are selected from pol 774, and pol 694, and pol 145, core 50, pol 385, env 339, and pol 501, and pol 420, and pol 412, and env 180, core 120, pol 96, and pol 618, pol 767 and pol 664;

(o) (k) multi-epitope constructs of each in (n), it also contains one or more septs between CTL epi-position and HTL epi-position or between the HTL epi-position;

(p) (a) multi-epitope constructs of each in (o), it also contains one or more I class MHC and/or II class MHC target sequence;

(q) multi-epitope constructs (p), wherein one or more target sequences are selected from: Ig κ burst, tissue plasminogen activator's burst, the insulin signaling sequence, endoplasmic reticulum burst, LAMP-1 lysosome target sequence, LAMP-2 lysosome target sequence, HLA-DM lysosome target sequence, the HLA-DM-relating sequence of HLA-DO, Ig-α cytoplasm domain, Ig-β cytoplasm domain, Ii albumen, influenza stromatin, the HBV surface antigen, HBV cAg and yeast Ty albumen;

(r) (a) multi-epitope constructs of each in (q), it was optimized CTL and/or the processing of HTL epi-position;

(s) (a) multi-epitope constructs of each in (r), wherein this CTL epi-position minimizes so that CTL and/or HTL connect the quantity of epi-position through screening;

(t) (k) multi-epitope constructs of each in (s), wherein this HTL epi-position minimizes so that CTL and/or HTL connect the quantity of epi-position through screening;

(u) (a) multi-epitope constructs of each in (t), it contains one or more flanking amino acid residues;

(the v) multi-epitope constructs of (u), wherein these one or more flanking amino acid residues are selected from: be positioned at the K of the C+1 position of CTL epi-position, R, N, Q, G, A, S, C, and T;

(w) (a) to (multi-epitope constructs of each v), wherein this HBV CTL epi-position connects with the order shown in Figure 27 A;

(x) (n) multi-epitope constructs of each in (w), wherein this HBV HTL epi-position connects with the order shown in Figure 28 A;

(y) (c) to (multi-epitope constructs of each v) or (x), wherein this HBV CTL epi-position connects with order shown in Figure 34;

(z) (a) multi-epitope constructs of each in (x), it contains the amino acid sequence shown in Figure 24 B or the table 13,14,18 or 19, or is made up of it;

(aa) multi-epitope constructs (z), it is by being selected from following nucleic acid sequence encoding: the nucleotide of the nucleotide sequence in the table 13+1 is to 1248, the nucleotide of the nucleotide sequence in the table 14+1 is to 1032, nucleotide sequence among Figure 24 C, the nucleotide of the nucleotide sequence in the table 18+1 to 2292 and table 19 in nucleotide+1 of nucleotide sequence to 2232;

(bb) (c) to (multi-epitope constructs of each v) or (x) or (y), it contains the amino acid sequence shown in table 23 or 24, or is made up of it;

(cc) multi-epitope constructs (bb), it is by being selected from following nucleic acid sequence encoding: the nucleotide of the nucleotide sequence in the table 23+1 is to 618, or the nucleotide of the nucleotide sequence in the table 24+1 is to 657.

In other embodiment, the invention provides the cell that contains above-mentioned polynucleotides and/or polypeptide; The composition that contains these polynucleotides and/or polypeptide and/or cell; Prepare these polynucleotides, polypeptide, cell and method for compositions; With the method (for example treating and/or preventing method) of using these polynucleotides and/or polypeptide and/or cell and/or composition immune response stimulating.Further describe the present invention below.

Definition

Definition below providing is so that those of ordinary skill in the art can understand the certain preferred embodiments of invention as herein described.But should be understood that these definition only are exemplary, should not be used to limit the scope of claims of the present invention.Those of ordinary skill in the art can change slightly to following definition, and utilizes the definition of such change to understand and implement invention disclosed herein.These changes are conspicuous for the ordinary skill in the art, and they can be applied in the claim described below, and will be understood that it is within the scope of the invention.

In this manual, " binding data " result is through being often expressed as " IC ₅₀".IC ₅₀Be in conjunction with experiment, observe suppress 50% with reference to peptide in conjunction with the time peptide concentration.Under given experiment condition (promptly limiting the peptide concentration of HLA albumen and mark), these are worth near K _DValue.The experiment write up that detects combination is in for example PCT publication number WO 94/20127 and WO 94/03205.Should be pointed out that if variation has taken place experiment condition, according to the concrete reagent that uses (for example HLA preparation etc.), IC ₅₀Value can change, and often is rapid.For example, the HLA molecule of excessive concentrations can increase the IC of the given part of apparent measurement ₅₀Perhaps, represent combination with respect to the reference peptide.Although along with concrete experiment becomes more responsive or more insensitive, the IC of the peptide of test ₅₀May have change to a certain degree, but can obviously not change with respect to the combination of reference peptide.For example, at the IC of reference peptide ₅₀Increase in the experiment of carrying out under 10 times the condition IC of experimental peptide ₅₀Also can change about 10 times.Therefore, for fear of ambiguous, whether be the judgement of good, medium, more weak or negative bond to peptide, normally based on its IC ₅₀, with respect to the IC of standard peptide ₅₀Can also use other detection system to detect combination, comprise use is following those: living cells (for example, Ceppellini etc., Nature 339:392,1989; Christnick etc., Nature 352:67,1991; Busch etc., Int.Immunol.2:443,19990; Hill etc., J.Immunol.147:189,1991; Del Guercio etc., J.Immunol.154:685,1995), the cell free system of use detergent pyrolysis product is (for example, Cerundolo etc., J.Immunol.21:2069,1991), the MHC of immobilized purifying is (for example, Hill etc., J.Immunol.152,2890,1994; Marshall etc., J.Immunol.152:4946,1994), the ELISA system (for example, Reay etc., EMBO J.11:2829,1992), surface plasma resonance (for example, Khilko etc., J.Biol.Chem.268:15425,1993); Solvable mensuration (for example, Ljunggren etc., Nature 346:476,1990 of detecting (Hammer etc., J.Exp.Med.180:2353,1994) and I class MHC stabilisation or assembling mutually of high flux; Schumache r etc., Cell 62:563,1990; Townsend etc., Cell 62:285,1990; Parker etc., J.Immunol.149:1896,1992).

Residue site in the epi-position in called after " c-terminus " or " c-terminus site " is meant that in the residue site of the end of the epi-position of the c-terminus that approaches peptide most it uses the conventional nomenclature that defines below to name." C+1 " is meant that this residue or site followed by the C-end residue of epi-position, are meant that promptly this residue is adjacent to the C-end of epi-position.The epi-position " c-terminus site " that occurs at the c-terminus of multi-epitope constructs can be in fact corresponding or not corresponding the carboxy terminal of polypeptide.In preferred embodiments, the epi-position of using in the multi-epitope constructs of optimizing is the epi-position of carrying motif, and the c-terminus of this epi-position defines with respect to basic anchor residue (corresponding to specific motif).

Residue site in the epi-position in called after " aminoterminal " or " aminoterminal site " is meant that in the residue site of the end of the N-terminal epi-position that approaches peptide most it uses the conventional nomenclature that defines below to name." N-1 " is meant that this residue or site are being close to the epi-position of the aminoterminal end of epi-position (position period 1).The epi-position " aminoterminal site " that occurs at the aminoterminal of multi-epitope constructs can be in fact corresponding or not corresponding the aminoterminal end of polypeptide.In preferred embodiments, the epi-position of using in the multi-epitope constructs of optimizing is the epi-position of carrying motif, and the aminoterminal of this epi-position defines with respect to basic anchor residue (corresponding to specific motif).

" calculator " or " computer system " generally includes: processor; At least one information storage/extraction element, for example hard disk, disc driver or magnetic tape station, at least one input unit, for example keyboard, mouse, touch-screen or microphone; And display unit.In addition, calculator can comprise the communication channel that is connected with network, makes the long-distance user can be by network and this calculator communication, to carry out multi-epitope constructs optimizational function as herein described.Such calculator can comprise greater or less than above-mentioned these.This network can be Local Area Network, wide area network (WAN) or World Wide Web, for example World Wide Web (for example internet).

As used herein " construct " typically refers to absent variable synthetic (composition) in nature.Construct can be " polynucleotide constructs " or " polypeptide construct ".Can produce construct by synthetic technology, for example the recombinant DNA preparation of nucleic acid and amino acid and peptide and polypeptide and expression or chemical synthesising technology.Can also be by a kind of material interpolation or affinity to be produced construct to another kind, this form that obtains can not occur in nature.

When mentioning nucleic acid and polynucleotides, term " multi-epitope constructs " is equal to phrase with term " mini gene " and " multi-epitope nucleic acid vaccine " with other to be used interchangeably, and the multi-epitope nucleic acid that comprises the peptide epitopes of the random length of encoding, this peptide epitopes can be incorporated on the molecule that works in immune system, preferred I class HLA and T-cell receptors or II class HLA and T-cell receptors.Epi-position nucleic acid in the multi-epitope constructs can encode I class HLA epi-position and/or II class HLA epi-position.The epi-position nucleic acid of the I class of encoding HLA is called CTL epi-position nucleic acid, and the epi-position nucleic acid of the II class of encoding HLA is called HTL epi-position nucleic acid.Some multi-epitope constructs can have the subclass of the multi-epitope nucleic acid of the multi-epitope nucleic acid subclass of the I class HLA epi-position of encoding and another II class HLA epi-position of encoding.CTL epi-position nucleic acid preferably code length is lower than about 15 residues or length and is lower than about 13 amino acid or length and is lower than about 11 amino acid, preferably is about 8 to about 13 amino acid, more preferably is about 8 to about 11 amino acid (for example 8,9,10 or 11) with most preferably be about 9 or 10 amino acid whose epitope peptides.HTL epi-position nucleic acid can code length be lower than about 50 residues, usually by about 6 to about 30 residues, more generally by about 12 to 25, often about 15 to 20, preferably about 7 to about 23, preferably about 7 to about 17, more preferably about 11 to about 15 (for example 11,12,13,14 or 15) individual and most preferably about 13 epitope peptides that amino acid is formed.Multi-epitope constructs as herein described preferably comprises 5 or more, 10 or more, 15 or more, 20 or more or 25 or multi-epitope nucleic acid more.All epi-position nucleic acid in the multi-epitope constructs can be from an organism (for example, the nucleotide sequence of each epi-position nucleic acid may reside in HBV or the HIV bacterial strain), perhaps multi-epitope constructs can comprise and (for example be present in 2 or a plurality of different organism, the nucleotide sequence of the nucleotide sequence of some coding epi-positions is from HBV, some are from HIV, and/or some are from HCV) in the epi-position nucleotide sequence.Term " epigene " is used in reference to some multi-epitope constructs here.As mentioned below, the flank of one or more epi-position nucleic acid in the multi-epitope constructs can be interval nucleic acid and/or other nucleic acid as herein described or known in the art.

When mentioning polypeptide, term " multi-epitope constructs " is equal to phrase with term " mini gene construct " and " polyepitope vaccines " with other to be used interchangeably, and the peptide epitopes that comprises a plurality of random lengths, it can be incorporated on the molecule that works in immune system, preferred I class HLA and T-cell receptors or II class HLA and T-cell receptors.Epi-position in the multi-epitope constructs can be I class HLA epi-position and/or II class HLA epi-position.I class HLA epi-position is called the CTL epi-position, and II class HLA epi-position is called the HTL epi-position.Some multi-epitope constructs can have the subclass of I class HLA epi-position subclass and another II class HLA epi-position.The length of CTL epi-position is preferably lower than about 15 residues or is lower than about 13 amino acid or is lower than about 11 residues, preferably is about 8 to about 13 amino acid, more preferably is about 8 to about 11 amino acid (for example 8,9,10 or 11) with most preferably be about 9 amino acid.The length of HTL epi-position be lower than about 50 residues, usually by about 6 to about 30 residues, more generally by about 12 to 25, often about 15 to 20, preferably about 7 to about 23, preferably about 7 to about 17, more preferably about 11 form to about 15 (for example 11,12,13,14 or 15) individual and most preferably about 13 amino acid.Multi-epitope constructs as herein described preferably comprises 5 or more, 10 or more, 15 or more, 20 or more or 25 or multi-epitope more.All epi-positions in the multi-epitope constructs can be from an organism (for example, each epi-position may reside in HBV or the HIV bacterial strain), perhaps multi-epitope constructs can comprise and (for example be present in 2 or a plurality of different organism, some epi-positions are from HBV, some are from HIV, and/or some are from HCV) in epi-position.Term " epigene " is used in reference to some multi-epitope constructs here.As mentioned below, the flank of one or more epi-positions in the multi-epitope constructs can be intervening sequence and/or other sequence as herein described or known in the art.

" polyepitope vaccines " and " vaccine of multi-epitope " synonym are meant the vaccine that contains a plurality of epi-positions.

" cross reactivity combination " is meant that peptide is by the combination of a more than HLA molecule; Synonym is " degeneracy combination ".

" hide epi-position " can induce reaction by carrying out immunity with the peptide that separates, and still when with the complete whole albumen that contains this epi-position during as antigen, this is reflected at external is not cross reactivity.

" epi-position of dominance " is at the epi-position of carrying out the induction of immunity reaction of immunity back with whole native antigen (see, for example, Sercarz etc., Annu.Rev.Immunol.11:729-766,1993).This reaction and the peptide epitopes that separates external be cross reaction.

For concrete amino acid sequence, " epi-position " is the one group of amino acid residue that participates in the identification of specific immunoglobulins, perhaps in the context of T cell, these residues are that the identification of tcr protein and/or main histocompatibility complex (MHC) acceptor is necessary.Immune system occasion in external or body, epi-position is the global feature of molecule, for example one-level, secondary and three grades of peptide structures and electric charge, they form the recognition site of immunoglobulin, TXi Baoshouti or HLA molecule together.In this application, epi-position and peptide often use interchangeably.But should be appreciated that albumen separation or purifying or peptide molecule bigger than epi-position of the present invention and that contain epi-position of the present invention still are within the scope of the present invention.

" flank residue " is the adjacent residue of position and epi-position.The flank residue can be imported or is inserted into the position that N-holds or C-holds of closing on epi-position.

" immunogenic peptide " or " peptide epitopes " are the peptides that contains allelomorph-specific motif or hyper-base preface, so this peptide can be incorporated into the HLA molecule, and induce CTL and/or htl response.Therefore, immunogenic peptide of the present invention can be incorporated into appropriate H LA molecule, after this induces Cytotoxic t cell responses or helper cell reaction at antigen (immunogenic peptide is derived from this antigen).

" irregular analog (heteroclitic analog) " is defined as the peptide that specific T cell is had the effectiveness of raising in this article, as by the reaction of the enhancing of given dose measured, or the reaction by more needing to reach identical more on a small quantity.The advantage of irregular analog comprises, epi-position can more effective or more economical (because needing amount still less can reach identical effect).In addition, the epi-position of modification can overcome the T cell anergy (T cell tolerance) of antigentic specificity.

" human lymphocyte antigen " or " HLA " be people I class or the main histocompatibility complex of II class (MHC) albumen (see, for example, Stites, etc., IMMUNOLOGY, the 8th edition, LangePublishing, Los Altos, CA (1994)).

As used herein " super type of HLA or HLA family " described the classification of HLA molecule, and they are according to total peptide-binding specificity grouping.The HLA I quasi-molecule that the peptide that carries some amino acid motif is had similar binding affinity is included in the super type of such HLA.Term HLA superfamily, the super type of HLA family, HLA family and HLA xx-sample molecule (the wherein specific HLA type of xx representative) are synonyms.

As used herein, be defined as with 50nM or lower IC about " high affinity " of HLA I quasi-molecule ₅₀Or K _DValue is carried out combination; " medium affinity " about HLA I quasi-molecule is defined as with about IC of 50 to about 500nM ₅₀Or K _DValue is carried out combination." high affinity " about HLA II quasi-molecule is defined as with 100nM or lower IC ₅₀Or K _DValue is carried out combination; " medium affinity " about HLA II quasi-molecule is defined as with about IC of 100 to about 1000nM ₅₀Or K _DValue is carried out combination.

IC ₅₀Be in conjunction with experiment, observe suppress 50% with reference to peptide in conjunction with the time peptide concentration.Depend on given experiment condition (promptly limiting the peptide concentration of HLA albumen and mark), these values may be near K _DValue.

In the context of 2 or a plurality of peptide sequences, " identical " or percentage " homogeny " are meant, when the contrast of in contrast window, carrying out maximum correspondence and comparison, as use sequence to contrast algorithm or measured, 2 or a plurality of sequence or subsequence identical or that have identical specified amino acid residues percentage by manual comparison and visual inspection.

At the specific site of multi-epitope constructs, for example distolateral at the C-of the C-end that closes on epi-position, " importing " amino acid residue comprises the customization multi-epitope, makes the residue that needs at specific site, for example, closes on epi-position, perhaps makes harmful residue not close on the C-end of epi-position.This term also is included in specific site and inserts amino acid residue, preferably preferred or medium amino acid residue.Can also be by an amino acid residue be substituted by another, and amino acid residue is imported sequence.Preferably, such replacement is carried out according to the simulation principle, for example described in common unsettled U.S. serial of submitting on March 1st, 1,999 09/260,714 and the PCT application number PCT/US00/19774.

Phrase " separation " or " biological pure " are meant that this material in fact or be substantially devoid of common meeting follow its component when the native state of this material.Therefore, the peptide according to separation of the present invention does not preferably contain the material of following this peptide usually in its in situ environment.

" connection " is meant that this area is used for known any method of connection peptides on function, the fusion that includes but not limited to recombinate, covalent bonds, disulfide-bonded, ionic bond combination, hydrogen bond combination and static combination.

" main histocompatibility complex " or " MHC " are a string genes, and it works in the cell interaction of the responsible physiologic immunity reaction of control.In the mankind, the MHC compound is also referred to as the HLA compound.About the detailed description of MHC and HLA compound, see Paul, FUNDAMENTALIMMUNOLOGY, the 3rd edition, Raven Press, New York, 1993.

As used herein, " centre of peptide " is meant the position of the peptide of non-amino or carboxyl terminal.

As used herein " the connection epi-position of minimal amount " is meant that like this some connect epi-positions, and it is less than and uses selection criteria at random to create.

Term " motif " is meant the pattern of the residue in the peptide of given length, and for I class HLA motif, this peptide normally about 8 is to about 13 amino acid, and for II class HLA motif, this peptide normally about 6 is to about 25 amino acid, and it can be by specific HLA molecular recognition.For every kind of albumen of every kind of people HLA allelomorph coding, peptide motif is normally different, and on the pattern of main and less important anchor residue difference is arranged.

" negative in conjunction with residue " or " harmful residue " is such amino acid, if be present in some position (typically not being main anchor position point) of peptide epitopes, it can reduce the binding affinity of this peptide and the corresponding HLA molecule of this peptide.

Phrase " is operably connected " and is meant such connection, and wherein nucleotide sequence is connected to this sequence (or a plurality of sequence) in the mode of the function that can change another nucleotide sequence (or a plurality of sequence).For example, may be operably coupled to the nucleic acid of regulating and controlling sequence (for example promotor/operator) or multi-epitope nucleic acid construct knows from experience expression with nucleic acid or construct and places the influence of regulating and controlling sequence or control down.For 2 nucleotide sequences (sequence of an encoding proteins for example, with a promoter region sequence that is connected to 5 of this coded sequence ' end), if inducing of promoter function causes transcribing of albumen coded sequence mRNA, if and the character (1) of the connection between 2 nucleotide sequences does not cause the generation of frameshift mutation, (2) do not stop expression regulation sequence to instruct the expression of mRNA or albumen yet, say that then these 2 nucleotide sequences are operably connected.Therefore, if promotor can influence transcribing of nucleotide sequence, then this promoter region is to be operably connected to this nucleotide sequence.

" optimization " is meant by following method raising to have the immunogenicity or the antigenicity of the right multi-epitope constructs of at least one epi-position: the screening epi-position is so that connect minimizing of epi-position, insertion is positioned at the C-end of epi-position or the flank residue of the distolateral wing of N-, and residue is connected the generation of epi-position or the flank residue is provided with further prevention with inserting at interval.Optimize the multi-epitope constructs that parameter makes up with respect to not being based on, use experiment known to those skilled in the art, presenting on for example immunogenicity detection in the HLA transgenic mice, ELISPOT, interferon-release experiment, tetramer dyeing, chromium release experiment and the dendritic cells detected immunogenicity or antigenic raising of the multi-epitope constructs of optimizing.

Term " peptide " uses in this manual interchangeably with " oligopeptides ", is meant a series of residues that are connected with each other, and L-amino acid is typically continuous by the alpha-amido and the peptide bond between the carboxyl of adjacent amino acid typically.CTL-of the present invention induces the length of peptide to be lower than about 15 residues, preferably length is 13 residues or still less, preferably long for about 8 to about 13 amino acid, more preferably long for about 8 to about 11 amino acid (for example 8,9,10 or 11) with most preferably longly be about 9 amino acid.Preferred HTL-induces the length of oligopeptides to be lower than about 50 residues, usually by about 6 to about 30 residues, more generally by about 12 to 25, often form by about 15 to 20 residues, and can encode long for about 7 to about 23, preferably about 7 to about 17, more preferably about 11 to about 15 (for example 11,12,13,14 or 15) individual and most preferably about 13 amino acid whose epitope peptides.Multi-epitope constructs as herein described preferably comprises 5 or more, 10 or more, 15 or more, 20 or more or 25 or multi-epitope nucleic acid more.

The nomenclature that is used to describe peptide, polypeptide and proteinate is in accordance with conventional practice, and wherein amino is positioned at the left side (N-end) of each amino acid residue, and carboxyl is positioned at right side (C-end).When mentioning the site of amino acid residue, according to the direction from amino to carboxyl numbering, wherein site 1 is near the site of the aminoterminal end of epi-position or peptide or polypeptide or albumen (this epi-position may be its part) with them.In the formula of the particular of the present invention that representative is selected, although not special the demonstration, amino-and carboxyl-end all be form in imaginary physiological pH value, except as otherwise noted.In the amino acid structure formula, each residue is usually by 3 letters or the single-letter name expression of standard.The amino acid residue of L-form is represented to have the 3 letter sign expressions of those amino acid whose D-forms of D-form by the single-letter or the small letter of small letter by the single-letter of capitalization or the capitalization initial of 3 letter signs.Glycine does not have asymmetric carbon atoms, is called for short and makes " Gly " or G.Amino acid whose sign is as follows.

The single-letter sign	3 letter signs	Amino acid
			??A	??Ala	Alanine
??C	??Cys	Cysteine
			??D	??Asp	Aspartic acid
??E	??Glu	Glutamic acid
			??F	??Phe	Phenyl alanine
??G	??Gly	Glycine
			??H	??His	Histidine
??I	??Ile	Isoleucine
			??K	??Lys	Lysine
??L	??Leu	Leucine
			??M	??Met	Methionine
??N	??Asn	Asparagine
			??P	??Pro	Proline
??Q	??Gln	Glutamine
			??R	??Arg	Arginine
??S	??Ser	Serine
			??T	??Thr	Threonine
??V	??Val	Valine
			??W	??Trp	Tryptophan
??Y	??Tyr	Tyrosine

Amino acid whose " chemical feature " is defined as: aromatics (F, W, Y); Aliphatic series-hydrophobic (L, I, V, M); Little polarity (S, T, C); High polarity (Q, N); Acid (D, E); Alkalescence (R, H, K); Proline; Alanine; And glycine.

Term " PanDR binding peptide ", " PanDR is in conjunction with epi-position ", " PADRE ^_Peptide " and " PADRE ^_" be meant the type of htl peptide, this peptide is can be in conjunction with the member of the molecule family of a more than HLA II class DR molecule to epi-position.PADRE ^_Toplink is in conjunction with most of HLA-DR molecules, and stimulates people's helper T lymphocyte (HTL) reaction in vitro and in vivo.Definition PADRE ^_The pattern of the molecule of family can be thought HLA II class hyper-base preface.For example, PADRE ^_Peptide can comprise formula aKXVAAWTLKAAa (SEQ ID NO:1), wherein " X " is Cyclohexylalanine, phenyl alanine or tyrosine, and " a " is D-alanine or L-alanine, have been found that they can be in conjunction with most of HLA-DR allelomorph, and stimulate auxiliary lymphocytic reaction from the T of most of individualities, regardless of their HLA type.PADRE ^_The replacement scheme of epi-position comprises all " L " natural amino acids, and it can be the form of peptide/polypeptide and the nucleic acid form of this epi-position of coding, for example, and in multi-epitope constructs.This paper also discloses PADRE ^_The instantiation of peptide.

" pharmaceutically acceptable " is meant compatible composition on general nontoxic, inertia and/or the physiology.

" be presented to HLA I class processing approach " and be meant that multi-epitope constructs is imported in the cell, make them mainly by the processing of HLA I class processing approach.Typically, use can be encoded the expression vector of multi-epitope constructs with in the multi-epitope constructs transfered cell.HLA II class epi-position by such multi-epitope constructs coding also can be presented to the II quasi-molecule, it be unclear that although this epi-position enters the mechanism of II class processing approach.

" main anchor residue " or " main MHC anchor " is the amino acid on the specific site of peptide sequence, and it can provide the contact point between immunogenic peptide and the HLA molecule.Usually 1～3 in the peptide of given length, common 2 main anchor residues are defined as " motif " of immunogenic peptide.Think that the closely peptide binding groove of contact HLA molecule of these residues, their side chain are embedded in the specificity bag in conjunction with ditch self.In one embodiment, for example, the main anchor residue of HLA I class epi-position is positioned at site 2 (from aminoterminal site meter) and the c-terminus site according to 9-residue peptide epi-position of the present invention.The main anchor position point of each motif and hyper-base preface is documented in the Table I and III of PCT/US00/27766 for example or PCT/US00/19774.The preferred amino acids that can be used in the anchor of most of II class epi-positions is made up of V, M, S, T, A and C in the M in the site 1 and F and the site 6.The tolerance amino acid that can occupy these sites of most of II class epi-positions is made up of P, L and I in the L in the site 1, I, V, W and Y and the site 6.These amino acid that exist in the site 1 of II class epi-position and site 6 have defined HLA-DR1,4,7 hyper-base prefaces.By 1 L, I, V, M, F, Y and A from the site, the D in site 4, E, N, Q, S and T, preferred amino acids among the K in site 6, R and the H has defined the HLA-DR3 binding motif.Other amino acid also may tolerate in these sites, but they are not preferred.

And, by changing having or not having of specific residue in these main anchor position points, promptly replace it, can create similar peptide.Such analog is used to regulate and control to comprise the binding affinity of the peptide of specific motif or hyper-base preface.

In the environment of various HLA molecules, when different peptides is discerned by identical T cell clone, take place " identification that mixes ".It is synonym that identification that mixes or combination combine with cross reactivity.

" immune response of protectiveness " or " curative immune response " is meant CTL and/or the htl response at the antigen that is derived from infectious agent or tumour antigen, and it prevents in some way or stops disease symptoms, side effect or process at least in part.Immune response can also comprise antibody response, and it is promoted by the stimulation of helper cell.

" regulating and controlling sequence " is meant polynucleotide sequence, and it helps the nucleic acid or the expression of nucleic acid construct in the specific host organism that are operably connected, or should express necessary.Be applicable to that procaryotic regulating and controlling sequence comprises promotor for example, optional operator sequence and internal ribosome binding site (IRES).The known genuine nucleus can utilize promotor, polyadenylation signal and enhancer.Promotor can be CMV promotor other promotor as herein described or known in the art.Regulating and controlling sequence comprises IRES.Other instantiation of regulating and controlling sequence is described in this article, or known in the art.

Term " residue " is meant by amido link or amido link analog and is incorporated into amino acid or amino acid analogue in peptide or the albumen.

" less important anchor residue " is the amino acid that is positioned at the main anchor position point site in addition of peptide, and it can influence the peptide combination.With by desired the comparing of amino acid random distribution in a site, less important anchor residue produces in the peptide of combination with obvious higher frequency.It is said that less important anchor residue occurs in " less important anchor position point ".Less important anchor residue can be differentiated to be such residue, it is present in higher frequency in the binding peptide of higher or medium affinity, and is perhaps relevant with the combination of higher or medium affinity.For example,, promptly replace it, can create similar peptide by changing having or not having of specific residue in these less important anchor position points.Such analog is used for fine regulating and control to comprise the binding affinity of the peptide of specific motif or hyper-base preface.Term " peptide of repairing (fixedpeptide) " is used in reference to peptide analogues sometimes.

" screening epi-position " is meant the order of determining or specifying the epi-position in the multi-epitope constructs.

" sept " is meant the sequence between 2 epi-positions that are inserted in multi-epitope constructs, to prevent the connecting generation of epi-position and/or to improve working (machining) efficiency.Multi-epitope constructs can have one or more nucleic acid at interval.Nucleic acid can be arranged in the flank of each epi-position nucleic acid of construct at interval, perhaps at interval the ratio of nucleic acid and epi-position nucleic acid can be about 2 to 10, about 5 to 10, about 6 to 10, about 7 to 10, about 8 to 10 or about 9 to 10, has wherein recorded about 8 to 10 ratio regular meeting some constructs are produced result preferably.

Nucleic acid one or more amino acid of can encoding at interval.The length of the interval nucleic acid of the I class HLA epi-position flank in the multi-epitope constructs is preferably 1 to about 8 amino acid.The length of the interval nucleic acid of the II class HLA epi-position flank in the multi-epitope constructs is preferably more than 5,6,7 or more a plurality of amino acid, more preferably is 5 or 6 amino acid whose length.

Can select number, the amino acid whose number in the sept and the amino acid composition of sept of the sept in the construct, to optimize epi-position processing and/or to minimize the connection epi-position.Preferably, by optimizing epi-position processing simultaneously and being connected motif and selecting sept.This paper has described the suitable amino acid that is used to optimize epi-position processing.In addition, this paper has described the suitable amino acid spacing of the number that is connected epi-position that minimizes in the construct for I class and II class HLA.For example, the sept of II class HLA epi-position flank preferably comprises G, P and/or N residue because usually known they be not main anchor residue (see, for example, PCT/US00/19774).The particularly preferred sept of II class HLA epi-position flank comprises G and P residue alternately, for example (GP) _n, (PG) _n, (GP) _nG, (PG) _nP etc., wherein n is 1～10 integer, is preferably 2 or about 2, wherein the instantiation of this sept is GPGPG (SEQ ID NO:2).A preferred sept particularly for I class HLA epi-position, contains 1,2,3 or a plurality of continuous alanine (A) residue (seeing that for example, Figure 23 A, it has described the sept with 3 continuous alanine residues).

In some multi-epitope constructs, the identical amino acid sequence of each interval nucleic acid coding is enough.In the multi-epitope constructs of interval nucleic acid with 2 identical amino acid sequences of encoding, the interval nucleic acid of encoding those sept can have identical or different nucleotide sequences, wherein different nucleotide sequences can be preferably, to reduce the possibility of undesirable recombination event when multi-epitope constructs is inserted in the cell.

In other multi-epitope constructs, one or more are nucleic acid different amino acid sequence of can encoding at interval.Identical amino acid sequence although many intervals nucleic acid can be encoded in the multi-epitope constructs, 1,2,3,4,5 or a plurality of intervals nucleic acid different amino acid sequence of can encoding, all in also may multi-epitope constructs are the different amino acid sequence of nucleic acid codings at interval.As described herein, whether can maximize epi-position processing and/or minimize the connection epi-position by the assay intervals sequence, can be optimized the epi-position nucleic acid of interval nucleic acid with respect to their side joints.

Whether more can optimize epi-position processing or minimize the connection epi-position according to the sept in the construct than other construct, multi-epitope constructs can be distinguished from each other, preferably, when the epi-position processing of optimizing construct simultaneously with is connected epi-position above other the time, can distinguish construct.This paper has described the epi-position processing that is used to detect construct and has been connected the motif whether computer-aid method of optimised mistake and the laboratory method in external and the body.

" epi-position of subdominance (subdominant epitope) " only causes less reaction or unresponsive epi-position after with the complete antigen immune that contains this epi-position, but can obtain reaction by the epi-position immunity that separates, and in intact proteins is used for external or body, can detect this reaction (not resembling the situation of hiding epi-position) during arousal response.

" hyper-base preface " is the amino acid sequence of peptide, and it provides the total binding specificity of HLA molecule by 2 or a plurality of HLA allelomorph coding.Preferably, 2 or a plurality of HLA antigen can carry the peptide of hyper-base preface with higher or medium affinity (as herein defined) identification.

" synthetic peptide " is meant and is not spontaneous and is to use the made peptides of method such as chemosynthesis or recombinant DNA technology.

" TCR contact residues " or " TXi Baoshouti contact residues " is that think can be by the amino acid residue in the epi-position of TXi Baoshouti combination; They are defined as the MHC anchor that is not main here.The TXi Baoshouti contact residues is defined as the one or more sites in the peptide, and wherein Ce Shi all analogs can both be induced the T-cell recognition, its with induce with wild type peptide relevant.

As used herein, term " autoploidy " is meant the complementary degree between 2 nucleotide sequences.When nucleic acid had the nucleotide sequence identical with other nucleic acid, speech " homogeneity " can substitute speech " autoploidy ".Can also detect sequence homology and sequence homogeneity by the hybrid experiment under higher stringency and/or low stringency, disclosed herein be low stringency or under high stringency can with the nucleic acid of multi-epitope constructs hybridization.In addition, by using algorithm known in the art and computer program analysis sequence, can detect sequence homology and sequence homogeneity.Herein disclosed is the method that whether identical with multi-epitope constructs such being used to detect nucleic acid or homology.The present invention partly relate to nucleotide sequence with multi-epitope constructs disclosed herein have 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more or 99% or the nucleotide sequence of more homogeneity.

As used herein, term " stringent condition " is meant the condition of the hybridization between the nucleotide sequence that allows nucleotide sequence and disclosed multi-epitope constructs.Can define suitable stringent condition, for example pass through before the hybridization and the salt in the hybridization solution or the concentration of formamide, or by hybridization temperature, it be well known in the art.More specifically, can improve stringency by following method: reduce salinity, increase the concentration of formamide, or the rising hybridization temperature.For example, the hybridization under the high stringency can about 37 ℃ to 42 ℃, in about 50% formamide, carry out.More specifically, hybridization can carried out under the high stringency: 42 ℃, 50% formamide, 5x SSPE, 0.3%SDS and 200 μ g/ml that shear with salmon sperm dna sex change in, or 42 ℃, containing in the solution of salmon sperm dna 50% formamide, 5x SSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x DenhardtShi solution, 10% dextran sulfate and 20 μ g/ml sex change, that shear, subsequently about 65 ℃, in 0.1xSSC, wash filter membrane.Hybridization can be carried out under the stringency that reduces: in about 35% to 25% formamide, at about 30 ℃ to 35 ℃.For example, the stringency of reduction can occur in 35 ℃, 35% formamide, 5xSSPE, 0.3%SDS and 200 μ g/ml that shear with salmon sperm dna sex change in.By calculating the purine of target nucleic acid: pyrimidine ratio, and the corresponding temperature of adjusting, can further dwindle and the specific corresponding temperature range of stringency level.The variation of well known above-mentioned scope and condition.

Except using hybrid experiment to estimate sequence homogeneity or the sequence homology, known computer program also can be used to detect specific nucleic acid whether with multi-epitope constructs homology disclosed herein.The example of such program is a Bestfit program (Wisconsin sequence analysis bag, Version 8 for Unix, Genetics Computer Group, University ResearchPark, 575 Science Drive, Madison, WI 53711), other sequence alignment program is known in the art, can be used for detecting whether homology of 2 or a plurality of nucleotide sequence.Bestfit uses Smith and Waterman, and local homology's algorithm of Advances in Applied Mathematics2:482-489 (1981) is to find the best autoploidy part between 2 sequences.When using Bestfit or any other sequence alignment program to detect particular sequence when for example 95% ground is identical with reference sequences, parameter can be set for as follows: the total length according to the reference nucleotide sequence is calculated homogeneity percentage, allows to exist in the nucleotide sum of reference sequences the autoploidy gap up to 5%.

Abbreviation used herein is as follows:

APC: antigen presenting cell

CD3:Pan T cell marking

CD4: helper T lymphocyte mark

CD8: cytotoxic T lymphocyte mark

CEA: carcinomebryonic antigen

CFA: complete Freund's adjuvant

CMV: human cytomegalovirus

CTL: Cytotoxic T lymphocyte

Cardiotoxin: 60 natural amino acid whose peptides, it can cause local muscle to destroy (inhibitors of protein kinase C)

DC: dendritic cells.DC by stimulating cytokine from the release of CTL system, be delivery cell as effective antigens and work, described CTL system is specific to the model peptide that is derived from hepatitis type B virus (HBV).External flow to first experiment mice after, use the CTL immune response that has promotion with the experiment in vitro of the DC of the stripped impulse stimulation of HBV peptide epitopes.

DMSO: dimethyl sulfoxide (DMSO)

DNA: DNA (deoxyribonucleic acid)

EBV:EB virus

ELISA: enzyme-linked immunosorbent assay

The method of ELISPOT:ELISA-sample, it detects the detectable cell factor as the difference on the culture membrane of each emiocytosis

Epigene: Polyepitope DNA construct

E: T: effector molecules: the ratio of target

FACS: the cell sifter of fluorescence-activation

FCS: hyclone

G-CSF: granulocyte colony stimulating factor

GM-CSF: granulocyte-huge is had a liking for cell (monocyte)-colony stimulating factor

HBV: hepatitis type B virus

HER2/Neu：c-erbB-2

HIV: human immunodeficiency virus

HLA: human lymphocyte antigen

HLA-DR: human lymphocyte antigen II class

HPLC: high performance liquid chromatography

HTC: helper cell

HTL: helper T lymphocyte

ID: homogeneity

IFA: incomplete Freund's adjuvant

IFN γ: interferon gamma

IL-4: interleukin-4 cell factor

IRES: internal ribosome entry site

IV: intravenous

LU _30%: (E: T) ratio reached the required Cytotoxic activity of 30% cracking at 100: 1

MAb: monoclone antibody

MAGE: melanoma-associated antigen

MHC: main histocompatibility complex

MLR: the lymphocyte reaction of mixing

MNC: monocyte

PADRE ^TM: the PanDR binding peptide

PATR：Pan?Troglodytes

PB: peripheral blood

PBL: peripheral blood lymphocyte

PBMC: peripheral blood lymphocytes

SC: subcutaneous

SDS: lauryl sodium sulfate

S.E.M.: the standard error of mean value

SU: secretory units

QD: administration once a day

TAA: the antigen that tumour is relevant

The TCR:T cell receptor

TNF: TNF

WBC: white blood corpuscle

The U.S.S.N.09/189 that the application and on November 10th, 1998 submit to, 702 is relevant, the latter is the U.S.S.N.08/205 that submitted on March 4th, 1994,713 part continuation application, the latter is the U.S.S.N.08/159 that has now abandoned that submitted on November 29th, 1993,184 part continuation application, the latter is the U.S.S.N.08/073 that has now abandoned that submitted on June 4th, 1993,205 part continuation application, the latter is the U.S.S.N.08/027 that has now abandoned that submitted on March 5th, 1993,146 part continuation application.The application goes back and U.S.S.N.09/226, and 775 is relevant, and the latter is U.S.S.N.08/815,396 part continuation application, and the latter requires the present U.S.S.N.60/013 that has abandoned, 113 priority.And the application goes back and U.S.S.N.09/017, and 735 is relevant, and the latter is the U.S.S.N.08/589 that abandons, 108; U.S.S.N.08/753,622, U.S.S.N.08/822,382, the U.S.S.N.60/013 that abandons, 980, U.S.S.N.08/454,033, U.S.S.N.09/116,424, and U.S.S.N.08/349,177 part continuation application.The application goes back and U.S.S.N.09/017, and 524, U.S.S.N.08/821,739, the U.S.S.N.60/013 that abandons, 833, U.S.S.N.08/758,409, U.S.S.N.08/589,107, U.S.S.N.08/451,913, U.S.S.N.08/186,266, U.S.S.N.09/116,061, and U.S.S.N.08/347,610 is relevant, the latter is U.S.S.N.08/159,339 part continuation application, the latter is the U.S.S.N.08/103 that abandons, 396 part continuation application, the latter is the U.S.S.N.08/027 that abandons, 746 part continuation application, the latter is the U.S.S.N.07/926 that abandons, 666 part continuation application.The application goes back and U.S.S.N.09/017, and 743, U.S.S.N.08/753,615; U.S.S.N.08/590,298, U.S.S.N.09/115,400, and U.S.S.N.08/452,843 is relevant, and the latter is U.S.S.N.08/344,824 part continuation application, the latter is the U.S.S.N.08/278 that abandons, 634 part continuation application.The application also with interim U.S.S.N.60/087,192 and U.S.S.N.09/009,953 is relevant, the latter is the U.S.S.N.60/036 that abandons, 713 and the U.S.S.N.60/037 that abandons, 432 part continuation application.In addition, the application goes back and U.S.S.N.09/098, and 584, and U.S.S.N.09/239,043 is relevant.The application also with the U.S.S.N.09/583 that submits to common unsettled 30 days Mays in 2000,200, the U.S.S.N.09/260 that on March 1st, 1999 submitted to, the U.S. Provisional Application of submitting on October 6th, 714 and 2000 number 60/239, the U.S. Provisional Application of submitting on November 18th, 008 and 1999 number 60/166,529 is relevant.In addition, the application also relates to: the U.S. Provisional Application of submitting on October 6th, 2000 of abandoning now number 60/239,008; Common unsettled U. S. application number 10/130,548, it is the PCT/US00/31856 that submitted on November 20th, 2000 and is disclosed as the American National phase application of WO 01/36452 May 25 calendar year 2001; With the common unsettled U. S. application of submitting on April 5th, 2002 number 10/116,118.All top applications are incorporated by reference here.

Description of drawings

Fig. 1 has illustrated the data of 3 kinds of different multi-epitope constructs, and every kind contains 20 to 25 different CTL epi-positions.

Fig. 2 has illustrated that (Fig. 2 a), wherein first kind of construct contains the synthetic altogether epi-position of 4 different linearities to 2 kinds of different synthetic polypeptide, and the 2nd construct contains GPGPG (SEQ ID NO:2) sept.Fig. 2 b has illustrated that these different constructs of 2 nanomoles are at IA ^bCause ability in the positive mouse, with the reacting phase ratio of inducing with one group of identical peptide (every kind of peptide 3 μ g) of equimolar amounts at the breeder reaction of various epi-positions.

Fig. 3 has illustrated the structure of Polyepitope DNA construct.The HLA restriction is presented on each epi-position, and the A*0201 epi-position is a runic.HLA binding affinity (IC ₅₀NM) under each epi-position.(a) figure of HIV-FT of order of the epi-position of coding is described.(b) figure of the specific construct of HBV-.With the C+1 amino acid of arrow indication with respect to core 18.C in core 18 ₁The specific construct of HBV-that the site has 1 aminoacid insertion is expressed as HBV.1X.

Fig. 4 has illustrated at HLA-A*0201/K ^bThe immunogenicity of the HLA-A*0201 epi-position of HIV-FT in the transgenic mice.(a) at the representational ctl response of epi-position Pol 498 (circle), Vpr 62 (triangle), Gag 386 (square).(filled symbols) arranged or do not having under the situation of (open symbols) every kind of peptide existence, ⁵¹Detected Jurkat-HLA-A*0201/K in the Cr release experiment ^bThe cytotoxicity of target cell.(b) summed up at HLA-A*0201/K ^bThe immunogenic ctl response of HIV-FT in the transgenic mice.Band is represented the geometric mean ctl response of positive culture.Also indicated the frequency of positive CTL culture.

Fig. 5 has shown that C+1 amino acid is to the immunogenic influence of epi-position.Analyzed the database that comprises from the ctl response of multiple multi-epitope constructs (representing 94 amino acid whose combinations of epi-position/C+1), to determine the frequency (%) of the situation that particular combinations is relevant with best ctl response.If at least 30% detection culture,, think that then ctl response is best greater than 100SU or 20LU.The number of times of observing given epi-position/C+1 amino acid combination also is provided.

Fig. 6 has shown the ctl response to the specific construct of HBV-, (a) dna immunization HLA-A*0201/K ^bBehind the transgenic mice, to the ctl response of core 18 epi-positions.(b) with construct to HLA-A*0201/K ^bAfter transgenic mice carried out dna immunization, to the ctl response of HBV core 18 epi-positions, the difference of described construct was in the C+1 site of core 18 aminoacid insertion to be arranged.

Fig. 7 has shown the level that the HBV core 18 in HBV.1 (shaded bar) and HBV.1K (hollow strips) cells transfected system is presented.Use that peptide-specific CTL system carried out that epi-position presents quantitatively.The presenting of HBV Pol 455 that shows is used to contrast purpose.

Fig. 8 has illustrated the 221A2K with the transfection of HIV-FT epigene construct ^bThe data of target cell.Having analyzed these cells transfected is the ability of presenting epi-position to being derived from the HLA transgenic mice and the CTL epi-position that various HIV-originate being had specific CTL.In order to proofread and correct the antigen sensitivity difference that different CTL are, the cell that uses untransfected has carried out the peptide dose titration abreast as APC.

Fig. 9 has shown the HIV multi-epitope constructs that uses method of the present invention to optimize.

Figure 10 has illustrated and has been used for the computer system of Automatic Optimal according to the multi-epitope constructs of one embodiment of the invention.

Figure 11 A-B has illustrated according to one embodiment of the invention and has contained the exemplary input text file that is useful on user's input parameter of carrying out Junctional Analyzer program.

Figure 12 has illustrated the flow chart that is used to differentiate the software program of best multi-epitope constructs according to one embodiment of the invention.

Figure 13 A-D has illustrated the exemplary output text that contains the output result of JunctionalAnalyzer program according to one embodiment of the invention.

Figure 14 A has illustrated the ctl response of EP-HIV-90 with respect to each inducing peptide among the IFA, and Figure 14 B has illustrated PfCTL.1, PfCTL.2 and the PfCTL.3 ctl response with respect to each inducing peptide.

Figure 15 has shown the influence of the htl response that GPGPG (the SEQ ID NO:2) sept in II class epi-position construct HIV 75 aggressiveness and HIV 60 aggressiveness causes defined epitope.

Figure 16 has illustrated at EP-HIV-1043-PADRE ^_The htl response of the defined epitope in the construct.

Figure 17 has illustrated at HIV 75 aggressiveness, EP-HIV-1043 and EP-HIV-1043-PADRE ^_Epi-position in the construct.

Figure 18 A-N has shown the amino acid sequence and the nucleotide sequence of some multi-epitope constructs.

Figure 19 A-D has shown the amino acid sequence of the epi-position in some multi-epitope constructs.

Figure 20 A-20F has shown the HBV CTL epi-position that is used to make up 3 relevant epigene construct HBV-2, HBV-2A and HBV-2B, the order of epi-position in the epigene construct, the immune response of in HLA-A2 or HLA-A3/11 transgenic mice, inducing and the amino acid and the nucleotide sequence of epigene construct.In Figure 20 B, the burst in HBV-2, HBV-2A and HBV-2B is an Ig κ common signal sequence, although can also use other burst.

Figure 21 A-21E has shown the HBV CTL epi-position of the epigene construct HBV-21A and the HBV-21B that are used to make up 2 21CTL epi-positions, the order of epi-position in the epigene construct, the immune response of in HLA-A2 or HLA-A3/11 transgenic mice, inducing and the amino acid and the nucleotide sequence of epigene construct.

Figure 22 A-22E has shown the HBV CTL epi-position of the epigene construct HBV-30B and the HBV-30C that are used to make up 2 30CTL epi-positions, the order of epi-position in the epigene construct, the immune response of in HLA-A2 or HLA-A3/11 transgenic mice, inducing and the amino acid and the nucleotide sequence of epigene construct.

Figure 23 A-23C has shown the modification to the sept of the CTL epi-position flank of 2 HLA-A2 restrictions in the HBV-30C epigene construct.Modification is used to increase processing and the efficient of presenting subsequently, thus and the immunogenicity of enhancing epi-position.Use HLA-A2 or HLA-A3/11 transgenic mice to detect immunogenicity, and write down the amino acid and the nucleotide sequence of epigene construct.In Figure 23 A, lysine (K) sept of the 18 epi-position flanks of core among the HBV-30C is modified to 3 alanine residues (AAA) that comprise among the HBV-30CL.An asparagine (N) sept of env 183 epi-position flanks also is modified to 3 alanine residues (AAA) that comprise among the HBV-30CL among the HBV-30C.

Figure 24 A-24C has shown HTL epi-position and their the allelic binding affinities of HLA-DR to selecting that is used to make up polyepitope vaccines, and described vaccine contains the HTL epi-position of being separated by GPGPG (SEQ IDNO:2) amino acid sept.The nucleotide sequence of polyepitope vaccines and the amino acid sequence of this nucleic acid coding are shown in Figure 24 C.

Figure 25 A-B has shown colony's coverage of the CTL epi-position that contains among the GCR-5835.In the combination population of the gene frequency of Figure 25 A. in being derived from Asian, Black people, European white people and North America white people population, the individual percentage (secret note) of the HLA-A/B-epi-position of quantity shown in presenting combination.The cumulative chart (open circles) that on right axle, has also shown colony's coverage percentage.Figure 25 B. has summed up colony's coverage accumulative perception of planning in Asian, Black people, European white people and North America white people population, it is the function of quantity of the epi-position of HLA allelomorph combination.

Figure 26 A-26B has shown colony's coverage of the epi-position in the tabulation.In the combination population of the gene frequency of Figure 26 A. in being derived from Asian, Black people, European white people and North America white people population, the individual percentage (secret note) of the HLA-DR-epi-position of quantity shown in manifesting combination.The cumulative chart (open circles) that on right axle, has also shown colony's coverage percentage.Figure 26 B. has summed up colony's coverage accumulative perception of planning in Asian, Black people, European white people and North America white people population, it is the function of quantity of the epi-position of HLA allelomorph combination.

Figure 27 A-27B has shown the schematic diagram of (A) HBV30K and (B) the super type restriction of HLA of component epi-position.The immunogenicity of vaccine 30 epi-position epigene constructs.With 100 μ g vaccine HBV epigene plasmid HBV30K or the immunity of prototype HBV vaccine HBV2 intramuscular HLA-A2 or-the A11 transgenic mice.After the immunization 11 days, be used in the epi-position stimulated in vitro splenocyte of encoding in the vaccine.Cultivate after 6 days, use original position IFN-γ ELISA experiment to detect epi-position-specific ctl response.

Figure 28 A-28B has shown HBV HTL vaccine constructs and its immunogenic schematic diagram.Figure 26 A. has indicated GPGPG (the SEQ ID NO:2) sept that imports between the epi-position.Figure 28 B. is with each peptide intramuscular immunity H2bxd mouse of 100 μ g vaccine HBV HTL epigene constructs or emulsification in CFA.After the immunization 11 days,, use elementary IFN-γ ELISPOT experiment to detect htl response from splenocyte purifying cd4 t cell.

Figure 29 A-29B has shown HBV vaccine plasmid configuration and their relative immunity originality.The two CMV promoter plasmids of Figure 29 A. schematic diagram (i); The plasmid that (ii) contains IRES; (iii) CTL+HTL epigene construct merges.The relative immunity originality of the different vaccine configurations of Figure 29 B..With 100 μ g HBV30K (contrast of CTLepigene construct), HBV30K.H1 (two CMV promoter plasmid), HBV30K.H3 (plasmid that contains IRES) or HBV30K/HTL (GCR-5835; The CTL+HTLepigene construct merges) intramuscular immunity HLA-A2-transgenic mice.After the immunization 11 days, be used in the epi-position stimulated in vitro splenocyte of encoding in the vaccine.Cultivate after 6 days, use original position IFN-γ ELISA experiment to detect epi-position-specific ctl response.

Figure 30 has shown the relative immunity originality of GCR-5835 and GCR-3697.With 50 μ g or 5 μ g GCR-5835 or GCR-3697 intramuscular immunity HLA-A2-transgenic mice.After the immunization 11 days,, use IFN-γ ELISPOT experiment to detect epi-position-specific ctl response from splenocyte separation of C D8+ cell.

Figure 31 has contrasted the PVP preparation, exposed and CT GCR-5835.GCR5835 immunity HLA-A2.1/K that prepare in PVP with 100 μ g, exposed or that in cardiotoxin (CT) pre-treated animal, expose ^bTransgenic mice once.Body is interior after 11 days, with the external splenocyte that stimulates again of peptide that indicates.After 6 days, use original position ELISA experiment to detect the IFN-γ that peptide causes.The data of positive culture are expressed as geometrical mean ± standard deviation of secretory units (SU).The frequency of the positive culture of test/total culture be indicated in each bar above.

Figure 32 has contrasted GCR-5835 and lipopeptid vaccine.With 100 μ g in cardiotoxin (CT) pre-treated animal GCR 5835 or 100 μ g lipopeptid vaccine immunities HLA-A2.1/K ^bTransgenic mice.Body is interior after 11 days, separation of C D8+ splenocyte, the IFN-γ (A) that the peptide that detection indicates in the ELISPOT experiment causes.Data are expressed as equalization point and form cell (SFC)/10 ⁶The splenocyte of individual coating.Perhaps, with the external splenocyte that stimulates again of peptide that indicates.After 6 days, use original position ELISA experiment to detect the IFN-γ (B) that peptide causes.The data of positive culture are expressed as geometrical mean ± standard deviation of secretory units (SU).

Figure 33 A-33B has summed up from each immunogenicity in mice data.HLA-A2.1/K ^bTransgenic mice is immunity, or with the GCR-5835 immunity of 100 μ g PVP-preparation once (A), or with immune 2 times (B) in 7 days interval.Back 11 days of last immunity uses the peptide aggregate that is indicated to stimulate splenocyte from every mouse outward again.After 6 days, the IFN-γ that the set of each peptide of detection and all peptides causes in the ELISPOT experiment.Data are expressed as equalization point and form cell (SFC)/10 ⁶The splenocyte of individual coating.

Figure 34 has shown the schematic diagram of HBV AOSIb and HBV AOSIb2 construct.HBV AOSIb2 construct has the processing of the amino acid (indicating with the arrow on the figure) of extra interpolation with the enhancing proteasome, and HBV AOSIb construct does not have extra residue.

Figure 35 A-35E has shown the result behind transient transfection people 293 cells under the situation that is with or without proteasome inhibitor MG132 existence.After transfection 24 hours, add 5 μ M proteasome inhibitor MG132.Added behind the proteasome inhibitor 24 hours, and used flow cytometry method and fluorescence microscopy to detect fluorescence (except as otherwise noted) in the living cells.(A) flow cytometry method (FACS) result of the time course of usefulness plasmid AOSIb cells transfected.(B) with flow cytometry method (FACS) result of plasmid HBV AOSIb cells transfected in the time of 24 hours.(C) with flow cytometry method (FACS) result of plasmid HBV AOSIb2 cells transfected in the time of 24 hours.(D) with figured data, contrasted fluorescence intensity.(E) between control plasmid, HBV AOSIb and HBV AOSIb2, contrasted the relative raising of the fluorescence intensity of above-mentioned experiment.

Figure 36 shown behind adding proteasome inhibitor lactacystin (25uM) or the MG132 (5uM) can detected albumen amount.Prepared full cell lysate from cells transfected, and transferred on the blotting membrane.Use at the antibody test of fusion partner albumen albumen.Arrow has indicated the prediction size of total length fusion.

Figure 37 A-37B has shown detected epi-position-specific t cell responses in the HLA transgenic mice of GCR-3697 immunity, uses the splenic lymphocyte that obtained in 11-14 days after immunity.In tibialis anterior with the immunity of 100ug dna double side ground 6-9 HLA-transgenic mice group.DNA carries in PBS or PVP preparation; Under the situation of PBS preparation, with cardiotoxin parenteral solution preliminary treatment injection site.

Figure 38 has contrasted the fluorescence intensity by detected 3 plasmids of facs analysis: the multi-epitope HBV AOSIb2 that multi-epitope HBV AOSIb that no epi-position construct (having only fluorescin), fluorescence are puted together or fluorescence are puted together.With plasmid transfection people's 293 cells, after transfection, added 5 μ M proteasome inhibitor MG 132 in 24 hours.Added behind the proteasome inhibitor 24 hours, and detected the fluorescence in the living cells by FACS.

Figure 39 has shown with no epi-position construct (having only fluorescin), the fluorescence microscopy image of the multi-epitope HBV AOSIb2 cultured cells that multi-epitope HBV AOSIb that fluorescence is puted together or fluorescence are puted together.With plasmid transfection people's 293 cells, after transfection, added 5 μ M proteasome inhibitor MG132 in 24 hours.Added behind the proteasome inhibitor 24 hours, and detected the fluorescence in the living cells by fluorescence microscope.

Detailed Description Of The Invention

Describe the present invention in detail below with reference to accompanying drawing, wherein similar element is marked with similar numeral in the text.

The invention provides the effect of optimizing polyepitope vaccines, preferably minimize the number that connects epi-position and maximization or improve immunogenicity and/or the method for enhancing antigenicity and the system of polyepitope vaccines at least.The present invention also provide can encode a plurality of CTL and/or HTL epi-position the multi-epitope nucleic acid construct and by such construct encoded polypeptides and contain such construct and/or polypeptide cell, contain the composition of such construct, polypeptide and/or cell and use the immunoreactive method of such construct and/or polypeptide and cytositimulation (for example methods of treatment).

In one embodiment of the invention, the Computerized method that is used for designing the multi-epitope constructs with a plurality of epi-positions comprises the steps: a plurality of input parameters are stored in that the memory of computer system, this input parameter comprise a plurality of epi-positions, at least one is used to differentiate at least one weighted value (enhancement weight value) of the motif, a plurality of aminoacid insertion things and each insert that connect epi-position; It is right to generate a tabulation position from described a plurality of epi-positions; On the basis of at least one weighted value of at least one motif, a plurality of insert and each insert, be that each epi-position is to determining at least one best of breed of aminoacid insertion thing; With at least one optimal arrangement that identifies a plurality of epi-positions, wherein each at least one best of breed of aminoacid insertion thing all is each junction that is inserted in 2 epi-positions, so the multi-epitope constructs of optimizing is provided.In preferred embodiments, the step of at least one optimal arrangement of discriminating epi-position can be finished by thorough search, wherein estimated all arrangements of a plurality of epi-positions, perhaps finished, wherein only estimated the subclass of all arrangements of a plurality of epi-positions by random search.

In another embodiment, this method is by calculating the function value (F) of every kind of right possible insert combination of each epi-position, for each epi-position to having determined at least one best of breed of aminoacid insertion thing, the scope of the insert number in the combination can be from 0 maximum insert number (MaxInsertions) value to user's input, according to equation F=(C+N)/J (when the J＞0) computing function value, when J=0, this equation becomes F=2 (C+N), wherein C equals the weighted value of C+1 flanking amino acid, N equals the weighted value of N-1 flanking amino acid, and J equals at the number based on the connection epi-position of every kind of combine detection of the insert of the epi-position centering of described at least one motif.

In another embodiment of the invention, the computer system that is used to design the multi-epitope constructs with a plurality of epi-positions comprises: be used to store the memory of a plurality of input parameters, for example a plurality of epi-positions, at least one is used to differentiate at least one weighted value of the motif, a plurality of aminoacid insertion things and each insert that connect epi-position; Be used for generating the right processor in a tabulation position from the parameter of memory calls input and from described a plurality of epi-positions; Wherein this processor on the basis of at least one weighted value of at least one motif, a plurality of insert and each insert, is that each epi-position is to determining at least one best of breed of aminoacid insertion thing also; This processor can also identify at least one optimal arrangement of a plurality of epi-positions, and wherein each in the best of breed of aminoacid insertion thing all is each junction that is inserted in 2 epi-positions, so that the multi-epitope constructs of optimization to be provided; With the monitor that is connected to processor, be used to the user to show at least one optimal arrangement of a plurality of epi-positions.

In another embodiment, the invention provides the data storage device that has the computer program that is used to design multi-epitope constructs with a plurality of epi-positions, when this computer program is controlled by computer system, can carry out the process comprise following step: call a plurality of input parameters from the memory of computer system, this input parameter comprises for example a plurality of epi-positions, at least one is used to differentiate at least one weighted value of the motif, a plurality of aminoacid insertion things and each insert that connect epi-position; It is right to generate a tabulation position from described a plurality of epi-positions; On the basis of at least one weighted value of at least one motif, a plurality of insert and each insert, be that each epi-position is to determining at least one best of breed of aminoacid insertion thing; With at least one optimal arrangement that identifies a plurality of epi-positions, wherein each at least one best of breed of aminoacid insertion thing all is each junction that is inserted in 2 epi-positions, so the multi-epitope constructs of optimizing is provided.

In another embodiment, the invention provides the method and system that design contains the multi-epitope constructs of a plurality of epi-positions.This method comprises following step: (i) sieve a plurality of epi-positions, to minimize the number that connects epi-position; (ii) import the flanking amino acid residue in the C+1 site that will be included in the epi-position in the multi-epitope constructs; (iii) import one or more amino acid residue at interval between 2 epi-positions of multi-epitope constructs, wherein this interval residue can prevent to connect the generation of epi-position; (iv) select one or more multi-epitope constructs, its amino acid with minimum connection epi-position number, minimal amount at interval residue and maximum number at flanking amino acid residue with respect to the C+1 site of each epi-position.In some embodiment, residue is independently selected from the residue of the main anchor residue of HLA II class that is not known at interval.In specific embodiments, import the generation that the interval residue can stop the HTL epi-position.Such sept often contains at least 5 amino acid residues, and they are independently selected from G, P and N.In some embodiment, sept is GPGPG (SEQ ID NO:2).

In some embodiment, import the generation that the interval residue can stop the CTL epi-position, and wherein this sept is 1,2,3,4,5,6,7 or 8 amino acid residue, it is independently selected from A and G..The flank residue often is to import in the C+1 site of CTL epi-position, and is selected from K, R, N, G and A.In some embodiment, the flank residue closes on intervening sequence.Method of the present invention can also comprise: with the N-end residue of the residue displacement epi-position that is selected from K, R, N, G and A, this N-end residue closes on the C-end of adjacent epi-position in the multi-epitope constructs.

In some embodiment, method of the present invention can also comprise following step: the structure of prediction multi-epitope constructs, also select to have one or more constructs of max architecture, promptly they can be by the processing of HLA processing approach, is included in all epi-positions in this construct with production.In some embodiment, multi-epitope constructs coding RP-HIV-1090, HIV-CPT as shown in Figure 9 or HIV-TC as shown in Figure 9 as shown in Figure 9.

In another embodiment of the invention, the system that is used to optimize multi-epitope constructs comprises computer system, it has a processor (for example central processing unit) and at least one is connected to the memory of processor, the data that the latter is used to store the instruction of processor execution and will be handled (promptly handling) by processor.This computer system also comprises input unit (for example keyboard) and at least one memory that is connected to processor, so that user's input will be by the desired parameters and the information of processor access.This processor can be independent CPU or be incorporated into many different treating apparatus/loop in the integrated circuit chip.Perhaps, this processor can be to connect or set by data/address bus discrete treating apparatus/loop optionally connected to one another by direct electric wire/lead.Similarly, described at least one memory can be a big storage device (for example EPROM) or many discrete storage devices optionally connected to one another (for example, EEPROM, EPROM, RAM, DRAM, SDRAM, Flash etc.) set, it is used for optionally storing data and/or program information (i.e. the instruction of being carried out by processor).Those of ordinary skill in the art can easily realize required Computer Systems Organization, to carry out operation disclosed herein and function.

In one embodiment, computer system comprises display, and it is used for demonstration information, instruction, image, figure etc.This computer system can be accepted user's input by keyboard.Number, the peptide that will process, each amino acid whose C+1 and the N-1 weights that the parameter of these users input can comprise insert (being flank residue and interval residue) for example be used to search for the motif that is connected epi-position.Based on these input value/parameters, computer system is carried out " JunctionalAnalyzer " software program, it automatically detects the number of the right connection epi-position of each peptide, also calculates " weighting " value of each combination of the flank residue can be inserted into the right junction of each peptide and sept.Then the result of Junctional Analyzer program is used for completely or search utility at random, it can determine whole group peptide " the best " combination or connect to have the minimum connection epi-position number and the multi-epitope polypeptide or the nucleic acid of maximum function (for example immunogenicity) value with foundation.

In one embodiment, if be lower than 14 by the number of the peptide of computer system processor, then carry out search utility completely by computer system, it detects all arrangements of the peptide of forming polypeptide, to find to have the arrangement of " best " or " the best " function value.In one embodiment, function value be to use equation (Ce+Ne)/J calculate (when J greater than 0 the time), perhaps use equation 2* (Ce+Ne) to calculate (when J equals 0), wherein Ce is the amino acid whose weighted value in the C+1 of peptide site, Ne is the amino acid whose weighted value in the N-1 of peptide site, and J is included in the number by the connection epi-position in the polypeptide of multi-epitope nucleic acid sequence encoding.Thereby, make the maximization of this function value, can identify that to have a minimum peptide that connects epi-position number and maximum flank residue weighted value right.If the number of peptide to be processed is 14 or more a plurality of, computer system is carried out the random search program, and its use " Monte Carlo " technology detects many zones of alignment area, with the best estimate (for example having the maximum function value) of the optimal arrangement of finding peptide.

In another embodiment, computer system allows user's input parameter value, and its regulation or restriction are thoroughly or the output result of random search program.For example, the result with identical function value (" MaxDuplicateFunctionValue=X ") that the user can import maximum number limits the number of arrangement, and this number produces as Search Results.Because many arrangements that search utility can find to produce the identical function value it is desirable to prevent that output file is filled a large amount of equivalents.In case realized this restriction, just can not report more result, up to finding bigger or " better " function value.As another example, the user can import the maximum number of " the value of hitting " of each search (probe) in random search procedure.This parameter can prevent that the random search program from once producing too much output in the search.In preferred embodiments, by several factors limit number of detected arrangement in search once: at input file be the quantity of the time of each search setting; The value of the speed of calculator and parameters " MaxHitsPerProbe " and " MaxDuplicateFunctionValues ".The parser that is used for producing and selecting to arrange can be according to the recursive algorithm of knowing that can find at many computer science teaching materials.For example, 6 kinds of arrangements of once getting 3 key elements can produce following order: ABC; ACB; BAC; BCA; CBA; CAB.As another example of input parameter, the user can import how to carry out random search, for example randomly, statistics ground or other methodology; The maximum duration (for example 5 minutes) that each search allows; Number with the search of carrying out.

The invention also discloses multi-epitope constructs by method design top and described below.Multi-epitope constructs comprises the interval nucleic acid between the subclass of epi-position nucleic acid or all epi-position nucleic acid.At interval nucleic acid can encoding amino acid sequence for one or more, and amino acid sequence of nucleic acid coding is different at interval with other for they, to optimize epi-position processing and to minimize existing of connection epi-position.

The present invention relates to design the method and system of immunogenic polyepitope vaccines with optimization.In preferred embodiments, this vaccine comprises CTL and HTL epi-position.Can provide significant colony's coverage of not having race's deflection according to vaccine of the present invention, preferably concentrate on different virus or other antigenic separator in conservative epi-position.By method and system disclosed herein, can optimize the magnitude and the width of the reaction of vaccine, and the simplest epi-position configuration can be provided.At last, provide the immunogenic conventional method of in human body, estimating polyepitope vaccines.

Method of the present invention comprises, designs multi-epitope constructs on the basis of principle as herein described.On the one hand, the invention provides and use single promotor multi-epitope constructs to induce reaction simultaneously at specific CTL and HTL epi-position.This construct can contain many different epi-positions, is preferably more than 10, often greater than 20,25,30,35,40,45,50,55,60,65,70 or or a plurality of.

In preferred embodiments, computer system is by carrying out following function and/or analyzing and differentiate the multi-epitope constructs that one or more are best:

(i) screening will be integrated into the epi-position of multi-epitope constructs, with the order of number that the connection epi-position that energy minimization forms is provided.Discussed this method for sieving in more detail with reference to Figure 11 and 12 below.Preferably, the less important Consideration when epi-position is sorted is placed to epi-position as follows, can promote that the residue of N-end of the immunogenic epi-position of CTL is arranged side by side with the C-end of another CTL epi-position.

(ii) can the flank site that immunogenic flank residue is inserted into epi-position will can be improved.In specific embodiments, the flank residue is the C+1 site that is inserted in the CTL epi-position.

(iii) intervening sequence can be inserted between the epi-position, to prevent to connect the generation of epi-position.In specific embodiments, intervening sequence can also comprise at the N-of attachment end can promote immunogenic residue, makes this residue be positioned at the flank of CTL epi-position C-end.

Preventing that HTL from connecting in the specific embodiments of epi-position, using the sept of forming with any known corresponding amino acid residue of HLA II class anchor residue by not, for example, between 2 HTL epi-positions, comprising the G and the P residue (GP sept) that replace.

Another aspect of the present invention, top factor (ii) comprises the particular amino acid residue that imports or replace the flank site that is positioned at epi-position, this site for example is close to the C-end of epi-position, produces thus with the construct (i.e. the multi-epitope constructs of not optimizing) that does not contain at the specific residue of this site importing or replacement and compares antigenicity and immunogenic multi-epitope constructs with raising.The method of optimizing multi-epitope constructs is included in the step that the C+1 site of epi-position (promptly being close to the site of epi-position C-end) imports the flank residue, preferred K, N, G, R or A.In an alternate embodiment, replaced and to have reduced immunogenic residue, promptly electronegative residue, for example residue of the non-tryptophan of the residue (I, L, M, V) of D, aliphatic series or aromatics.Provide favourable flank residue by placing suitable epi-position, or, can import the flank residue by inserting specific residue.

Pointed as the background technology part, the coding nearly multi-epitope constructs (mini gene) of 10 epi-positions has been used to induce the reaction that resists many different epi-positions.Data (Ishioka etc., J Immunol, Vol.162 (7): 3915-25 (1999)) about experimental multi-epitope constructs pMin.1 are disclosed.Disclosed herein is the parameter that is used to design and estimate the immunogenic multi-epitope constructs with optimization, it is applicable to countless target disease indications.

On the basis of many researchs, differentiated design parameter.In the preliminary assessment of multi-epitope constructs, the data about 3 different multi-epitope constructs have been proposed, each all contains 20 to 25 different CTL epi-positions (Fig. 1).1 construct is based on the epi-position (HIV-1) that HIV-derives, and other 2 then contain the epi-position (being respectively HCV1 and HCV2) that HCV-derives.In A2 or A11 HLA transgenic mice, detected the immunogenicity (not estimating the epi-position of A1, A24 and B7 restriction) of these different multi-epitope constructs.

Thereby, behind the single intramuscular injection dna vaccination 11 days, after single 6 days external stimulates again, estimated reaction at 8 to 14 different representational epi-positions, use experiment to detect CTL activity (chromium discharges or original position IFN generates, and is as described herein).Under the situation of HIV-1, HCV1 and HCV2, can confirm to cause the epitope specificity CTL of 6/8 (75%), 10/14 (72%) and 13/14 (93%) test epi-position.Thereby, can easily design the multi-epitope constructs that can cause simultaneously at the ctl response of a large amount of epi-positions.But should emphasize, not detect the CTL of some epi-positions is caused that in some of 36 examples investigating, reaction is rare, and the variation of at least 3 orders of magnitude (1000 times) is arranged on magnitude.These results effectively confirm, need more carefully to analyze and optimize multi-epitope constructs.

The suboptimum performance of the initiation of some epi-position may be relevant with the size of multi-epitope constructs, and this possibility is also tested.In fact, most of disclosed reports have been described the multi-epitope constructs of 10 epi-positions at the most, reported the construct of 20 epi-positions in a few cases, but have only detected the activity at 2 or 3 epi-positions.In order to study this possibility, synthetic and detected 2 littler epigene constructs (HIV-1.1 and HIV-1.2), each comprises 10 epi-positions, and corresponding with half of HIV-1epigene construct.Detected reaction at 4 representational epi-positions.

As if table 1. immunogenicity do not rely on epigene construct size

	Ctl response to different epigene constructs
								HIV 1 (20 aggressiveness)		HIV 1.1 (10 aggressiveness)		HIV 1.2 (10 aggressiveness)
The CTL epi-position	Frequency 1)	Magnitude 2)	Frequency	Magnitude	Frequency	Magnitude
							??Pol?774	??0/8	??*	??0/4	??*	??NA 3)	??NA
??Pol?498	??18/19	??46.7	??4/4	??16.4	??NA	??NA
							??Gag?271	??4/13	??4.0	??NA	??NA	??0/4	??*
??Env?134	??5/8	??16.1	??NA	??NA	??4/4	??14.8

1) representative produces the independent culture part of positive reaction

2) lytic unit (LU)

3) inapplicable

Finding that the reaction that littler epigene construct is induced is comparable, if what difference is arranged, then is to be lower than those (tables 1) of being induced by the construct of 20 epi-positions.Therefore, as if can not explain the suboptimum initiating power of observed some epi-position, thereby use other parameter disclosed herein to design effective multi-epitope constructs about the factor of epigene construct size.

Connect minimizing of motif

One of Consideration when the design multi-epitope constructs is the connection epi-position that by mistake produces when epi-position is placed each other with closing on.The existence meeting appreciable impact performance of this epi-position in multi-epitope constructs.Herein disclosed is guidance strategy, to be used to develop polyepitope vaccines at these undesired effects.At first can minimize the connection epi-position, to identify the order that the number that wherein connects epi-position has been minimized by the screening epi-position.Such method for sieving can use a computer or finish by eyes, if necessary, perhaps depends on the number that will be included in the epi-position in the multi-epitope constructs.

For example, according to one embodiment of the invention can use can recognition mode computer program, Panorama for example, by ProVUE Development, Huntington Beach, California, U.S.A produces.When designing specific multi-epitope constructs, can consider very a large amount of different epi-position arrangements.Computer program is accepted the particular group of input epi-position to be considered and the motif that will scan, and to estimate whether the connection epi-position of carrying these motifs is arranged.For example, program can be simulated and set up multi-epitope constructs, and checks that in heuristic computational algorithm epi-position is right, to avoid or to minimize the generation that connects motif.Program can for example estimate 6 * 10 ⁵(half of about 1,000,000) individual multi-epitope constructs configuration/second.

Use the construct of 10 epi-positions of the described computer program exhaustive analysis of leading portion need check 10 factorial ≌ 3.6 * 10 ⁶Individual combination, and can in 6 seconds, finish.Can be in 2 days the construct of 14 epi-positions of exhaustive analysis.Thereby along with the increase of the construct of investigating, very rapidly increase analysis time.But, be not always to need exhaustive analysis, and program can be moved the time span of any needs.In both cases, computer system of the present invention can both be differentiated with providing and have minimum at least one configuration that is connected the epi-position number.

The result's of these class methods example is as shown in table 2.This table has shown the number of the connection motif in 10 times of identical epi-position different random assortments, and described identical epi-position is included among the HCV1epigene, and the latter is contained 25 epi-positions, and is the result of Computer Analysis in 2 days.In the classification of not optimizing, a large amount of HLA-A2, A11 and K have been found ^bMotif, about 25 to 38, average 31.Through contrast, only 2 such connection motifs are present in the classification of HCV1epigene construct.As a result, computer program can be used for minimizing effectively the number of the connection motif of multi-epitope constructs.

Table 2. connects the generation of epi-position

The epigene construct	Selection criteria	Connect motif
			??HCV.a	At random	??33
??HCV.b	At random	??26
			??HCV.c	At random	??28
??HCV.d	At random	??27
			??HCV.e	At random	??30
??HCV.f	At random	??26
			??HCV.g	At random	??38
??HCV.h	At random	??33
			??HCV.i	At random	??33
??HCV.j	At random	??34
			??HCV.l	Minimized	??2

Eliminate the body internal reaction that the II class connects epi-position and the restriction of test I I class

As eliminating other key element that connects epi-position, intervening sequence can be inserted into side by side the time and can form between 2 epi-positions that connect epi-position.

In one embodiment,, between 2 epi-positions, insert sept in order to proofread and correct the connection epi-position problem of HTL epi-position, for example, 5 amino acid whose length.Being incorporated into amino acid residue in such sept, to be preferably known be not the amino acid residue of the main anchor residue of HLA II class binding motif arbitrarily.Such residue comprises G, P and N.In preferred embodiments, the sept that will have a sequence GPGPG (SEQ ID NO:2) is inserted between 2 epi-positions.Work in the past is verified, and the GP sept can disturb II class binding interactions (Sette etc., J.Immunol., 143:1268-73 (1989)) especially effectively.People II class binding motif and mouse IA that all are known ^b(the II class that the HLA transgenic mice is expressed) can not tolerate and be positioned at G or the P that (it is at a distance of 4 residues) are put in main anchor position.In fact this method has verified that the epi-position of II class restriction can not form the connection epi-position.

In the embodiment of this design viewpoint of checking, we have synthesized the polypeptide that includes the HTL epi-position that HIV-derives.These epi-positions are that the HLA DR of cross reactivity widely is in conjunction with epi-position.Confirmed that then these epi-positions also can be effectively in conjunction with mouse IA ^bThe II quasi-molecule.The figure that these 2 different synthetic polypeptide of investigating are described is shown in Fig. 2 a.

First construct contains 4 linearly aligned different epi-positions, and the 2nd construct contains GPGPG (SEQ ID NO:2) sept.Also synthesized and 3 potential corresponding synthetic peptides of epi-position that are connected.

These different constructs of 2 nanomoles have been tested at IA ^bCause ability in the positive mouse, and contrast with reaction that the described same peptide set (every kind of peptide 3 μ g) of equivalent is induced at the breeder reaction of various epi-positions.More specifically, injected the CFA emulsion of peptide for the groups of 3 mouse, injected back 11 days, their lymph-node cell of culture in vitro 3 days detected the integration of thymidine at last 24 hours that cultivate.Found that (Fig. 2 b), as high affinity IA at them ^bDesired on the basis of binding ability, four all epi-positions have all been induced breeder reaction preferably.When these peptides are injected together, observed 4.9 to 17.9 stimulation index (SI) value.But, when test pack contains the linear polypeptide of identical epi-position, do not have reaction at Pol335.This is with relevant at the appearance of the reaction that is connected epi-position of having crossed over Gag 171 and Pol 335.This problem has been eliminated in the application of GPGPG (SEQ ID NO:2) sept, may be to connect epi-position by destroying, and has recovered Pol 335 reactions.The magnitude of observed reaction is similar to the peptide set that separates viewed.

These results confirm, can induce the reaction of the II class epi-position of deriving at a plurality of HIV-simultaneously, have also illustrated how to use IA ^b/ DR cross reaction Journal of Sex Research comprises the immunogenicity of the various constructs of HTL epi-position.At last, they have confirmed that the suitable interval thing can be used for destroying effectively the II class and connect epi-position, and these connect epi-position otherwise can hinder effective vaccine immunogenicity.

Under the situation of the reaction of I class restriction, people such as McMichael (Tussey etc., Immunity, Vol.3 (1): 65-77 (1995)) have proposed the inhibition of the reaction of the situation of connection epi-position of natural generation and epitope specificity subsequently.In order to solve the connection epi-position problem of I class, carried out similar analysis.For example, mouse motif and the great majority selected by screening are total to somebody I class HLA A and B motif, use specific computer program to differentiate the connection epi-position of potential I class restriction.

Can also use intervening sequence to prevent that CTL from connecting epi-position similarly.Often, very little residue (for example A or G) is preferred residue at interval.G also relatively low frequency ground as the preferred main anchor residue of HLAI class binding motif (see, for example, PCT/US00/24802).The length of these septs can be different, and for example the length of intervening sequence 1,2,3,4,5,6,7 or 8 amino acid residue typically is longer sometimes.Because the physical restriction when producing multi-epitope constructs, littler length often is preferred.

Flanking region is to the immunogenic influence of CTL multi-epitope constructs

Another factor that will consider when the design multi-epitope constructs is to be inserted with in the site of the distolateral wing of the C-of CTL epi-position and to be beneficial to immunogenic residue.

Research disclosed herein can identify residue, and it can improve immunogenicity, and therefore is inserted in the multi-epitope constructs to optimize immunogenicity.

The branch subenvironment of expressing epi-position often influences frequency and/or the magnitude that causes the CTL of this epitope specificity significantly in the HLA transgenic mice.Table 3 has shown 2 embodiment.

The difference of the T cell efficiency of initiation of the specificity epitope in the different epigene construct of table 3.

			Flanking sequence	Epi-position	Flanking sequence	Immune response	Immune response
								Epi-position	The Epigene construct	??SEQ ??ID ??NO：	(N end)	Sequence	(C-end)	Frequency	Magnitude 1)
??Core?18	??HBV.1	??3	??TLKAAA	??FLPSDFFPSV	??FLLSLG	??6/6	??5.5
								??pMin1	??4	??TLKAAA	??FLPSDFFPSV	??KLTPLC	??6/6	??1074.5
	??Core?132	??HCV1	??5	??ILGGWV	??DLMGYIPLV	??YLVAYQ	??2/12								??107.7
??HCV2								??6	??VPGSRG	??DLMGYIPLV	??AKFVA	??17/18	??929.2

1) IFN γ secretory units

The immunogenic magnitude of HBV core 18 epi-positions of expressing in the pMin5epigene construct is hanged down about 200 times than observed under the situation of pMin1epigene construct.Similarly, the immunogenicity of HCV core 132 epi-positions of expressing in the background of HCV1epigene construct is very little, and significant T cell causes only 2 middle susceptible of proofs in 12 that carry out different independently CTL experiment/cultivations.The experiment of these 2 positives has produced the reaction of about 100SU IFN γ.But, when under the background of HCV2epigene construct, expressing identical epi-position, in 17/18 experiment, observed positive reaction, its average magnitude has raise 5 times approximately.

The immunogenicity of HIV-FT in the HLA-A*0201/Kb transgenic mice

(Fig. 3 is the CTL epi-position that 20 HIV-derive of encoding a) for HIV Polyepitope DNA vaccine HIV-FT.In these 20 epi-positions, 8 by HLA-A*0201 restriction, and 9 by the HLA-A*1101 restriction, and 3 are limited by HLA-B*0702.All epi-positions are attached on their the relevant restriction element with higher or medium affinity.The epi-position of all HLA-A*0201 restriction with roughly similarly affinity in conjunction with the HLA-A*0201 molecule of purifying, its IC ₅₀(Fig. 3 a) in the scope of 19-192nM for value.The HLA-A*0201 epi-position of selecting that is included among the HIV-FT can be discerned in the individuality that HIV-1 infects, when with IFA emulsification and be used to cause HLA-A*0201/K ^bDuring transgenic mice, it also is being highly effective aspect ctl response that causes Memorability.Construct is used to the continuous epi-position of encoding, thereby without any inserting intervening sequence, simultaneously common Ig κ burst is fused to 5 of construct ' end between them, to promote the transhipment (Ishioka etc. of antigen in endoplasmic reticulum of coding, J.Immunol.162:3915-3925,1999).

Pass through HLA-A*0201/K ^bThe muscle of transgenic mice is interior immune, has estimated the ability of the interior ctl response of body of HIV-FT initiation Memorability.Every kind of HLA-A*0201 epi-position that use is encoded in HIV-FT, the splenocyte that has stimulated the animal of the 100 μ g HIV-FT plasmid DNA immunity of using by oneself is cultivated and has been tested peptide-specific CTL activity after 6 days.At the representational ctl response of 3 kinds of epi-positions among the HIV-FT shown in Fig. 4 a.In order to edit the result who obtains from difference experiment more expediently, the percentage cytotoxicity values of every kind of spleen cell cultures thing is represented (Vitiello, etc., J.Clin.Invest 95:341-349,1995) with lytic unit.In the epi-position that 8 kinds of HLA-A*0201 of HIV-FT coding limit, Pol 498, Env 134, Pol 448, Vpr 62, Nef 221 and Gag 271 have caused ctl response (Fig. 4 b) behind dna immunization.The variation of the magnitude of ctl response has surpassed 10 times scope, and high value approaches the 50LU of Pol 498, and low value is the 4LU of Nef 221 and Gag 271.Similarly, the frequency of the ctl response of Memorability is also different between epi-position, Pol 498 epi-positions induced reaction in 94% experiment, and only in 31% experiment, detected ctl response at Gag 271.In a word, the dna immunization of HIV-FT (its continuous programming code does not contain the amino acid whose epi-position in any interval) has been induced the ctl response at the Memorability of most of epi-positions of testing.But the magnitude of reaction and frequency have very big difference between epi-position.

The epi-position immunogenicity is presented the related of level with the HIV-FT epi-position in cells transfected system

Tested the differentiated immunogenicity of the HLA-A*0201 epi-position among the HIV-FT then.Can get rid of differentiated MHC binding affinity, (Fig. 3 a) in conjunction with HLA-A*0201 because all epi-positions are all with high affinity.In addition, can get rid of at HLA-A*0201/K ^bLack suitable TCR specificity inventory (repertoire) in the transgenic mice, because all epi-positions have all produced the ctl response that can contrast behind the peptide immunity HLA transgenic mice with best pretreated emulsification in IFA.The variation that is the relative quantity of each epi-position of passing the T cell recognition can be explained the immunogenic difference of epi-position.

In order to test this point, be used in the HIV-FT transfection expressed in the episome carrier and can have expressed HLA-A*0201/K ^bThe human T-cell of gene is Jurkat cell (Vitiello etc., J Exp.Med.173,1007-1015,1991).Selected the human cell line to eliminate any possible artificial illusion, described illusion may be relevant with the potential difference of working ability between people and the mouse.This cells transfected system can mate the people MHC with human antigen's working ability and presents, and can support the exploitation based on the dna vaccination of CTL epi-position that is used for human body subsequently.

Presenting in the target of transfection of 4 kinds of HLA-A*0201 epi-positions (Pol 498, and Env 134, Pol 448 and Nef 221) of encoding detected by peptide-specific CTL system in HIV-FT.For the production of quantitative every kind of epi-position and the level of presenting, will hatch together the every kind of epi-position or the peptide of the CTL of various epitope specificities system and the target of untransfected, different amounts.These CTL dose-effect curves are as calibration curve, induce peptide concentration with observed identical IFN γ secretion level in the target cell of HIV-FT transfection with detection.This value is called " peptide is equal to dosage ", and is used as the relative tolerance of the epi-position amount of presenting on cells transfected.

Table 4 has been summed up the analysis result of 8 HLA-A*0201 epi-positions of encoding in HIV-FT.Peptide is equal to the peptide equivalent that be lower than 0.4ng/ml of dosage from the high value 33.3ng/ml of Nef 221 to epi-position Gag 271, Gag 386 and Pol 774.These results show that together in the human cell line with the HIV-FT transfection, there is at least 100 times variation in the level of presenting of the epi-position of different HLA-A*0201 restrictions.In antigenicity experiment, also all be immunogenic in the body with all epi-positions of can detected level presenting.Unique immunogenic but nonantigenic epi-position is Gag 271.In this case, with HIV-FT immunity HLA-A*0201/K ^bTransgenic mice has been induced more weak ctl response in being less than 1/3rd test cultures.Showing other 2 the epi-position Gag 386 and the Pol 774 that are lower than the susceptibility limit in the antigenicity experiment is non-immunogenicities.In a word, these results show, can be presented owing to the epi-position of suboptimum at least in part by the heterogeneity in the ctl response of HIV-FT immune induction.

Table 4:HIV-FT immunogenicity and antigenic contrast

Epi-position	The HIV-FT immunogenicity		The HIV-FT antigenicity
						Magnitude 1	Frequency 2	The peptide equivalent 3	??n 4
??Pol?498 ??Env?134 ??Pol?448 ??Vpr?62 ??Nef?221 ??Gag?271 ??Gag?386 ??Pol?774	??58.8(22) ??16.1(5.0) ??15.7(2.6) ??9.9(1.9) ??4.4(1.3) ??4.0(1.4) ??0 ??0	??94％(16/17) ??63％(5/8) ??54％(7/13) ??83％(10.12) ??78％(7/9) ??31％(4/13) ??0％(0/17) ??0％(0/8)	??23.8(2.0) ??6.2(1.2) ??24.7(3.9) ??ND ??333(6.0) ??＜0.4 ??＜0.4 ??＜0.4	??4 ??3 ??3 ??- ??3 ??6 ??3 ??1

1 is expressed as the magnitude of LU (ref); Coefficient R+0.44 with respect to the peptide equivalent

The frequency of 2 positive cultures (culture number＞2LU/ total number measured); Coefficient R+0.8 with respect to the peptide equivalent.

3 magnitudes of representing with ng/ml

The number of 4 independent experiments

Flanking amino acid influences the interior immunogenicity of body of the CTL epi-position after the immunity

As described herein, the specific amino acids of each CTL epi-position flank can influence or improve the working (machining) efficiency of epi-position by changing antigen to the susceptibility of proteolysis shearing.In order to detect flanking amino acid to the immunogenic influence of epi-position, from with the HLA-A*0201 of many incoherent experimental Polyepitope DNA constructs (CTL epi-position that its coding is minimum does not have insetion sequence) immunity ,-A*1101 and-the B*0701 transgenic mice obtained the immunogenicity data.Edited the database of representing the combination of 94 kinds of different epi-positions/flank residue, with the flanking amino acid of determining the next-door neighbour to the immunogenic possible influence of epi-position.Given epi-position and flanking amino acid combination are only used once, with the artificial deviation that prevents to cause analysis owing to redundant.If in the culture of at least 30% test,, think that then the epi-position immunogenicity in the HLA transgenosis is best greater than 100SU or 20LU.Typically ctl response is divided into 4 classes: (+++), significantly---surpass 200LU or 1000SU; (++), better--20-200LU or 100-1000SU; (+), medium--2 to 20LU or 10 to 100SU; (+/-), more weak or negative---be lower than 2LU or 10SU.According to the amino acid whose chemical type in the flank site, the number with reaction suboptimum the best is classified, and use X ²The significance of difference has been determined in check.

The N-terminal amino acid type of epi-position and any relation between the immunogenicity are not found in this analysis.But having identified carboxyl-distolateral wing residue is the remarkable effect of C+1 residue.The ctl response with the best is relevant the most continually for positively charged amino acid K or R, and frequency is 68% (Fig 5).In 55.5% test experiments, the amino acid N that is positioned at the C+1 residue is relevant with the Q also ctl response with strong; When the C+1 site of epi-position flank was N, they can induce best ctl response in 3/4 situation.Usually, little residue for example C, G, A, T and S can promote medium ctl response, and it induces strong reaction in 54% the combination that can be used for analyzing.On the contrary, its flank is that aromatics and aliphatic amino acid whose epi-position only can be induced best body internal reaction respectively in 36% and 17% situation.Electronegative residue D can produce the ctl response of suboptimum.Use X ²Check (P＜0.03) finds that immunogenic influence is significant on the statistics to C+1 amino acid to epi-position.When holding residue similarly to analyze, do not observe the immunogenic appreciable impact of epi-position to C-away from the C+1 site.

Directly estimate the C1 residue to the immunogenic influence of epi-position

In order directly to estimate preferred type and amino acid whose influence harmful type in C+1 flank site, estimated 2 multi-epitope constructs, they are called HBV.1 and HBV.2 (Fig 3b).As HIV-FT, these HBV constructs are encoded and are not inserted the continuous epitope of sept in its sequence.By replacing the HIV-1 epi-position among the pMin1 (experimental multi-epitope constructs characterized (Ishioka, the same) with the epi-position that similarly is derived from HBV in the past), HBV.1 and HBV.2epigene have been generated.

For HBV.1, the HIV-1 epi-position that is close to HBV core 18 epi-positions of high immunogenicity is replaced with HBV Pol 562 epi-positions.This becomes F with the C+1 residue from K.By between HBV core 18 and Pol 562 epi-positions, inserting extra epi-position HBV Pol 629, generated the 2nd construct HBV.2; This variation is that C+1 amino acid is replaced with the K residue.When at HLA-A*0201/K ^bWhen estimating the immunogenicity of core 18 epi-positions in these different background in the transgenic mice, detect core 18 epi-positions and in HBV.1, be actually non-immunogenicity, but be that immunogenic by force (Fig. 6 a) in HBV.2.Immunogenic reduction is as predicting by the analysis before us in the body of this epi-position.

For further test C+1 flanking amino acid has been estimated one group of construct to the immunogenic influence of CTL epi-position, the difference of itself and HBV.1 is to insert an amino acid (Fig. 3 b) with respect to the C+1 site of core 18 epi-positions.When by W, Y or L during, observe less or not at the ctl response (Fig 6b) of core 18 epi-positions at C+1 site side joint.On the contrary, inserting a K residue can the remarkable ctl response that increases core 18.This reaction can with observed comparing in HBV.2, the flank of core 18 epi-positions is Pol 629 epi-positions (Pol 629 has K at N-end) in the latter.Also observed and inserted the core 18CTL reaction that R, C, N or G strengthen.The effect of these inserts is specific, because the immunogenicity of other epi-position in these constructs does not show significant change (data not shown) in ctl response.In a word, these data show that C+1 amino acid can influence the immunogenicity of epi-position consumingly.

The immunogenic variation of CTL epi-position is relevant with the amount of presenting

If the immunogenic variation of the core 18 relevant with different C+1 residues is results of differentiated susceptibility that proteolysis is sheared, should in different constructs, can detect so epi-position present level than big-difference.In order to test this point, with the episome carrier transfection that can express HBV.1 or HBV.1K the Jurkat cell, it can be expressed and the identical HLA-A*0201/K that expresses in transgenic mice ^bGene.As K during in the C+1 site, height＞10 when there is F in the level of core 18 epi-positions than in identical site ⁵(Fig. 7).This species diversity that core 18 is presented can not be owing to the difference of the gene expression between the target cell system, because the variation of presenting of Pol 455 is lower than 10 times.These data acknowledgements a significant effect, promptly the amino acid in C+1 site can be given the efficient that the epi-position in the Polyepitope DNA vaccine is presented.Thereby these data show, influence the factor of the level that epi-position presents by design, can optimize the immunogenicity of the CTL epi-position in the dna vaccination.This class optimization is applicable to the vaccine based on epi-position that other form of use (for example viral vectors and other expression vector known to those skilled in the art) is carried, because this effect is just given after antigen is transcribed and translated.

In a word, for the flank residue, find very little residue (for example A, C or G) or bigger residue (for example Q, W, K or R) generally with preferably or noticeable response relevant.Because the disappearance of the C+1 residue that the terminator in the multi-epitope constructs causes or the existence of medium sized residue (for example S or T) are relevant with the pattern of medium reaction.At last, under the situation of (V, I, L, the M) of electronegative residue D, aliphatic series or the non-trp residue (Y, F) of aromatics, observed relatively poor relatively reaction.These results show that the specific residue of the distolateral wing of epi-position C-can influence response frequency and magnitude significantly.Flank residue in the C+1 site can also import with intervening sequence.Thereby, comprised and helped the flank residue of immunogenic residue, preferred K, R, N, A or G as sept.

Screening and optimization multi-epitope constructs

In order to use the present invention to develop multi-epitope constructs, use the parameter screening of this paper definition and optimized the epi-position that is used for being included in multi-epitope constructs.Can use a computer and sieve and optimize,, then not use a computer for a spot of epi-position.

Typically can followingly carry out computerized optimization.The example of a computerized system is provided below, and it can differentiate and optimize the epi-position combination, for example provides the connection epi-position of minimal amount and the flank residue of maximum number for the epi-position combination.Figure 10 has illustrated the computer system 100 according to one embodiment of the invention, and it is used to optimize multi-epitope constructs.Computer system 100 can be the calculator of general type, it comprises treatment loop, central processing unit (CPU) for example, memory, for example hard disk drive (ROM), random-access memory (ram), cache memory and other component typically see device and loop (not shown) in the modern computer.In preferred embodiments, except other component and device, computer system 100 also comprises the hard disk of Macintosh G3 333MHz processor, 6G (GB), the RAM of 96M and the cache memory of 512K (KB), and its per second can search for 600,000 to 700,000 arrangement.Computer system 100 also comprises and is used for showing the display 102 of text, figure and out of Memory and allowing to be used for keyboard 104 to computer system 100 input data, order and out of Memory to the user.

As shown in figure 10, in one embodiment, computer system 100 can be by computer network 160 and one or more remote computer 150 communications, so the user of registration can carry out the optimization method of connection analysis as herein described and multi-epitope constructs a long way off by land and provide password or the ID that needs at remote computer 150.Computer network 160 can be Local Area Network, wide area network (WAN) or World Wide Web (being the internet).The network of these types is well known in the art, and therefore, this paper does not discuss these networks and their communications protocol.

In preferred embodiments, computer system 100 has been stored software program in the hard disk drive storage of computer system 100, for example object code.This object code is carried out by CPU, to realize function as herein described.The component or the module of software program can realize following function: analyze the epi-position that is connected with the peptide junction that is identified in polypeptide (by multi-epitope nucleic acid construct coding), and estimate the sept in these are connected and the combination of flank residue.This software module is called " Junctional Analyzer " module or program in this article.In preferred embodiments, Junctional Analyzer is according to other standard described below, data and operational factor analysis with handle peptide by user's input.

Figure 11 A-B (being called Figure 11 hereinafter) has illustrated the exemplary input text file 200 that contains user input data and parameter, and according to one embodiment of the invention, it is used by Junctional Analyzer program.As shown in figure 11, various types of input data are provided for program.At first, the user can import one group of peptide to be processed or epi-position 202.When it was positioned at C+1 and N-1 site, the weights 204 of one group of every seed amino acid also were input in the text by the user.In one embodiment, analyzing immunogenicity or the antigenicity " amplification " that experimental result determines that weights, described experimental result have reacted every seed amino acid (when it is positioned at the C+1 of polypeptide or N-1 site) by statistics ground or experience ground acts on.But, can be by the methodology of any amount, comprise the research in external and the body, according to the ideal standard that is used to detect weights, be that every seed amino acid distributes weights, this is obvious for the ordinary skill in the art.Some embodiment of such experiment or research have been described in further detail below.

In preferred embodiments, on the immunogenic basis of epi-position, the database that contains different epi-positions/flank residue combination has been carried out layering, sieved number best and reaction suboptimum, with arrays of amino acid and assign weights.Text also contains one group and is used to detect the motif 206 that connects epi-position.In preferred embodiments, the user can also import the amino acid (sept and flank residue) between every pair of peptide (MaxInsertions) 208 of will being inserted in of maximum number, and it works as sept and/or flank residue.Can also input control program other parameter, value or the instruction (here be called together " parameters ") of operation, for example: " OutputToScreen (Y/N) " 210; " OutputToFile (Y/N) " 212; Can be accepted as effective result's minimum function value (" MinimumAccepted ") 214; Maximum number (" MaxDuplicateFunctionValue ") 216 with result of identical function value; In several minutes, allow the maximum times (" SearchTime ") 218 of search; Whether need thorough search (" Exhaustive=Y/N ") 220; The number of times of random search (" NumStochasticProbes ") 222; The maximum number (" MaxHitsPerProbe ") 224 of the value of hitting that each search allows in random search procedure; With the beginning of each search should be at random or other (" RandomProbeStart (Y/N) ") 226.These parameters are only used for task of explanation.As conspicuous for those of ordinary skill in the art, can input control program other parameter of operation and output format.

Motif 206 in the text 200 provides and has been used to differentiate " mask (mask) " or the structural model that connects epi-position.For example, first motif 206a shown in Figure 11, XXXX (F or Y) XX (L, I, M or V), having defined length is 8 amino acid whose epi-positions.This site that value " X " is illustrated in epi-position can be an amino acid arbitrarily.Value " (F or Y) " expression F amino acid or Y amino acid can be in the 5th sites of epi-position.Similarly, any among amino acid L, the I, M or the V that list of " (L, I, M or V) " expression can be in the 8th site of epi-position.Therefore, can satisfy top motif standard, then it be differentiated to connecting epi-position if comprise 8 amino acid whose sequences of the bonding pad of 2 peptides.

Figure 12 has illustrated the flow chart of an embodiment of Junctional Analyzer program.In step 301, program is accepted user's input and order, carries out to connect analysis operation.In preferred embodiments, program uses input text file 200 as shown in figure 11 to come input parameter 202-226.As known in the art, such text can be from, Microsoft Excel for example ^TMThe spreadsheet file or document is for desirable input parameter (for example epi-position, motif, flank residue weights, maximum life median numbers, maximum search time etc.) is specified in its operation.In step 303, it is right that Junctional Analyzer program produces all epi-positions of row.For example, if the user has imported 10 epitope sequences, have then that altogether the individual epi-positions in 90 (10 * 9) (peptide) are right.Then, in step 305,, for every pair of peptide or epi-position, program detects the connection epi-position can cause minimal amount and/or from one group of insert of the maximum effect of the C+1 of interval residue and N-1 contribution.In order to carry out this detection, this program for each peptide to calculating the function value of each possible sept combination, wherein the number of sept in 0 to MaxInsertions208 scope (Figure 11) can be considered known or predetermined amino acid whose any arrangement.In preferred embodiments, equation computing function value below using: F=(C+N)/J, wherein C is the weighted value of flanking amino acid that is positioned at the C+1 site of epi-position, and N is the weighted value of flanking amino acid that is positioned at the N-1 site of epi-position, and J is the number of the connection epi-position that exists.Because a plurality of motifs can satisfy a peptide to connecting, J can be the numeral greater than 1.When J=0, F=2 (C+N).Select the reason of the 2nd equation to be, for fixing (C+N) value, when J when 2 become 1, function value F doubles, when J when 1 becomes 0, function value F can double once more.But should be understood that top equation only is exemplary, can easily increase be used to estimate other right equation of peptide to program at any time.Those of ordinary skill in the art can easily understand, according to the ideal standard that is used to emphasize or estimate, can improve or change top equation.In step 307, program is the best of breed of every pair of peptide output insert (sept and/or flank residue) and the maximum function value of every pair of peptide.In preferred embodiments, in step 307, the output of this program is to produce as the text of output, and it has listed the insert that can produce the maximum function result for every pair of peptide.

Figure 13 A-D (being called Figure 13 hereinafter) has illustrated exemplary output text 400, and it makes up having listed the sept with maximum function value for each peptide.In the embodiment shown in fig. 13, handled 11 peptides, be labeled as A-K 202 (Figure 11), motif 206 is used to detect the flank epi-position, has used the weighted value of each potential flank residue 204, and MaxInsertions 208 is set at 4.The operation of control Junctional Analyzer program and other parameter of form are set at as parameter and are provided with shown in 402.Purpose for convenience in preferred embodiments, has repeated these input parameters in output text 400.Output text 400 comprises output table 404, and it contains 305 result (Figure 12) in steps.First right peptide of first row (Col.1) expression of output table 404.The 2nd row (Col.2) of output table have been listed first aminoacid insertion thing, and it plays sept and C+1 flanking amino acid simultaneously.The 3rd has listed the 2nd amino acid at interval.The 4th has listed the 3rd amino acid at interval.The 5th has listed the 4th at interval amino acid, and it also is the N-1 flanking amino acid of the 2nd the right peptide listed in the 6th row.The 7th has listed the weighted value of the C+1 flanking amino acid of listing in the 2nd row.The 8th has listed the weighted value of the N-1 flanking amino acid of listing in the 6th row 412.The 9th has listed the summation of C+1 and N-1 weighted value.The 10th has listed the number of the connection epi-position of peptide centering, and the 11st classifies peptide as to having listed the maximum function value, and it is based on above-mentioned equation.For example, first row of output table 404 shows, peptide VLAEAMSQV (SEQ ID NO:7)-ILKEPVHGV (SEQ ID NO:8) of peptide to(for) correspondence is to A-B, and the combination of the sept of 3 amino acid CAL can be eliminated all connection epi-positions, and maximum function value 8.80 is provided.But should be understood that according to the present invention the output selection that also can make other.For example, analysis according to the level of detail and/or user's needs, output table 404 can show 32 best results for every pair of peptide, is that all possible insert shows each result according to the order of estimating perhaps, perhaps carries out the motif search procedure and produces bigger output file.

In preferred embodiments, the information that is included in the output table 404 is used for carrying out " J search (Exhaustive J Search) completely " or " J search (StochasticJ Search) at random ", to differentiate the polypeptide construct that connects all 11 peptides, comprise best sept combination.For 11 peptides, for example, will have 10 connections.Therefore, the arrangement that will produce the maximum summation of the function value of having considered all 10 connections differentiates to be " the best " arrangement of multi-epitope constructs.In one embodiment, for user's convenience, output text 400 also contains following source list: peptide s/ epi-position 202, the weights 204 that use, motif 206 that uses and MaxInsertion value 208 and input other parameter in the input text file 200 of Figure 11 are provided with 402.

" J search completely " checks all arrangements of peptide, and selection has the maximum function summation that.But because order of a permutation takes advantage of character, along with the increase of the number of peptide to be processed, " J search completely " the required time of finishing almost increases exponentially.For example, use the Macintosh 333MHz calculator of standard, be about 2.9 hours the running time that 13 peptides are estimated, then is about 40 hours for 14 peptides." J search at random " is used to search for many zones of collating sequence, rather than whole alignment area, and reports the best-of-breed functionality summation that it is found.By only reporting the arrangement of satisfying or surpassing present maximum function summation, the more extensive region that can search for collating sequence.This technology has been successfully used to nearly 20 peptides.It is about 1.3 * 10 that estimation is thoroughly searched for the required time to 20 peptides ⁵Year.

With reference to Figure 12, in step 309, the program decision still is " J search at random " to the execution " J search completely " of may arranging from the polypeptide of exporting text 400 again.In preferred embodiments, made by the user in the decision of step 309, it imports this search will be completely or at random, as representing (Figure 11) by input parameter Exhaustive (Y/N) 220.In other embodiment, program can according to the number of peptide to be processed select automatically at random or search completely.For example, if comprise and be less than 14 epi-positions, program is selected " J search completely " approach automatically.Search utility inspection completely constitutes all of epi-position of multi-epitope constructs arranges, for the summation of the right optimizational function of all epi-positions finds to have that of optimum value.This can guarantee to find " the best " to arrange, because checked all.If multi-epitope constructs will comprise 14 or more a plurality of epi-position, then use " J search at random ".In preferred embodiments, " at random J search " uses MonteCarlo technology known to those skilled in the art, checks many zones of alignment area, with the best estimate of the optimal arrangement of finding peptide.But, also can realize other stochastic search methods according to the present invention.For example, in each random search, be not to select initial arrangement randomly, but program may require each search from that different arrangement of peptide of user's input as starting point.For example, if 3 peptide A, B and C are just arranged, 3 search will be from for example ABC, BAC and CBA.This method has covered possible arrangement equitably, equably.

If selected " J search at random ", then, in step 311, program is by starting search beginning random search.Then, in step 313, program determines whether to have surpassed the maximum search time of each search.If also do not reach the maximum search time, then, in step 315, program determines whether once search has has met or exceeded the maximum " hits " of each search.In one embodiment, during the maximum function summation of registration before the function value summation of arranging is equal to or greater than (for the arrangement of analyzing before one or more), then register the search hit value.If also do not reach the maximum hits of each search, so, in step 317, current random search is estimated next the arrangement or one group of arrangement, and this process turns back to step 313.If determine to have met or exceeded the maximum hits of each search in step 315, so, program proceeds to step 319, and wherein program determines whether to have carried out the search of maximum number.In addition, if determine to have met or exceeded the maximum time limit of each search in step 313, program proceeds to step 319, determines whether to have finished the search of maximum number.If in the definite search that does not reach maximum number of step 319, this program turns back to step 311, starts new search.If in the definite search of having carried out maximum number of step 319, program proceeds to step 323, it exports the preferably group of the optimal arrangement that identified here before this point.Should " preferably group " can be only rearrange by those with the highest function summation, perhaps, can be by having rearranging of preceding 3 function summations the highest, for example, or any other outputting standard of needing of user.

In a preferred embodiment, if search has reached the maximum hits of the appointment of each search, any untapped time of this time search will determine to give remaining each search how long to distribute divided by remaining search.In other words, if once search is owing to found the too many value of hitting and premature termination, so remaining search can be assigned with more time.The those of ordinary skill in computer programming field can easily be realized this function.

If selected search completely in step 309, so, start search completely in step 321, it analyzes each arrangement, as mentioned above.When finishing exhaustive analysis, program proceeds to step 323, and it exports " the preferably group " of the optimal arrangement of discovery here.As mentioned above, should can comprise " preferably group " those and arrange, and maybe can satisfy the arrangement (for example, having 30 arrangements of high function value) of any ideal standard of user's appointment with the highest general function value or preceding 3 general function values the highest.

For each deciding step discussed above (for example step 313,315 and 319), program can be arranged in execution of the periodic time interval (for example per 5 seconds) puts question to, perhaps, program can be arranged in by analysis the intact arrangement (for example 5) that specifies number or intact by analysis each arrange the back and carry out and put question to.Those of ordinary skill in the art can easily realize and adjust these operations and timing scheme.

Program output provides the best epi-position of row to arrange.Because many arrangements can have identical Function of Evaluation value, therefore generated several so that when selecting optimal arrangement, can consider other factors.The example that uses the multi-epitope constructs that above-mentioned computerized technology produces as shown in Figure 9.The exemplary flow chart of finishing by method and system of the present invention is provided above.Can understand easily that as those of ordinary skill in the art other factors (for example analysis in distribution of charges, hydrophobic/hydrophilic zone or folding prediction) also can be incorporated in the functional evaluation, with further optimization multi-epitope constructs.In addition, optimize the multi-epitope constructs of (by 1 time or repeatedly identical and similar processing) by processing, can further optimize multi-epitope constructs.In processing bout subsequently, can contrast the parameter of in first optimization bout, using and revise one or more parameters.The example of the multi-epitope constructs of optimizing in 2 bouts is the HBV-30CL construct.

Can also optimize multi-epitope constructs by the macromolecular structure of considering to obtain.Macromolecular structure for example polypeptide structure can be described according to the tissue of varying level.General discussion about this tissue, see, for example, Alberts etc., Molecular Biology of the Cell (the 3rd edition, 1994) and Cantor and Schimmel, Biophysical Chemistry Part I:The Conformation of Biological Macromolecules (1980)." primary structure " is meant the amino acid sequence of particular peptide." secondary structure " is meant the three-dimensional structure of local order in polypeptide.The so-called domain of these structures.Domain is the polypeptide portion that forms the tight functional unit of polypeptide.Typical domain being combined to form by secondary structure (for example, beta sheet and alpha-helix)." tertiary structure " is meant the complete three-dimensional structure of polypeptide monomer." quaternary structure " is meant by three grades of three-dimensional structures that the unit is connected to form independently non-covalently.

Use sequence analysis program known to those skilled in the art, can carry out structure prediction, for example distribution of charges, hydrophobic/hydrophilic regional analysis or folding prediction, for example, can identify hydrophobic and hydrophilic domain (see, for example, Kyte ﹠amp; Doolittle, J.Mol.Biol.157:105-132 (1982) and Stryer, Biochemistry (the 3rd edition, 1988); Be also shown in the sequence analysis program based on the internet of any amount, for example those that find at dot.imgen.bcm.tmc.edu.

Can also generate the 3 d structure model of multi-epitope constructs.This generally is that amino acid sequence input by will analyzing can produce in the predictive computer system of model and finishes.Amino acid sequence has been represented the primary sequence or the subsequence of albumen, the structural information of its encoding proteins.Use software known to those skilled in the art then,, generated the 3 d structure model of albumen by the cooperation of computer system.

Amino acid sequence has been represented primary structure, and its coding forms secondary, three grades and the required information of quaternary structure of target protein.Software is investigated some parameter by the primary sequence coding, to produce structural model.These parameters are called " energy term (energy term) ", mainly comprise electrostatic potential, hydrophobic potential, solvent accessible surface and hydrogen bond combination.The secondary energy term comprises the Van der Waals gesture.Biomolecule forms the structure of the energy term of energy minimization accumulating form.Therefore, computer program uses and creates the secondary structure model by primary structure or amino acid sequences encoded these.On the basis of the energy term of secondary structure, form the tertiary structure of secondary structure encoded protein then.The user can import other variable, and for example albumen is membrane-bound or soluble, its position in vivo, and its cell position, for example cytoplasmic, surperficial or nuclear.The energy term of these variablees and secondary structure is used to form the tertiary structure model.When the tertiary structure modeling, computer program is with the hydrophobic surface and similar coupling of secondary structure, with the hydrophilic surface and the similar coupling of secondary structure.Those the easiest multi-epitope constructs have been selected then near the HLA machine component.

The immunogenic judgement of polyepitope vaccines

The PDT R﹠D Representative of multi-epitope constructs great challenge, this is because in conjunction with the species specificity of the peptide of MHC.Tend in conjunction with on the same group peptide (Rammensee etc., Immunogenetics, Vol.41 (4): 178-228 (1995)) not from different MHC types not of the same race.As a result, can not in the laboratory animal of routine, test the construct of forming by people's epi-position.Usually can obtain to overcome the replacement scheme of this restriction.They comprise: 1) construct like the test class, and it contains the epi-position of non--people MHC restriction; 2) rely on the contrast epi-position, it is by non--people MHC restriction; 3) cross reactivity between dependence people and the non--people MHC; 4) use the HLA transgenic animal; With 5) experiment of body endoantigen, its end user's cell.Analysis antigenicity and immunogenic Progress in technique have been summed up below simply.

I class HLA transgenic mice

Set up the HLA transgenic mice and differentiated (Sette etc., JImmunol, Vol.153 (12): 5586-92 (1994) to be used for epi-position; Wentworth etc., Int Immunol, Vol.8 (5): 651-9 (1996); Engelhard etc., J Immunol, Vol.146 (4): 1226-32 (1991); Man etc., Int Immunol, Vol.7 (4): 597-605 (1995); Shirai etc., J.Immunol, Vol.154 (6): 2733-42 (1995)) and the application of vaccine development (Ishioka etc., J Immunol, Vol.162 (7): 3915-25 (1999)) purpose.Many disclosed reports after deliberation HLA A2.1/K ^bThe application of mouse, but should be pointed out that B*27 and B*3501 mouse also are available.And, also produced HLA A*11/K ^bMouse (Alexander etc., J Immunol, Vol.159 (10): 4753-61 (1997)) and HLA B71K ^bWith HLA Al/K ^bMouse.

Data have been analyzed, to determine A2.1/K from 38 different potential epi-positions ^bOverlapping level (Wentworth etc., Eur J Immunol, Vol.26 (1): 97-101 (1996)) between the CTL inventory of-transgenic mice and A2.1+ people's A2.1-restriction.In people and mouse, MHC peptide binding affinity threshold value and the peptide of about 500nM causes that the ability of ctl response in the body is relevant.For the peptide of 85% high-bond, 58% medium bond and 83% low/negative bond, in human body, observed the uniformity of higher level in data and the mouse body between the data.Also obtained similar result (Alexander etc., J Immunol, Vol.159 (10): 4753-61 (1997)) with HLAA11 and HLA B7 transgenic mice.Therefore, because existence is overlapping widely between the TXi Baoshouti inventory of HLA transgenic mice and the people CTL, transgenic mice can be used to estimate the immunogenicity of multi-epitope constructs as herein described.

The different specificity that relates to the transhipment of HLA A11 mouse can not stop the application of HLA-A11 transgenic mice in estimating immunogenicity.Although mouse and people TAP can both transport the peptide with hydrophobic end effectively, only reported that people TAP can transport the peptide with positively charged C end effectively, for example by those of other member's combination of A3, A11 and the super type of A3.This problem is not suitable for A2, A1 or B7, because mouse and people TAP can transport comparably by the peptide of A2, B7 or A1 combination.Consistent with this understanding, Vitiello (Vitiello etc., J Exp Med, Vol.173 (4): 1007-15 (1991)) and Rotzschke (Rotzschke O, Falk K., Curr Opin Immunol, Vol.6 (1): 45-51 (1994)) think that mouse is similar with the processing in people's cell, and Cerundolo (Rotzschke O, Falk K., Curr OpinImmunol, Vol.6 (1): 45-51 (1994)) think that mouse and the people's cell that can express HLA A3 molecule there are differences.But, use HLA A11 transgenosis, body has been observed the expression of the HLA molecule on T and the B cell interiorly, and this disadvantageous specificity that shows the mouse TAP of report does not stop A11/K ^bStabilisation and transhipment (Alexander etc., J.Immunol, Vol.159 (10): 4753-61 (1997)) in the body of molecule.These data and in the past observed Toplink wash-out consistent (Maier etc., Immunogenetics from the mouse cell of using the transfection of A11 molecule with charged C-end; Vol.40 (4): 306-8 (1994)).Also detected of the reaction of HLA A11 mouse, the most important thing is reaction (Ishioka etc., JImmunol, Vol.162 (7): 3915-25 (1999)) by the epi-position of the A11 restriction of multi-epitope constructs coding to complex antigen (for example influenza).Therefore, the TAP problem peripheral issue of transgenic mice seemingly.

Another potential relevant issues of using the HLA transgenic mice are β2Wei Qiudanbai possible influences to HLA expression and binding specificity.As everyone knows, people β 2 can with than the higher affinity of mouse β2Wei Qiudanbai and stability in conjunction with people and mouse MHC (Shields etc., MolImmunol Vol.35 (14-15): 919-28 (1998)).Also well-known, the more stabilized complex of MHC heavy chain and β 2 helps the external source of I class MHC and loads (Vitiello etc., Science, Vol.250 (4986): 1423-6 (1990)).By generating HLA/K ^bWith the mouse of people β 2 double transgenics, we after testing the potential impact of this variable.At HLA B7/K ^bUnder the situation of mouse, the expression of people β 2 is favourable, and is to realize that good expression institute is definitely necessary under the situation of HLA A1 transgenic mice.Therefore, the present inventor raises and has used HLA/K at large and routinely ^bWith β 2 double transgenic mouse.Thereby the HLA transgenic mice can be used to imitate the identification of the HLA-restriction of 4 main HLA specificitys (being A2, A11, B7 and A1), and the specific transgenic mice of HLA that can develop other is as estimating immunogenic appropriate model.

The antigenicity test of I class epi-position

Several separate experiment system shows that the density of the I class/peptide complexes on the cell surface may cause relevant with the T cell.Thereby the level of the epi-position that detection produces on the surface of APC and presents can provide and estimate the effectiveness of multi-epitope nucleic acid vaccine to external people's cell indirectly.As to using replenishing of HLA I class transgenic mice, this method has the advantage (Ishioka etc., J Immunol, Vol.162 (7): 3915-25 (1999)) of the processing in can scrutineer's cell.

Can obtain several possible scheme of the quantitative finished peptide of sample plot.By detecting from the amount of the peptide of APC surface wash-out, the quantitatively amount of the peptide on the cell surface (Sijts etc., JImmunol, Vol.156 (2): 683-92 (1996); Demotz etc., Nature, Vol.342 (6250): 682-4 (1989)).Perhaps, the amount that cracking of inducing by target cell that detect to infect or transfection or lymphokine discharge, determine that the cracking of same levels such as obtaining or lymphokine discharge required peptide concentration then, can estimate the number (Kageyama etc. of peptide-MHC compound, J.Immunol, Vol.154 (2): 567-76 (1995)).

Similar scheme has been used for detecting the epi-position of multi-epitope nucleic acid-cells transfected system and has presented.More specifically, be that immunogenic multi-epitope constructs also has been processed into best epi-position by the people's cell with identical construct transfection in the HLA transgenic mice, the magnitude of observed reaction and the relevant (Ishioka etc. of the observed antigenicity of people target cell that use transfection in transgenic mice, J Immunol, Vol.162 (7): 3915-25 (1999)).

The experiment of use antigenicity can be advanced many relevant constructs (variant on epi-position order or flank residue) transfection APC, and can estimate the influence that aforementioned variable is presented epi-position.When a large amount of relatively different construct of needs assessment, this can be preferred test macro.Although it needs a large amount of epi-position-specific CTL, (the Alexander-Miller etc. of CTL system that can produce hypersensitivity have been developed, Proc Natl Acad Sci USA, Vol.93 (9): 4102-7 (1996)) and with them be proliferated into larger amt (Greenberg P.D., Riddell S.R., Science, Vol.285 (5427): 546-51 (1999)) scheme is to overcome this potential problem.

Being used for cells transfected and can not reflecting the cell of carrying out function in the APC body of selecting should also be noted that if can obtain misleading result.The cell (it is also referred to as " specialty " APC) that typically uses B cell-line is as the transfection acceptor.In the practice in this field, can accept the application of the B cell of such transfection.And, had been noted that the good association between the result in the people-B cells in vitro data of using transfection and the body that uses the HLA transgenic mice, as described in more detail.

Detect htl response

In preferred embodiments, optimized the immune response that vaccine constructs induces the II class to limit.A kind of method that evaluation comprises the multi-epitope constructs of II class epi-position is to use the HLA-DR transgenic mice.Several seminar have produced and have characterized HLA-DR transgenic mice (Taneja V., David C.S., Immunol Rev, Vol.169:67-79 (1999)).

Also have replacement scheme, it depends on the cross reactivity between the specific MHC molecule that some people MHC molecule and laboratory animal express.People such as Bertoni (Bertoni etc., J Immunol, Vol.161 (8): 4447-55 (1998)) have been noted that the considerable cross reactivity between some PATR molecule that can confirm some super type of HLA I class and chimpanzee expression.II quasi-molecule (Geluk etc., J Exp Med, Vol.177 (4): 979-87 (1993)) and I quasi-molecule (Dzuris, etc., J.Immunol., July 1999) people of level and the cross reactivity between the macaque have also been had been noted that.At last, it is further noted that motif that the super type of people HLA B7 identifies basically with mouse I class L ^dIdentical (Rammensee etc., Immunogenetics, Vol.41 (4): the 178-228 (1995)) that identifies.About the epi-position of the HLA DR restriction in the test mouse, Wall etc. (Wall etc., J.Immunol., 152:4526-36 (1994)) have confirmed DRl and IA ^bMotif have similitude.We have raised our transgenic mice routinely, to utilize this lucky similitude.And we are also verified, and our most of Toplink are in conjunction with IA ^bSo we use these mouse to study CTL and HTL immunogenicity.

The immune response of detection and quantitative clinical sample

A key element estimating the vaccine performance is to estimate immunoreactive ability in its inductor.This area is often used and is known at immunogene with at the CTL of common Memorability antigen and the analysis of htl response.The experiment of adopting comprises chromium release, lymphokine secretion and lymphocytic hyperplasia experiment.

More responsive technology, for example ELISPOT experiment, intracellular cytokine dyeing and tetramer dyeing have been that this area is available.Estimate that these method for updating are than common CTL and responsive 10 to the 100 times of (Murali-Krishna etc. of HTL experiment, Immunity, Vol.8 (2): 177-87 (1998)), because traditional method only detects the subclass of the T cell of energy in-vitro multiplication, in fact only represented a part (the Ogg G.S. of memory T cell compartment, McMichael A.J., Curr Opin Immunol, Vol.10 (4): 393-6 (1998)).More specifically, under the situation of HIV, these technology have been used to detect antigen-specific ctl response of patient, and the technology before it uses can not detect (Ogg etc., Science, Vol.279 (5359): 2103-6 (1998); Gray etc., J Immunol, Vol.162 (3): 1780-8 (1999); Ogg etc., J Virol, Vol.73 (11): 9153-60 (1999); Kalams etc., J Virol, Vol.73 (8): 6721-8 (1999); Larsson etc., AIDS, Vol.13 (7): 767-77 (1999); Corne etc., J Acquir Immune Defic SyndrHum Retrovirol, Vol.20 (5): 442-7 (1999)).

Except the only a few exception, be difficult to confirm the direct activity (Ogg G.S., McMichael A.J., Curr Opin Immunol, Vol.10 (4): 393-6 (1998)) of new isolated cells by traditional experiment.But the hypersensitivity of the technology of renewal has made the researcher can detect from the people of infection or reaction (Murali-Krishna etc., Immunity, Vol.8 (2): the 177-87 (1998) of the new isolated cells of laboratory animal; Ogg G.S., McMichael A.J., Curr Opin Immunol, Vol.10 (4): 393-6 (1998)).The availability of the experiment of these sensitivities (it does not rely on external stimulation step again) has greatly promoted the research of the CTL function in natural infection and the cancer.On the contrary, the ultimate experiment that is used as the validity of judgment experiment vaccine is carried out (Ogg G.S., McMichael A.J., Curr Opin Immunol, Vol.10 (4): 393-6 (1998)) with one or more external stimulation step more usually.In fact, except only a few exception (Hanke etc., Vaccine, Vol.16 (4): 426-35 (1998)), be difficult to confirm to be used to cause that immunity that the experimental vaccine of ctl response carries out can cause the CD8+T cell effect of the new I class-restriction that separates.The application of sensitive experiment (for example ELISPOT or original position IFN γ ELISA) with the combination of stimulation step again, to reach maximum susceptibility; The MHC tetramer also is used for this purpose.

People such as Altman have described the MHC tetramer first in 1996.They have produced soluble HLA-A2 I quasi-molecule, and it folds with the specific peptide of HIV-that contains the CTL epi-position, and complexing becomes to have the fluorescently-labeled tetramer.They are used for the T cell mass that can discern described epi-position (Ogg G.S., McMichael A.J., CurrOpin Immunol, Vol.10 (4): 393-6 (1998)) of mark from the individuality of HIV-infection.By these cells of flow cytometry standard measure, provide frequency measurement then to the T cell of this epitope specificity.This technology has been popularized (Ogg G.S., MeMichael A.J., Curr Opin Immunol, Vol.10 (4): 393-6 (1998) very much in HIV research and other infectious diseases; Ogg etc., Science, Vol.279 (5359): 2103-6 (1998); Gray etc., J Immunol, Vol.162 (3): 1780-8 (1999); Ogg etc., J Virol, Vol.73 (11): 9153-60 (1999); Kalams etc., J Virol, Vol.73 (8): 6721-8 (1999)).But the HLA polymorphism can limit the general applicability of this technology, because tetramer technology depends on definite HLA/ peptide combination.But verified, under the background of the different members of A2, A3 and the super type of B7, peptide-specific CTL system can discern multiple peptide, comprises peptide (Threlkeld etc., J Immunol, Vol.159 (4): 1648-57 (1997) that HIV-derives; Bertoni etc., J Clin Invest, Vol.100 (3): 503-13 (1997)).Comprehensive these conclusions can confirm, the TXi Baoshouti (TCR) of given MHC/ peptide combination has the identical peptide from the different MHC molecular presentations of identical super type can detected affinity.

Inducing under the relevant situation of the main and lasting anamnestic reaction of the effect of preventative vaccine, stimulation test can be to monitor vaccine-induced immunoreactive optimum and responsive method again.On the contrary, under the situation of therapeutic vaccine, active main immunology association is inducing of effector T cell function, the most suitably detects by basic experiment.Thereby the immunologic surveillance that is applied as efficacy of vaccines of sensitive experiment provides only method of testing.

The antigenicity of multi-epitope constructs in the people APC of transfection

Carrying out antigenicity tests the epi-position in appraiser's cell to process and present.Use can with the multi-epitope nucleic acid vaccine effectively the episome carrier of transfection people target cell carry out such analysis.

For example, with the transfection of HIV-1epigene vaccine 221A2K ^bTarget cell.221A2K ^bTarget cell can be expressed A2K ^bGene, it is expressed in the HLA transgenic mice, but does not express endogenous I class (Shimizu Y, DeMars R, J Immunol, Vol.142 (9): 3320-8 (1989)).Having tested these cells transfected is the ability of antigen-presenting to CTL, and this CTL system has specificity from the HLA transgenic mice and to the CTL epi-position that various HIV-derive.In order to proofread and correct the antigen sensitivity difference that different CTL are, the cell that uses untransfected has carried out the peptide dose titration abreast as APC.Representational data as shown in Figure 8.Under the situation of the specific CTL of HIV Pol 498-, the target cell of transfection is induced the IFN γ that has discharged 378pg/ml.The inspection of peptide dose response shows that the IFN γ that reaches similar level discharges needs outer seedbed to add the 48ng/ml peptide.These results show that cells transfected has been presented more substantial relatively Pol 498 epi-positions, are equivalent to the peptide that the 48ng/ml external source adds.

Table 5.HIV-1 mini gene in people's cell of transfection antigenicity and the contrast between the immunogenicity in the HLA transgenic mice
					Epi-position	Antigenicity		Immunogenicity
The peptide equivalent 1)	?n 2)	The % reaction 3)	Magnitude 4)
				??HIV?Pol?498		??30.5	?(6)	??95％	??46.7
??HIV?EnV?134	??6.2	?(3)	??62％		??16.1
				??HIV?Nef??221		??2.1	?(5)	??82％	??3.8
??HIV?Gag?271	??＜0.2	?(6)	??31％		??4
				1) ng/ml; 2) number of independent experiment; 3) % of the CTL culture of generation positive findings; 4) lytic unit

By contrast, use CTL to be checked through the IFN γ that is lower than 25pg/ml to Gag 271 epitope specificities.Use the control peptide titration of the target cell of untransfected to show, this negative findings can not be owing to the relatively poor susceptibility of the specific CTL system that uses because can detect be low to moderate 0.2pg/ml " peptide equivalent " (PE).As if thereby Gag 271 epi-positions can not be processed effectively and present in the target cell of HIV-1 transfection.Use " peptide equivalent " figure quantitatively approximate as working (machining) efficiency, the target cell that can estimate transfection has been presented and has reduced by 200 times Gag 271 than Pol 498 epi-positions at least.

Table 5 has been listed the result of each independent detection that is included in 4 different epi-positions among the HIV-FT.The scope of the amount of each epi-position that the HIV-FT cells transfected generates from the 30.5PE of Pol 498 to be low to moderate Gag 271 less than 0.2PE.2 epi-position Env 134 and Nef 221 are relevant with 6.1 and the medium value of 2.1PE respectively.

Then immunogenicity value in the body of observed each epi-position after these results and the immunity of HIV-FT construct is associated.As the prediction, Pol 498 epi-positions also be have most immunogenic.Env 134 and Nef 221 epi-positions (to having observed medium immunogenicity in its body) external also be by the processing of people's cell mid-efficiency of transfection ground.At last, Gag 271 (it is not observed detectable external processing) is also relevant with immunogenicity in the body of suboptimum aspect frequency and magnitude.

These data have several important hints.At first, they show that the different epi-positions that are included in the given construct can and present with differentiated efficient processing.Secondly, they show that the amount of finished epi-position of immunogenicity and generation is proportional.At last, these results provide important checking for the application of transgenic mice in optimizing the human polyepitope vaccines.

III. the preparation of multi-epitope constructs

As described in application PCT/US00/27766 of PCT for example or the PCT/US00/19774, the epi-position that is used for being included in multi-epitope constructs is typically carried HLA I class or II class binding motif.Can be according to for example Ishioka etc., J.Immunol. (1999) 162 (7): the described method of 3915-3925, preparation multi-epitope constructs.

A plurality of HLA II classes or I class epi-position in the multi-epitope constructs can be derived from identical antigen or different antigen.For example, multi-epitope constructs can contain one or more HLA epi-positions, and this epi-position can be derived from 2 of identical virus not synantigens or be derived from 2 of different virus not synantigens.The epi-position that is used for being included in multi-epitope constructs can be selected by those skilled in the art, for example, selects to contain the epi-position of HLA allelomorph-specific motif or hyper-base preface by using a computer.Multi-epitope constructs of the present invention one or more cross reactivity combination or general widely HLA II class epi-positions of can also encoding, for example, PADRE ^_(Epimmune, San Diego CA) (are documented in for example U.S. Patent number 5,736,142 to epi-position; In 6,413,935 and 5,679,640) or PADRE ^_Family molecule.

General HLA II class epi-position can advantageously make up other HLA I class and II class epi-position, with the number of increase by the cell of given antigenic activation, and provides HLA-allelic wider colony's coverage of reaction.Therefore, multi-epitope constructs of the present invention can comprise the specific HLA epi-position of antigen, general HLA II class epi-position or the combination of specific HLA epi-position and the HLA II class epi-position that at least one is general.

The length of HLA I class epi-position generally is to be lower than about 15 residues, be preferably 13 residues or shorter length, be preferably about 8 to about 13 amino acid whose length, more preferably be about 8 to about 11 (for example 8,9,10 or 11) amino acid whose length, be most preferably about 9 amino acid whose length.HLA II class epi-position generally is the length that is lower than about 50 residues, often by about 6 to about 30 residues, more frequent about 12 to 25, often form, and can code length be about 7 to about 23, preferably about 7 to about 17, more preferably about 11 to about 15 (for example 11,12,13,14 or 15) and most preferably about 13 amino acid whose epitope peptides by about 15 to 20 residues.HLA I class or II class epi-position can be derived from any desirable target antigen.Target antigen can be viral antigen, surface receptor, tumour antigen, oncogene, enzyme or any pathogene, need produce immunoreactive cell or molecule to it.Can select epi-position in conjunction with the allelic ability of one or more HLA based on them.Also can be included in the multi-epitope constructs as herein described with the similar epi-position of native sequences.Similar peptide like this is documented in, and for example, PCT applies for PCT/US97/03778, and the common unsettled U.S.S.N.09/260 that PCT/US00/19774 and on March 1st, 1999 submit to is in 714.

Based on the method for the sept between optimization epi-position configuration as herein described and the epi-position, those skilled in the art can be included in any HLA epi-position in the multi-epitope constructs as herein described.Fig. 2,3,9,17,18A-18N, 27A, 28A, 29A and table 13,14,18 and 19 have been described the exemplary multi-epitope constructs that uses the listed epi-position of Figure 19 A-19E.Exemplary construct also is presented at Figure 20 B, 20D, 20E, and 20F (epi-position is listed in Figure 20 A); Figure 21 B, 21D, and 21E (epi-position is listed in Figure 21 A); Figure 22 B, 22D, and 22E (epi-position is listed in 22A); Figure 23 C; In Figure 24 B and 24C (epi-position is listed in Figure 24 A).Multi-epitope constructs can comprise 5 or more or 6,7,8,9,10,11,12,13,14,15,20,25 or 30 or more a plurality of in the epi-position described in Figure 19 A-19E, 20A, 21A, 22A and the 24A.Can use method optimization as herein described to comprise the multi-epitope constructs of any combination of these epi-positions, also can optimize sept.

Can use method well known in the art to produce multi-epitope constructs.For example, can synthesize and connect and compose the polypeptide of multi-epitope constructs.Typically, use recombinant DNA technology to make up multi-epitope constructs.

IV. the expression vector of multi-epitope constructs and structure

Multi-epitope constructs of the present invention typically is expression vector, and it contains the nucleic acid of the multi-epitope polypeptide of encoding.The structure of such expression vector for example is documented among the PCT/US99/10646.This expression vector contains at least 1 energy is expressed transcriptional units (its code nucleic acid) in suitable biological cell element, so antigen is expressed and target appropriate H LA molecule.For example, in order to use to the people, the promoter element that can work in people's cell is integrated in the expression vector.

In preferred embodiments, the present invention uses the routine techniques in genetic recombination field.The basic article that discloses the conventional method of the present invention's use comprises Sambrook etc., MolecularCloning, A Laboratory Manual (the 2nd edition, 1989); Kriegler, GeneTransfer and Expression:A Laboratory Manual (1990); With CurrentProtocols in Molecular Biology (Ausubel etc. compile 1994); Oligonucleotide Synthesis:A Practical Approach (Gait compiles 1984); Kuijpers, Nucleic Acid Research 18 (17): 5197 (1994); Dueholm, J.Org.Chem.59:5767-5773 (1994); Methods in MolecularBiology, volume 20 (Agrawal compiles); And Tijssen, LaboratoryTechniques in Biochemistry and MolecularBiology--Hybridization with Nucleic Acid Probes, for example, Part I, chapter 2 " Overview of principles of hybridization and thestrategy of nucleicacid probe assays " (1993)).

Fitted in the construct according to will the encode nucleic acid of epi-position of standard technique.Usually, use Oligonucleolide primers to separate or can the encode nucleotide sequence of multi-epitope polypeptide of chemosynthesis by amplification technique.When suitable, can also use the recombinant clone technology.The oligonucleotide sequence of selecting can increase (when using PCR to assemble construct) or coding (when using synthetic oligonucleotides to assemble construct) target epi-position.

The amplification technique of use primer typically is used for from DNA or RNA increases and the sequence of the epi-position that the separation energy coding is selected (is seen United States Patent (USP) 4,683,195 and 4,683,202; PCRProtocols:A Guide to Methods and Applications (Innis etc. compile 1990)).Can use polymerase chain reaction (PCR) (PCR) and ligase chain reaction methods such as (LCR) to come directly from mRNA, cDNA, genomic library or cDNA storehouse amplification epi-position nucleotide sequence.The restriction endonuclease site can be inserted in the primer.Multi-epitope constructs by PCR reaction amplification can be from the Ago-Gel purifying, and the clone advances in the suitable carriers.

Synthetic oligonucleotides also can be used to make up multi-epitope constructs.This method is to use a series of overlapping oligonucleotides to finish, and they have represented the sense strand and the nonsense strand of gene.Then these dna fragmentations are annealed, connected and clone.According to Beaucage ﹠amp; Caruthers, the solid phase phosphoramidite three ester methods that Tetrahedron Letts.22:1859-1862 (1981) at first describes, use as Van Devanter etc., the described automatic synthesizer of Nucleic Acids Res.12:6159-6168 (1984), the oligonucleotides that can not obtain on can chemosynthesis market.By as Pearson ﹠amp; Reanier, described natural acrylamide gel electrophoresis of J.Chrom.255:137-149 (1983) or anion exchange HPLC come purification of oligonucleotides.

Typically the epi-position subclone with multi-epitope constructs advances in the expression vector, and the latter is contained strong promoter and other regulating and controlling sequence, for example enhancer and the polyadenylation site that guidance is transcribed.Suitable promotor is well known in the art, and is documented in for example Sambrook etc. and Ausubel etc.The eukaryotic expression system that is used for mammalian cell is well known in the art, and can obtain from the market.Such promoter element for example comprises, cytomegalovirus (CMV), Rous sarcoma virus long terminal repeat (RSV LTR) and simian virus 40 (SV40).

Expression vector typically contains transcriptional units or expression cassette, and it contains expresses all required other elements of multi-epitope constructs in host cell.Thereby typical expression cassette contains the promotor that is operably connected to multi-epitope constructs and the required signal of polyadenylation effectively of transcript.Other element of box can comprise enhancer and intron, and it has function splicing donor and acceptor site.

Except promoter sequence, expression cassette can also contain the transcription termination region that is positioned at the structural gene downstream, so that effective termination to be provided.The terminator can obtain from the gene identical with promoter sequence, perhaps also can obtain from different genes.

The particular expression carrier of cell is not vital especially to be used for transporting hereditary information into.Can use and be used for any conventional carrier of expressing at eukaryotic.Typically use the expression vector contain from the regulating element of eucaryon virus as carrier for expression of eukaryon, for example, SV40 carrier, CMV carrier, papillomatosis poisonous carrier and be derived from the carrier of Epstein-Barr virus.

Multi-epitope constructs of the present invention can be expressed from variety carrier, comprises plasmid vector and viral vectors or bacteria carrier.The example of virus expression carrier comprises the virus host of attenuation, for example cowpox or fowl pox.As an example of this scheme, express the nucleotide sequence of the peptide of the present invention of encoding as carrier with vaccinia virus.After the host of tumour was carried in importing, the vaccinia virus of reorganization can be expressed immunogenic peptide, caused host's CTL and/or htl response thus.The cowpox carrier and the method that are used for immunization protocol are documented in for example U.S. Patent number 4,722,848.

Those skilled in the art can understand multiple other carrier of using of being used for the treatment of property or immunity, for example adenovirus and adeno-associated virus vector, retroviral vector, the non-virus carrier anthrax toxin carrier etc. of BCG (tuberculosis vaccines), typhoid bacillus carrier, detoxification for example.

Immunogenicity and the antigenicity of having estimated multi-epitope constructs as described herein.

The target sequence

Expression vector of the present invention can be encoded, and one or more are operably connected to the MHC epi-position of MHC target sequence, are called " target nucleic acid " or " target sequence " here.Compare with the antigen that conveying is independent, the application of MHC target sequence can strengthen the immune response to antigen, it is directed to site and the transporte to cells surface that MHC divides sub-assemblies with peptide epitopes, increases the available number that is used for the MHC molecule-peptide epitopes compound of combination and activated T cell thus.

I class MHC target sequence can be used for the present invention, for example, I class MHC epitope peptide can be navigated to those sequences (see, for example, Rammensee etc., Immunogenetics 41:178-228 (1995)) of cytosol approach or endoplasmic reticulum.For example, the cytosol approach can be processed the endogenous antigen at cell inner expression.Though do not wish to be subjected to the constraint of any concrete theory, think that cytosol albumen is subjected to the degraded of proteasome endopeptidase activity at least in part, and be transported to endoplasmic reticulum by TAP molecule (the transhipment thing relevant) with processing.In endoplasmic reticulum, antigen can be in conjunction with I class MHC molecule.The endoplasmic reticulum burst is walked around cytosol processing approach, and directly endogenous antigen is navigated to endoplasmic reticulum, and proteolysis is a fragments of peptides there.Such I class MHC target sequence is well known in the art, comprises for example burst, for example from those of Ig κ, tissue plasminogen activator or insulin.Preferred signal peptide is a people Ig κ chain-ordering.The endoplasmic reticulum burst can also be used for II class MHC epitope mapping is arrived endoplasmic reticulum, i.e. the site of I class MHC molecule assembling.II class MHC target sequence also can be used for the present invention, for example peptide can be navigated to those of endocytic pathway.These target sequences typically instruct born of the same parents' exoantigen to enter endocytic pathway, and this causes antigen to be transferred to the lysosome compartment, and cut into by proteolysis can be in conjunction with the antigenic peptides of II class MHC molecule for antigen there.In the normal process of exogenous antigen, II class MHC epi-position can be guided to the pinosome (endosome) of endocytic pathway and/or subsequently to the sequence of lysosome (II class MHC epi-position can in conjunction with II class MHC molecule) there be II class MHC target sequence.For example, one group of useful in the present invention II class MHC target sequence is a lysosome target sequence, and it can navigate to lysosome with polypeptide.Because typically in conjunction with the antigenic peptides of the proteolysis processing that is derived from the endocytosis antigen in the lysosome, lysosome target sequence can be used as II class MHC target sequence and works II class MHC molecule.Lysosome target sequence is well known in the art, is included in the sequence of finding among lysosomal protein LAMP-1 and the LAMP-2, and of (U.S. Patent numbers 5,633,234 that on May 27th, 1997 authorized) such as August, it is incorporated by reference here.

Other lysosomal protein that contains lysosome target sequence comprises HLA-DM.HLA-DM is endosome/lysosomal albumen, and it is promoting that antigenic peptides works in the combination of II class MHC molecule.Because it is arranged in lysosome, HLA-DM has the lysosome target sequence (it is incorporated by reference for Copier etc., J.Immunol.157:1017-1027 (1996)) that can play the molecular targeted sequence effect of II class MHC here.

Resident lysosomal protein HLA-DO also can play lysosome target sequence.Different with the resident lysosomal protein LAMP-1 of the motif (it navigates to lysosome with albumen) of the specific Tyr of containing that can encode with HLA-DM, HLA-DO is by navigating to lysosome (Liljedahl etc. with combining of HLA-DM, EMBO J 15:4817-4824 (1996)), it is incorporated by reference here.Therefore, can cause combine with HLA-DM and subsequently HLA-DO be displaced to lysosomal HLA-DO sequence and can be used as II class MHC target sequence.Similarly, the mouse homologue H2-DO of HLA-DO can be used to provide II class MHC target sequence.II class MHC epi-position can be fused to HLA-DO or H2-DO, and is positioned to lysosome.

In another embodiment, the cytoplasm domain of B-cell receptor subunit I g-α and Ig-β can mediate the internalization of antigen and improve the efficient of antigen presentation, and as Bonnerot etc., Immunity 3:335-347 (1995) is described.Therefore, the cytoplasm domain of Ig-α and Ig-β albumen can play II class MHC target sequence, and it can be with II class MHC epitope mapping to endocytic pathway, to process and in conjunction with II class MHC molecule.

Another example that can instruct II class MHC epi-position to enter the II class MHC target sequence of endocytic pathway is the sequence that can instruct the polypeptide secretion, and wherein this polypeptide can enter the endosome approach.These can instruct II class MHC target sequences of polypeptide secretion can imitate normal approach, external source, the outer antigen of born of the same parents is processed into by this approach can be in conjunction with the peptide of II class MHC molecule.Can instruct polypeptide to pass endoplasmic reticulum and any burst of finally being secreted can be used as II class MHC target sequence, as long as can enter endosome/lysosomal approach and be sheared can be in conjunction with the peptide of II class MHC molecule for the polypeptide of secreting.

In another embodiment, Ii albumen can be in conjunction with the II class MHC molecule in the endoplasmic reticulum, and it can prevent that peptide in the endoplasmic reticulum is in conjunction with II class MHC molecule there.Therefore, the fusion physical efficiency of II class MHC epi-position and Ii albumen arrives endoplasmic reticulum and II class MHC molecule with II class MHC epitope mapping.For example, can remove the CLIP sequence of Ii albumen, and replace with II class MHC epitope sequences, make II class MHC epi-position can navigate to endoplasmic reticulum, epi-position can be in conjunction with II class MHC molecule there.

In some situation, antigen self can be used as II class MHC or I class target sequence, and can merge with general II class MHC epi-position, with immune response stimulating.Although normally as processing and present with the compound of I class MHC molecule, the cytoplasmic protein of long life (for example influenza stromatin) can enter II class MHC molecule processing approach to the kytoplasm viral antigen, as Gu é guen ﹠amp; Long, Proc.Natl.Acad.Sci.USA 93:14692-14697 (1996) is described.Therefore, the cytoplasmic protein of long life can play I class MHC and/or II class MHC target sequence.For example, can the encode expression vector of the influenza stromatin that merges mutually with general II class MHC epi-position can be advantageously used in influenza antigens and general II class MHC epitope mapping to I class MHC and II class MHC approach, to stimulate the immune response to influenza.

Other example that can play the antigen of II class MHC target sequence effect comprises the polypeptide that can spontaneously form particle.This polypeptide is to come out from the emiocytosis that can produce them, and spontaneously forms particle, and it is taken in by endocytosis (for example receptor mediated endocytosis) by antigen presenting cell, perhaps eats by phagocytosis.Enter endosome/lysosomal approach after, particle is cut into antigenic peptides by proteolysis.

A kind of such polypeptide that can spontaneously form particle is HBV surface antigen (HBV-S), as Diminsky etc., and Vaccine 15:637-647 (1997) or Le Borgne etc., Virology240:304-315 (1998) is described.Other polypeptide that can spontaneously form particle is the HBV cAg, and as Kuhr_ber etc., International Immunol.9:1203-1212 (1997) is described.Other polypeptide that can spontaneously form particle is a yeast Ty albumen, and as Weber etc., Vaccine 13:831-834 (1995) is described.For example, the expression vector that contains the HBV-S antigen that merges mutually with general II class MHC epi-position can be advantageously used in HBV-S antigen and general II class MHC epitope mapping to II class MHC approach, to stimulate the immune response at HBV.

Use in the body

The present invention also provides by use the method that expression vector of the present invention comes immune response stimulating to individuality.Using expression vector of the present invention, to come immune response stimulating be favourable, because expression vector of the present invention can be with MHC epi-position target to the MHC molecule, thereby increases the CTL of antigenic activation of expression vector codes and the number of HTL.

At first, expression vector of the present invention is to screen in mouse, to detect the expression vector that has optimum activity in stimulating desirable immune response.Therefore carry out initial research, when feasible, used the little musculus cdna of MHC target sequence.The method that detects the activity of expression vector of the present invention is well known in the art, comprises for example detecting the T cell activation ³The absorption of H-thymidine and can detect the CTL activity ⁵¹Cr discharges, as described in following example II and III.Carried out with at those the similar experiments described in the EXAMPLE IV, the expression vector that has the immune response stimulating activity with detection.Also in the people, tested and had active expression vector.Have active expression vector in order to prevent the secondary immune response of potential mouse sequence to coding, to have modified, make I class MHC or II class MHC target sequence source from people's gene.For example, expression vector of the present invention has been inserted in the replacement of zone similarity of people's homologue that will contain the gene of various I class MHC or II class MHC target sequence.In human body, tested the activity of the expression vector immune response stimulating that contains people I class MHC or II class MHC target sequence, for example those described in the example I below.

The invention still further relates to pharmaceutical composition, it contains pharmaceutically acceptable carrier and expression vector of the present invention.Pharmaceutically acceptable carrier is well known in the art, comprise water-based or nonaqueous solution, suspension and emulsion, comprise physiological buffer salt solution, alcohol/aqueous solution or other solvent or medium, for example for example olive oil or injectable organic ester of ethylene glycol, glycerine, oil.

Pharmaceutically acceptable carrier can contain physiologically acceptable compound, and it for example can the stably express carrier or strengthens the absorption of expression vector.Physiologically acceptable compound so for example comprises, carbohydrate, for example glucose, sucrose or glucan, antioxidant is ascorbic acid or glutathione for example, chelating agent, low-molecular-weight polypeptide, antimicrobial, inert gas or other stabilizing agent or excipient.Expression vector in addition can be compound with other component, for example peptide, polypeptide and carbohydrate.Expression vector also can with can use (for example using the vaccine rifle) and give individual particle or pearl compound.According to the approach of for example using expression vector, those skilled in the art can select pharmaceutically acceptable carrier, comprises physiologically acceptable compound.

The invention still further relates to the method that the pharmaceutical composition that comprises expression vector of the present invention comes immune response stimulating of using.Expression vector is to use by method well known in the art, as (Ann.Rev.Immunol.15:617-648 (1997)) such as Donnelly; Felgner etc. (U.S. Patent number 5,580,859 that on December 3rd, 1996 authorized); Felgner ((U.S. Patent number 5,703,055 that on December 30th, 1997 authorized); ((U.S. Patent number 5,679,647 that on October 21st, 1997 authorized) is described with Carson etc.In one embodiment, multi-epitope constructs is to use as naked nucleic acid.

Can use the pharmaceutical composition that contains expression vector of the present invention by different approach comes in the immune response of object moderate stimulation, for example comprise, per os ground, ground in the vagina, rectum ground or stomach other places, ground in the intravenous ground, muscle, hypodermically, in ground, the pond ground in ground, the peritonaeum in ground, the capsule in the socket of the eye for example, or, use for example skin patch or transdermal iontophoretic therapy by Transdermal absorption passive or promotion.And said composition can or be used partly by injection, intubate, and the latter can be passive, for example passes through directly to use ointment or powder, or initiatively, for example use nasal spray or inhalant.Expression vector can also be used as local spray, and in this case, a kind of component in the composition is suitable propellant.Pharmaceutical composition also can be integrated in liposome, microballoon or other the polymer substrate when needed, as Felgner etc., U.S. Patent number 5,703,055; Gregoriadis, LiposomeTechnology, I to III volume (the 2nd revised edition, 1993) is described.For example, the liposome of being made up of phosphatide or other lipid is nontoxic, physiologically acceptable and metabolizable carrier, its preparation with use all relative simple.

Expression vector of the present invention can be transported to the systemic intercellular space of animal, as Felgner etc., and U.S. Patent number 5,580,859 and 5,703,055 is described.It is especially effective medication that expression vector of the present invention is transported to muscle, comprises intracutaneous and subcutaneous injection and cutaneous penetration.Cutaneous penetration (for example passing through ionotherapy) also is the effective ways that expression vector of the present invention are transported to muscle.Can also adopt the epidermis administration of expression vector of the present invention.The epidermis administration comprises the outermost layer that physically or chemically stimulates epidermis, to cause the immune response (Carson etc., U.S. Patent number 5,679,647) at this stimulation.

Use expression vector of the present invention and come other effective ways of immune response stimulating to comprise mucosa delivery, as Carson etc., U.S. Patent number 5,679,647 is described.For mucosa delivery, the most effective medication comprises that intranasal administration contains the suitable aerosol of expression vector and pharmaceutical composition.Suppository and topical formulations also can be transported to expression vector mucosal tissues phallic, vagina and sites eyes effectively.In addition, expression vector can be compound with particle, and use by the vaccine rifle.

The dosage of using depends on medication, usually at about 0.1 μ g between about 200 μ g.For example, dosage can be from about 0.05 μ g/kg to about 50mg/kg, particularly about 0.005-5mg/kg.Can determine effective dose, for example use immune response behind the expression vector by detection.For example,, comprise ELISA or other immunization experiment, can detect generation by the antibody of the II class MHC epi-position of expression vector codes or I class MHC epitope specificity by method well known in the art.In addition, by method well known in the art, comprise and for example can detect the T cell activation ³The absorption of H-thymidine and can detect the CTL activity ⁵¹Cr discharges (example II of face and III as follows), can detect the activation or the ctl response of t helper cell.

For preventative or curative purpose, the pharmaceutical composition that contains expression vector of the present invention can be administered to mammal, particularly the people.Use the example of the disease that expression vector of the present invention can treat or prevent to comprise infection and prostate cancer, kidney, cervical carcinoma, lymphoma, condyloma acuminatum and the acquired immune deficiency syndrome (AIDS) (AIDS) of HBV, HCV, HIV and CMV.

In therapeutic was used, expression vector of the present invention was to be administered to suffer from cancer, autoimmunity disease or infected viral individuality.Be in latent period or acute stage disease the patient can treat with expression vector of the present invention (comprising those that can express all general purpose I I class MHC epi-positions), can separate with other treatment when appropriate or combine application.

In therapeutic and prophylactic use, use the pharmaceutical composition that contains expression vector of the present invention to the patient, present in an amount at least sufficient to cause effective immune response to antigen, perhaps improve the sign or the symptom of disease.The amount that being enough to of using improved the expression vector of the sign of disease or symptom is defined as treatment and goes up effective dosage.Be enough to reach treatment and go up that the amount of the expression vector of effective dose depends on the pharmaceutical composition that comprises expression vector of the present invention, administering mode, situation and the order of severity, patient's body weight and general health and prescription doctor's the judgement of the disease that will treat.

Embodiment

The following examples are used to explain rather than the invention of requirement for restriction protection.Be understood that, embodiment as herein described and embodiment only are used for task of explanation, those skilled in the art can know various improvement or variation on its basis, and they are also included within the scope of the application's spirit and scope and appended claim.

Embodiment 1-9 provides the immunogenicity of checking multi-epitope constructs and the example of antigenic experiment.

The experiment of embodiment 1 antigenicity

Can generate the peptide of high affinity-specific CTL system from the splenocyte of using the transgenic mice that DNA, peptide/IFA or lipopeptid cause.Tout court, stimulate splenocyte with 0.1 μ g/ml peptide and LPS mother cell (blasts) from transgenic mice.Stimulate back 10 days first, and after this use LPS mother cell irritation cell more weekly, middle with 0.1 μ g/ml peptide stimulation 1 hour.Stimulated test CTL system in above-mentioned original position IFN γ ELISA back 5 days.Perhaps, can use and be derived from the CTL system that has for example infected the target pathogene or had the patient of target disease (for example cancer).According to the special knowledge of this area, can produce the specific CTL system that can not obtain from the PBMC of normal donor from transgenic mice or patient.

For the CTL system that is derived from transgenic mice, the target cell that can be used for these experiments is to use A2.1/K ^b, A11/K ^b, A1/K ^bOr B7/K ^bThe Jurkat of transfection or .221 cell.These all cell-lines can obtain at present (Epimmune Inc., San Diego, CA).In the situation of people CTL system, used .221 cell with suitable people HLA allelomorph transfection.We have the .221 cell with A2 and A1 transfection now, are producing A11, A24 and B7 transfectant.In an alternate embodiment, if unforeseen problem occurred aspect target cell, what then LPS mother cell and EBV-are transformed is to be respectively applied for mouse and people CTL system.

For test antigen, with the CTL and 10 of serial dilution ⁵Individual target cell hatches together, and having used scope in hatching is 1 to 10 ^-6A plurality of peptide concentrations of μ g/ml.In addition, also CTL is hatched with the target cell of free radical because of carrier (containing the target multi-epitope constructs) transfection.Episome carrier (episomal vector) is known in the art.

Following relative quantity of quantitatively in the APC of multi-epitope nucleic acid-transfection, processing the peptide that produces naturally.Write down the amount that CTL ties up to the IFN γ that produces behind the target cell of identification transfection.The calibration curve that produces when hatching abreast with the peptide of concentration known with identical CTL system obtains producing the amount of the required synthetic peptide of the IFN γ of same amount by interpolation method (interpolated).

Embodiment 2 mouse, immunization and cell culture

Having described the deriving of transgenic mice of using in this research is HLA-A2.1/K ^b(Vitiello etc., J Exp Med, Vol.173 (4): 1007-15 (1991)) and A11/K ^b(Alexander etc., J Immunol, Vol.159 (10): 4753-61 (1997).HLA B7K ^bTransgenic mice be can autotrophy obtain (Epimmune Inc., San Diego, CA).HLA DR2, DR3 and DR4 transgenic mice are available from C.David (Mayo Clinic).Not genetically modified H-2 ^bMouse is available from Charles River Laboratories or other commercial supplier.Carry out immunization as (Ishioka etc., J Immunol, Vol.162 (7): 3915-25 (1999)) is described.All cells is all grown in the medium of being made up of RPMI 1640 medium and HEPES (GibcoLife Technologies), and this HEPES has added 10%FBS, 4mM L-glutaminate, 50 μ M 2-ME, 0.5mM Sodium Pyruvate, 100 μ g/ml streptomycins and 100U/ml penicillin.

HLA transgenic mice and antigenicity experiment are used to test and optimize the purpose of ctl response.HLA-DR and IA ^bBetween natural cross reactivity also can be used to test htl response.This evaluation provides about the antigenicity of multi-epitope constructs and immunogenic judgement.

Embodiment 3 proliferation experiments

In order to estimate the ability of HTL epi-position induction of immunity reaction, often carry out methods of testing such as proliferation experiment.For example, use DynaBeads Mouse CD4 (L3T4) (Dynal), from spleen single-cell suspension liquid immune magnetic isolated mouse CD4T lymphocyte.Tout court, with 2 * 10 ⁷Individual splenocyte and 5.6 * 10 ⁷Individual magnetic beads was hatched 40 minutes together at 4 ℃, washed then 3 times.Use separately magnetic beads of DetachaBead Mouse CD4 (Dynal).In 96 flat hole microtiter plates, with the CD4T lymphocyte (2 * 10 that separates ⁵Cells/well) with 5 * 10 ⁵The splenocyte of (3500 rad) homology of individual irradiation is cultivated together in triplicate.The peptide of purifying is added in the hand-hole, and final concentration is 20,1,0.05 and 0 μ g/ml, cultured cell totally 4 days.Gathering in the crops precontract 14 hours, and in each hole, adding 1 μ Ci's ³H-thymidine (ICN).Use Filtermate Harvester (Packard) results hole, to Unifilter GF/B flat board (Packard).Use TopCount ^TMMicroplate scintillation counter (Packard) detects by liquid scintillation counting (LSC) ³The integration of H-thymidine.

Embodiment 4 ⁵¹The chromium release experiment

The described experiment that is used to detect the CTL activity is well known in the art.By detect from ⁵¹The target group of Cr-mark discharge ⁵¹The percentage of Cr, described experiment is the lytic activity of T cell colony (Brunner etc., Immunology, Vol.14 (2): 181-96 (1968)) quantitatively.Usually will be expressed as CTL frequency/10 from the data that the chromium release experiment obtains ⁶Cell (limiting dilution analysis, LDA; (Current Protocols in Immunology, Vol 1, John Wiley﹠amp; Sons, Inc., USA 1991 Chapter 3, Manual of Clinical LaboratoryImmunology, the 15th edition, ASM Press, 1997Section R), or the quantitative assessment (lytic unit of the not hell to pay by whole CTL activity; The LU experiment).In LU experiment, will ⁵¹The E of the standard that obtains in the Cr-release experiment: T ratio and cytotoxicity percentage data and curves have changed into lytic unit (LU)/10 ⁶The effector molecules cell, 1LU is defined as and reaches 30% the required lytic activity (Wunderlick of target cell cracking, J., Shearer, G., and Livingston, A.In:J.Coligan, A.Kruisbeek, D.Margulies, E.Shevach, and W.Strober (volume), Current Protocols in Immunology, Vol 1, " Assays for T cell function:induction and measurement ofcytotoxic Tlymphocyte activity. " John Wiley ﹠amp; Sons, Inc., USA, p.3.11.18).Lu calculates can quantitative reaction, thereby easily contrasts different experimental values.

Embodiment 5 original position IFN γ ELISA

Developed original position IFN γ ELISA experiment, and the splenocyte that stimulates again with peptide of new separation has been carried out optimizing (see, for example, McKinney etc., J.Immunol.Meth.237 (1-2): 105-117 (2000)).This experiment is based on the ELISPOT experiment, but has been to use soluble chromagen, makes it can easily be applicable to high throughput analysis.In experiment first and that stimulate again, this technology all than traditional supernatant ELISA or ⁵¹The Cr release experiment is more responsive, because observed among the ELISA in position to be reflected in these other experiment be can not be detected.Based on each cell, the susceptibility of original position ELISA is about 1 IFN γ secretory cell/10 ⁴The cell of individual coating.

(rat anti-mouse IFN α MAb, clone R4-6A2 Pharmingen) cover 96 hole ELISA flat boards, spend the night at 4 ℃, and the 10%FBS that is used among the PBS in room temperature sealed 2 hours then with anti--IFN γ.Former generation splenocyte or CTL and peptide and 10 with serial dilution ⁵JurkatA2.1/K ^bCells/well is at 37 ℃, 5%CO ₂Under cultivated 20 hours.Next day, washed cell, (rat anti-mouse IFN γ mAb, Clone XMG1.2 Pharmingen) detect the IFN γ that secretes, and detect the amount that has been secreted into the IFN γ in the hole in sandwich ELISA to use biotinylated α-IFN γ.According to manufacturer's explanation, use the strepavidin (Zymed) and the TMB (ImmunoPure of HRP-coupling ^_TMB Substrate Kit Pierce) develops the color.On Labsystems Multiskan RC ELISA plate reader, read absorbance at 450nm.Based on the number of cell of the IFN γ (causing) of secretion 100pg,, original position IFN γ ELISA data have been estimated with secretory units (SU) in the background amount that does not have to have proofreaied and correct under the situation of peptide IFN by particular peptide.

Embodiment 6ELISPOT experiment

By detecting each cell was produced and discharged specific lymphokine (often being IFN γ) by inducing ability, the ELISPOT experiment can be quantitatively to the frequency of the T cell of given peptide specific.The hypersensitivity of ELISPOT experiment has made the researcher can detect from the people of infection or reaction (Murali-Krishna etc., Immunity, Vol.8 (2): the 177-87 (1998) of the new isolated cells of laboratory animal; Ogg etc., Science, Vol.279 (5359): 2103-6 (1998)).Carry out ELISPOT as the step of above-mentioned IFN γ ELISA and test step to the last, at this moment use ExtrAvidin-AP (Sigma, dilution in 1: 500) to replace the HRP-streptavidin.According to manufacturer's explanation, use substrate 5-BCIP (BioRad) colour developing.Use phase contrast microscope to calculate the spot number.Perhaps, use Zeiss KS ELISPOT reader to calculate the spot number.In this case, use BCIP/NBT (Zymed) substrate.

The ELISPOT experiment is used for quantitative immune response routinely.The spot number can hand computation, still, in preferred pattern, has used the KS ELISPOT reader from Zeiss, it be have the software that is designed for identification especially and calculates the spot number based on microscopical system.

The dyeing of embodiment 7 tetramers

Tetramer dyeing is the flow cytometry technology, and it can detect epi-position-specific people CD8+T-lymphocyte, and this is based on peptide epitopes/I class antigen with to the interaction between the specific T-cell receptors of epi-position.This experiment can fast quantification the people CD8+T-lymphocyte of epitope specificity in new separate blood sample.Can obtain being used for the MHC tetramer of various HIV peptides/HLA combination, for example from the NIH storage vault (Tetramer Core Facility:http: //www.miaid.nih.gov/reposit/tetramer/index.html).For mark epi-position-specific cell, with 1 * 10 of 100 μ l volumes ⁶PBMC (can obtain the form of different fluorescent dye combinations from the commercial channel with the monoclone antibody of the suitable tetramer of 5 μ g/ml+can discern people CD3 and CD8 in the dark, comprise PharMingen, San Diego, CA or BioSource, Camarillo CA) is hatched 40 minutes together.Washed cell, (Becton DickinsonImmunocytometry Systems, San Jose CA) fix with paraformaldehyde before the analysis using FACSan or FACSCalibur flow-cytometer.

Embodiment 8 is from the experiment of clinical sample

Can use kinds of experiments to estimate freezing from the specific CD8+CTL activity in patient or volunteer's the PBMC sample.ELISPOT, chromium release, original position IFN γ release, propagation and tetramer experiment all are used for estimating the reaction from various experimental models (for example those of mouse and/or primate source).

Described the experimental technique about mouse in these experiments above, these are applicable to robot system, as (Livingston etc., J Immu7101, Vol.159 (3): 1383-92 (1997); Heathcote etc., Hepatology, Vol.30 (2): 531-6 (1999); Livingston etc., J Immunol, Vol.162 (5): 3088-95 (1999)) described, those of ordinary skill in the art can expect it is further improved.Calculating to the amount of finishing the essential freezing PBMC sample of this experiment also is documented among the embodiment 14 in more detail.

Embodiment 9 transgenic animal

The dosage of use in 10-100 μ l volume is up to DNA or the peptide of 100 μ g, in preceding shin bone muscle in the muscle or at the immune transgenic mouse (HLA-A2.1/K hypodermically of root of the tail portion ^bH2 ^bHLA-A11/K ^bHLA-B7/K ^b).DNA is formulated in the salt solution, and peptide is in IFA.After 11-21 days, use CO ₂Suffocate and put to death animal, take out their spleen, as the source of the cell of vitro detection CTL function.Typically, each experimental group is used 3-6 mouse.In addition, use from the spleen of non-immune mouse source as the APC that is used for stimulating again the CTL culture.Used the male and female animal in 8-12 age in week.

Embodiment 10 induces the confirmation at the reaction of a plurality of CTL and HTL epi-position simultaneously

The structure and the test of CTL epi-position string

This embodiment provides the example of testing a plurality of CTL and HTL epi-position.For example, synthesized the epi-position string that comprises 10-12 the different CTL epi-positions under same promotor control, and be integrated into standard plasmid pcDNA 3.1 (Invitrogen, San Diego).These constructs comprise the burst of standard and general HTL epi-position, PADRE ^_Epi-position.Select every group of epi-position that colony's coverage of balance is provided.In order to help test and optimization, the epi-position that has comprised balance is presented, and verified this epi-position is immunogenic in transgenic mice and/or is antigenic in the people.

As described herein, by the program of using a computer, selected the certain order of these CTL epi-positions, connect motif to minimize the I class.If after having considered order to greatest extent, potential connection epi-position still is present in according in the construct of the present invention, and then synthetic corresponding peptide monitors the ctl response at this epi-position in the HLA transgenic mice.Usually, it is successful with enough connecting minimizing of motif.But if detected reaction at any connection epi-position, these connect epi-positions and are separated by the sept of 1-2 short residue, for example K, AK, KA, KK or A, they can with the proteolysis shearing preferences compatibility of expection previously discussed.

Because the final use of the construct of optimizing is people's vaccine, used people's codon of optimizing.Similarly, if this construct will be expressed in bacterium or S19 cell, the use that then can improve codon is to provide the expression in these systems.But in order to help the optimizing process in the HLA transgenic mice, whenever possible, just screening carefully is best people's codon to mouse also.It is very similar that the codon of people and mouse is selected.See, for example, table 21 and 22.

Can be used for the antigenicity experiment carried out abreast with body build-in test at the HLA transgenic mice with people's cell of various multi-epitope nucleic acid vaccine construct transfection.By the use of these two different experimental systems, overcome because the potential difference between the polyepitope vaccines effect that differentiated codon utilization is caused.

Typically, the antigenicity and the immunogenicity test of plasmid construction body have been carried out abreast.Carried out the body build-in test of transgenic mice for A2, A11 and B7HLA transgenic mice.Improve the scheme of setting up according to our laboratory, the every kind of plasmid of 100 μ g of having given the intramuscular injection of cardiotoxin pretreatment of mice, post-evaluation in 11 days reaction (Ishioka etc., Immunol, Vol.162 (7): 3915-25 (1999)).Experiment comprises the ELISPOT of new isolated cells, the interferon gamma of stimulated cells culture discharges and cytotoxicity chromium release experiment again.All above-mentioned technology all are well known in the art.The reaction that detects simultaneously at a plurality of epi-positions is unchallenged, because set up the transgenic mice of large group for these HLA types " self ground (in house) ".In repeatedly reading was tested, the group of 4-6 mouse was enough to detect the reaction at the different epi-positions of 6-10 kind.Typically in HLA A2 transgenic mice, test HLA A2-epi-position restriction, that HIV-derives.But if encounter problems, the test of the antigenicity of end user APC can be used as alternative, perhaps can be used for replenishing the transgenic mice experiment.

Illustrate as the research institute of reporting here, for the immunogenicity of expanding transgenic animal and the association between the antigenicity, use antigenicity to test the reaction of estimating at epi-position, for example Pol498, Env 134, Nef 221, Gag 271, the CTL system that has prepared high affinity for these epi-positions.In order to produce other appropriate C TL system, the HLA transgenic mice that directly has been used in the peptide of emulsification in the adjuvant or lipopeptid immunity, as described herein, this mouse is used in our laboratory routinely, with what generation was used for the antigenicity experiment is.

The antigenicity experiment also is used to read the main results of the infeasible epi-position of optimization experiment in the body.These epi-positions comprise the epi-position of A24 and possible A1 restriction and the immunogenic any epi-position of right and wrong in the HLA transgenic mice.Under any such situation, we have used the people CTL system that generates from the individuality that is exposed to pathogene.Perhaps, the dendritic cells peripheral blood lymphocyte of using GMCSF/IL4-to induce, we have produced and have been used for the CTL system (Celis etc., Proc Natl Acad Sci USA, Vol.91 (6): 2105-9 (1994)) that external CTL induces.

Produced the episome carrier of the multi-epitope constructs of encoding, and transfection advanced among the suitable human cell line, to produce target cell.For example, can end user T cell-line Jurkat, but also successfully used lymphoblastoid cell lines.Source and the target cell of the HLA-that characterizes preferably EBV cell-line that mate, that isozygoty as the MHC of purifying often used in the experiment that the CTL that originates for the end user is, also is used as the recipient of multi-epitope nucleic acid transfection.Be derived from the experiment of the CTL system of HLA transgenic mice for use, use HLA/K with coupling ^bSet (Shimizu Y, DeMars R, J Immunol, Vol.142 (9): 3320-8 (1989)) the I class feminine gender of chimeric construct body transfection, cell-line .221 that EBV-transforms, sudden change is as the recipient of multi-epitope nucleic acid transfection.Such cell can be presented peptide antigen effectively to CTL system (Celis etc., Proc Natl Acad Sci USA, Vol.91 (6): 2105-9 (1994)).

The structure and the test of HTL epi-position string

Synthesize the epi-position string that comprises 3-20 the different HTL epi-positions under same promotor control, and be integrated into standard plasmid pcDNA3.1 (Invitrogen, San Diego).In order to help test and to optimize,, known to IA to represent evenly for given construct has been selected every group of epi-position ^bIt in the mouse immunogenic epi-position.In addition, synthesized and be connected corresponding all peptides, and tested and IA ^bCombination, the combination with one group 14 different DR molecules has been tested on most important ground, they have represented prevailing in the world DR allelomorph (Southwood etc., J Immunol, Vol.160 (7): 3363-73 (1998)).Thereby, in these plasmids, do not create the not HTL epi-position of definite object antigen.But, if detected have good DR in conjunction with potentiality (therefore, immunogenicity in the body of potential DR restriction) bonding pad, then import GPGPG (SEQ ID NO:2) uniformly-spaced thing eliminate them.In all constructs, also will minimize the number that the I class connects motif, as described herein.

Use the mouse of HLA DR transgenic mice and/or H2b haplotype, the immunogenicity of having tested the experimental vaccine plasmid.Detected the generation (IL5, IFN γ) of propagation and/or cell factor.In typical method, inject every kind of plasmid of 100 μ g in the mouse muscle of handling to cardiotoxin, post-evaluation in 11 days immune response (Ishioka etc., J Immunol, Vol.162 (7): 3915-25 (1999)).

Interaction between test CTL and the HTL epi-position

Above-mentioned activity has produced the little functional block of epi-position, the reaction/antigenicity when they are used to confirm all analyzable epi-positions.These pieces are objects of further optimizing, as described in the following examples.Use these identical constructs, estimated immunodominance.More specifically, all CTL epi-position constructs is admixed together, maybe that all HTL epi-position constructs is admixed together.To compare with the result who obtains from the identical construct that separates injection from the result that described construct group obtains then.

These constructs also are used to detect the influence of HTL epi-position to the reaction of CTL epi-position.More specifically, compiled the plasmid that contains HTL and CTL, and be expelled in the mouse, CTL and the htl response of having tested the epi-position of selecting as described herein.Often whether test case can be strengthened to ctl response as the HTL epi-position that is derived from target antigen and surpass the reaction level that one group of DR reaches in conjunction with the plasmid of epi-position (for example, PADRE_ peptide or PADRE_ family molecule) that contains that uses in the CTL epi-position construct.Typically, also test the PADRE_ peptide and whether can suppress or increase reaction to the HTL epi-position of target antigen-derive, perhaps on the contrary, whether the HTL epi-position that is derived from target antigen can suppress or increase PADRE ^_The reaction of peptide.

Use this methodology can reach reaction to a large amount of epi-positions.Compile construct and may suppress reaction at some more weak epi-positions.In this case, after optimization, repeat to compile experiment.

The optimization of embodiment 11CTL and HTL multi-epitope constructs

This embodiment has described the optimization of embodiment 10 described CTL and HTL construct.Also in optimizing the mini gene construct, estimated the flank residue to antigenicity and immunogenic potential impact.These researchs relate to comprising of flank residue, and the synonym of this flank residue is " sept ", have been used to promote effective processing.

Can followingly carry out such analysis.At first, analyzed the test result of embodiment 10 described CTL multi-epitope constructs, with obtain activity and in the trend between the existing of the specific residue at 3 residue places of the epi-position N-and the distolateral wing of C-with related.The epi-position (it is being a suboptimum aspect the magnitude of reaction) of having observed the CTL initiation of suboptimum is the target that flanking region is optimized.For every kind of CTL multi-epitope nucleic acid vaccine of 10-12 the different CTL epi-position of encoding, produced " second generation " multi-epitope nucleic acid vaccine of configuration with optimization.

In one embodiment, first optimal design is to import alanine (a) or lysine (K) residue at the site of all epi-positions with sub-optimal performance C+1.Second optimal design is that the C+1 site imports the natural residue in the target antigen (for example HIV) relevant with antigenicity.

For the epi-position of selecting, also imported other modification.More specifically, also studied the effect that imports other residue sept at epi-position C-and N-end.According to the analysis result of embodiment 10 described multi-epitope nucleic acid vaccines, the residue of research can comprise for example G, Q, W, S and T in addition.If set up the connection epi-position by these modifications, the epi-position order that has reasonably designed other then as described herein, this order can be eliminated and connect epi-position.As described herein, the antigenicity and the immunogenicity of having tested all second generation constructs.

As the result of these modifications, identified variation with the corresponding activity of specific modification of multi-epitope constructs.Find that some modification has general beneficial effect.In order to confirm this point, produce and tested other multi-epitope nucleic acid vaccine, wherein all epi-positions (just show acceptable antigenicity with immunogenic those) have been carried out identical modification.In some cases, observed the activity of the rising of some epi-positions, other then do not have, and perhaps not too wish ground, and some modification has increased the activity of some epi-positions, but has reduced the activity of other epi-position.Under these circumstances, design and tested other multi-epitope nucleic acid vaccine, to keep useful modification, getting rid of verified simultaneously is those variations that be harmful to or adiaphorous.

Selected these multi-epitope nucleic acid vaccines, made: the connection epi-position that a) has minimum prediction; And b) in the former multi-epitope nucleic acid vaccine non-functional epi-position now in more effective background.

For the HTL multi-epitope constructs, checked the data that obtain from " first generation " construct epi-position site and the trend of sept (for example GPGPG (SEQ ID NO:2) sept) aspect closing on during connecting epi-position, construct.If detected particular tendency, then based on these trend design second generation constructs.Perhaps, under the situation of the multi-epitope constructs that can produce the suboptimum activity, reappraised the potential validity of other target strategy (for example based on Ii and LAMP those), and contrasted with targeting sequencing target non-target and simple.

When the bigger variation that detected in the activity of described CTL of this part or HTL multi-epitope constructs, the result is consistent with influence, for example influence the construct activity conformation or " long-range " act on.By present molecule and cytobiology technology, can analyze these variations.For example, analyze or primer extension assay by Northern, can analyze the mRNA expression that is with various multi-epitope constructs cells transfected and stability (Current Protocols inMolecular Biology, Vol 3, John Wiley ﹠amp; Sons, Inc.USA 1999).

In all multi-epitope nucleic acid vaccines, can also comprise antibody labeling, for example MYC/his.This mark is used for the test proteins expression.Also be used for the comprising of MYC/his mark (Manstein etc., Gene, Vol.162 (1): 129-34 (1995)) detecting the stability of expressed products by pulse chase experiment.Contrast the result of these result of experiment and antigenicity and immunogenicity experiments then.Studied the association between the different variablees whether these results exist trend and general rule and test.

Embodiment 12 detects the simplest plasmid configuration of the epi-position that can carry selection effectively

Embodiment 11 and 12 described experiments are used to solve the variable about the design of multi-epitope nucleic acid vaccine.Ideally, in whole proposal, use the carrier that can be used for the people, but also can use a dna vaccination plasmid that is used for vaccine epi-position optimization research, transferred to the carrier that is applicable to human then.Actual carrier selects to depend on several variablees.For example, the availability of carrier, be suitable for human, reliable source is arranged, for example national genophore laboratory (University ofMichigan) is a factor.

In the present embodiment, also connected the epi-position that the construct of optimizing forms bulk.Under the situation of CTL multi-epitope constructs, all constructs all are preferably used for integrating PADRE ^_Peptide and targeting sequencing target.More specifically, connect 2 pairs of 10-12CTL epi-position constructs, produced 2 20-24CTL epi-position constructs.Connection in epi-position has produced under the situation more active than (reduction) of littler construct suboptimum, has studied the alternative combinations and the order that connect.Connect the specific right of the 20-24CTL epi-position construct that can produce optimum activity then, and estimated the activity of the construct that comprises all CTL epi-positions that obtains.Nearly 2 alternating directions have been studied once more.Because this construct is relatively large, confirmed the specific effect of target sequence, because the targeting sequencing target may be more effective to undersized construct, and large-sized construct may be located most effectively by the ubiquitin signal.More specifically, produced a construct that does not have any specific target sequence, and contrasted with the construct that is used for by adding ubiquitin molecule is degraded.

Similarly strategy also is applicable to HTL.2 pairs of 3-5HTL epi-position constructs have been connected, to produce 2 7-9HTL epi-position constructs.Once more, produced under the active situation of (reduction) of suboptimum, studied the alternative combinations and the order that connect in the connection of these epi-positions.Connect the specific right of the 7-9CTL epi-position construct that can produce optimum activity, and estimated the activity of the construct that comprises all HTL epi-positions that obtains.Nearly 2 alternating directions have been studied once more.

Based on these results, for the clinical testing assessment has selected to carry effectively one group of for example plasmid configuration of the optimization of HIV epi-position.Certainly, can be individually or use epi-position in combination from any target antigen (infective or relevant) with disease.This configuration needs one or more HTL epi-position constructs and one or more CTL epi-position constructs.Most preferably can carry the long CTL of epi-position of all codings and the combination of a long HTL epi-position construct effectively, because it has simplified the further clinical research of vaccine.When common injection, can observe under 2 undesirable interactions situations between the construct, can test the different plasmid of injection in same animal, but inject in different injection site or at different time points.Perhaps, if do not identify the CTL construct and the HTL construct of all desirable epi-positions of encoding, then consider further research and establishment body group.

The evaluation and the sign of the CD8+ lymphocyte reaction that embodiment 13 induces after with the polyepitope vaccines immunity

The detection of CD8+ lymphocyte reaction depends on the ELISPOT technology usually.The ELISPOT experiment is known in the art, and is used in routinely in our laboratory.As described herein, also used automatic Zeiss ELISPOT reader.The experiment that is used to detect the CD8+ reaction mainly is to test in new isolated cells and the IFN γ ELISPOT that carries out on the external stimulated cells again with peptide.In addition, under selected situation, used the chromium release experiment.The result who obtains is associated with observed result in the ELISPOT experiment.Also to selected peptide/MHC combination having carried out tetramer dyeing.

Carry out and verified clinical testing.The timing of this activity is consistent with the time period after having selected clinical vaccine epigene construct, and prior to the availability of the actual sample that obtains from the individuality of participating in clinical testing.Can set up based on the experience of this area and be used for the experiment that CTL estimates, for example set up experience (Livingston etc., J Immunol, Vol.159 (3): the 1383-92 (1997) of I phase of experimental HBV vaccine and the II phase CTL evaluation experimental in testing; Heathcote etc., Hepatology, Vol.30 (2): 531-6 (1999); Livingston etc., J Immunol, Vol.162 (5): 3088-95 (1999)).More specifically, can use the Ficoll purifying be derived from normal subjects and for example be derived from not vaccinated volunteer's PBMC.As previously mentioned, can use other antigenicity target according to the present invention.

The design based on the vaccine constructs of DNA of the multi-epitope that embodiment 14 optimizes

Designed the construct of optimizing under the help of above-mentioned computer-aid method, it has minimized simultaneously and has connected the formation of epi-position, and has optimized the C+1 working (machining) efficiency.Be used to make connection to minimize following motif: mouse K ^b(XXXX (F or Y) X _2-3(L, I, M or V)); D ^b(XXXXNX _2-3L, I, M or V)); People A2 (X (L or M) X _6-7V); People A3/A11 (X (L, I, M or V) X _6-7(K, R or Y)); With people B7 (XPX _6-7(L, I, M, V or F)).From data computation shown in Figure 6 the C+1 propensity value, as follows: K=2.2; N=2; G=1.8; T=1.5; A, F, S=1.33; W, Q=1.2; R=1.7; M, Y=1; I=0.86; L=0.76; V, D, H, E, P=0.Allow to insert maximum 4 amino acid.The example of the construct by this method and other method as herein described design as shown in figure 18.Characterized many such constructs in the immunogenicity research in vitro and in vivo, as mentioned below.Figure 19 has listed the amino acid epitope sequences by some nucleic acid sequence encoding in the multi-epitope constructs.

The immunogenicity test of embodiment 15 multi-epitope CTL constructs and the influence of flanking amino acid

The HLA transgenic mice is used for the immunogenicity test of different multi-epitope constructs.By with 50 μ l, 10 μ M cardiotoxin bilaterals be injected into tibialis anterior meat, preliminary treatment one group of mouse, after 4 or 5 days, the 100 μ g DNA construct that will dilute in PBS are administered to identical muscle.In another group, be used in every mouse of peptide injection of emulsification among the CFA, wherein this peptide correspondence the epi-position in the DNA construct that is administered to mouse in DNA injection group.Splenocyte is reclaimed from the animal of dna immunizationization and the animal of peptide immunization in immunity back 11-14 days, uses one in the several experiments that comprise following experiment to detect the CTL activity: standard ⁵¹The Cr-release experiment; The ELISPOT experiment, it does not wherein carry out peptide epitopes-specific stimulation again by the generation of the CD8+T-lymphocyte detection γ-IFN of purifying; With original position ELISA, it comprises with peptide epitopes and carries out epi-position-specific external stimulation step again.The example of the CTL activity of being induced by the EP-HIV-1090 construct after peptide epitopes stimulates is shown in Figure 14 A, and the CTL activity of being induced by PfCTL.1, PfCTL.2 and PFCTL.3 construct after peptide epitopes stimulates as shown in Figure 14B.

By inserting different amino acid, directly estimated effect at the different aminoacids in C+1 flank site in C+1 site with respect to core 18 epi-positions of HBV.1 construct.The immunogenicity data have confirmed that clearly these core 18 epi-positions have the immunogenicity (Fig. 6 b) of reduction when being W, Y or L in the C+1 site.On the contrary, K residue of insertion has increased the ctl response at core 18 significantly.Use R, C, N or G in the C+1 site, also observed the enhancing of ctl response.These data clearly illustrate that the design that can promote multi-epitope constructs is optimized in C+1 processing.

The immunogenicity test of embodiment 16 multi-epitope HTL constructs and the influence of intervening sequence

Develop the general sept formed by GPGPG (SEQ ID NO:2) with HTL epi-position separately, thereby interrupted the connection epi-position.The logic that designs this sept is that any known mouse or people I class MHC or II class MHC molecule all can not use G or P as main anchor, i.e. site 1 and 6 in the core space of htl peptide epi-position.5 amino acid that imported by this sept have separated the epi-position of closing on, so the amino acid of 2 epi-positions physically can not be as the anchor in 1 and 6 sites.The synthetic peptide that use is made up of 4 HIV-1 epi-positions (the sept that do not have with 3 septs and other, known can be in conjunction with mouse IA ^b), the effectiveness of having tested GPGPG (SEQ ID NO:2) sept.HIV 75 aggressiveness are the constructs with 3 GPGPG (SEQ ID NO:2) sept, and HIV 60 aggressiveness are the constructs that do not contain 3 septs.Under the situation that does not have sept, induced at 3 htl response in 4 epi-positions with the peptide among CFA immunity CB6F1 mouse, but epi-positions all are immunogenic (Figure 15) existing under the situation of sept all.This evidence shows that sept can improve the performance of multi-epitope constructs.

By using EP-HIV-1043-PADRE ^_The immune interiorly H2 of construct muscle ^BXdMouse has been estimated the ability based on htl response in the multi-epitope HTL construct inductor of DNA.EP-HIV-1043-PADRE ^_Construct as shown in figure 18, EP-HIV-1043-PADRE ^_Difference between construct and the EP-HIV-1043 is, the former comprises C-end GPGPG (SEQ ID NO:2) sept, and its back is PADRE ^_Sequence A KXVAAWTLKAAA (SEQ ID NO:1).Reinforced immunological is not carried out in back 11 days of immunity, is purified into cd4 t cell from spleen, has detected the htl response of peptide specific in elementary γ-IFNELISPOT experiment.The example of the HTL activity of being induced by the construct of the HIV epi-position of encoding as shown in figure 16.Generally, carry out with multi-epitope HIV HTL construct that htl response that dna immunization induces is substantially equal to or greater than the magnitude of the reaction of peptide immune induction.

Embodiment 17 is based on the HBV immunotherapeutical developing vaccines of epi-position

1. brief introduction

The natural association of virus sweep

The relevant cell immune response of the removing of infecting with acute HBV naturally be wide spectrum with polyspecific.This reaction comprises CTL and HTL, and they point to the epi-position (Chisari, F.V. and Ferrari, C.Annu.Rev.Immunol.13:29-60 (1995)) from multiple viral gene product.Immune system is difficult to solve chronic HBV infection, but when it takes place, reduction relevant (Guidotti, the L.G. and the Chisari of the raising of removing and the CTL activity of virus, ALT increase (flares) and viral load, F.V., Annu.Rev.Immunol.19:65-91 (2001)).The removing of virus also can produce in the individuality of accepting IFN-α treatment of significant proportion (10-15%), and this is similar to spontaneous removing, and this effect is relevant with the enhanced cell immune response.

In several researchs, investigated the magnitude that infects relevant cell immune response with control HBV.For sake of comparison, following value (mean value and scope) has been represented the number of antigen-specific cell in per 1,000,000 CD8+ cells.People such as Lohr use the ELISPOT experiment to come acute phase specific reaction of detected HBV-in peripheral blood lymphocyte (PBL) (Lohr, H.F. etc., Liver 18:405-413 (1998)) of quantitatively infecting.They have reported that having 400-2800 spot at HBV core 18-27 forms cell (SFC) (average 1400).Maini etc. have used tetramer dyeing, it is reported that it is than the responsive about 4 times of (Tan of ELISPOT experiment, L.C. etc., J.Immunol.162:1827-1835 (1999)), for core 18-27 epi-position has detected 80-14,000 tetramer positive cells, mean value is 4,000 (Maini, M.K. etc., Gastroenterology 117:1386-1396 (1999)).Consider the sensitivity differences of experiment, convert thereof into 20 to 3500 ELISPOT positive cells of estimation range, 1000 specific cells of average out to.

Use identical experiment, Webster etc. have reported 7000 tetramer positive cells at core 18-27 epi-position (1750 ELISPOT positive cells), 200 cells at env335 (50 ELISPOT positive cells) and 1200 cells at pol 562 epi-positions (300 ELISPOT positive cells) (Webster, G.J. etc., Hepatology.32:1117-1124 (2000)).In the situation of 2 other epi-positions analyzing, observed at 200 tetramer positive cells of average out to (scope of 80-6000) of env 335 with at 220 cells of average out to (scope of 80-3200) (Maini of pol 562 epi-positions, M.K. etc., Gastroenterology 117:1386-1396 (1999)).These reactions about rough estimate of ELISPOT cell are, are 50SFC (scope of 20-1500) and are 55SFC (scope of 20-800) at the mean value of pol 562 at the mean value of env 335.These data can be compared with the data of using the LDA experiment to obtain, and the latter's susceptibility is than low about 40-50 times (Murali-Krishna, K. etc., the Adv.Exp.Med.Biol.452:123-142 (1998)) of ELISPOT experiment.For example, people such as Rehermann estimate that 15 cells are specific to env 335, and 18 cells are specific (Rehermann, B. etc., J.Clin.Invest.97:1655-1665 (1996)) to pol 445.Suppose that these experiments have 45 times sensitivity differences, then these values respectively corresponding the ELISPOT positive cell of 675 and 810 epitope specificities.Other data are from people such as Lohr, and they use the ELISPOT experiment to come the specific reaction of HBV-(Lohr H.F. etc., Liver 18:4-5-413 (1998)) among the patient that quantitative IFN-α treatment (it causes the removing of virus) causes.This research in, reported average 600 to the specific SFC of HBV core 18-27 (scope is 200-1300).

In a word, in the process of removing HBV virus, in PBL, detected CTL to various HBV epitope specificities.Frequency range by the detected functioning cell of ELISPOT be 20-400 cell/1,000,000 CD8+ cells (low value) to 820-3500SFC/ 1,000,000 CD8+ cells (high value), average response is 50-1000SFC/ 1,000,000 CD8+ cells.

Use the HBV-transgenic mice directly to confirm the importance of the specific CTL of HBV-.More specifically, different viral antigen (comprising env, core and pol antigen) is had specific and is subjected to the clone's of mouse MHC molecule restriction the succession of CTL to shift the expression (Tsui that can eliminate viral antigen, L.V. etc., Proc.Natl.Acad.Sci.USA.92:12398-12402 (1995); Guidotti, L.G. etc., Immunity.4:25-36 (1996)).These data have clearly proved the importance that ctl response infects control HBV.

The magnitude of the htl response in the HBV course of infection is usually less than CTL's.Use holoantigen and ELISPOT experiment, people such as Lohr have observed sum frequency 47 ± 5.2SFC/ 1,000,000 CD4+ cells that IFN-α treatment causes in the patient, 42 ± 12SFC/, 1,000,000 CD4+ cells (Lohr, H.F. etc., Liver 18:405-413 (1998)) in acute infection, have been observed.People such as Webster report has detected 2,900 the tetramer positive cells/1,000,000 CD4+ cells (Webster, G.J. etc., Hepatology.32:1117-1124 (2000)) at cAg among the patient in 10 weeks after infection.

As a result, these data provide the method for the immunogenicity level that detects the therapeutic HBV vaccine that is used to induce ctl response.

B. immunological tolerance is relevant with chronic HBV infection

HBV epi-position-specific immunological tolerance relevant (Chisari, F.V. and Ferrari, C.Annu.Rev.Immunol.13:29-60 (1995) with chronic HBV infection; Alexander, J. etc., Immunol.Res.18:79-92 (1998); Milich, D.R., Can.J.Gastroenterol.14:781-787 (2000); Hilleman, M.R. etc., Vaccine.19:1837-1848 (2001); Jung, M.C. etc., Lancet Infect.Dis.2:43-50 (2002)).In the individuality that infects, think that high-caliber viremia virusemia is the reason of this immune tolerance state.Can cause the unbalance and general peripheral tolerance of general Th1/Th2 although this effect is so remarkable, it can not eliminate the specific CTL precursor of HBV-(Rossol, S. etc., B.J.Clin.Invest.99:3025-3033 (1997); Chen etc., Immunity 12:83-93 (2000); Sette, A.D. etc., J.Immunol.166:1389-1397 (2001)).In fact, the research in the HBV-transgenic mice is verified, uses the vaccine based on epi-position can " destroy " tolerance (Livingston, B.D. etc., J.Immunol.159:1383-1392 (1997) with the HTL epi-position that is derived from the optimization of non-pathogene; Alexander, J. etc., Immunol.Res.18:79-92 (1998); Sette, A.D. etc., J.Immunol.166:1389-1397 (2001)).From the process that infects at spontaneous elimination HBV and the data that obtain of the patient's sample that in process, obtains to IFN-α therapeutic response show that also this defective is reversible.Other data of supporting this hypothesis are from the research of using the antiviral drugs Lamivudine, and are as described below.

HBV immunization therapy clinical testing in the past

The clinical research of the lipopeptid vaccine that use is made up of HTL epi-position that mixes and HBV core 18CTL epi-position provides immunogenic data (Livingston, B.D. etc., the Hum.Immunol.60:1013-1017 (1999) of each epi-position that proves normal volunteer; Livingston, B.D. etc., J.Immunol.159:1383-1392 (1997); Vitiello, A. etc., J.Clin.Invest.95:341-349 (1995)).The CTL level of in health objects, inducing can with detected comparing in the individuality of acute infection, this individuality spontaneously or as the result of IFN-α treatment has been removed virus.But the follow-up test of carrying out in the Chronic HBV patient is disappointing: the level of the CTL that induces in these patients is starkly lower than observed level in normal subjects, does not also observe the reduction of viral load.Importantly, when carrying out these clinical testings, the antiviral drugs therapy can not obtain.Thereby, have no idea to reduce the viremia virusemia relevant with immunological tolerance.

D. the antiviral drugs therapy is to the influence of hbv replication, integration and immune system tolerance

Chronic HBV infection is with high-caliber viremia virusemia, and average about 2.2 * 10 ¹¹Individual virion/3L serum, this is equivalent to average total body burden (Nowak, M.A. etc., Proc.Natl.Acad.Sci.USA.93:4398-4402 (1996)).Think that the existence of a large amount of virions in the serum is (being at least in part) reasons (Schlaak, J.F. etc., J.Hepatology 30:353-358 (1999)) of detected immunological tolerance in the Chronic HBV patient.Nucleoside analog Lamivudine (Epivir-HBV) (NC 27709 for GlaxoSmithKline, Research Triangle Park) is a reverse transcriptase inhibitors, and initial exploitation is used for the treatment of HIV.It also is approved for the treatment chronic HBV infection, known it hbv replication is had effective inhibitory action, and can promptly reduce the production (Nowak, M.A. etc., Proc.Natl.Acad.Sci.USA.93:4398-4402 (1996)) of new infectious viral particle in the patient.In a plurality of researchs, in using the Most patients of lamivudine therapy, HBV DNA becomes and can not detect (Dienstag, J.L. etc., Hepatology 30:1082-1087 (1999); Boni, C. etc., Hepatology.33:963-971 (2001)).In preceding 6 middle of the month of treatment, the viremia virusemia level has had remarkable decline, and should descend in long-term treatment continues always.HBsAg and HBeAg level descend in Most patients in time, although its speed and magnitude are lower than virion observed.Great majority with lamivudine therapy 6 months or patient more of a specified duration in, the liver enzyme is also reduced to normal level (Dienstag, J.L. etc., Hepatology 30:1082-1087 (1999); Boni, C. etc., Hepatology.33:963-971 (2001)).Lamivudine can not suppress the generation of virus protein fully, because the HBV DNA of DNA of covalently closed circle (cccDNA) and integration can support the generation of virus protein in the long time.

As if in addition, lamivudine therapy can overcome or reduce at least the hypoergia of specific CTL of HBV-and HTL, typically be hypoergia (Boni, C. etc., the B.J.Clin.Invest.102:968-975 (1998) of chronic HBV infection; Boni, C. etc., Hepatology.33:963-971 (2001)).What is interesting is, after viremia virusemia begins sharply to descend, the bounce-back that behind the beginning lamivudine therapy, the T-cytoactive just occurred in 1 month.But, when suspending lamivudine therapy, according to the duration of treatment, virus replication in the shortest 1 week, just rebound (Dienstag, J.L. etc., N.Eng.J.Med.333:1657-1661 (1995); Dienstag, J.L. etc., Hepatology 30:1082-1087 (1999)).In addition, reported the rapid appearance of drug-fast HBV mutant strain.Thereby Lamivudine is limited from the validity in treatment chronic HBV infection aspect.

E. the design of immunotherapeutical vaccine

Can the inducing cell immune response design and the evaluation of therapeutic vaccine of (its magnitude is that the removing of control hbv replication and final mediation virus is required) have very big clinical importance.Vaccine is used to induce specific CTL of HBV-and htl response, and has carried out clinical trial in the volunteer of health and chronically infected patient.In the back in one group, the patient only limits to successfully treat those of minimum 6 months with Lamivudine or similar antiviral agent.

Epi-position is selected

From HLA-A2 ,-A3 and-the CTL epi-position of the super type of B7 family

When according to the overlapping of them and when independently peptide divides into groups in conjunction with the feature of inventory, most of HLA I quasi-molecules can be included into the super type of main HLA I class (table 6A-B) of relative minority.By select can be in conjunction with the great majority in the given super type or whole epi-positions of HLA molecules, the number of can the effective polyepitope vaccines of limit production required epi-position.Select the super type of modal HLA to help being used for the treatment of design (Bertoni, R., J. etc., the J.Clin.Invest.100:503-513 (1997) of the vaccine of the individuality that HBV infects; Sette, A. etc., Immunogenetics.50:201-212 (1999); Sette, A. etc., Curr.Opin.Immunol.10:478-482 (1998)).

The phenotypic frequency of table 6A.HLA I class

Super type	HLA allelomorph	Phenotypic frequency (%)
						The Asian	Black people	The Europe white people	The North America white people
		? ? ??A2	??A *0201 ??A *0202 ??A *0203 ??A *0206 ??A *6802	??15.8 ??0.2 ??8.7 ??10.8 ??0.2	??19.6 ??8.7 ??0.2 ??0.6 ??9.6					??45.1 ??1.5 ??0.2 ??0.2 ??1.3	??32.0 ??3.7 ??4.1 ??7.8 ??2.2
? ? ??A3	??A *0301 ??A *1101 ??A *3101 ??A *3301 ??A *6801					??1.3 ??35.3 ??8.2 ??5.2 ??0.5	??14.6 ??1.1 ??1.3 ??4.0 ??7.0	??26.8 ??11.5 ??5.1 ??1.8 ??6.0	??25.9 ??12.4 ??4.5 ??1.6 ??5.0
		? ??A1	??A *0101 ??A *2902 ??A *3002	??1.5 ??0.5 ??2.2	??7.0 ??5.1 ??30.7					??30.7 ??6.3 ??4.7	??29.4 ??5.7 ??4.9
? ? ??A24	??A *2402 ??A *2301 ? ??A *2902 ??A *3002					??49.5 ??0.2 ? ??0.5 ??2.2	??4.2 ??17.9 ? ??5.1 ??30.7	??16.5 ??3.2 ? ??6.3 ??4.7	??15.6 ??4.5 ? ??5.7 ??4.9
		? ? ??B7	??B *0702 ??B *3501 ??B *5101 ??B *5301 ??B *5401	??5.6 ??9.3 ??12.2 ??0.2 ??8.6	??13.8 ??9.0 ??4.6 ??19.4 ??0.1					??24.9 ??16.0 ??10.7 ??0.6 ??0.1	??25.6 ??17.4 ??9.3 ??1.2 ??0.1

The phenotypic frequency of table 6B.HLA II class

Antigen	Phenotypic frequency (%)
					The Asian	Black people	The Europe white people	The North America white people
	??DR1 ??DR2w2B1 ??DR3 ??DR4w4 ??DR4w14 ??DR4w15 ??DR5w11 ??DR6w19 ??DR7 ??DR8w2 ??DR9 ??DR5w12	??6.0 ??34.7 ??5.2 ??0.9 ??1.7 ??16.0 ??7.7 ??10.5 ??4.2 ??18.6 ??23.5 ??15.3	??13.1 ??29.2 ??22.4 ??3.3 ??0.7 ??1.0 ??23.1 ??39.9 ??14.8 ??10.7 ??3.9 ??9.6	??19.3 ??27.6 ??24.7 ??14.3 ??4.3 ??1.5 ??18.2 ??21.6 ??25.5 ??5.4 ??2.0 ??3.4					??22.5 ??27.3 ??21.0 ??14.8 ??7.5 ??1.7 ??18.5 ??22.0 ??23.4 ??6.7 ??2.0 ??2.2

Identified one group of CTL epi-position that is derived from HBV, they can be in conjunction with the super type allelomorph of a plurality of HLA (table 7).

Table 7.HBV vaccine HLA-A2 ,-A 3﹠amp;-B7 CTL epi-position

The super type of HLA	Epi-position	Sequence	??SEQ?ID ??NO：	Conservative (%) 1	Prototype allelomorph		Immunogenicity
									In conjunction with (IC 50nM) 2	??XRN 3	The people 4	Mouse 5
					??A2 ??A2 ??A2 ??A2 ??A2 ??A2 ??A2 ??A2 ??A2	Core	18 env 183 env 335 env 338 env 378 pol 455 pol 538 pol 773 pol 562	??FLPSDFFPSV ??FLLTRILTI ??WLSLLVPFV ??LLVPFVQWFV ??LLPIFFCLWV ??GLSRYVARL ??YMDDVVLGV ??ILRGTSFVYV ??FLLSLGIHL					??9 ??10 ??11 ??12 ??13 ??14 ??15 ??16 ??17	??45 ??80 ??100 ??95 ??100 ??55 ??90 ? ??95	??3.5 ??9.8 ??5.4 ??5.7 ??158.9 ??55.9 ??6.4 ? ??7.8	??5 ??4 ??4 ??5 ??1 ??3 ??5 ? ??3	??+ ??+ ??+ ? ? ??+ ??+ ? ??+	??+ ??+ ??+ ? ? ??+ ??+ ? ??+
??A2	??pol?642	??ALMPLYACI	??18	??95					??12.9	??4
					??A2 ??A3 ??A3 ??A3 ??A3 ??A3 ??A3 ??A3/A1 ??A3	Env 338 cores 141 pol 149 pol 150 pol 388 pol 47 pol 531 pol 629 pol 665	??GLSPTVWLSV ??STLPETTVVRR ??HTLWKAGILYK ??TLWKAGILYK ??LVVDFSQFSR ??NVSIPWTHK ??SAICSVVRR ??KVGNFTGLY ??QAFTFSPTYK	??19 ??20 ??21 ??22 ??23 ??24 ??25 ??26 ??27					? ??95 ??100 ??100 ??100 ??100 ??95 ??95 ??95	? ??735/4.5 *??15.4/15.6 ??2.1/33 ??6875/17 ??174/117 ??2189/29 ??58/365 ??249/8	? ??4 ??5 ??2 ??3 ??3 ??3 ??2 ??3	? ??+ ??+ ? ??+ ??+ ??+ ? ??+	? ??+ ??+ ? ??+ ??+ ??+ ? ??+
??B7 ? ??B7 ??B7 ??B7 ??B7 ??B7 ??B7 ??B7 ??B7	Core	19 env 313 pol 354 pol 429 pol 640 pol 541 pol 530 pol 640 pol 640	??LPSDFFPSV ? ??IPIPSSWAF ??TPARVTGGVF ??HPAAMPHLL ??YPALMPLYA ??FPHCLAFSY ??FPHCLAFSYM ??YPALMPLY ??YPALMPLYACI	??28 ? ??29 ??30 ??31 ??32 ??33 ??34 ??35 ??36					??45 ? ??100 ??90 ??100 ? ? ??95 ? ??95	??3026.8 ? ??42.3 ??13.2 ??56.6 ? ? ??58.5 ? ??1393.4	??4 ? ??4 ??2 ??4 ? ? ??5 ? ??3	??+ ? ??+ ??+ ??- ? ? ??- ? ??-						??- ? ??+ ??- ??+ ? ? ??+ ? ??+

1. the sequence homogeneity of 20 strain HBV (comprising adr, adw, ayr and ayw separator).

2. the prototype allelomorph of each super type is A2:A*0201, A3:A*0301/A*1101, B7:B*0702.

3. with the allelic number of super type of≤500nm combination.

4. the ctl response of the Memorability among the patient that chronic or acute HBV infects.

5. after being used in the peptide immunity of emulsification among the IFA, the ctl response of in the HLA-transgenic mice, inducing.

* respectively in conjunction with HLA-A*0301 and-A*1101.

HLA-A2 ,-A3 and-the super type epi-position of B7 respectively selects 6 to be used for vaccine development.The intercepting value (cutoff) of the binding affinity of investigating is 500nM, because the binding affinity of this level relevant with CTL immunogenicity and antigenicity (Sette, A. etc., J.Immunol.153:5586-5592 (1994)).These all epi-positions are all guarded in the most general HBV bacterial strain.Core 18 epi-positions are guarded in the HBV sequence of suitable relatively 45% test, contain the conservative variant that replaces (isoleucine replacement leucine) but the most of sequences that do not contain this defined epitope can be coded in epi-position C-end.18 all except that one selected epi-positions can both be in conjunction with at least 3 among 5 members the most common of given super type.These epi-positions are derived from env, pol and cAg, and the immunoreactive target of pointing to a plurality of viral antigens with our generation is consistent, thereby have imitated removing naturally of HBV.

Use the CTL experiment of Memorability and, confirmed the human immune system identification (Bertoni, R., J. etc., J.Clin.Invest.100:503-513 (1997)) of these epi-positions from the PBL that suffers from acute or chronically infected individuality.PBL has confirmed that to the immunity identification of these epi-positions epi-position is to produce in the HBV of nature course of infection, suitable TCR is present in people's inventory.Except the epi-position exception of 3 HLA-B7-restrictions, discerned whole group of vaccine epi-position (table 7) from the CD8+T-lymphocyte that HBV patient obtains.

Also use the HLA-transgenic animal tested HLA-A2 ,-A3 and-immunogenicity of B7 epi-position.After being used in the synthetic peptide immunity of emulsification among the IFA, use original position IFN-γ ELISA experiment to detect ctl response (Vitiello, A. etc., J.Clin.Invest.95:341-349 (1995)).In order to estimate, the data transaction that will obtain in this experiment becomes secretory units (SU) (McKinney, D.M. etc., J.Immunol.Methods.237:105-117 (2000)).SU be as to the reaction of particular peptide, can secrete the number of the cell of 100pg IFN-γ, and the background amount of the IFN-γ that generates when not having peptide is proofreaied and correct.Last row data presented at table 7 has been summed up these result of experiment.Great majority in these epi-positions are that immunogenic this fact is important in the HLA-transgenic mice, use small animal model to estimate the method for the effectiveness of polyepitope vaccines because it provides.

In a word, be fit to be included in based on being used for vaccine development in the vaccine of epi-position and by one group of epi-position of 3 super types restrictions of common HLA I class.

From HLA-A1 and-the CTL epi-position of the super type of A24

In order to use the vaccine therapy patient and to improve the multiplicity of the determinant in the epi-position bag be included in us, differentiated can in conjunction with HLA-A1 and-a plurality of members' of the super type of A24 epi-position (table 8).

Table 8.HBV vaccine HLA-A1 ﹠amp;-A24 CTL epi-position

HBV vaccine HLA-A1 ﹠amp;-A24 CTL epi-position

The super type of HLA	Epi-position	Sequence	??SEQ?ID ??NO：	Conservative (%) 1	Prototype allelomorph
							In conjunction with (IC 50nM) 2	??XRN 3
					??A1 ??A1 ??A1 ??A1 ??A1 ??A1 ??A1 ??A1 ??A24 ??A24 ??A24 ??A24 ??A24 ??A24 ??A24 ??A24 ??A24 ??A24	Env 359 cores 419 cores 137 pol 149 pol 166 pol 415 pol 580 env 249 env 236 pol 392 env 332 env 332 core 10l cores 117 pol 167 pol 529 pol 639 pol 745			??WMMWYWGPSLY ??DLLDTASALY ??LTFGRETVLEY ??HTLWKAGILY ??ASFCGSPY ??LSLDVSAAFY ??YSLNFMGY ??ILLLCLIFLL ??RWMCLRRFII ??SWPKFAVPNL ??RFSWLSLLVPF ??RFSWLSLLVPF ??LWFHISCLTF ??EYLVSFGVW ??SFCGSPYSW ??AFPHCLAF ??GYPALMPLY ??KYTSFPWLL	??37 ??38 ??39 ??40 ??41 ??42 ??43 ??44 ??45 ??46 ??47 ??47 ??48 ??49 ??50 ??51 ??52 ??53	??85 ??75 ??75 ??100 ??100 ??95 ??85 ??100 ??95 ??95 ??100 ??100 ??85 ??90 ??100 ??95 ??95 ??85	??16.3 ??2.3 ??80.0 ??381.0 ??247.0 ??6.0 ??382.0 ??192.0 ??11.0 ??2.1 ??12.0 ??12.0 ??6.7 ??16.0 ??146.0 ??78.0 ??280.0 ??1.0	??3 ??3 ??3 ??3 ??3 ??3 ??3 ??1 ??3 ??2 ??2 ??2 ??3 ??2 ??3 ??3 ??2 ??3

1. the sequence homogeneity of 20 strain HBV (comprising adr, adw, ayr and ayw separator).

2. the prototype allelomorph of each super type is A1:A*0101, A24:A*2402

3. with the allelic number of super type of≤500nm combination.

In the peptide of over one hundred the motif positive that identifies, based on they to the HLA-A1 of purifying or-A24 in conjunction with feature and relevant super type molecule, selected 24 peptides to be used for further research, select to HLA-A1 or HLA-A24 restriction each 6 as vaccine; 3 relevant allelomorph be used to define HLA-A1 and-the super type of A24 family.In these epi-positions, reported independently core 117 and pol 745 can the origin self-infections CTL identification (Sobao, Y. etc., J.Hepatol.34:922-929 (2001)) of individuality of HBV.

Can be discerned by people CD8+T-lymphocyte for the evidence that other is provided confirms the epi-position of selecting, the PBL that we use the normal donor of infection never to obtain has induced elementary ctl response.This elementary CTL induce experiment used by leukopheresis from HLA-A1 or-PBL that the male and female donor of the A24 positive obtains.This PBL is as dendritic cells (DC), antigen presenting cell and the lymphocytic source of CD8+T-(Keogh, E. etc., J.Immunol.167:787-796 (2001)).In order to induce precursor CTL to be divided into mature cell, under the situation that has 10ng/ml recombined human IL-7 to exist, the CD8+ cell of purifying is cultivated altogether with producing DC cell factor, peptide-pulse.This incubation step has induced the activation of precursor CTL and beginning ripe, but needs to stimulate and enlarged culture enlarges their number again, tests being used for.At the 7th and 14 day, stimulate again with the adhesion monocyte of peptide pulse.Stimulated the CTL activity of culture of having used original position ELISA or ELISPOT experiment test for the 2nd time back 7 days.If the reaction that detects be at least 2 times of background expression (using irrelevant peptide to detect) and 〉=the 50pg/ hole, think that then culture is positive.Positive reaction has confirmed that suitable TCR exists in the people.

Use HLA-A1 and-data that the A24 epi-position obtains are summarised in the table 9.

Table 9.HLA-A1﹠amp; The main immunogenic of-A24HBV CTL epi-position

The super type of HLA	Epi-position	Sequence	??SEQ?ID ??NO：	Donor+/ test sum 1	The sum of+hole/test 2	Average SI 3	Clean IFN-γ (pg/well) 4
								??A1 ??A1 ??A1 ??A1 ??A1 ??A1 ??A24 ??A24 ??A24 ??A24 ??A24 ??A24	Env 359 cores 419 cores 137 pol 166 pol 415 env 249 env 236 env 332 cores 101 cores 117 pol 392 pol 745	??WMMWYWGPSLY ??DLLDTASALY ??LTFGRETVLEY ??ASFCGSPY ??LSLDVSAAFY ??ILLLCLIFLL ??RWMCLRRFIIF ??RFSWLSLLVPF ??LWFHISCLTF ??EYLVSFGVWI ??SWPKFAVPNL ??KYTSFPWLL	??37 ??38 ??39 ??41 ??42 ??44 ??45 ??47 ??48 ??49 ??46 ??53	??1/4 ??1/3 ??3/3 ??2/4 ??1/4 ??3/3 ??0/2 ??1/2 ??1/2 ??1/2 ??0/3 ??2/2	??1/192 ??3/144 ??3/144 ??3/192 ??1/192 ??7/144 ??0/96 ??1/96 ??2/96 ??1/96 ??0/144 ??10/96	??23.0 ??29.0 ??27.3 ??41.2 ??57.0 ??21.0 ? ??2.5 ??2.8 ??2.3 ? ??108.0	??175 ??67 ??100 ??60 ??56 ??93 ? ??186 ??248 ??158 ? ??144

1. the number that has the donor of positive ctl response in Ce Shi the donor sum.

2. the number that has the culture of positive ctl response in Ce Shi total culture.

3. the average stimulation index of the ctl response that calculates from following formula: peptide exists the IFN-γ secretion/peptide under the situation not have IFN-γ secretion under the situation.

4. clean IFN-γ produces, and carries out adjustment with respect to the irrelevant peptide of contrast.

Data being expressed as the average stimulation index of the positive number of perforations in total hole of test, positive culture and the clean IFN-γ of positive culture discharges.For epi-position that is included in all HLA-A1 restrictions in this HBV vaccine research and 4/6 HLA-A24 epi-position, induced significant ctl response.

HLA-A1 and-the A24 transgenic mice is unavailable at present.But, at HLA-A24 epi-position and mouse I class K ^dBinding motif between have the very similitude of high level.Therefore, we have tested the external K in conjunction with purifying of 4 vaccine HLA-A24 epi-positions ^dThe ability of molecule, and estimated immunogenicity.We find that one in these epi-positions at H2 ^BxdIn the mouse immunogenic (table 10).Do not test the combination of 2 other epi-positions, but when usefulness peptide/IFA emulsion immunity after at H2 ^BxdWhen testing in the mouse, confirm that they are immunogenic.The scope of external post-stimulatory again IFN-gamma reaction is 158.7 to 339.6SU.This activity level is similar to known contrast K ^dThe observed level of epi-position of-restriction (Romero, etc., Nature 341:323. (1989)).Thereby, can under the situation that does not have the HLA-A24 transgenic mice, use H2 ^Bxd

Table 10.HLA-A24 epi-position and K ^dCross reactivity

Epi-position	Sequence	??SEQ?ID ??NO：	Conservative (%) 1	??K dIn conjunction with (IC 50nM	The mouse immunogenicity 2(SU)
						Env 236 pol 392 env 332 env 332 cores 101 cores 117 pol 167 pol 529 pol 639 pol 745	??RWMCLRRFII ??SWPKFAVPNL ??RFSWLSLLVPF ??RFSWLSLLVPF ??LWFHISCLTF ??EYLVSFGVW ??SFCGSPYSW ??AFPHCLAF ??GYPALMPLY ??KYTSFPWLL	??45 ??46 ??47 ??47 ??48 ??49 ??50 ??51 ??52 ??53	??95 ??95 ??100 ??100 ??85 ??90 ??100 ??95 ??95 ??85	??NT ??NT ? ??- ??- ??- ? ? ? ??77.5	??339.6 ??261.0 ? ??0.0 ??0.0 ??0.0 ? ? ? ??158.7
??CS?252 3	??SYIPSAEKI	??54	??NA	??9.2	??49.2

1. the sequence homogeneity of 20 strain HBV (comprising adr, adw, ayr and ayw separator).

2.CTL reaction is with peptide/IFA immunity H2 ^BxdDetect in the ELISA experiment (McKinney etc. 2000) in position behind the mouse.

3. contrast A24 epi-position (Romero etc. 1991).

NT: do not detect

NA: inapplicable

Colony's coverage of the vaccine of C. estimating of forming by the CTL epi-position

Based on the phenotypic frequency of the HLA type of HLA factory (workshop) definition and epi-position in conjunction with feature, detected the coverage (Gjerston of colony of the vaccine of forming by selected epi-position, D.W. and Terasaki, P.I.HLA.American society forHistocompatibility and Immunogenetics.Lenexa, Kans (1998)).

Be included in modal HLA molecule in each of 5 selected super types of HLA I class and they in the distribution among the common race shown in table 6A.Contain can in conjunction with shown in the phenotypic frequency of calculating of individuality of HLA type of epi-position of I class-restriction of number and expection can be hereditarily the epi-position of selecting be carried out the cumulative frequency of the individuality that immunology responds shown in Figure 25 A-25B.In having the Utopian compound population of average HLA frequency, can identify average 11.1 epi-positions-HLA and make up (Figure 25 A).The analysis showed that of colony's coverage to the expectation among the main race do not have significantly race's deflection (Figure 25 B).

The selection of D.HTL epi-position

Based on epi-position-peptide combination, the HLA-DR type can be divided into 2 main super types, be defined as HLA-DR-1,4,7 and-the super type of DR3 (Wilson, C.C. etc., J.Virol.75:4195-4207 (2001); Doolan, D.L. etc., J.Immunol.165:1123-1137 (2000); Southwood, S. etc., J Immunol.160:3363-3373 (1998)).Use is similar to the method that is used to differentiate the CTL epi-position, has differentiated one group of super type epi-position of HLA-DR that is derived from HBV, and based on they in conjunction with feature selecting 16 be used for further research (table 11).

Table 11.HBV vaccine HTL epi-position

The super type of HLA	Epi-position	Sequence	?(SEQ?ID ??NO：)	Conservative (%) 1	HLA-DR binding ability (IC 50nM)														??#DR ??bound 2
																				??DRB1 ? *0101	??DRB1 ? *1501	??DRB1 ? *0301	??DRB1 ? *0401	??DRB1 ? *0405	??DRB1 ? *1101	??DRB1 ? *1201	??DRB1 ? *1302	??DRB1 ? *0701	??DRB1 ? *0802	??DRB1 ? *0901	??DRB5 ? *0101	??DRB3 ? *0101	??DRB4 ? *0101
					??DR	Pol 412 pol 664 env 180 pol 774 cores 120 pol 145 env 339 pol 501 pol 523 pol 618 pol 767 cores 50	LQSLTNLLSSNLSWL KQAFTFSPTYKAFLC AGFFLL bamboo ILTIPQS GTSFVYVPSALNPAD VSFGVWIRTPPAYRPPNAPI RHYLHTLWKAGILYK LVPFVQWFVGLSPTV LHLYSHPIILGFRKI PFLLAQFTSAICSVV KQCFRKLPVNRPIDW AANWILRGTSFVYVP PHHTALRQAILCWGELMTLA	??(55) ??(56) ??(57) ??(58) ??(59) ??(60) ??(61) ??(62) ??(63) ??(64) ??(65) ??(66)	??90 ??60 ??80 ??80 ??90 ??100 ??95 ??80 ??95 ??45 ??70 ??90	??2.0 ??10 ??1 ??15 ??27 ??17 ??408 ??248 ??27 ??3.0 ??55 ??810	??21 ??41 ??217 ??748 ??43 ??4.0 ??14 ??558 ??359 ??4370 ??386 ??8.0	??- ??- ??- ??- ??- ??- ??- ??- ??- ??- ??- ??-	??10.0 ??88 ??9 ??119 ??58 ??2271 ??315 ? ??560 ??40 ??966 ??326	??47 ??181 ??258 ??94 ??220 ??1499 ??28 ??244 ??246 ??34 ??1634 ??-	??303 ??82 ??6 ??443 ??11 ??42 ??54 ??492 ??1749 ??1617 ??1520 ??458	??397 ??- ??4229 ??- ??817 ??149 ??452 ??9462 ??- ??- ??802 ??-	??143 ??190 ??9 ??- ??565 ??766 ??2330 ??- ??59 ??821 ??143 ??-	??173 ??90 ??8 ??94 ??78 ??61 ??2744 ??- ??328 ??62 ??44 ??676																??598 ??416 ??189 ??818 ??76 ??36 ??60 ??800 ??940 ??872 ??214 ??210	??791 ??142 ??56 ??220 ??1773 ??133 ??31 ??1551 ??1373 ??5175 ??299 ??952	??1067 ??144 ??1158 ??400 ??7 ??35 ??1516 ??560 ??4764 ??1246 ??3276 ??124	??1837 ??4848 ??4374 ??- ??6454 ??- ??1661 ??- ??- ??- ??- ??575	??4179 ??322 ??696 ??- ??395 ??782 ??22 ??102 ??1347 ??3060 ??6553 ??48	??10 ??11 ??10 ??9 ??8 ??10 ??9 ??8 ??7 ??6 ??8 ??7
??DR3	??pol?694 ??pol?385 ??pol?96 ??pol?420	??LCQVFADATPTGWGL ??ESRLVVDFSQFSRGN ??VGPLTVNEKRRLKLI ??SSNLSWLSLDVSAAF	??(67) ??(68) ??(69) ??(70)	??95 ??45 ??60 ??85															??7470 ??7372 ??8415 ??38	??5009 ??1368 ??4153 ??3089	??67 ??36 ??43 ??62	??490 ??208 ??3916 ??168	??1203 ??251 ??1908 ??17	??- ??- ??6666 ??4923	??- ??- ??- ??1859	??2022 ??946 ??4461 ??36	??- ??- ??- ??5063	??- ??- ??5354 ??1065	??- ??- ??- ??7126	??- ??- ??4330 ??-	??1808 ??2525 ??- ??5	??1044 ??8711 ??8121 ??7	??2 ??3 ??1 ??4

1. the sequence homogeneity of 20 strain HBV (comprising adr, adw, ayr and ayw separator).

2. with the allelic number of DR of IC50≤1000nM combination.

In HBV patient and mouse, estimated the immunogenicity (table 12) of vaccine HTL epi-position.

The immunogenicity of table 12.HBV vaccine HTL epi-position

The super type of HLA HLA	??Alt?pep	With ... overlapping	??Ag	Epi-position	Sequence	Core frequency (X/20)	??SEQ?ID ??NO：	Immunogenicity
										HBV patient 1	??H2 bxdMouse 2
								??DR	? ??1186.13				? ??HBV ? ??HBV	? ??pol?412 ? ??pol?664	??LQSLTNLLSS ??NLSWL ??KQAFTFSPT ??YKAFLC ??AGFFLLTRIL	? ??18 ? ??19	? ??55 ? ??56	? ??+ ? ??+	? ??+ ? ??+
??830.01	??1280.08	??HBV ? ??HBV	??env?180 ? ??pol?774	??TIPQS ??GTSFVYVS ??ALNPAD ??VSFGVWIRT ??PPAYRPPNA	??16 ? ??18	??57 ? ??58	??+ ? ??+			??+ ? ??+
									??1186.25			??HBV ? ??HBV ? ??HBV ? ??HBV	??core?120 ? ??pol?145 ? ??env?339 ? ??pol?501	??PI ??RHYLHTLW ??KAGILYK ??LVPFVQWFV ??GLSPTV ??LHLYSHPIIL ??GFRKI ??PFLLAQFTSA	? ? ??20 ? ??19 ? ??16	??59 ? ??60 ? ??61 ? ??62	??+ ? ??+ ? ??+ ? ??+	??+ ? ??+ ? ??- ? ??+
??1186.19	??1186.26	??HBV ? ??HBV ? ??HBV	??pol?523 ? ??pol?618 ? ??pol?767	??ICSVV ??KQCFRKLPV ??NRPIDW ??AANWILRGT ??SFVYVP ??PHHTALRQA ??ILCWGELMT	??19 ? ??16 ? ??16	??63 ? ??64 ??65	??+ ? ??+ ? ??+			??+ ? ??- ? ??-
									??F039.01			??HBV	??core?50	??LA		??66	??+	??-
? ??DR3	? ??35.0100	? ? ? ? ? ? ? ??1186.18	? ??HBV ? ??HBV ? ??HBV ? ??HBV	? ??pol?694 ? ??pol?385 ? ??pol?96 ? ??pol?420	??LCQVFADAT ??PTGWGL ??ESRLVVDFS ??QFSRGN ??VGPLTVNEK ??RRLKLI ??SSNLSWLSL ??DVSAAF	? ??19 ? ??20 ? ? ? ??20	? ??67 ? ??68 ? ??69 ? ??70			? ??+ ? ??+ ? ??- ? ??-									? ??+ ? ??+ ? ??+ ? ??+

1. the Memorability ctl response in the patient that chronic or acute HBV infects

With after peptide/CFA emulsion immunity at H2 ^BXdThe htl response of inducing in the mouse

Except 2 HLA-DR3 epi-positions, in the people that HBV-infects, all epi-positions have been identified.Also use H2 ^BXdMouse has characterized the immunogenicity of HTL epi-position.The preferences of epi-position-peptide combination is similar to HLA-DR1 and IA ^b, this allows the contrast experiment (Wall, K.A. etc., J.Immunol.152:4526-4536 (1994)) in the non-transgenic mouse.12 HTL epi-positions are immunogenic in these mouse, and are measured as the fresh ELISPOT experiment of carrying out in 11-14 days with synthetic peptide (table 12) the immunity back of 25 μ g purifying.

In a word, these data have identified one group and have been applicable to the HTL epi-position that is included in the HBV vaccine constructs.

Colony's coverage in the expectation of HTL epitope levels

The HTL epi-position of selecting is derived from core, pol and env antigen, thereby the chance that produces the polyspecific reaction in the individuality of immunity is provided.These epi-positions also provide high-caliber the most common race's the prediction colony coverage that contained.Table 6B has summed up HLA type and their distributions in common race that is included in the analysis.Have can in conjunction with shown in number II class-restriction epi-position the HLA type individuality calculating phenotypic frequency and predict and can make the cumulative frequency of immunoreactive individuality shown in Figure 26 A-26B to the epi-position of selection hereditarily.We predict that average 17.2 epi-positions-HLA combination can identify in Utopian population, this population is formed (Figure 26 A) by observed average HLA frequency in main race.The average number of Shi Bie epi-position-combination is greater than 16 (sums of epi-position), because the binding ability of epi-position heterozygosity and height degeneracy potentially.Main race's analysis has confirmed colony's coverage (Figure 26 B) very widely.

3. the design of mini gene construct

Background

Our research concentrates on the vaccine constructs that exploitation is made up of a plurality of epi-positions.Many different breadboard studies confirm that can be constructed multi-epitope constructs by in the mode of " pearl string " epi-position being contacted each other.But the immunogenicity of each the CTL epi-position in this construct often is an alterable height.Variation can be owing to the efficiency variance of the cell processing that produces epi-position.We find to guarantee with suitable amino acid sept that the effective protein proteins body is sheared can the processing of balance epi-position and immunogenicity (Velders, M.P. etc., J.Immunol.166:5366-5373 (2001); Livingston, B.D. etc., Vaccine.19:4652-4660 (2001)).

Considered to set up the artificial epi-position possibility of (being called " connecting epi-position ").Connecting epi-position may arrange or change the Direction of Reaction in inappropriate mode, and/or may be with people's (self) thus sequence homology is induced anti-self reaction.Designed computer program, right for each epi-position, it selects to optimize the sept composition that proteosome is sheared, and minimizes the generation of epi-position motif as sept by the amino acid that adds other.Thereby our epigene construct design software has been estimated different epi-position and has been arranged, and the proteosome of having selected to have best prediction is sheared and minimum be connected those of motif generation rate.

It is unessential that optimization HLA-DR shears in conjunction with the proteosome of epi-position, remains main design consideration although avoid connecting epi-position.Because the motif of HLA II quasi-molecule identification is defined more widely, we have designed based on the strategy that uses the general sept of being made up of GPGPG (SEQ ID NO:2) J.Immunol.168:5499-5509 (2002) such as () Livingston.This sept has the ability of the combination of interrupting whole or most of modal HLA-DR types, because compatible relatively poor (Livingston, B. etc., the J.Immunol.168:5499-5506 (2002)) of it and most of people and mouse II class binding motif.

The N-end of epigeneCTL construct comprises sequence MGMQVIQSLFLLLLWVPGS RG (for example, the amino acid/11 of SEQ ID NO:72-22).This is based on the common sequences of Ig κ secretion signal, and is used for the I class MHC target of auxiliary polypeptide product.

Another important elements of vaccine design strategy is comprise (Alexander, J. etc., the Immunity 1:751-761 (1994)) of general HTL epi-position.This non-natural epi-position is used for high affinity in conjunction with modal HLA molecule, and realizes best immunogenicity by maximization TCR contact residues.This HTL epi-position can be induced htl response, to support inducing and increasing (Alexander, J. etc., Immunity 1:752-761 (1994) of ctl response; Alexander, J. etc., Immunol.Res.18:79-92 (1998)).Using is not that the HTL epi-position that is derived from HBV can be provided in the distinct advantages that the CTL that supports in the chronic disease induces the aspect, because the tolerance relevant with chronic infection can partly weaken the specific htl response of HBV-.In fact, verified this HTL epi-position can make immune system overcome the specific T cell tolerance of HBV-(Livingston, B.D. etc., Hum.Immunol.60:1013-1017 (1999) in the transgenic mice of expressing HBV antigen; Livingston, B.D. etc., J.Immunol.162:3088-3095 (1999); Alexander, J. etc., Immunol.Res.18:79-92 (1998); Sette, A.D. etc., J.Immunol.166:1389-1397 (2001)).This HTL epi-position is also contained in the HTL vaccine constructs, because it can strengthen the reaction that other HTL epi-position is induced.Be somebody's turn to do " to assisting of other co-factor " notion with disclosed observed result in the CD40 system is consistent recently, the latter shows that dendritic cells permission (being defined as the accessory molecule on the incremental adjustments dendritic cells that HTL-induces) also can be applied to htl response (Gerloni, M.S. etc., Proc.Natl.Acad.Sci.USA.97:13269-13274 (2000); Van Mierlo, G.J. etc., Proc.Natl.Acad.Sci.USA.99:5561-5566 (2002)).

B. design energy coding source is from the mini gene construct of the CTL of HBV epi-position

First prototype vaccine construct HBV1 comprise 17 HLA-A2 ,-A3 and-the B7 epi-position, and disappearance amino acid sept.Modified this construct (HBV2) to integrate the immunogenicity of suitable interval thing and the many component epi-positions of enhancing.HBV2 has induced the ctl response at the wide spectrum epi-position, the latter generally with those quite (data not shown) of the peptide immune induction of emulsification in IFA.This contrast make people can estimate each defined epitope without any under the situation of processing constraint can detected activity, thereby and the standardization of factors such as the availability of the epi-position-specific TCR inventory in the various mouse strains that are provided for estimating before clinical and size.

Many new epigene constructs be used to comprise HLA-A1 and-the A24 epi-position, so that bigger colony's coverage to be provided.Made up 4 epigene constructs that comprise 21 and 30 CTL epi-positions, and tested immunogenicity, concentrated on the HLA-A2 epi-position.4 all constructs have been induced widely, effective ctl response (data not shown).Because the construct of 30 epi-positions should provide bigger epi-position to cover redundancy in the patient population of expection, other research concentrates on these bigger epigene constructs.The construct HBV30C of 30 specific epi-positions induced at HLA-A3 and-the strong ctl response of A24 epi-position (uses H2 ^BxdMouse has detected the latter).Although the immunogenicity of 2 HLA-A2 epi-positions (core 18 and env 183) in this construct is relatively poor, further sept optimization has recovered the immunogenicity of these epi-positions.The schematic diagram of CTL vaccine HBV30K and amino acid sequence are shown in Figure 27 A and table 13.The example of polynucleotide sequence of HBV 30K of encoding is as shown in table 13.

HLA-A2 and-immunogenicity of HBV 30K in the A11 transgenic mice is shown in Figure 27 B.As a comparison, also shown the CTL activity of inducing with after effective HBV2 prototype building body and the peptide/IFA immunity.In a word, the ctl response that causes of HBV 30K is the same with HBV2 strong.In fact, HBV30K has induced at being the ctl response of immunogenic all components epi-position in the HLA transgenic animal, and typically the reacting phase of inducing after these ctl responses and the peptide immunity is worked as.The result of these data is to select HBV 30K as leading CTL vaccine.

Table 13.HBV30K construct

C. design energy coding source is from the mini gene construct of the HTL of HBV epi-position

Designed an epigene construct of 16 the HTL epi-positions of encoding, it has integrated the general sept of GPGPG (SEQ ID NO:2).The schematic diagram of this HBV HTL construct and amino acid sequence are shown in Figure 28 A and table 14.The example of polynucleotide sequence of HBV HTL construct of encoding is as shown in table 14.At H2 ^BxdDetect the immunogenicity (Figure 28 B) of this construct in the mouse, used the ELISPOT experiment to detect the lymphocytopoietic IFN-γ of CD4+T-.The reaction of 50% test epi-position (6/12) is the same strong with the inducing peptide of emulsification in CFA.In remaining 6 epi-positions, only 2 epi-positions can not induced reaction after using the immunity of HTL vaccine constructs.

Table 14.HBV HTL construct

Minimize the content that connects epi-position effectively

After having defined the epigene construct of CTL and HTL vaccine constructs, we have carried out more deep sign.At first, we have examined computer based epigene construct design effort and have minimized the existence that connects epi-position effectively.Use motif scanning to detect the content that is connected epi-position of CTL and HTL component, and contrast with identical CTL and two groups of randomized arrangement of HTL epi-position.The result is as shown in Table 15.

Table 15. minimizes the example that connects epi-position in vaccine constructs

Construct	Scheme	?SEP?ID?NO：	The CTL motif that connects 4
				??HBV?30k′	Minimized		??1
??HBV?30R1	At random 2		??84
				??HBV?30R2	At random		??99
??HBV?HTL′	??GPGPG	??2	??12
				??HBV?HTL?NS1	No sept 3		??42
??HBV?HTL?NS2	No sept		??37

1. vaccine CTL and HTLepigene construct.

2. to processing the randomized arrangement of the CTL epi-position of optimizing.

3. the HTLepigene construct that does not have sept.

4. carry HLA-A1 ,-A2 ,-A3 ,-number of the connection epi-position of A24 or B7 motif.

Compare with randomized arrangement, the decreased number of the connection epi-position in the CTL epiposition vaccine of optimal design about 100 times.Although the HTL component can not minimize the connection CTL epi-position of existence specifically, use GPGPG sept (SEQ ID NO:2) is eliminated the HTL functional epitope in the specific HTL epi-position of the HBV-string, can make the connection CTL epi-position of existence reduce about 4 times really.When analyzing CTL epi-position string, do not consider to connect the HTL epi-position, because this epi-position that exists in the CTLepigene construct only is used to stimulate nonspecific auxiliary, great majority are with the mode (Alexander identical with above-mentioned general HTL epi-position, J. etc., Immunity 1:751-761 (1994)).

Carried out blast search, to estimate the autoploidy potentiality of the bonding pad in HBV CTL and the HTLepigene construct.As list entries, we have studied 47 sequences being made up of following key element: 4 C-end residues of epi-position, 4 N-end residues of intervening sequence itself (if existence) and following epi-position.About the blast search parameter, we have used search option " Short nearly exact matches ".In order to carry out the search of minimum strict standard, we have used the default setting on the webpage; Expected value is 20,000, and the word string size is set at 2.Biological hurdle is defined as homo sapiens (Homos apiens).Table 16 has been listed the result.

There is not bonding pad with anyone sequence 100% homology.The highest autoploidy is 78%, and minimum is 54% (average 63 ± 7.4).In order to contrast, the chance sample of 7 CTL and 4 HTL HBV epi-positions carried out the autoploidy search (table 17) that is equal to.

Detected best autoploidy is 67%, and the poorest is 30% (average 54 ± 13).

Table 16. connects people's autoploidy Search Results of motif based on the epigene construct

The bonding pad	The source	??SEQ?ID ??NO	Sequence	The % autoploidy	Catalog number (Cat.No.)
						??1	The agnoprotein of pol 562-NAAA-pol 745 MGC:20975	??75 ? ? ? ??76	??GIHLNAAAKYTS ? ? ? ??GIHLN *AA ****	??-- ? ? ? ??58	? ? ? ? ??AAH14187
??2	Pol 745-NAAA-env 332 sulfonylureas acceptors 1	??77 ? ??78	??PWLLNAAARFSW ? ??PWLLNA ******	??-- ? ??50	? ??AAC36724.1
						??3	Env 332-NAA-pol 530 KIAA1219 albumen	??79 ??80	??LVPFNAAFPHC ? ***F+AAF *HC	??-- ??55	? ??BAA86533.2
??4	The albumen of pol 530-KAA-pol 388 supposition	??81 ??82	??FSYMKAALVVD ??FSYMKAA ****	??-- ??63	??XP_073807.1
						??5	The albumen FLJ22313 of the albumen supposition of pol 388-GA-env 249 supposition	??83 ??84 ? ??85	??QFSRGAJLLL ??QFS *GAIL **? ? *FSR *AILL *	??-- ??70 ? ??70	? ??XP_066589.1 ? ??NP_071768.2
? ??6	The albumen of env 249-NAAA-pol 149 supposition	? ??86 ? ??87	? ??IFLLNAAAHTLW ? ?? **LLNA **H *LW	? ??-- ? ??58	? ? ? ??XP_060325.1
						??7	The albumen FLJ14753 of pol 149-KA-env 359 nebulins supposition	??88 ??89 ? ??90	??ILYKKAWMMW ??JLYK *AW ***? ? *****AWMMW	??-- ??60 ? ??50	? ??AAB02622.1 ? ??AAH21093
??8	??pol?149-KA-pol?640 ??Intergrase?interactor ??1	??91 ??92	??PSLYKAYPAL ? *SLYK *YP+L	??-- ??70	? ??AAA81905.1
						??9	??pol?640-GAA-env?335	??93	??YACIGAAWLSL	??--

	Steroids 18-hydroxylase CGI-67 albumen	? ??94 ??95	? ? **C+ *A *WLSL ??YA *I *AAWL+L	? ??55 ??73	? ??AAB34642.1 ??AAD34062.1
						??10	The albumen KIAA1742 albumen of env 335-NAAA-env 183 supposition	? ??96 ? ??97 ??98	? ??VPFVNAAAFLLT ? ? *PFVNA **FL **? *PFVN *AA *LL *	? ??-- ? ??58 ??67	? ? ? ??CAD38882.1 ??BAB21833.2
??11	??env?183-NAAA-env ? ??313 ??hRANKL2	? ??99 ? ??100	? ??ILTINAAAIPIP ? ? *LTINA **IP **	? ??-- ? ??58	? ? ? ??AAC517.62.1
						??12	Env 313-KAAA-core 117 AP-2heta transcription factors	? ??101 ? ? ??102	? ??SWAFKAAAEYLV ? ? ? ****KAAAEYL *	? ??-- ? ? ??58	? ? ? ? ??CAC04182.1
??13	The albumen of core 117-N-core 19 supposition	??103 ??104	??FGVWNLPSD ? *GVWNL *SD	??-- ??78	? ??CAD38975.1
						??14	Core 19-KAAA-core 18 newborn polypeptide related complexes	? ??105 ? ??106	? ??FPSVKAAAFLPS ? ? *PS *KAAAFL **	? ??-- ? ??67	? ? ? ??XP_061543.1
??15	Core 18-KAAA-core 419 zinc finger protein 64s	? ??107 ? ??108	? ??FPSVKAAADLLD ? ? **SVKAA++LL *	? ??-- ? ??58	? ? ? ??XP_087479.4
						??16	Core 419-N-pol 392 immunoglobulin kappa VLJ districts	??109 ??110	??SALYNSWPK ? ***YN+WPK	??-- ??55	? ??AAM46537.1
??17	Pol 392-KAAA-pol 531 DNA poly.epsilon catalytic subunits	??111 ? ??112	??VPNLKAAASAIC ? ? **NLKAAAS ***	??-- ? ??58	? ? ??AAA15448.1
						??18	The albumen of pol 531-K-pol 415 supposition	??113 ??114	??VVRRKLSLD ??V+RRK+SLD	??-- ??78	??XP_064183.1
??19	Pol 415-NAA-padre potassium voltage-gated channel	??115 ??116	??AAFYNAAAKFV ? *AFYN *A+KF *	??-- ??64	? ??AAH27932.1
						??20	Padre-KAA-pol 47 laminin β precursors	??117 ??118	??KAAAKAANVSI ??KAA *KAA+ **	??-- ??64	??AC005048.1
??21	The albumen of pol 47-GAA-pol 455 supposition	??119 ??120	??WTHKGAAGLSR ??WTHKG+ *GL+R	??-- ??73	??XP_117843.1
						??22	The albumen of pol 455-NAAA-core 141 solute carrier families 39 (zinc transporter) supposition	??121 ? ? ??122 ??123	??VARLNAAASTLP ? ? ??VARE+AAA ****??VA *L *AAA+TL *	??-- ? ? ??58 ??67	? ? ? ??NP_060237.1 ??XP_120525.1
??23	The albumen of core 141-K-pol 429 supposition	??124 ??125	??WRRKHPAA ??VRRKHP *A *	??-- ??78	? ??XP_117855.1
						??24	The albumen of pol 429-KAAA-env 236 supposition	??126 ? ??127	??PHLLKAAARWMC ? ? **LL *AA *RW *C	??-- ? ??58	? ? ??XP_105701.1
??25	??env?236-N-pol?166	??128	??RFIINASFC	??--

	The albumen of supposing	??129	??RFII+A *F *	??67	??XP_072766.5
						??26	The albumen of pol 166-KAA-pol 538 supposition	??130 ??131	??GSPYKAAYMDD ? **PY **AYMD *	??-- ??54	??AAH01463
??27	The albumen of the albumen supposition of pol 538-NA-core 101 supposition	??132 ??133 ??134	??VLGVNALWFH ? **GV+ALWF *??VL *+ *ALWFH	??-- ??60 ??70	? ??XP_118305.1 ??XP_059358.1
						??28	Core 101-KAAA-pol 354 KIAA1853protein α 1 type XIII collagen	??135 ? ??136 ? ??137	??CLTFKAAATPAR ? ? **TFKA *ATP **? ? **T *KAAAT *AR	??-- ? ??58 ? ??67	? ? ??BAB47482.1 ? ??NP_542994.1
??29	Pol 354-KAAA-core 137 the unknowns	??138 ? ??139	??GGVFKAAALTFG ? ? *GV **AA+LTFG	??-- ? ??67	? ? ??AE006639.1
						??30	Albumin X-linked mental retardation cand. the gene of core 137-K-pol 665 supposition	??140 ??141 ? ??142	??VLEYKQAFT ??VL+YKQ *F *? ??VL *YKQ *FT	??-- ??67 ? ??78	? ??XP_101671.1 ? ??CAA65075.1
??31	Pol 665-GPGPG-pol 774 sialyltransferases 1 N2B-Titin isoform	??143 ? ??144 ??145	??PTYKGPGPGGTSF ? ? **YKGPGPG ****? **YK *PGP *GT *F	??-- ? ??54 ??61	? ? ??CAA35111.1 ??CAD12455.1
						??32	Pol 774-GPGPG-pol 694 Gorky's antigens	??146 ? ??147	??NPADGPGPGLCQV ? ??NPAD *PGPG ****	??-- ? ??61	? ? ??AAC06338.1
??33	Pol 694-GPGPG-pol 145 L-myc-l proto-oncogene proteins	??148 ? ??149	??GWGLGPGPGRHYL ? ? *WGLGPG *G ****	??-- ? ??54	? ? ? AAA59879.1
						??34	Pol 145-GPGPG-core 50 sialyltransferases 1	??150 ? ??151	??ILYKGPGPGPHHT ? ? **YKGPGPG ****	??-- ? ??54	? ? ??CAA35111.1
??35	The albumen mitogen-activated protein kinase of core 50-GPGPG-pol 385 supposition	??152 ? ? ??153 ? ??154	??MTLAGPGPGESRL ? ? ? **LAGPGPG *SR *? ? ****GPG *GESRL	??-- ? ? ??69 ? ??61	? ? ? ??XP_069591.1 ? ??XP_027237.1
						??36	Pol 385-GPGPG-pol 523 protein kinase C mu CD1-alpha-3 antigens	??155 ? ??156 ??157	??SRGNGPGPGPFLL ? ? **G+GPGP *PFL *??SRG **PGPG **LL	??-- ? ??61 ??69	? ? ??CAA53384.1 ??AAA51935.1
??37	Pol 523-GPGPG-env 339 induction type nitric oxide synthases	??158 ? ? ??159	??CSWGPGPGLVPF ? ? ??C *++GPG *G+VPF	??-- ? ? ??61	? ? ? ??AAL02120.1
						??38	??env?339-GPGPG-pol ??501 ??Atrophin-1	??160 ? ??161	??SPTVGPGPGLHLY ? ??SPTVGPGP *****	??-- ? ??61	? ??S50832
??39	The albumen of the albumen supposition of pol 501-GPGPG-pol 420 supposition	? ??162 ??163 ??164	??FRKIGPGPGSSNL ? *RKI *PGPG ****? *RKI **G *GSSN *	??-- ? ??54 ??61	? ? ??XP_069589.1 ??XP_169769.1

??40	Pol 420-GPGPG-pol 412 Epsin 2b albumen	??165 ? ??166	??SAAFGPGPGLQSL ? ??S *+FGPGPGr++S+	??-- ? ??61	? ? ??AAC78609.1
						??41	The protein product that the albumen of pol 412-GPGPG-env 180 supposition is named	? ??167 ??168 ? ??169	? ??LSWLGPGPGAGFF ??LSWLGPG *G ****? ? ***LGPGP **GFF	? ??-- ??61 ? ??61	? ? ??XP_097563.1 ? ??BAC05301.1
??42	Env 180-GPGPG-core 120 transmembrane proteins	??170 ? ??171	??IPQSGPGPGVSFG ? ? **PQ+GPGPGV **	??-- ? ??61	? ? ??AAC64943.1
						Protein product neuregulin 2 isotypes 4 that 43 core 120-GPGPG-pol 96 are unnamed		??172 ? ? ??173 ??174	??NAPIGPGPGVGPL ? ? ? ****GPGPG *GPL ? *AP *GPGPG *GP *	??-- ? ? ??61 ??69	? ? ? ??BAC05043.1 ??AAF28851.1
??44	Pol 96-GPGPG-pol 618 agnoproteins	??175 ? ??176	??LKLIGPGPGKQCF ? ??LKL *GPGPG ****	??-- ? ??61	? ? ??AF318376.1
						??45	The albumen of pol 618-GPGPG-pol 767 supposition	??177 ? ??178	??PIDWGPGPGAANW ? ? **DWGPGPG ****	??-- ? ??54	? ? ??XP_066062.1
??46	Pol 767-GPGPG-pol 664 TAF4RNA polymerase IIs	??179 ? ??180	??VYVPGPGPGKQAF ? ? ***PGPGPGK *A *	??-- ? ??61	? ??XP_036470.2
						47 pol 664-GPGPG-padre POLYCYSTIC KIDNEY DISEASE, 1 albumen		??181 ??182	??AFLCGPGPGAKFV ? **LCGP *PGA ***	??-- ??54	??AAC37576.1

1. use 4 interval groups that close on residue containing from adjacent epi-position as search sequence.

2.BLAST search parameter: desired value 20000, word string size 2, matrix PAM30, comparison number 250

3. at first shown sequences match as a result with minimum E value.

● the sequence that does not identify has E value＜1.

4., then also show them if identified other sequence with bigger autoploidy.

5. unmatched residue represented in asterisk (*); The residue that plus sige (+) expression has similar chemical composition.

People's immunology Search Results that table 17. uses random table precedence preface to carry out

Bonding pad Region	The source	??SEQ ??ID ? ??NO：	Sequence	The % autoploidy	Catalog number (Cat.No.)
						??4	Pol 530 endothelin receptor B delta 3	??183 ??184	??FPHCLAFSYM ? **HCLAFS **	??-- ??60	??AF114165.1
??8	The albumen FLJ12389 of pol 149 supposition	??185 ? ??186	??WMMWYWGPSLY ? ??WMMW *W *****	??-- ? ??45	? ? ??NP_076417.1
						??11	The protein D KFZ of env 183 supposition	??187 ??188	??FLLTRILTI ? *LLTR+LT *	??-- ??67	??AAH30825.1
??13	Core 177 Robison ester translocases	??189 ? ??190	??EYLVSFGVW ? ? *YLV *FGV *	??-- ? ??67	? ? ??CAA75608.1
						??15 ??T	Core	18 cell-stimulating NFKB sample albumen	??191 ??192	??FLPSDFFPSV ??FLP *DF+P **	??-- ??60	??NP_116110.2
??18	Pol 531 strides film 4-domain (MS4A8B) albumen	??193 ? ??194	??SAICSVVRR ? ??SAICS *V **	??-- ? ??67	? ? ??AF237905.1
						??24	The albumen of pol 429 supposition	??195 ??196	??HPAAMPHLL ??H *AAMPH **	??-- ??67	??XP_120541.1
??35	Core 50 cysteine dioxygenases	??197 ? ? ??198	??PHHTALRQAILCWGE ??LMTLA ? *********ILCWGE ****? ? *	??-- ? ? ??30	? ? ? ??BAA12873.1
						??36	Pol 385 B/K albumen	??199 ??200	??ESRLVVDFSQFSRGN ? ****VVDF *+FSR **	??-- ??47	??NP_057608.1
??38	Env 339 cytochrome B5s 61	??201 ??202	??LVPFVQWFVGLSPTV ? ***FVQW *VG *S ***	??-- ??47	??AAC50212.1
						??47	The albumen FLJ23441 of pol 664 supposition	??203 ? ??204	??KQAFTFSPTYKAFLC ? ? *Q *FTF *PT++A ***	??-- ? ??47	? ??AAH07800

1. use 4 interval groups that close on residue containing from adjacent epi-position as search sequence.

2.BLAST search parameter: desired value 20000, word string size 2, matrix PAM30, comparison number 250

3. at first shown sequences match as a result with minimum E value.

● do not identify sequence (except the human hepatitis B virus capsid) with E value＜1.

4. unmatched residue represented in asterisk (*); The residue that plus sige (+) expression has similar chemical composition.

In a word, vaccine bonding pad and human sequence's autoploidy degree and other sequence do not have difference, and the latter is HBV epi-position itself for example, its by immune system think right and wrong-self, and relevant with autoimmune disorder.

E. effective optimization that antigen is processed and epi-position is presented

For making up physical efficiency, the epigene that confirms to optimize effectively epi-position is transported in the immune system, with the vaccine constructs transfection people's lymph matricyte system, the APC (Livingston, B.D. etc., Vaccine.19:4652-4660 (2001)) that it is tested as antigenicity subsequently.These experiments provide the method that detects the relative quantity of the epi-position that produces as processing result.Use these experiments, Livingston has confirmed that sept optimization can improve the output of specificity epitope nearly thousand times (Livingston, B.D. etc., Vaccine.19:4652-4660 (2001)).Use various vaccine constructs, comprise GCR-5835 (as follows), produced transformant, as construct based on full HBV gene.These results show, are efficiently based on the processing optimization method of specific interval residue.And, be the people source owing to be used for cells transfected system, these data provide important checking in robot system for the The above results that obtains in the HLA-transgenic mice.

4. the configuration of vaccine, preparation and conveying

The vaccine configuration

Design independently and optimized HTL and CTLepigene construct.But, think that in the unique DNA vaccine transport of H TL and CTL component are best altogether.3 vaccine replacement schemes comprise: (1) uses 2 CMV promotors of separating; (2) use is with the CMV promotor of IRES; (3) can in single reading frame, the encode construct (Figure 29 A) of CTL+HTL component.About the example of the 3rd alternative, see Table 18 and 19 (for example GCR-5835 and GCR-3697).Use the HLA-A2 transgenic mice to estimate the immunogenicity of these Different Strategies; The result is shown in Figure 29 B.Obviously, every kind of configuration has all been induced the basic ctl response at all HLA-A2 epi-positions that equates.In a word, the performance of the construct of fusion and other vaccine configuration quite or better than it in view of its simplicity, think that at present it is leading vaccine configuration.

In order to set up the vaccine that is more suitable for human and to improve immunogenicity potentially, modified the nucleotide sequence (table 18) of GCR-5835, with match people codon frequency, raising mRNA stability and minimizing mRNA secondary structure.In the HLA-A2 transgenic mice, will be called the immunogenicity of construct and the contrasting of GCR-5835 of the modification of GCR-3697 (table 19).GCR-3697 that optimizes with 5 μ g or 50 μ g people or the immunity of GCR-5835 intramuscular animal, and use elementary IFN-γ ELISPOT experiment to detect ctl response (Figure 30).When 50 μ g dosage, in animal, average 5 times have been improved at the magnitude of the ctl response of epi-position core 18, env 183 and pol 538 with the GCR-3697 immunity.The htl response that these 2 constructs are induced roughly is (data not shown) that is equal to.Because as if the GCR-3697 vaccine constructs has the effectiveness of stronger CTL immunogenicity aspect, and may have the immunogenic character that can strengthen the people, selecting it is rational as main vaccine.

Epigene fusion constructs in the table 18.GCR-5835 plasmid

Epigene fusion constructs in the table 19.GCR-3697 plasmid

The vaccine ingredients

Do not confirm that as yet it is best (Wang, etc., Science 282:476 (1998)) that naked DNA vaccine is carried vaccine immunogens in human body.Therefore, we have selected alternative ingredients, and it is based on the use of polymeric surfactant polyvinylpyrrolidone (PVP).Tissue distribution, protection DNA that the induction system of this non-condensation is used to improve DNA avoid degraded and strengthen cell taking in.Usually as the pharmaceutical dosage form excipient, it is nontoxic to PVP, and is approved for people's clinical practice.As if the character of PVP and the mechanism of action closely similar with non-ionic block copolymer C RL1005.Finished safety, toxicity and bio distribution/removing experiment, be applied to the HIV-1 vaccine to support it.As if these data have not only been supported the safety of ingredients, and the absorption of the cell of DNA has also improved above tens of times (alog) than naked DNA.Thereby available data can be supported the application of this induction system.

Use HIV-1 vaccine and several HBV vaccine constructs, we have estimated the immunogenic influence of PVP to CTL and HTL epi-position.Use data acknowledgement that the HIV-1 epi-position of epigene construct coding obtains PVP can improve the immunogenicity of epi-position, immunogenicity relatively poor (data not shown) when this epi-position is carried in naked DNA vaccine.3 kinds of different ingredients, promptly in the pre-background that causes of PVP, naked DNA and cardiotoxin (CT), estimated the immunogenicity of GCR-5835.The CT preliminary treatment is the experimental technique that strengthens the validity of naked DNA injection in being usually used in laboratory animal.CT can destroy meat fiber, and they take in DNA (Davis, H.L. etc., Mol.Genet.2:1847-1851 (1993)) then when regeneration.The result as shown in figure 31.Although the CT preliminary treatment is being the most effective aspect the reaction that causes high-magnitude, this method can not be used clinically.Compare with naked DNA, the DNA of PVP-preparation has improved 2 reaction magnitude in 6 epi-positions that detect, and makes the frequency gets higher of 5 positive reaction in 6 epi-positions simultaneously.These data show, carry with naked DNA and compare, and the PVP ingredients can improve the effectiveness of vaccine.

The method of administration of vaccine and conveying

Can carry the DNA plasmid vaccine of PVP preparation in the interior ground of muscle (i.m.).Intramuscular method of administration is generally used for dna vaccination.In preliminary experiment, we have used HBV prototype epigene construct pMin1 to estimate various DNA transport way (table 20).In these experiments, intramuscular pin carried and the needleless of the DNA of the PVP preparation by Biojector is carried and the bombardment conveying of the golden particulate/DNA by PowderJect contrasts.In a word, the performance that intramuscular pin is carried and other carrying method of test quite or better, although other carrying method also can use.

Improvement to the naked DNA vaccine technology

Verified naked DNA vaccine is relative relatively poor immunogene in inhuman primate and people, but the research of finishing so far all is based on the epi-position that conveying can be encoded the complete genome of full-length proteins or be optimized through sept.As if although have suitable relatively people's immunogenicity, the naked DNA immunity is " initiation " ctl response (Ramshaw, J.A. and Ramsay, A.J., Immunol.Today 21:163-165 (2000)) very effectively.

The absorption that the design of use epigene construct and the adding of PVP improve DNA.The epigene construct can comprise miniplasmids dna backbone and little vaccine insert, compares with the bigger construct of test clinically, and its cell that can improve DNA is taken in.

Table 20. is used to induce the contrast of the DNA transport way of ctl response

Relatively be used to induce the DNA transport way of ctl response
				Carry	Immunogenicity (SU) 1
??HBVcore18	??HIV?pol?476	??HBV?pol?455
			?IM ?ID ?Biojector		??1342.9(1.8) ??740.1(1.5) ??44.7(3.9)	??1133.3(1.3) ??0 ??103.2(1.4)	??879.5(2.1) ??0 ??44.8(3.1)

Carry	Immunogenicity (SFC/10 6Cd8 cell) 2
							??HBV?core?18	??HIV?pol?476	??HBV?pol?455	??HIV?env?120	??HBV?pol?551	??HBV?env?335
	IM Gene?Gun	??285.0(17.4) ??287.5(23.8)	??147.5(19.8) ??146.7(24.2)	??155.0(15.0) ??25.8(7.5)	??60.0(15.6) ??0.8(5.5)	??485.0(24.9) ??35.5(7.6)							??68.3(8.2) ??35.8(16.2)

1. the immunogenicity of the pMin1 when using different transport way among the HLA-A2.Use original position ELISA experiment to detect ctl response (McKinney, D.M. etc., J.Immunol.Methods.237:105-117 (2000)).

2. the immunogenicity of the pMin1 when using the immunity of IM pin or particle gun among the HLA-A2.Use elementary IFN-γ ELISPOT to detect ctl response.

Think use earlier dna vaccination re-use albumen or viral vectors strengthen the initiation-reinforcement scheme of the allos of reaction be gene vaccine immunogenic scheme (Ramshaw arranged at present most, J.A. and Ramsay, A.J., Immunol.Today 21:163-165 (2000)). the initiation of allos-reinforcement scheme can be as the part of HBV vaccine delivery.

5. the effectiveness of vaccine and sign

A. obtained immunogenic related levels at the HLA-transgenic mice

In the HLA-A2-transgenic mice, estimated the magnitude of using the reaction that the GCR-5835 vaccine obtains, and contrasted with reaction with experimental lipopeptid vaccine CY-1899 immune induction.Selected this lipopeptid vaccine to be used for this evaluation,, and can in healthy people, cause effective ctl response (Livingston, B.D. etc., J.Immunol.159:1383-1392 (1997)) because core 18 epi-positions are present among vaccine constructs and the CY-1899.The reaction of inducing in mouse shown in figure 32.The splenocyte of the GCR-5835 construct mice immunized of using by oneself has generated the IFN-gamma reaction at the epi-position of all 6 HLA-A2-restrictions of encoding in construct; Use the ELISPOT experiment to carry out detecting (Figure 32 A).Also observed reaction, but magnitude is starkly lower than the core 18 epi-positions reaction of using the GCR-5835 vaccine constructs to induce at 18 epi-positions of the core among the CY-1899.But, stimulate 6 days again with peptide after, these 2 multi-form vaccine-induced cores 18 reactions closely similar (Figure 32 B).

Those that find that the magnitude of the reaction that the epi-position with other A2-restriction obtains can mediation remove the HBV infection with known are compared.We have observed from about 100SFC/10 ⁶CD8+ cell (env 335) is extremely greater than 300SFC/10 ⁶The elementary ELISPOT reaction of CD8+ cell (env 183) is just in the scope of detected other reaction in as the described acute infection of 1A part.

B. Fan Ying quality

The removing of HBV is by a series of comprehensive molecular events mediations, comprises the direct cracking of the incident of indirect lymphokine mediation and infected cell (particularly carrying those of virus of integration).Detected the generation of IFN-γ in described all experiments so far, IFN-γ is correlated with, because this lymphokine has participated in removing (Chisari, F.V. and Ferrari, C.Annu.Rev.Immunol.13:29-60 (1995) that HBV infects; Guidotti, L.G. etc., Immunity.4:25-36 (1996)).Verified dna immunization can be induced the CTL (Ishioka, G.Y. etc., J.Immunol.162:3915-3925 (1999)) with lytic activity.The HLA-transgenic mice also can be used GCR-5835 and/or GCR-3697 immunity.

Use has usually obtained described immunization experiment result from the mixed product of the splenocyte of 3-6 mouse.Carried out other experiment, to detect the reaction of whether in each animal, having induced at a plurality of epi-positions.With the interval in a week, be used in the GCR-5835 for preparing among the PVP, immune HLA-A2 transgenic mice 1 time or 2 times.Collect splenocyte from each animal respectively, stimulate again with 6 HLA-A2 epitope peptide mixtures of encoding in the vaccine.Use the ELISPOT experiment to detect the IFN-γ secretion that each peptide causes then.Behind the single immunization, all mouse all reacts at least one epi-position, and average response rate is 4.2 ± 2.0 epi-positions/mouse (Figure 33 A).After the 2nd immunity, the average number of the epi-position that identifies is increased to 5.6 ± 0.5 (Figure 33 B).Consider the data of recent immunodominance aspect, these data have special relevance (Rodriguez, F. etc., J.Virol.76:4251-4259 (2002)), and show immunogene optimization and repeat the immunity narrow reaction (Chen that immunodominance causes that can be used to contend with, M. etc., J Virol.74:7587-7599 (2000); Yewdell, J.W. etc., Annu.Rev.Immunol.17:51-88 (1999)).

6. sum up and conclusion

Multi-epitope CTL/HTLepigene makes up physical efficiency immunization therapy chronic HBV infection effectively, also can be used for the treatment of, chronically infected individuality treatment through antiviral agent.

Described above and be used to differentiate the CTL that is applicable to vaccine design and the method for HTL epi-position.Colony's coverage of the expectation that these epi-position groups in different ethnic backgrounds are provided is consistent with width and many specific phases of the reaction of the solution of following HBV to infect naturally with the immune response redundancy.Be used to assemble optimization (CTL epi-position) and minimize (the HTL epi-position) that is connected motif that the vaccine design method of multi-epitope constructs needs proteosome to shear.

Produced the particular vaccine construct, it can be induced at great majority in the HLA-transgenic mice or all test the effective ctl response of epi-position.The magnitude of the HBV epi-position that vaccine constructs is induced in transgenic mice-specific CTL level and the response class that uses CY-1899 vaccine (known its be immunogenic in human body) to induce seemingly, and with to solve in the HBV course of infection in human body the level of observed ctl response similar.

In addition, we have confirmed that different vaccine configurational energies carries CTL and HTL epi-position effectively simultaneously.The epigene construct can contain HTL and CTL epi-position, and they are synthetic from single hereditary insert conllinear ground (co-linearly), and vaccine can easily be produced like this, and is stable.

Compare with naked DNA, relevant based on the DNA preparation of PVP with the activity of enhancing.Similarly, intramuscular conveying is seemingly the most feasible in the system of research, and its activity is the same good with other carrying method (Biojector or particle gun) at least.Can also use following combination: the epigene construct that is used in the optimization of preparing among the PVP causes, and strengthens with viral vectors subsequently.

Table 21: the codon utilization table of people's gene (homo sapiens)

Amino acid	Codon	Number	Frequency
				??Phe	??UUU	?326146	??0.4525
??Phe	??UUC	?394680	??0.5475
				Amount to		?720826
				??Leu	??UUA	?139249	??0.0728
??Leu	??UUG	?242151	??0.1266
				??Leu	??CUU	?246206	??0.1287
??Leu	??CUC	?374262	??0.1956
				??Leu	??CUA	?133980	??0.0700
??Leu	??CUG	?777077	??0.4062
				Amount to		?1912925
				??Ile	??AUU	?303721	??0.3554
??Ile	??AUC	?414483	??0.4850
				??Ile	??AUA	?136399	??0.1596
Amount to		?854603
??Met	??AUG	?430946	??1.0000
				Amount to		?430946
				??Val	??GUU	?210423	??0.1773
??Val	??GUC	?282445	??0.2380
				??Val	??GUA	?134991	??0.1137
??Val	??GUG	?559044	??0.4710
				Amount to		?1186903
				??Ser	??UCU	?282407	??0.1840
??Ser	??UCC	?336349	??0.2191
				??Ser	??UCA	?225963	??0.1472
??Ser	??UCG	?86761	??0.0565
				??Ser	??AGU	?230047	??0.1499
??Ser	??AGC	?373362	??0.2433
				Amount to		?1534889
				??Pro	??CCU	?333705	??0.2834
??Pro	??CCC	?386462	??0.3281
				??Pro	??CCA	?322220	??0.2736
??Pro	??CCG	?135317	??0.1149
				Amount to		?1177704
				??Thr	??ACU	?247913	??0.2419
??Thr	??ACC	?371420	??0.3624

Amino acid	Codon	Number	Frequency
				??Thr	??ACA	?285655	??0.2787
??Thr	??ACG	?120022	??0.1171
				Amount to		?1025010
				??Ala	??GCU	?360146	??0.2637
??Ala	??GCC	?551452	??0.4037
				??Ala	??GCA	?308034	??0.2255
??Ala	??GCG	?146233	??0.1071
				Amount to		?1365865
				??Tyr	??UAU	?232240	??0.4347
??Tyr	??UAC	?301978	??0.5653
				Amount to		?534218
				??His	??CAU	?201389	??0.4113
??His	??CAC	?288200	??0.5887
				Amount to		?489589
				??Gln	??CAA	?227742	??0.2541
??Gln	??CAG	?668391	??0.7459
				Amount to		?896133
				??Asn	??AAU	?322271	??0.4614
??Asn	??AAC	?376210	??0.5386
				Amount to		?698481
				??Lys	??AAA	?462660	??0.4212
??Lys	??AAG	?635755	??0.5788
				Amount to		?1098415
				??Asp	??GAU	?430744	??0.4613
??Asp	??GAC	?502940	??0.5387
				Amount to		?933684
				??Glu	??GAA	?561277	??0.4161
??Glu	??GAG	?787712	??0.5839
				Amount to		?1348989
				??Cys	??UGU	?190962	??0.4468
??Cys	??UGC	?236400	??0.5532
				Amount to		?427362
				??Trp	??UGG	?248083	??1.0000
Amount to		?248083
??Arg	??CGU	?90899	??0.0830

Amino acid	Codon	Number	Frequency
				??Arg	??CGC	?210931	??0.1927
??Arg	??CGA	?122555	??0.1120
				??Arg	??CGG	?228970	??0.2092
??Arg	??AGA	?221221	??0.2021
				??Arg	??AGG	?220119	??0.2011
Amount to		?1094695
??Gly	??GGU	?209450	??0.1632
				??Gly	??GGC	?441320	??0.3438
??Gly	??GGA	?315726	??0.2459
				??Gly	??GGG	?317263	??0.2471
Amount to		?1283759
Terminator	??UAA	?13963
				Terminator	??UAG	?10631
Terminator	??UGA	?24607

Table 22: the codon utilization table of little musculus cdna (Mus musculus)

Amino acid	Codon	Number	Frequency
				??Phe	??UUU	??150467	??0.4321
??Phe	??UUC	??197795	??0.5679
				Amount to		??348262
				??Leu	??UUA	??55635	??0.0625
??Leu	??UUG	??116210	??0.1306
				??Leu	??CUU	??114699	??0.1289
??Leu	??CUC	??179248	??0.2015
				??Leu	??CUA	??69237	??0.0778
??Leu	??CUG	??354743	??0.3987
				Amount to		??889772
				??Ile	??AUU	??137513	??0.3367
??Ile	??AUC	??208533	??0.5106
				??Ile	??AUA	??62349	??0.1527
Amount to		??408395
??Met	??AUG	??204546	??1.0000
				Amount to		??204546
				??Val	??GUU	??93754	??0.1673
??Val	??GUC	??140762	??0.2513
				??Val	??GUA	??64417	??0.1150

Amino acid	Codon	Number	Frequency
				??Val	??GUG	??261308	??0.4664
Amount to		??560241
??Ser	??UCU	??139576	??0.1936
				??Ser	??UCC	??160313	??0.2224
??Ser	??UCA	??100524	??0.1394
				??Ser	??UCG	??38632	??0.0536
??Ser	??AGU	??108413	??0.1504
				??Ser	??AGC	??173518	??0.2407
Amount to		??720976
??Pro	??CCU	??162613	??0.3036
				??Pro	??CCC	??164796	??0.3077
??Pro	??CCA	??151091	??0.2821
				??Pro	??CCG	??57032	??0.1065
Amount to		??535532
??Thr	??ACU	??119832	??0.2472
				??Thr	??ACC	??172415	??0.3556
??Thr	??ACA	??140420	??0.2896
				??Thr	??ACG	??52142	??0.1076
Amount to		??484809
??Ala	??GCU	??178593	??0.2905
				??Ala	??GCC	??236018	??0.3839
??Ala	??GCA	??139697	??0.2272
				??Ala	??GCG	??60444	??0.0983
Amount to		??614752
??Tyr	??UAU	??108556	??0.4219
				??Tyr	??UAC	??148772	??0.5781
Amount to		??257328
??His	??CAU	??88786	??0.3973
				??His	??CAC	??134705	??0.6027
Amount to		??223491
??Gln	??CAA	??101783	??0.2520
				??Gln	??CAG	??302064	??0.7480
Amount to		??403847
??Asn	??AAU	??138868	??0.4254
				??Asn	??AAC	??187541	??0.5746
Amount to		??326409

Amino acid	Codon	Number	Frequency
				??Lys	??AAA	??188707	??0.3839
??Lys	??AAG	??302799	??0.6161
				Amount to		??491506
				??Asp	??GAU	??189372	??0.4414
??Asp	??GAC	??239670	??0.5586
				Amount to		??429042
				??Glu	??GAA	??235842	??0.4015
??Glu	??GAG	??351582	??0.5985
				Amount to		??587424
				??Cys	??UGU	??97385	??0.4716
??Cys	??UGC	??109130	??0.5284
				Amount to		??206515
				??Trp	??UGG	??112588	??1.0000
Amount to		??112588
??Arg	??CGU	??41703	??0.0863
				??Arg	??CGC	??86351	??0.1787
??Arg	??CGA	??58928	??0.1220
				??Arg	??CGG	??92277	??0.1910
??Arg	??AGA	??101029	??0.2091
				??Arg	??AGG	??102859	??0.2129
Amount to		??483147
??Gly	??GGU	??103673	??0.1750
				??Gly	??GGC	??198604	??0.3352
??Gly	??GGA	??151497	??0.2557
				??Gly	??GGG	??138700	??0.2341
Amount to		??592474
Terminator	??UAA	??5499
				Terminator	??UAG	??4661
Terminator	??UGA	??10356

The external proteasome processing of embodiment 18 hepatitis type B virus multi-epitope gene products

Brief introduction

Adopted and be used for the method based on CTL epi-position of design at the vaccine of chronic HBV (HBV).The synthetic gene that has prepared a series of 16 epi-positions of encoding, wherein epi-position by the amino acid sept that is used to strengthen proteolysis processing separately.The external translation of this HBV multi-epitope mini gene and the transient expression in the human cell line have caused the quick degraded of polyprotein, as desired to the unsettled gene outcome of proteasome activity.This HBV multi-epitope (AOSIb) directly is fused on the fluorescin, to be easy to detection.In the culture of transfection, add proteasome-specific inhibitor and significantly improved the amount of the fusion in the cell, as judging by facs analysis, fluorescence microscopy and Western blotting.The ability of the processing of proteasome inhibitor blocking-up multi-epitope gene product and immunogenicity in the body of the pathogene in the DNA plasmid-specific epi-position confirmed that together the amino acid sept can be guaranteed the processing of I class effectively.Prepared HBV multi-epitope constructs (AOSIb.2) subsequently, it contains the amino acid additive that several expections can improve proteasome processing.The result shows that the intervening sequence that uses can promote the proteasome processing of polypeptide expressed and effective CTL epi-position to present in this HBV multi-epitope plasmid.

2. experimental technique

Designed the DNA expression cassette, wherein HBV multilist bit string is fused on the fluorescence labeling, is beneficial to Protein Detection and external quantitative.Add on the construct having the different septs of forming, to estimate potential improvement epi-position processing in the born of the same parents.In plasmid-cells transfected, added proteasome inhibitor, to stop the proteasome degraded of cytosol albumen.Detect by fluorescence labeling, monitor the existence of fusion through FACS, fluorescence microscopy or Western blotting.The amount of taking in the fluorescence in the cell is carried out quantitatively, to seek the variation in the polyprotein processing.Also be that 2 kinds of plasmids have detected immunogenic influence in the body in the HLA-A2 transgenic mice, to determine whether the amino acid sept has beneficial effect.

3.HBV the composition of multi-epitope constructs

The epi-position that HBV AOSIb and HBV AOSIb2 carry the virus-specific of optimizing.This construct coding HLA-A2, HLA-A3 and the super type epi-position of HLA-B7, totally 16 epi-positions.The HBVAOSIb2 construct has the amino acid that is used to strengthen proteasome processing of extra interpolation, and HBV AOSIb does not then have extra residue.The schematic diagram of CTL construct HBV AOSIb and HBV AOSIb2 and amino acid sequence are shown in Figure 34 and Biao 23-24.Can encode HBV AOSIb and HBV AOSIb2 polynucleotide sequence example as the table 23-24 shown in.

Table 23. is by the epigene of HBV AOSIb construct coding

Table 24. is by the epigene of HBV AOSIb2 construct coding

4. external protein expression and detection method

With can coding fluorescence-multi-epitope HBV AOSIb that puts together or the plasmid of HBV AOSIb.2 or do not contain epi-position fluorescence report plasmid transient transfection people's 293 cell-lines.After the transfection 24 hours, add the irreversible proteasome inhibitor MG132 of 5 μ M.In 24 hours of adding proteasome inhibitor, by the fluorescence in flow cytometry method (FACS) or the fluorescence microscopy detection living cells.Detected in the increase that is with or without under the situation of proteasome inhibitor with the number of the fluorecyte of plasmid HBVAOSIb transfection, shown in Figure 35 A, it is the function with the incubation time of inhibitor.Figure 38 has contrasted the number of hatching (passing through FACS) detected fluorecyte after 24 hours with the cell of 3 kinds of different plasmid transfections and inhibitor.Plasmid HBV AOSIb.2 to sept-optimization has observed more far-reaching effect.Also the fluorescence microscopy by living cells has detected the number that can express the cell of various fluorescent fusion proteins, as shown in figure 39.Carried out the Western blotting detection by following step: prepare full cell lysate from cells transfected,, transfer on the blotting membrane and use antibody detection protein at fusion partner albumen by the gel electrophoresis protein isolate.Detected behind adding proteasome inhibitor lactacystin (25 μ M) or the MG132 (5 μ M) increase of amount that can detected albumen then.The result as shown in figure 36.

5. mouse immune originality experimental technique

Injected can the encode plasmid of HBVAOSIb or HBV AOSIb2 multi-epitope of 100 μ g for genetically modified HLA-A2 mouse muscle interiorly.Put to death mouse after 14 days, their spleen is homogenized, collect T lymphocyte and APC.Corresponding to the peptide of various HBV epi-positions at culture moderate stimulation cell.Detect the secretion (to detect secretory units) of IFN-γ by improved ELISA method.The result is summarised in the table 25.

The HLA-A2Tg mouse immune originality of table 25. plasmid AOSIb and AOSIb.2

	HBV AOSIb (100ug dosage)		HBV AOSIb2 (100ug dosage)
					Epi-position	Magnitude	Frequency	Magnitude	Frequency
??core18	??102.7 (1.8)	??6/6	??480.6 (1.4)	??6/6
					??pol562	??-	??0/6	??260.2 (2.1)	??6/6
??pol538	??2643.8 (1.3)	??6/6	??2332.3 (1.4)	??6/6
					??pol455	??2234.6 (1.3)	??6/6	??334.3 (1.3)	??6/6
??env183	??877.5 (1.3)	??6/6	??962.8 (1.3)	??6/6
					??env335	??6.1	??1/6	??44.9 (1.6)	??6/6
??pol642	??1859.8 (1.6)	??6/6	??1819.0 (1.6)	??6/6

6. result's general introduction

The HBV DNA construct carries the epi-position of the virus-specific of the box that is arranged in optimization that can cause ctl response, other amino acid has been imported between the epi-position of a construct, presents with the I class that strengthens proteasome processing and antigen thus potentially.

2 kinds of HBV-fluorescin fusions are all more unstable than independent fluorescin, show that the HBV multi-epitope is easy to degraded, and drive the degraded of whole fusion product.Proteasome inhibitor helps detecting more substantial fluorescence fusion product, but does not influence for the fusion partner of single expression, shows that this is actually the cytoplasmic protein enzyme body activity that multi-epitope strengthens.Proteasome inhibitor is compared the more remarkable of HBV AOSIb fusion to the influence of the HBV AOSIb2 product of sept-optimization, shows that the processing site of adding on the HBV AOSIb2 molecule has the ideal role that improves its processability." optimization " HBV AOSIb2 plasmid that studies confirm that in the HLA-A2 transgenic mice has improved with the immunogenicity that HBV AOSIb compares its several epi-positions.

Embodiment 19 is detected epi-position-specific t cell responses in the HLA transgenic mice of GCR-3697 immunity

The splenic lymphocyte of using the immunity back to obtain in 11-14 days has detected the epi-position-specific t cell responses (Figure 37) in the HLA transgenic mice of GCR-3697 immunity.With 100 μ g DNA in tibialis anterior meat bilateral ground immunity the group of 6-9 HLA-transgenic mice.DNA carries in PBS or PVP preparation; Under the situation of PBS preparation, with cardiotoxin parenteral solution preliminary treatment injection site.With the cycle of 100 μ g DNA with 4 days, twice immunity use preparation mice immunized based on PVP.

Based on the generation of IFN-γ, use original position ELISA experiment, detected ctl response.Following experimentizing: at 37 ℃, 5%CO ₂In, with splenocyte (2.5 * 10 ⁷) with the splenocyte (10 of lipopolysaccharides (the LPS)-activation of peptide (1 μ g/ml) and irradiation ⁷) in the RPMI medium, cultivated 6 days.Stimulate after 6 days, under the situation that is with or without peptide (1 μ g/ml), the splenocyte and 10 of serial dilution ⁵The Jurkat target cell of HLA-coupling was cultivated 20 hours.(Costar, Corning test on NY) being coated with ELISA flat board to the specific rat monoclonal antibody of mouse IFN-γ (Clone RA-6A2, BD Biosciences/Pharmingen) in advance.Next day,, use the ELISA of sandwich form to detect the amount of the IFN-γ of secretion by removing cell with the PBS washing flat board that contains 0.05% Tween-20.Use biotinylated rat monoclonal antibody (clone XMG1.2, BD Biosciences/Pharmingen) to detect the IFN-γ that catches.According to manufacturer's explanation, use horseradish peroxidase-streptavidin (Zymed) and 3,3 ', 5,5 ' tetramethyl benzidine and H ₂O ₂(ImmunoPure TMB Substrate Kit Pierce) develops the color.On Labsystems Multiskan RC ELISA plate reader, read absorbance at 450nm.Become secretory units (SU) to estimate (McKinney etc. 2000) original position ELISA data transaction.

In a word, GCR-3697 has induced the ctl response at a plurality of epi-positions of the super type restriction of multiple HLA-.The magnitude of the reaction of inducing and width with it has been generally acknowledged that the immunoreactive character that belongs to the treatment value is consistent.

The whole content of every piece of document quoting in background technology, definition, detailed Description Of The Invention and embodiment (comprise registration in patent, patent application, periodical, summary, laboratory manual, books, the sequence library or other open) is incorporated by reference here.

It will be understood by those skilled in the art that or only use normal experiment just can determine many equivalents of the specific embodiments of invention as herein described.These equivalents also should be included in the following claim.

Sequence table

<110>Epimmune?Inc.

Genencor?International，Inc.

＜120〉multi-epitope constructs of You Huaing and application thereof

<130>2060.020PC03

<150>US?60/415.463

<151>2002-10-03

<150>US?60/419,973

<151>2002-10-22

<160>479

<170>PatentIn?version?3.2

<210>1

<211>13

<212>PRT

＜213〉the unknown

<220>

＜223〉PADRE peptide, HLA II class hyper-base preface example

<220>

<221>MISC_FEATURE

<222>(1)..(1)

＜223〉can be D-or L-alanine

<220>

<221>MISC_FEATURE

<222>(3)..(3)

＜223〉Xaa can be Cyclohexylalanine, phenylalanine or tyrosine

<220>

<221>MISC_FEATURE

<222>(13)..(13)

＜223〉can be D-or L-alanine

<400>1

Ala?Lys?Xaa?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala

1???????????????5???????????????????10

<210>2

<211>5

<212>PRT

＜213〉the unknown

<220>

＜223〉spacer peptide

<400>2

Gly?Pro?Gly?Pro?Gly

1???????????????5

<210>3

<211>22

<212>PRT

＜213〉the unknown

<220>

＜223〉CTL multi-epitope constructs

<400>3

Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val

1???????????????5???????????????????10??????????????????15

Phe?Leu?Leu?Ser?Leu?Gly

20

<210>4

<211>22

<212>PRT

＜213〉the unknown

<220>

＜223〉CTL multi-epitope constructs

<400>4

Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val

1???????????????5???????????????????10??????????????????15

Lys?Leu?Thr?Pro?Leu?Cys

20

<210>5

<211>15

<212>PRT

＜213〉the unknown

<220>

＜223〉CTL multi-epitope constructs

<400>5

Ile?Leu?Gly?Gly?Trp?Val?Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val

1???????????????5???????????????????10??????????????????15

<210>6

<211>20

<212>PRT

＜213〉the unknown

<220>

＜223〉CTL multi-epitope constructs

<400>6

Val?Pro?Gly?Ser?Arg?Gly?Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val?Ala

1???????????????5???????????????????10??????????????????15

Lys?Phe?Val?Ala

20

<210>7

<211>9

<212>PRT

＜213〉the unknown

<220>

＜223〉artificial peptide

<400>7

Val?Leu?Ala?Glu?Ala?Met?Ser?Gln?Val

1???????????????5

<210>8

<211>9

<212>PRT

＜213〉the unknown

<220>

＜223〉artificial peptide

<400>8

Ile?Leu?Lys?Glu?Pro?Val?His?Gly?Val

1???????????????5

<210>9

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>9

Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val

1???????????????5???????????????????10

<210>10

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>10

Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile

1???????????????5

<210>11

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>11

Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val

1???????????????5

<210>12

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>12

Leu?Leu?Val?Pro?Phe?Val?Gln?Trp?Phe?Val

1???????????????5???????????????????10

<210>13

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>13

Leu?Leu?Pro?Ile?Phe?Phe?Cys?Leu?Trp?Val

1???????????????5???????????????????10

<210>14

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>14

Gly?Leu?Ser?Arg?Tyr?Val?Ala?Arg?Leu

1???????????????5

<210>15

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>15

Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val

1???????????????5

<210>16

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>16

Ile?Leu?Arg?Gly?Thr?Ser?Phe?Val?Tyr?Val

1???????????????5???????????????????10

<210>17

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>17

Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu

1???????????????5

<210>18

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>18

Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

1???????????????5

<210>19

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>19

Gly?Leu?Ser?Pro?Thr?Val?Trp?Leu?Ser?Val

1???????????????5???????????????????10

<210>20

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>20

Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val?Arg?Arg

1???????????????5???????????????????10

<210>21

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>21

His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys

1???????????????5???????????????????10

<210>22

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>22

Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys

1???????????????5???????????????????10

<210>23

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>23

Leu?Val?Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg

1???????????????5???????????????????10

<210>24

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>24

Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys

1???????????????5

<210>25

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>25

Ser?Ala?Ile?Cys?Ser?Val?Val?Arg?Arg

1???????????????5

<210>26

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>26

Lys?Val?Gly?Asn?Phe?Thr?Gly?Leu?Tyr

1???????????????5

<210>27

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>27

Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr?Lys

l???????????????5???????????????????10

<210>28

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>28

Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val

1???????????????5

<210>29

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>29

Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe

1???????????????5

<210>30

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>30

Thr?Pro?Ala?Arg?Val?Thr?Gly?Gly?Val?Phe

1???????????????5???????????????????10

<210>31

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>31

His?Pro?Ala?Ala?Met?Pro?Hi?s?Leu?Leu

1???????????????5

<210>32

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>32

Tyr?Pro?Ala?Leu?Met?Pro?Leu?Tyr?Ala

1???????????????5

<210>33

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>33

Phe?Pro?His?Cys?Leu?Ala?Phe?Ser?Tyr

1???????????????5

<210>34

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>34

Phe?Pro?His?Cys?Leu?Ala?Phe?Ser?Tyr?Met

1???????????????5???????????????????10

<210>35

<211>7

<212>PRT

＜213〉hepatitis type B virus

<400>35

Tyr?Pro?Ala?Leu?Met?Leu?Tyr

1???????????????5

<210>36

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>36

Tyr?Pro?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

1???????????????5???????????????????10

<210>37

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>37

Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr

1???????????????5???????????????????10

<210>38

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>38

Asp?Leu?Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr

1???????????????5???????????????????10

<210>39

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>39

Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr

1???????????????5???????????????????10

<210>40

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>40

His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

1???????????????5???????????????????10

<210>41

<211>8

<212>PRT

＜213〉hepatitis type B virus

<400>41

Ala?Ser?Phe?Cys?Gly?Ser?Pro?Tyr

1???????????????5

<210>42

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>42

Leu?Ser?Leu?Asp?Val?Ser?Ala?Ala?Phe?Tyr

1???????????????5???????????????????10

<210>43

<211>8

<212>PRT

＜213〉hepatitis type B virus

<400>43

Tyr?Ser?Leu?Asn?Phe?Met?Gly?Tyr

1???????????????5

<210>44

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>44

Ile?Leu?Leu?Leu?Cys?Leu?Ile?Phe?Leu?Leu

1???????????????5???????????????????10

<210>45

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>45

Arg?Trp?Met?Cys?Leu?Arg?Arg?Phe?Ile?Ile

1???????????????5???????????????????10

<210>46

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>46

Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu

1???????????????5???????????????????10

<210>47

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>47

Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe

1???????????????5???????????????????10

<210>48

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>48

Leu?Trp?Phe?His?Ile?Ser?Cys?Leu?Thr?Phe

1???????????????5???????????????????10

<210>49

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>49

Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp

1???????????????5

<210>50

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>50

Ser?Phe?Cys?Gly?Ser?Pro?Tyr?Ser?Trp

1???????????????5

<210>51

<211>8

<212>PRT

＜213〉hepatitis type B virus

<400>51

Ala?Phe?Pro?His?Cys?Leu?Ala?Phe

1???????????????5

<210>52

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>52

Gly?Tyr?Pro?Ala?Leu?Met?Pro?Leu?Tyr

1???????????????5

<210>53

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>53

Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu

1???????????????5

<210>54

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>54

Ser?Tyr?Ile?Pro?Ser?Ala?Glu?Lys?Ile

1???????????????5

<210>55

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>55

Leu?Gln?Ser?Leu?Thr?Asn?Leu?Leu?Ser?Ser?Asn?Leu?Ser?Trp?Leu

1???????????????5???????????????????10??????????????????15

<210>56

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>56

Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr?Lys?Ala?Phe?Leu?Cys

1???????????????5???????????????????10??????????????????15

<210>57

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>57

Ala?Gly?Phe?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Pro?Gln?Ser

1???????????????5???????????????????10??????????????????15

<210>58

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>58

Gly?Thr?Ser?Phe?Val?Tyr?Val?Pro?Ser?Ala?Leu?Asn?Pro?Ala?Asp

1???????????????5???????????????????10??????????????????15

<210>59

<211>20

<212>PRT

＜213〉hepatitis type B virus

<400>59

Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg?Pro?Pro

1???????????????5???????????????????10??????????????????15

Asn?Ala?Pro?Ile

20

<210>60

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>60

Arg?His?Tyr?Leu?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys

1???????????????5???????????????????10??????????????????15

<210>61

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>61

Leu?Val?Pro?Phe?Val?Gln?Trp?Phe?Val?Gly?Leu?Ser?Pro?Thr?Val

1???????????????5???????????????????10??????????????????15

<210>62

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>62

Leu?His?Leu?Tyr?Ser?His?Pro?Ile?Ile?Leu?Gly?Phe?Arg?Lys?Ile

1???????????????5???????????????????10??????????????????15

<210>63

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>63

Pro?Phe?Leu?Leu?Ala?Gln?Phe?Thr?Ser?Ala?Ile?Cys?Ser?Val?Val

1???????????????5???????????????????10??????????????????15

<210>64

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>64

Lys?Gln?Cys?Phe?Arg?Lys?Leu?Pro?Val?Asn?Arg?Pro?Ile?Asp?Trp

1???????????????5???????????????????10??????????????????15

<210>65

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>65

Ala?Ala?Asn?Trp?Ile?Leu?Arg?Gly?Thr?Ser?Phe?Val?Tyr?Val?Pro

1???????????????5???????????????????10??????????????????15

<210>66

<211>20

<212>PRT

＜213〉hepatitis type B virus

<400>66

Pro?His?His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu?Leu

1???????????????5???????????????????10??????????????????15

Met?Thr?Leu?Ala

20

<210>67

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>67

Leu?Cys?Gln?Val?Phe?Ala?Asp?Ala?Thr?Pro?Thr?Gly?Trp?Gly?Leu

1???????????????5???????????????????10??????????????????15

<210>68

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>68

Glu?Ser?Arg?Leu?Val?Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Gly?Asn

1???????????????5???????????????????10??????????????????15

<210>69

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>69

Val?Gly?Pro?Leu?Thr?Val?Asn?Glu?Lys?Arg?Arg?Leu?Lys?Leu?Ile

1???????????????5???????????????????10??????????????????15

<210>70

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>70

Ser?Ser?Asn?Leu?Ser?Trp?Leu?Ser?Leu?Asp?Val?Ser?Ala?Ala?Phe

1???????????????5???????????????????10??????????????????15

<210>71

<211>1251

<212>DNA

＜213〉hepatitis type B virus

<400>71

atgggaatgc?aggtgcaaat?acagtctctc?ttccttttgc?ttctctgggt?tccaggatca????60

cggggcttct?tgcttagctt?gggcatccac?ctaaatgctg?ctgcaaaata?cacatctttt????120

ccttggctcc?ttaatgccgc?cgctaggttt?tcatggctga?gtctgctagt?acctttcaat????180

gcggctttcc?cacattgcct?agcttttagc?tatatgaaag?ctgctttagt?cgtggacttt????240

tcacagttta?gcagaggagc?aatcctgctg?ctatgtctga?tattccttct?aaacgcagca????300

gcccacacac?tctggaaagc?tggtatcctt?tacaagaaag?cctggatgat?gtggtattgg????360

ggacccagcc?tctacaaagc?ataccctgcc?ctgatgccac?tatacgcatg?cattggcgcg????420

gcagcctggt?tatccctttt?agtaccgttt?gtcaacgccg?cagcgggatt?tctattaacc????480

agaatcctga?cgattaatgc?tgccgccatt?ccgatcccaa?gttcctgggc?attcaaagca????540

gccgcggagt?atctggtttc?atttggcgta?tggaacctgc?caagcgactt?ctttccttct????600

gttaaggccg?ctgctttcct?cccctccgat?ttctttccat?cggtgaaagc?cgctgccgac????660

ctccttgata?ccgcgagcgc?tctgtacaac?tcgtggccaa?aattcgcagt?tccaaaccta????720

aaagccgccg?ccagtgccat?ttgttccgtg?gtaaggagaa?aattatcact?cgacgtgtcc????780

gcagcatttt?ataacgctgc?tgcaaagttt?gtcgcagcat?ggacattgaa?ggctgcagcg????840

aaagcagcaa?atgtatcaat?accctggacc?cacaagggtg?cagccgggct?gtctaggtat????900

gtggcgaggc?taaacgccgc?cgcctcaaca?ctgcctgaga?ctactgtcgt?gagacgcaaa????960

caccctgccg?caatgcccca?cctgctgaaa?gcagccgcac?gatggatgtg?cctcagaaga????1020

ttcataataa?acgcttcttt?ctgtgggtca?ccctacaaag?ccgcttacat?ggacgatgtg????1080

gtcctcggag?tgaatgccct?ctggttccat?atcagctgcc?tgacattcaa?ggcagccgcc????1140

acccccgctc?gtgtgacagg?aggtgtcttc?aaagccgcgg?cactgacttt?cggtcgggaa????1200

actgtattgg?aatataagca?ggccttcaca?ttctccccaa?catacaagtg?a?????????????1251

<210>72

<211>416

<212>PRT

＜213〉hepatitis type B virus

<400>72

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu?Asn

20??????????????????25??????????????????30

Ala?Ala?Ala?Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Ala?Ala?Ala

35??????????????????40??????????????????45

Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala?Phe?Pro

50??????????????????55??????????????????60

His?Cys?Leu?Ala?Phe?Ser?Tyr?Met?Lys?Ala?Ala?Leu?Val?Val?Asp?Phe

65??????????????????70??????????????????75??????????????????80

Ser?Gln?Phe?Ser?Arg?Gly?Ala?Ile?Leu?Leu?Leu?Cys?Leu?Ile?Phe?Leu

85??????????????????90??????????????????95

Leu?Asn?Ala?Ala?Ala?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys

100?????????????????105?????????????????110

Lys?Ala?Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Tyr

115?????????????????120?????????????????125

Pro?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile?Gly?Ala?Ala?Ala?Trp?Leu

130?????????????????135?????????????????140

Ser?Leu?Leu?Val?Pro?Phe?Val?Asn?Ala?Ala?Ala?Gly?Phe?Leu?Leu?Thr

145?????????????????150?????????????????155?????????????????160

Arg?Ile?Leu?Thr?Ile?Asn?Ala?Ala?Ala?Ile?Pro?Ile?Pro?Ser?Ser?Trp

165?????????????????170?????????????????175

Ala?Phe?Lys?Ala?Ala?Ala?Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Asn

180?????????????????185?????????????????190

Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Phe?Leu?Pro

195?????????????????200?????????????????205

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Asp?Leu?Leu?Asp?Thr

210?????????????????215?????????????????220

Ala?Ser?Ala?Leu?Tyr?Asn?Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu

225?????????????????230?????????????????235?????????????????240

Lys?Ala?Ala?Ala?Ser?Ala?Ile?Cys?Ser?Val?Val?Arg?Arg?Lys?Leu?Ser

245?????????????????250?????????????????255

Leu?Asp?Val?Ser?Ala?Ala?Phe?Tyr?Asn?Ala?Ala?Ala?Lys?Phe?Val?Ala

260?????????????????265?????????????????270

Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Ala?Ala?Asn?Val?Ser?Ile?Pro

275?????????????????280?????????????????285

Trp?Thr?His?Lys?Gly?Ala?Ala?Gly?Leu?Ser?Arg?Tyr?Val?Ala?Arg?Leu

290?????????????????295?????????????????300

Asn?Ala?Ala?Ala?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Lys

305?????????????????310?????????????????315?????????????????320

His?Pro?Ala?Ala?Met?Pro?His?Leu?Leu?Lys?Ala?Ala?Ala?Arg?Trp?Met

325?????????????????330?????????????????335

Cys?Leu?Arg?Arg?Phe?Ile?Ile?Asn?Ala?Ser?Phe?Cys?Gly?Ser?Pro?Tyr

340?????????????????345?????????????????350

Lys?Ala?Ala?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Asn?Ala?Leu?Trp

355?????????????????360?????????????????365

Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Lys?Ala?Ala?Ala?Thr?Pro?Ala?Arg

370?????????????????375?????????????????380

Val?Thr?Gly?Gly?Val?Phe?Lys?Ala?Ala?Ala?Leu?Thr?Phe?Gly?Arg?Glu

385?????????????????390?????????????????395?????????????????400

Thr?Val?Leu?Glu?Tyr?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr?Lys

405?????????????????410?????????????????415

<210>73

<211>1035

<212>DNA

＜213〉hepatitis type B virus

<400>73

atgggaactt?cttttgtgta?tgtcccttcc?gctctgaacc?cagcagacgg?acccgggcct????60

ggcctgtgcc?aggtcttcgc?cgacgcaact?cccacagggt?gggggctggg?gccaggacca????120

ggcaggcact?acctgcatac?tctgtggaag?gcaggaatcc?tctataaagg?gcccggccca????180

ggccctcacc?acaccgccct?gaggcaggcc?atcctgtgct?ggggggagct?catgaccctg????240

gccggacctg?gacccgggga?gagcagactg?gtggtggatt?tcagccaatt?cagcagagga????300

aacggacccg?gccctgggcc?ttttctgctg?gctcagttta?catctgctat?ttgttctgtg????360

gtcggccccg?ggcccggact?cgtgcctttc?gtgcagtggt?tcgtgggact?gtcccctaca????420

gtcgggcccg?gcccagggct?gcatctgtac?tcccacccaa?tcatcctcgg?cttccgcaag????480

attggacccg?gcccaggctc?cagcaatctc?tcctggctct?ctctggacgt?gtctgccgcc????540

tttggccctg?gaccaggcct?gcaaagcctg?actaatctgc?tcagcagcaa?cctgtcctgg????600

ctgggacctg?gcccaggggc?tggcttcttt?ctgctcaccc?ggattctcac?aattccccag????660

tccggaccag?gaccaggagt?cagtttcggg?gtgtggatca?ggacccctcc?tgcttataga????720

ccacccaatg?ctccaatcgg?ccccggccct?ggcgtcgggc?cactgaccgt?gaatgagaag????780

cgccggctga?agctgatcgg?ccctggccct?ggcaagcagt?gctttcgcaa?actgcccgtg????840

aacagaccta?ttgattgggg?ccccggccct?ggagcagcca?actggattct?caggggaaca????900

agcttcgtct?acgtgcccgg?gcccggacca?gggaagcagg?cttttacctt?ctctcccact????960

tacaaggcct?tcctctgtgg?gccaggcccc?ggcgccaagt?ttgtggcagc?atggaccctc????1020

aaagccgctg?cctga?????????????????????????????????????????????????????1035

<210>74

<211>344

<212>PRT

＜213〉hepatitis type B virus

<400>74

Met?Gly?Thr?Ser?Phe?Val?Tyr?Val?Pro?Ser?Ala?Leu?Asn?Pro?Ala?Asp

1???????????????5???????????????????10??????????????????15

Gly?Pro?Gly?Pro?Gly?Leu?Cys?Gln?Val?Phe?Ala?Asp?Ala?Thr?Pro?Thr

20??????????????????25??????????????????30

Gly?Trp?Gly?Leu?Gly?Pro?Gly?Pro?Gly?Arg?His?Tyr?Leu?His?Thr?Leu

35??????????????????40??????????????????45

Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Pro?His?His

50??????????????????55??????????????????60

Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Gtu?Leu?Met?Thr?Leu

65??????????????????70??????????????????75??????????????????80

Ala?Gly?Pro?Gly?Pro?Gly?Glu?Ser?Arg?Leu?Val?Val?Asp?Phe?Ser?Gln

85??????????????????90??????????????????95

Phe?Ser?Arg?Gly?Asn?Gly?Pro?Gly?Pro?Gly?Pro?Phe?Leu?Leu?Ala?Gln

100?????????????????105?????????????????110

Phe?Thr?Ser?Ala?Ile?Cys?Ser?Val?Val?Gly?Pro?Gly?Pro?Gly?Leu?Val

115?????????????????120?????????????????125

Pro?Phe?Val?Gln?Trp?Phe?Val?Gly?Leu?Ser?Pro?Thr?Val?Gly?Pro?Gly

130?????????????????135?????????????????140

Pro?Gly?Leu?His?Leu?Tyr?Ser?His?Pro?Ile?Ile?Leu?Gly?Phe?Arg?Lys

145?????????????????150?????????????????155?????????????????160

Ile?Gly?Pro?Gly?Pro?Gly?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Ser?Leu?Asp

165?????????????????170?????????????????175

Val?Ser?Ala?Ala?Phe?Gly?Pro?Gly?Pro?Gly?Leu?Gln?Ser?Leu?Thr?Asn

180?????????????????185?????????????????190

Leu?Leu?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Gly?Pro?Gly?Pro?Gly?Ala?Gly

195??????????????????200?????????????????205

Phe?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Pro?Gln?Ser?Gly?Pro?Gly

210?????????????????215?????????????????220

Pro?Gly?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg

225?????????????????230?????????????????235?????????????????240

Pro?Pro?Asn?Ala?Pro?Ile?Gly?Pro?Gly?Pro?Gly?Val?Gly?Pro?Leu?Thr

245?????????????????250?????????????????255

Val?Asn?Glu?Lys?Arg?Arg?Leu?Lys?Leu?Ile?Gly?Pro?Gly?Pro?Gly?Lys

260?????????????????265?????????????????270

Gln?Cys?Phe?Arg?Lys?Leu?Pro?Val?Asn?Arg?Pro?Ile?Asp?Trp?Gly?Pro

275?????????????????280?????????????????285

Gly?Pro?Gly?Ala?Ala?Asn?Trp?Ile?Leu?Arg?Gly?Thr?Ser?Phe?Val?Tyr

290?????????????????295?????????????????300

Val?Pro?Gly?Pro?Gly?Pro?Gly?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

305?????????????????310?????????????????315?????????????????320

Tyr?Lys?Ala?Phe?Leu?Cys?Gly?Pro?Gly?Pro?Gly?Ala?Lys?Phe?Val?Ala

325?????????????????330?????????????????335

Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala

340

<210>75

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>75

Gly?Ile?His?Leu?Asn?Ala?Ala?Ala?Lys?Tyr?Thr?Ser

1???????????????5???????????????????10

<210>76

<211>12

<212>PRT

＜213〉homo sapiens

<400>76

Gly?Ile?His?Leu?Asn?Met?Ala?Ala?Gly?Ser?Gly?Val

1???????????????5???????????????????10

<210>77

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>77

Pro?Trp?Leu?Leu?Asn?Ala?Ala?Ala?Arg?Phe?Ser?Trp

1???????????????5???????????????????10

<210>78

<211>12

<212>PRT

＜213〉homo sapiens

<400>78

Pro?Trp?Leu?Leu?Asn?Ala?Thr?Val?Glu?Glu?Asn?Ile

1???????????????5???????????????????10

<210>79

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>79

Leu?Val?Pro?Phe?Asn?Ala?Ala?Phe?Pro?His?Cys

1???????????????5???????????????????10

<210>80

<211>11

<212>PRT

＜213〉homo sapiens

<400>80

Ser?Trp?Leu?Phe?Asp?Ala?Ala?Phe?Val?His?Cys

1???????????????5???????????????????10

<210>81

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>81

Phe?Ser?Tyr?Met?Lys?Ala?Ala?Leu?Val?Val?Asp

1???????????????5???????????????????10

<210>82

<211>11

<212>PRT

＜213〉homo sapiens

<400>82

Phe?Ser?Tyr?Met?Lys?Ala?Ala?Met?Thr?Pro?Ala

1???????????????5???????????????????10

<210>83

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>83

Gln?Phe?Ser?Arg?Gly?Ala?Ile?Leu?Leu?Leu

1???????????????5???????????????????10

<210>84

<211>10

<212>PRT

＜213〉homo sapiens

<400>84

Gln?Phe?Ser?Ser?Gly?Ala?Ile?Leu?Arg?Val

1???????????????5???????????????????10

<210>85

<211>10

<212>PRT

＜213〉homo sapiens

<400>85

Thr?Phe?Ser?Arg?Ala?Ala?Ile?Leu?Leu?Ser

1???????????????5???????????????????10

<210>86

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>86

Ile?Phe?Leu?Leu?Asn?Ala?Ala?Ala?His?Thr?Leu?Trp

1???????????????5???????????????????10

<210>87

<211>12

<212>PRT

＜213〉homo sapiens

<400>87

Ile?Thr?Leu?Leu?Asn?Ala?Arg?Asn?His?Lys?Leu?Trp

1???????????????5???????????????????10

<210>88

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>88

Ile?Leu?Tyr?Lys?Lys?Ala?Trp?Met?Met?Trp

1???????????????5???????????????????10

<210>89

<211>10

<212>PRT

＜213〉homo sapiens

<400>89

Ile?Leu?Tyr?Lys?Gly?Ala?Trp?Glu?Gly?Thr

1???????????????5???????????????????10

<210>90

<211>10

<212>PRT

＜213〉homo sapiens

<400>90

Gly?Phe?Gly?Ser?Gln?Ala?Trp?Met?Met?Trp

1???????????????5???????????????????10

<210>91

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>91

Pro?Ser?Leu?Tyr?Lys?Ala?Tyr?Pro?Ala?Leu

1???????????????5???????????????????10

<210>92

<211>10

<212>PRT

＜213〉homo sapiens

<400>92

Gly?Ser?Leu?Tyr?Lys?Arg?Tyr?Pro?Ser?Leu

1???????????????5???????????????????10

<210>93

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>93

Tyr?Ala?Cys?Ile?Gly?Ala?Ala?Trp?Leu?Ser?Leu

1???????????????5???????????????????10

<210>94

<211>11

<212>PRT

＜213〉homo sapiens

<400>94

Glu?Val?Cys?Val?Ala?Ala?Pro?Trp?Leu?Ser?Leu

1???????????????5???????????????????10

<210>95

<211>11

<212>PRT

＜213〉homo sapiens

<400>95

Tyr?Ala?Asp?Ile?Glu?Ala?Ala?Trp?Leu?Ala?Leu

1???????????????5???????????????????10

<210>96

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>96

Val?Pro?Phe?Val?Asn?Ala?Ala?Ala?Phe?Leu?Leu?Thr

1???????????????5???????????????????10

<210>97

<211>12

<212>PRT

＜213〉homo sapiens

<400>97

Ile?Pro?Phe?Val?Asn?Ala?Gly?Thr?Phe?Leu?Lys?Asn

1???????????????5???????????????????10

<210>98

<211>12

<212>PRT

＜213〉homo sapiens

<400>98

Phe?Pro?Phe?Val?Asn?Leu?Ala?Ala?Leu?Leu?Leu?Leu

1???????????????5???????????????????10

<210>99

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>99

Ile?Leu?Thr?Ile?Asn?Ala?Ala?Ala?Ile?Pro?Ile?Pro

1???????????????5???????????????????10

<210>100

<211>12

<212>PRT

＜213〉homo sapiens

<400>100

His?Leu?Thr?Ile?Asn?Ala?Thr?Asp?Ile?Pro?Ser?Gly

1???????????????5???????????????????10

<210>101

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>101

Ser?Trp?Ala?Phe?Lys?Ala?Ala?Ala?Glu?Tyr?Leu?Val

1???????????????5???????????????????10

<210>102

<211>12

<212>PRT

＜213〉homo sapiens

<400>102

Glu?Phe?Pro?Ala?Lys?Ala?Ala?Ala?Glu?Tyr?Leu?Cys

1???????????????5???????????????????10

<210>103

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>103

Phe?Gly?Val?Trp?Asn?Leu?Pro?Ser?Asp

1???????????????5

<210>104

<211>9

<212>PRT

＜213〉homo sapiens

<400>104

Asn?Gly?Val?Trp?Asn?Leu?Ser?Ser?Asp

1???????????????5

<210>105

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>105

Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Phe?Leu?Pro?Ser

1???????????????5???????????????????10

<210>106

<211>12

<212>PRT

＜213〉homo sapiens

<400>106

Gly?Pro?Ser?Thr?Lys?Ala?Ala?Ala?Phe?Leu?Gln?Arg

1???????????????5???????????????????10

<210>107

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>107

Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Asp?Leu?Leu?Asp

1???????????????5???????????????????10

<210>108

<211>12

<212>PRT

＜213〉homo sapiens

<400>108

Asp?Val?Ser?Val?Lys?Ala?Ala?Ser?Glu?Leu?Leu?Met

1???????????????5???????????????????10

<210>109

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>109

Ser?Ala?Leu?Tyr?Asn?Ser?Trp?Pro?Lys

1???????????????5

<210>110

<211>9

<212>PRT

＜213〉homo sapiens

<400>110

Cys?Gln?Gln?Tyr?Asn?Asn?Trp?Pro?Lys

1???????????????5

<210>111

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>111

Val?Pro?Asn?Leu?Lys?Ala?Ala?Ala?Ser?Ala?Ile?Cys

1???????????????5???????????????????10

<210>112

<211>12

<212>PRT

＜213〉homo sapiens

<400>112

Ser?His?Asn?Leu?Lys?Ala?Ala?Ala?Ser?Lys?Leu?Gly

1???????????????5???????????????????10

<210>113

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>113

Val?Val?Arg?Arg?Lys?Leu?Ser?Leu?Asp

1???????????????5

<210>114

<211>9

<212>PRT

＜213〉homo sapiens

<400>114

Val?Leu?Arg?Arg?Lys?Val?Ser?Leu?Asp

1???????????????5

<210>115

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>115

Ala?Ala?Phe?Tyr?Asn?Ala?Ala?Ala?Lys?Phe?Val

1???????????????5???????????????????10

<210>116

<211>11

<212>PRT

＜213〉homo sapiens

<400>116

Leu?Ala?Phe?Tyr?Asn?Asp?Ala?Ser?Lys?Phe?Asp

1???????????????5???????????????????10

<210>117

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>117

Lys?Ala?Ala?Ala?Lys?Ala?Ala?Asn?Val?Ser?Ile

1???????????????5???????????????????10

<210>118

<211>11

<212>PRT

＜213〉homo sapiens

<400>118

Lys?Ala?Ala?Glu?Lys?Ala?Ala?Asn?Ile?Leu?Leu

1???????????????5???????????????????10

<210>119

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>119

Trp?Thr?His?Lys?Gly?Ala?Ala?Gly?Leu?Ser?Arg

1???????????????5???????????????????10

<210>120

<211>11

<212>PRT

＜213〉homo sapiens

<400>120

Trp?Thr?His?Lys?Gly?Ser?Pro?Gly?Leu?Thr?Arg

1???????????????5???????????????????10

<210>121

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>121

Val?Ala?Arg?Leu?Asn?Ala?Ala?Ala?Ser?Thr?Leu?Pro

1???????????????5???????????????????10

<210>122

<211>12

<212>PRT

＜213〉homo sapiens

<400>122

Val?Ala?Arg?Leu?Ser?Ala?Ala?Ala?Val?Leu?Tyr?Leu

1???????????????5???????????????????10

<210>123

<211>12

<212>PRT

＜213〉homo sapiens

<400>123

Val?Ala?Ala?Leu?Gly?Ala?Ala?Ala?Thr?Thr?Leu?Glu

1???????????????5???????????????????10

<210>124

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>124

Val?Val?Arg?Arg?Lys?His?Pro?Ala?Ala

1???????????????5

<210>125

<211>9

<212>PRT

＜213〉homo sapiens

<400>125

Val?Arg?Arg?Lys?His?Pro?Asp?Ala?Asn

1???????????????5

<210>126

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>126

Pro?His?Leu?Leu?Lys?Ala?Ala?Ala?Arg?Trp?Met?Cys

1???????????????5???????????????????10

<210>127

<211>12

<212>PRT

＜213〉homo sapiens

<400>127

Leu?Pro?Leu?Leu?Thr?Ala?Ala?Thr?Arg?Trp?Arg?Cys

1???????????????5???????????????????10

<210>128

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>128

Arg?Phe?Ile?Ile?Asn?Ala?Ser?Phe?Cys

1???????????????5

<210>129

<211>9

<212>PRT

＜213〉homo sapiens

<400>129

Arg?Phe?Ile?Ile?Ser?Ala?Glu?Phe?Arg

1???????????????5

<210>130

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>130

Gly?Ser?Pro?Tyr?Lys?Ala?Ala?Tyr?Met?Asp?Asp

1???????????????5???????????????????10

<210>131

<211>11

<212>PRT

＜213〉homo sapiens

<400>131

Gln?Pro?Pro?Tyr?Asn?Pro?Ala?Tyr?Met?Asp?Ala

1???????????????5???????????????????10

<210>132

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>132

Val?Leu?Gly?Val?Asn?Ala?Leu?Trp?Phe?His

1???????????????5???????????????????10

<210>133

<211>10

<212>PRT

＜213〉homo sapiens

<400>133

Asp?Pro?Gly?Val?Ser?Ala?Leu?Trp?Phe?Lys

1???????????????5???????????????????10

<210>134

<211>10

<212>PRT

＜213〉homo sapiens

<400>134

Val?Leu?Phe?Ile?Pro?Ala?Leu?Trp?Phe?His

1???????????????5???????????????????10

<210>135

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>135

Cys?Leu?Thr?Phe?Lys?Ala?Ala?Ala?Thr?Pro?Ala?Arg

1???????????????5???????????????????10

<210>136

<211>12

<212>PRT

＜213〉homo sapiens

<400>136

Arg?Gly?Thr?Phe?Lys?Ala?Val?Ala?Thr?Pro?Arg?Pro

1???????????????5???????????????????10

<210>137

<211>12

<212>PRT

＜213〉homo sapiens

<400>137

Glu?Arg?Thr?His?Lys?Ala?Ala?Ala?Thr?Gly?Ala?Arg

1???????????????5???????????????????10

<210>138

<211>12

<212>PRT

＜213〉hepatitis type B virus

<400>138

Gly?Gly?Val?Phe?Lys?Ala?Ala?Ala?Leu?Thr?Phe?Gly

1???????????????5???????????????????10

<210>139

<211>12

<212>PRT

＜213〉homo sapiens

<400>139

Ser?Gly?Val?Leu?Gly?Ala?Ala?Ser?Leu?Thr?Phe?Gly

1???????????????5???????????????????10

<210>140

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>140

Val?Leu?Glu?Tyr?Lys?Gln?Ala?Phe?Thr

1???????????????5

<210>141

<211>9

<212>PRT

＜213〉homo sapiens

<400>141

Val?Leu?Gln?Tyr?Lys?Gln?Val?Phe?Leu

1???????????????5

<210>142

<211>9

<212>PRT

＜213〉homo sapiens

<400>142

Val?Leu?Leu?Tyr?Lys?Gln?Asp?Phe?Thr

1???????????????5

<210>143

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>143

Pro?Thr?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Gly?Thr?Ser?Phe

1???????????????5???????????????????10

<210>144

<211>13

<212>PRT

＜213〉homo sapiens

<400>144

Val?Ser?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Ile?Lys?Phe?Ser

1???????????????5???????????????????10

<210>145

<211>13

<212>PRT

＜213〉homo sapiens

<400>145

Tyr?Pro?Tyr?Lys?Glu?Pro?Gly?Pro?Pro?Gly?Thr?Pro?Phe

1???????????????5???????????????????10

<210>146

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>146

Asn?Pro?Ala?Asp?Gly?Pro?Gly?Pro?Gly?Leu?Cys?Gln?Val

1???????????????5???????????????????10

<210>147

<211>13

<212>PRT

＜213〉homo sapiens

<400>147

Asn?Pro?Ala?Asp?Glu?Pro?Gly?Pro?Gly?Ala?Pro?Ala?Pro

1???????????????5???????????????????10

<2l0>148

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>148

Gly?Trp?Gly?Leu?Gly?Pro?Gly?Pro?Gly?Arg?His?Tyr?Leu

1???????????????5???????????????????10

<210>149

<211>13

<212>PRT

＜213〉homo sapiens

<400>149

Pro?Trp?Gly?Leu?Gly?Pro?Gly?Ala?Gly?Asp?Pro?Ala?Pro

1???????????????5???????????????????10

<210>150

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>150

Ile?Leu?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Pro?His?His?Thr

1???????????????5???????????????????10

<210>151

<211>13

<212>PRT

＜213〉homo sapiens

<400>151

Val?Ser?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Ile?Lys?Phe?Ser

1???????????????5???????????????????10

<210>152

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>152

Met?Thr?Leu?Ala?Gly?Pro?Gly?Pro?Gly?Glu?Ser?Arg?Leu

1???????????????5???????????????????10

<210>153

<211>13

<212>PRT

＜213〉homo sapiens

<400>153

Gly?Leu?Leu?Ala?Gly?Pro?Gly?Pro?Gly?Gly?Ser?Arg?Val

1???????????????5???????????????????10

<210>154

<211>13

<212>PRT

＜213〉homo sapiens

<400>154

Glu?Asp?Ser?Glu?Gly?Pro?Gly?Ser?Gly?Glu?Ser?Arg?Leu

1???????????????5???????????????????10

<210>155

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>155

Ser?Arg?Gly?Asn?Gly?Pro?Gly?Pro?Gly?Pro?Phe?Leu?Leu

1???????????????5???????????????????10

<210>156

<211>13

<212>PRT

＜213〉homo sapiens

<400>156

Val?Pro?Gly?Ser?Gly?Pro?Gly?Pro?Ala?Pro?Phe?Leu?Ala

1???????????????5???????????????????10

<210>157

<211>13

<212>PRT

＜213〉homo sapiens

<400>157

Ser?Arg?Gly?Pro?Ser?Pro?Gly?Pro?Gly?Arg?Leu?Leu?Leu

1????????????????5???????????????????10

<210>158

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>158

Cys?Ser?Val?Val?Gly?Pro?Gly?Pro?Gly?Leu?Val?Pro?Phe

1???????????????5???????????????????10

<210>159

<211>13

<212>PRT

＜213〉homo sapiens

<400>159

Cys?Ile?Leu?Ile?Gly?Pro?Gly?Thr?Gly?Ile?Val?Pro?Phe

1???????????????5???????????????????10

<210>160

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>160

Ser?Pro?Thr?Val?Gly?Pro?Gly?Pro?Gly?Leu?His?Leu?Tyr

1???????????????5???????????????????10

<210>161

<211>13

<212>PRT

＜213〉homo sapiens

<400>161

Ser?Pro?Thr?Val?Gly?Pro?Gly?Pro?Leu?Pro?Pro?Ala?Gly

1???????????????5???????????????????10

<210>162

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>162

Phe?Arg?Lys?Ile?Gly?Pro?Gly?Pro?Gly?Ser?Ser?Asn?Leu

1???????????????5???????????????????10

<210>163

<211>13

<212>PRT

＜213〉homo sapiens

<400>163

Asn?Arg?Lys?Ile?Ala?Pro?Gly?Pro?Gly?Gly?Gln?Ser?Glu

1???????????????5???????????????????10

<210>164

<211>13

<212>PRT

＜213〉homo sapiens

<400>164

Gly?Arg?Lys?Ile?Glu?Ser?Gly?Leu?Gly?Ser?Ser?Asn?Gly

1???????????????5???????????????????10

<210>165

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>165

Ser?Ala?Ala?Phe?Gly?Pro?Gly?Pro?Gly?Leu?Gln?Ser?Leu

1???????????????5???????????????????10

<210>166

<211>13

<212>PRT

＜213〉homo sapiens

<400>166

Ser?Thr?Ser?Phe?Gly?Pro?Gly?Pro?Gly?Val?Glu?Ser?Met

1???????????????5???????????????????10

<210>167

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>167

Leu?Ser?Trp?Leu?Gly?Pro?Gly?Pro?Gly?Ala?Gly?Phe?Phe

1???????????????5???????????????????10

<210>168

<211>13

<212>PRT

＜213〉homo sapiens

<400>168

Leu?Ser?Trp?Leu?Gly?Pro?Gly?Arg?Gly?Cys?Gln?Ile?Cys

1???????????????5???????????????????10

<210>169

<211>13

<212>PRT

＜213〉homo sapiens

<400>169

Ser?Lys?Val?Leu?Gly?Pro?Gly?Pro?Asp?Thr?Gly?Phe?Phe

1???????????????5???????????????????10

<210>170

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>170

Ile?Pro?Gln?Ser?Gly?Pro?Gly?Pro?Gly?Val?Ser?Phe?Gly

1???????????????5???????????????????10

<210>171

<211>13

<212>PRT

＜213〉homo sapiens

<400>171

Pro?Gly?Pro?Gln?Ala?Gly?Pro?Gly?Pro?Gly?Val?Arg?Asp

1???????????????5???????????????????10

<210>172

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>172

Asn?Ala?Pro?Ile?Gly?Pro?Gly?Pro?Gly?Val?Gly?Pro?Leu

1???????????????5???????????????????10

<210>173

<211>13

<212>PRT

＜213〉homo sapiens

<400>173

Asn?Arg?Glu?Ala?Gly?Pro?Gly?Pro?Gly?Pro?Gly?Pro?Leu

1???????????????5???????????????????10

<210>174

<211>13

<212>PRT

＜213〉homo sapiens

<400>174

Pro?Ala?Pro?Pro?Gly?Pro?Gly?Pro?Gly?Pro?Gly?Pro?Gly

1???????????????5???????????????????10

<210>175

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>175

Leu?Lys?Leu?Ile?Gly?Pro?Gly?Pro?Gly?Lys?G1n?Cys?Phe

1???????????????5???????????????????10

<210>176

<211>13

<212>PRT

＜213〉homo sapiens

<400>176

Leu?Lys?Leu?Arg?Gly?Pro?Gly?Pro?Gly?Leu?Ala?Ala?Ala

1???????????????5???????????????????10

<210>177

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>177

Pro?Ile?Asp?Trp?Gly?Pro?Gly?Pro?Gly?Ala?Ala?Asn?Trp

1???????????????5???????????????????10

<210>178

<211>13

<212>PRT

＜213〉homo sapiens

<400>178

Cys?Leu?Asp?Trp?Gly?Pro?Gly?Pro?Gly?Thr?Gly?Glu?Gln

1???????????????5???????????????????10

<210>179

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>179

Val?Tyr?Val?Pro?Gly?Pro?Gly?Pro?Gly?Lys?Gln?Ala?Phe

1???????????????5???????????????????10

<210>180

<211>13

<212>PRT

＜213〉homo sapiens

<400>180

Gly?Pro?Gly?Pro?Gly?Pro?Gly?Pro?Gly?Lys?Pro?Ala?Gly

1???????????????5???????????????????10

<210>181

<211>13

<212>PRT

＜213〉hepatitis type B virus

<400>181

Ala?Phe?Leu?Cys?Gly?Pro?Gly?Pro?Gly?Ala?Lys?Phe?Val

1???????????????5???????????????????10

<210>182

<211>13

<212>PRT

＜213〉homo sapiens

<400>182

Pro?Cys?Leu?Cys?Gly?Pro?Ala?Pro?Gly?Ala?Ala?Cys?Arg

1???????????????5???????????????????10

<210>183

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>183

Phe?Pro?His?Cys?Leu?Ala?Phe?Ser?Tyr?Met

1???????????????5???????????????????10

<210>184

<211>10

<212>PRT

＜213〉homo sapiens

<400>184

Arg?Leu?His?Cys?Leu?Ala?Phe?Ser?Gln?Arg

1???????????????5???????????????????10

<210>185

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>185

Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr

1???????????????5???????????????????10

<210>186

<211>11

<212>PRT

＜213〉homo sapiens

<400>186

Trp?Met?Met?Trp?Asn?Trp?Met?Val?Ser?Leu?Leu

1???????????????5???????????????????10

<210>187

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>187

Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile

1???????????????5

<210>188

<211>9

<212>PRT

＜213〉homo sapiens

<400>188

Asp?Leu?Leu?Thr?Arg?Val?Leu?Thr?Trp

1???????????????5

<210>189

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>189

Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp

1???????????????5

<210>190

<211>9

<212>PRT

＜213〉homo sapiens

<400>190

Gly?Tyr?Leu?Val?Val?Phe?Gly?Val?Lys

1???????????????5

<210>191

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>191

Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val

1???????????????5???????????????????10

<210>192

<211>10

<212>PRT

＜213〉homo sapiens

<400>192

Phe?Leu?Pro?Pro?Asp?Phe?Tyr?Pro?Pro?Ser

1???????????????5???????????????????10

<210>193

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>193

Ser?Ala?Ile?Cys?Ser?Val?Val?Arg?Arg

1???????????????5

<210>194

<211>9

<212>PRT

＜213〉homo sapiens

<400>194

Ser?Ala?Ile?Cys?Ser?Ala?Val?Gly?Val

1???????????????5

<210>195

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>195

His?Pro?Ala?Ala?Met?Pro?His?Leu?Leu

1???????????????5

<210>196

<211>9

<212>PRT

＜213〉homo sapiens

<400>196

His?Ala?Ala?Ala?Met?Pro?His?Ser?Cys

1???????????????5

<210>197

<211>20

<212>PRT

＜213〉hepatitis type B virus

<400>197

Pro?His?His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu?Leu

1???????????????5???????????????????10??????????????????15

Met?Thr?Leu?Ala

20

<210>198

<211>20

<212>PRT

＜213〉homo sapiens

<400>198

Gln?Gly?Asn?Gly?Lys?Phe?Asn?Leu?Met?Ile?Leu?Cys?Trp?Gly?Glu?Gly

1???????????????5???????????????????10??????????????????15

His?Gly?Ser?Ser

20

<210>199

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>199

Glu?Ser?Arg?Leu?Val?Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Gly?Asn

1???????????????5???????????????????10??????????????????15

<210>200

<211>15

<212>PRT

＜213〉homo sapiens

<400>200

Leu?Leu?Leu?Thr?Val?Val?Asp?Phe?Asp?Lys?Phe?Ser?Arg?His?Cys

1???????????????5???????????????????10??????????????????15

<210>201

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>201

Leu?Val?Pro?Phe?Val?Gln?Trp?Phe?Val?Gly?Leu?Ser?Pro?Thr?Val

1???????????????5???????????????????10??????????????????15

<210>202

<211>15

<212>PRT

＜213〉homo sapiens

<400>202

Val?Leu?Tyr?Phe?Val?Gln?Trp?Leu?Val?Gly?Phe?Ser?Phe?Phe?Leu

1???????????????5???????????????????10??????????????????15

<210>203

<211>15

<212>PRT

＜213〉hepatitis type B virus

<400>203

Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr?Lys?Ala?Phe?Leu?Cys

1???????????????5???????????????????10??????????????????15

<210>204

<211>15

<212>PRT

＜213〉homo sapiens

<400>204

Thr?Gln?Val?Phe?Thr?Phe?Gly?Pro?Thr?Phe?Arg?Ala?Glu?Asn?Ser

1???????????????5???????????????????10??????????????????15

<210>205

<211>2315

<212>DNA

＜213〉hepatitis type B virus

<400>205

gaattcaggt?cgccgccacc?atgggaatgc?aggtgcaaat?acagtctctc?ttccttttgc????60

ttctctgggt?tccaggatca?cggggcttct?tgcttagctt?gggcatccac?ctaaatgctg????120

ctgcaaaata?cacatctttt?ccttggctcc?ttaatgccgc?cgctaggttt?tcatggctga????180

gtctgctagt?acctttcaat?gcggctttcc?cacattgcct?agcttttagc?tatatgaaag????240

ctgctttagt?cgtggacttt?tcacagttta?gcagaggagc?aatcctgctg?ctatgtctga????300

tattccttct?aaacgcagca?gcccacacac?tctggaaagc?tggtatcctt?tacaagaaag????360

cctggatgat?gtggtattgg?ggacccagcc?tctacaaagc?ataccctgcc?ctgatgccac????420

tatacgcatg?cattggcgcg?gcagcctggt?tatccctttt?agtaccgttt?gtcaacgccg????480

cagcgggatt?tctattaacc?agaatcctga?cgattaatgc?tgccgccatt?ccgatcccaa????540

gttcctgggc?attcaaagca?gccgcggagt?atctggtttc?atttggcgta?tggaacctgc????600

caagcgactt?ctttccttct?gttaaggccg?ctgctttcct?cccctccgat?ttctttccat????660

cggtgaaagc?cgctgccgac?ctccttgata?ccgcgagcgc?tctgtacaac?tcgtggccaa????720

aattcgcagt?tccaaaccta?aaagccgccg?ccagtgccat?ttgttccgtg?gtaaggagaa????780

aattatcact?cgacgtgtcc?gcagcatttt?ataacgctgc?tgcaaagttt?gtcgcagcat????840

ggacattgaa?ggctgcagcg?aaagcagcaa?atgtatcaat?accctggacc?cacaagggtg????900

cagccgggct?gtctaggtat?gtggcgaggc?taaacgccgc?cgcctcaaca?ctgcctgaga????960

ctactgtcgt?gagacgcaaa?caccctgccg?caatgcccca?cctgctgaaa?gcagccgcac????1020

gatggatgtg?cctcagaaga?ttcataataa?acgcttcttt?ctgtgggtca?ccctacaaag????1080

ccgcttacat?ggacgatgtg?gtcctcggag?tgaatgccct?ctggttccat?atcagctgcc????1140

tgacattcaa?ggcagccgcc?acccccgctc?gtgtgacagg?aggtgtcttc?aaagccgcgg????1200

cactgacttt?cggtcgggaa?actgtattgg?aatataagca?ggccttcaca?ttctccccaa????1260

catacaagaa?cgcaggaact?tcttttgtgt?atgtcccttc?cgctctgaac?ccagcagacg????1320

gacccgggcc?tggcctgtgc?caggtcttcg?ccgacgcaac?tcccacaggg?tgggggctgg????1380

ggccaggacc?aggcaggcac?tacctgcata?ctctgtggaa?ggcaggaatc?ctctataaag????1440

ggcccggccc?aggccctcac?cacaccgccc?tgaggcaggc?catcctgtgc?tggggggagc????1500

tcatgaccct?ggccggacct?ggacccgggg?agagcagact?ggtggtggat?ttcagccaat????1560

tcagcagagg?aaacggaccc?ggccctgggc?cttttctgct?ggctcagttt?acatctgcta????1620

tttgttctgt?ggtcggcccc?gggcccggac?tcgtgccttt?cgtgcagtgg?ttcgtgggac????1680

tgtcccctac?agtcgggccc?ggcccagggc?tgcatctgta?ctcccaccca?atcatcctcg????1740

gcttccgcaa?gattggaccc?ggcccaggct?ccagcaatct?ctcctggctc?tctctggacg????1800

tgtctgccgc?ctttggccct?ggaccaggcc?tgcaaagcct?gactaatctg?ctcagcagca????1860

acctgtcctg?gctgggacct?ggcccagggg?ctggcttctt?tctgctcacc?cggattctca????1920

caattcccca?gtccggacca?ggaccaggag?tcagtttcgg?ggtgtggatc?aggacccctc????1980

ctgcttatag?accacccaat?gctccaatcg?gccccggccc?tggcgtcggg?ccactgaccg????2040

tgaatgagaa?gcgccggctg?aagctgatcg?gccctggccc?tggcaagcag?tgctttcgca????2100

aactgcccgt?gaacagacct?attgattggg?gccccggccc?tggagcagcc?aactggattc????2160

tcaggggaac?aagcttcgtc?tacgtgcccg?ggcccggacc?agggaagcag?gcttttacct????2220

tctctcccac?ttacaaggcc?ttcctctgtg?ggccaggccc?cggcgccaag?tttgtggcag????2280

catggaccct?caaagccgct?gcctgaggat?cctga???????????????????????????????2315

<210>206

<211>763

<212>PRT

＜213〉hepatitis type B virus

<400>206

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu?Asn

20??????????????????25??????????????????30

Ala?Ala?Ala?Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Ala?Ala?Ala

35???????????????????40??????????????????45

Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala?Phe?Pro

50??????????????????55??????????????????60

His?Cys?Leu?Ala?Phe?Ser?Tyr?Met?Lys?Ala?Ala?Leu?Val?Val?Asp?Phe

65??????????????????70??????????????????75??????????????????80

Ser?Gln?Phe?Ser?Arg?Gly?Ala?Ile?Leu?Leu?Leu?Cys?Leu?Ile?Phe?Leu

85??????????????????90??????????????????95

Leu?Asn?Ala?Ala?Ala?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys

100?????????????????105?????????????????110

Lys?Ala?Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Tyr

115?????????????????120?????????????????125

Pro?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile?Gly?Ala?Ala?Ala?Trp?Leu

130?????????????????135?????????????????140

Ser?Leu?Leu?Val?Pro?Phe?Val?Asn?Ala?Ala?Ala?Gly?Phe?Leu?Leu?Thr

145?????????????????150?????????????????155?????????????????160

Arg?Ile?Leu?Thr?Ile?Asn?Ala?Ala?Ala?Ile?Pro?Ile?Pro?Ser?Ser?Trp

165?????????????????170?????????????????175

Ala?Phe?Lys?Ala?Ala?Ala?Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Asn

180?????????????????185?????????????????190

Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Phe?Leu?Pro

195?????????????????200?????????????????205

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Asp?Leu?Leu?Asp?Thr

210?????????????????215?????????????????220

Ala?Ser?Ala?Leu?Tyr?Asn?Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu

225?????????????????230?????????????????235?????????????????240

Lys?Ala?Ala?Ala?Ser?Ala?Ile?Cys?Ser?Val?Val?Arg?Arg?Lys?Leu?Ser

245?????????????????250?????????????????255

Leu?Asp?Val?Ser?Ala?Ala?Phe?Tyr?Asn?Ala?Ala?Ala?Lys?Phe?Val?Ala

260?????????????????265?????????????????270

Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Ala?Ala?Asn?Val?Ser?Ile?Pro

275?????????????????280?????????????????285

Trp?Thr?His?Lys?Gly?Ala?Ala?Gly?Leu?Ser?Arg?Tyr?Val?Ala?Arg?Leu

290?????????????????295?????????????????300

Asn?Ala?Ala?Ala?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Lys

305?????????????????310?????????????????315?????????????????320

His?Pro?Ala?Ala?Met?Pro?His?Leu?Leu?Lys?Ala?Ala?Ala?Arg?Trp?Met

325?????????????????330?????????????????335

Cys?Leu?Arg?Arg?Phe?Ile?Ile?Asn?Ala?Ser?Phe?Cys?Gly?Ser?Pro?Tyr

340?????????????????345?????????????????350

Lys?Ala?Ala?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Asn?Ala?Leu?Trp

355?????????????????360?????????????????365

Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Lys?Ala?Ala?Ala?Thr?Pro?Ala?Arg

370?????????????????375?????????????????380

Val?Thr?Gly?Gly?Val?Phe?Lys?Ala?Ala?Ala?Leu?Thr?Phe?Gly?Arg?Glu

385?????????????????390?????????????????395?????????????????400

Thr?Val?Leu?Glu?Tyr?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr?Lys

405?????????????????410?????????????????415

Asn?Ala?Gly?Thr?Ser?Phe?Val?Tyr?Val?Pro?Ser?Ala?Leu?Asn?Pro?Ala

420?????????????????425?????????????????430

Asp?Gly?Pro?Gly?Pro?Gly?Leu?Cys?Gln?Val?Phe?Ala?Asp?Ala?Thr?Pro

435?????????????????440?????????????????445

Thr?Gly?Trp?Gly?Leu?Gly?Pro?Gly?Pro?Gly?Arg?His?Tyr?Leu?His?Thr

450?????????????????455?????????????????460

Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Pro?His

465?????????????????470?????????????????475?????????????????480

His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu?Leu?Met?Thr

485?????????????????490?????????????????495

Leu?Ala?Gly?Pro?Gly?Pro?Gly?Glu?Ser?Arg?Leu?Val?Val?Asp?Phe?Ser

500?????????????????505?????????????????510

Gln?Phe?Ser?Arg?Gly?Asn?Gly?Pro?Gly?Pro?Gly?Pro?Phe?Leu?Leu?Ala

515?????????????????520?????????????????525

Gln?Phe?Thr?Ser?Ala?Ile?Cys?Ser?Val?Val?Gly?Pro?Gly?Pro?Gly?Leu

530?????????????????535?????????????????540

Val?Pro?Phe?Val?Gln?Trp?Phe?Val?Gly?Leu?Ser?Pro?Thr?Val?Gly?Pro

545?????????????????550??????????????????555????????????????560

Gly?Pro?Gly?Leu?His?Leu?Tyr?Ser?His?Pro?Ile?Ile?Leu?Gly?Phe?Arg

565?????????????????570?????????????????575

Lys?Ile?Gly?Pro?Gly?Pro?Gly?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Ser?Leu

580?????????????????585?????????????????590

Asp?Val?Ser?Ala?Ala?Phe?Gly?Pro?Gly?Pro?Gly?Leu?Gln?Ser?Leu?Thr

595?????????????????600?????????????????605

Asn?Leu?Leu?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Gly?Pro?Gly?Pro?Gly?Ala

610?????????????????615?????????????????620

Gly?Phe?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Pro?Gln?Ser?Gly?Pro

625?????????????????630?????????????????635?????????????????640

Gly?Pro?Gly?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr

645?????????????????650?????????????????655

Arg?Pro?Pro?Asn?Ala?Pro?Ile?Gly?Pro?Gly?Pro?Gly?Val?Gly?Pro?Leu

660?????????????????665?????????????????670

Thr?Val?Asn?Glu?Lys?Arg?Arg?Leu?Lys?Leu?Ile?Gly?Pro?Gly?Pro?Gly

675?????????????????680?????????????????685

Lys?Gln?Cys?Phe?Arg?Lys?Leu?Pro?Val?Asn?Arg?Pro?Ile?Asp?Trp?Gly

690?????????????????695?????????????????700

Pro?Gly?Pro?Gly?Ala?Ala?Asn?Trp?Ile?Leu?Arg?Gly?Thr?Ser?Phe?Val

705?????????????????710?????????????????715?????????????????720

Tyr?Val?Pro?Gly?Pro?Gly?Pro?Gly?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro

725?????????????????730?????????????????735

Thr?Tyr?Lys?Ala?Phe?Leu?Cys?Gly?Pro?Gly?Pro?Gly?Ala?Lys?Phe?Val

740?????????????????745?????????????????750

Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Gly?Ser

755?????????????????760

<210>207

<211>2235

<212>DNA

＜213〉hepatitis type B virus

<400>207

atgggcatgc?aggtgcagat?ccagagcctg?ttcctgctcc?tgctgtgggt?gccaggaagc????60

agaggctttc?tcctgtccct?gggcatccac?ctgaacgccg?ctgcaaagta?caccagcttc????120

ccctggctgc?tcaacgccgc?tgcccggttc?agctggctgt?ccctgctcgt?gcccttcaac????180

gcagccttcc?cccactgcct?ggccttcagc?tacatgaaag?cagccctggt?ggtcgacttc????240

tcccagttca?gccggggagc?catcctgctc?ctgtgcctga?tctttctgct?caacgccgct????300

gcccacaccc?tgtggaaggc?tggcatcctg?tacaagaaag?cctggatgat?gtggtactgg????360

ggacccagcc?tgtacaaggc?atatccagcc?ctgatgcccc?tgtacgcctg?catcggagct????420

gccgcatggc?tgagcctcct?ggtgcccttc?gtgaacgccg?ctgccgggtt?cctgctgaca????480

agaatcctga?ccatcaacgc?cgcagccatt?cctatcccct?ccagctgggc?cttcaaggca????540

gccgccgagt?acctggtgag?cttcggagtc?tggaacctgc?ccagcgactt?ctttcccagc????600

gtgaaagccg?cagccttcct?gccctccgac?ttctttccca?gcgtgaaggc?cgcagccgat????660

ctcctggaca?ccgctagcgc?cctgtacaac?agctggccca?agttcgccgt?gcccaacctg????720

aaggccgcag?ccagcgccat?ctgcagcgtg?gtcagacgga?agctgtccct?cgatgtgagc????780

gccgctttct?acaacgccgc?cgcaaagttc?gtggccgcct?ggaccctgaa?agccgctgcc????840

aaggcagcca?acgtgagcat?cccctggacc?cacaaaggag?ccgcaggact?gagccggtat????900

gtggccagac?tgaacgccgc?tgccagcacc?ctgcccgaga?ccacagtggt?cagacggaag????960

caccccgccg?ccatgcccca?cctgctgaag?gccgcagccc?ggtggatgtg?cctcagacgg????1020

ttcatcatca?acgcttcctt?ctgtggcagc?ccctacaagg?ccgcctacat?ggatgacgtg????1080

gtcctgggag?tgaacgccct?ctggttccac?atcagctgcc?tgaccttcaa?agccgctgcc????1140

acacccgcaa?gagtgaccgg?aggcgtgttc?aaggctgcag?ccctgacctt?cggccgggag????1200

accgtgctgg?agtacaagca?ggccttcacc?ttcagcccca?cctacaagaa?cgccggcacc????1260

agctttgtgt?acgtcccaag?cgccctgaat?cccgcagacg?gccccggccc?cggactgtgc????1320

caggtgttcg?ccgatgccac?accaaccgga?tggggcctgg?gccctggacc?cggcagacac????1380

tacctgcata?ccctgtggaa?ggcaggaatc?ctgtacaaag?gccccggccc?tggaccccat????1440

cacaccgctc?tgcggcaggc?catcctgtgc?tggggcgagc?tcatgactct?ggcaggaccc????1500

ggccccggcg?aatccaggct?ggtggtggac?tttagccagt?tctccagagg?caacggaccc????1560

ggcccaggac?ccttcctgct?cgcccagttc?accagcgcca?tctgcagcgt?ggtcggacct????1620

ggcccaggac?tggtgccctt?cgtgcagtgg?ttcgtcggcc?tcagccccac?cgtcggacct????1680

ggccccggcc?tgcacctcta?cagccaccct?atcattctgg?gcttcagaaa?gatcggacca????1740

ggccccggct?ccagcaacct?gtcctggctc?agcctggacg?tcagcgcagc?cttcggaccc????1800

ggccctggcc?tgcagagcct?gaccaacctg?ctcagcagca?acctcagctg?gctgggccca????1860

ggacccggcg?caggcttctt?tctgctcacc?agaatcctga?ccatccctca?gagcggcccc????1920

ggaccaggcg?tgagcttcgg?cgtgtggatt?cggactcctc?ccgcctacag?acccccaaat????1980

gcccccatcg?gcccaggacc?cggcgtcgga?cctctgactg?tgaacgagaa?gcggagactg????2040

aagctgatcg?gccccggacc?aggcaaacag?tgcttcagga?agctccctgt?gaacagacct????2100

atcgactggg?gccccggacc?cggcgcagcc?aactggattc?tgagaggcac?cagcttcgtg????2160

tacgtccctg?gacccggccc?tggcaagcaa?gccttcacct?tcagccccac?ctacaaggca????2220

ttcctgtgcg?gatag?????????????????????????????????????????????????????2235

<210>208

<211>744

<212>PRT

＜213〉hepatitis type B virus

<400>208

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu?Asn

20??????????????????25??????????????????30

Ala?Ala?Ala?Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Ala?Ala?Ala

35??????????????????40??????????????????45

Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala?Phe?Pro

50??????????????????55??????????????????60

His?Cys?Leu?Ala?Phe?Ser?Tyr?Met?Lys?Ala?Ala?Leu?Val?Val?Asp?Phe

65??????????????????70??????????????????75??????????????????80

Ser?Gln?Phe?Ser?Arg?Gly?Ala?Ile?Leu?Leu?Leu?Cys?Leu?Ile?Phe?Leu

85??????????????????90??????????????????95

Leu?Asn?Ala?Ala?Ala?His?Thr?Leu?Trp?Lys?Ata?Gly?Ile?Leu?Tyr?Lys

100?????????????????105?????????????????110

Lys?Ala?Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Tyr

115?????????????????120?????????????????125

Pro?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile?Gly?Ala?Ala?Ala?Trp?Leu

130?????????????????135?????????????????140

Ser?Leu?Leu?Val?Pro?Phe?Val?Asn?Ala?Ala?Ala?Gly?Phe?Leu?Leu?Thr

145?????????????????150?????????????????155?????????????????160

Arg?Ile?Leu?Thr?Ile?Asn?Ala?Ala?Ala?Ile?Pro?Ile?Pro?Ser?Ser?Trp

165?????????????????170?????????????????175

Ala?Phe?Lys?Ala?Ala?Ala?Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Asn

180?????????????????185?????????????????190

Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Phe?Leu?Pro

195?????????????????200?????????????????205

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Ala?Ala?Asp?Leu?Leu?Asp?Thr

210?????????????????215?????????????????220

Ala?Ser?Ala?Leu?Tyr?Asn?Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu

225?????????????????230?????????????????235?????????????????240

Lys?Ala?Ala?Ala?Ser?Ala?Ile?Cys?Ser?Val?Val?Arg?Arg?Lys?Leu?Ser

245?????????????????250?????????????????255

Leu?Asp?Val?Ser?Ala?Ala?Phe?Tyr?Asn?Ala?Ala?Ala?Lys?Phe?Val?Ala

260?????????????????265?????????????????270

Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Ala?Ala?Asn?Val?Ser?Ile?Pro

275?????????????????280?????????????????285

Trp?Thr?His?Lys?Gly?Ala?Ala?Gly?Leu?Ser?Arg?Tyr?Val?Ala?Arg?Leu

290?????????????????295?????????????????300

Asn?Ala?Ala?Ala?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Lys

305?????????????????310?????????????????315?????????????????320

His?Pro?Ala?Ala?Met?Pro?His?Leu?Leu?Lys?Ala?Ala?Ala?Arg?Trp?Met

325?????????????????330?????????????????335

Cys?Leu?Arg?Arg?Phe?Ile?Ile?Asn?Ala?Ser?Phe?Cys?Gly?Ser?Pro?Tyr

340?????????????????345?????????????????350

Lys?Ala?Ala?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Asn?Ala?Leu?Trp

355?????????????????360?????????????????365

Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Lys?Ala?Ala?Ala?Thr?Pro?Ala?Arg

370?????????????????375?????????????????380

Val?Thr?Gly?Gly?Val?Phe?Lys?Ala?Ala?Ala?Leu?Thr?Phe?Gly?Arg?Glu

385?????????????????390?????????????????395?????????????????400

Thr?Val?Leu?Glu?Tyr?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr?Lys

405?????????????????410?????????????????415

Asn?Ala?Gly?Thr?Ser?Phe?Val?Tyr?Val?Pro?Ser?Ala?Leu?Asn?Pro?Ala

420?????????????????425?????????????????430

Asp?Gly?Pro?Gly?Pro?Gly?Leu?Cys?Gln?Val?Phe?Ala?Asp?Ala?Thr?Pro

435?????????????????440?????????????????445

Thr?Gly?Trp?Gly?Leu?Gly?Pro?Gly?Pro?Gly?Arg?His?Tyr?Leu?His?Thr

450?????????????????455?????????????????460

Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Pro?His

465?????????????????470?????????????????475?????????????????480

His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu?Leu?Met?Thr

485?????????????????490?????????????????495

Leu?Ala?Gly?Pro?Gly?Pro?Gly?Glu?Ser?Arg?Leu?Val?Val?Asp?Phe?Ser

500?????????????????505?????????????????510

Gln?Phe?Ser?Arg?Gly?Asn?Gly?Pro?Gly?Pro?Gly?Pro?Phe?Leu?Leu?Ala

515?????????????????520?????????????????525

Gln?Phe?Thr?Ser?Ala?Ile?Cys?Ser?Val?Val?Gly?Pro?Gly?Pro?Gly?Leu

530?????????????????535?????????????????540

Val?Pro?Phe?Val?Gln?Trp?Phe?Val?Gly?Leu?Ser?Pro?Thr?Val?Gly?Pro

545?????????????????550?????????????????555?????????????????560

Gly?Pro?Gly?Leu?His?Leu?Tyr?Ser?His?Pro?Ile?Ile?Leu?Gly?Phe?Arg

565?????????????????570?????????????????575

Lys?Ile?Gly?Pro?Gly?Pro?Gly?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Ser?Leu

580?????????????????585?????????????????590

Asp?Val?Ser?Ala?Ala?Phe?Gly?Pro?Gly?Pro?Gly?Leu?Gln?Ser?Leu?Thr

595?????????????????600?????????????????605

Asn?Leu?Leu?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Gly?Pro?Gly?Pro?Gly?Ala

610?????????????????615?????????????????620

Gly?Phe?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Pro?Gln?Ser?Gly?Pro

625?????????????????630?????????????????635?????????????????640

Gly?Pro?Gly?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr

645?????????????????650?????????????????655

Arg?Pro?Pro?Asn?Ala?Pro?Ile?Gly?Pro?Gly?Pro?Gly?Val?Gly?Pro?Leu

660?????????????????665?????????????????670

Thr?Val?Asn?Glu?Lys?Arg?Arg?Leu?Lys?Leu?Ile?Gly?Pro?Gly?Pro?Gly

675?????????????????680?????????????????685

Lys?Gln?Cys?Phe?Arg?Lys?Leu?Pro?Val?Asn?Arg?Pro?Ile?Asp?Trp?Gly

690?????????????????695?????????????????700

Pro?Gly?Pro?Gly?Ala?Ala?Asn?Trp?Ile?Leu?Arg?Gly?Thr?Ser?Phe?Val

705?????????????????710?????????????????715?????????????????720

Tyr?Val?Pro?Gly?Pro?Gly?Pro?Gly?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro

725?????????????????730?????????????????735

Thr?Tyr?Lys?Ala?Phe?Leu?Cys?Gly

740

<210>209

<211>621

<212>DNA

＜213〉hepatitis type B virus

<400>209

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgtt?cctgctgtcc????180

ctgggaatcc?acctgtatat?ggatgacgtg?gtgctgggag?tgggactgtc?caggtacgtg????240

gctaggctgt?tcctgctgac?cagaatcctg?accatctcca?ccctgccaga?gaccaccgtg????300

gtgaggaggc?aggccttcac?ctttagccct?acctataagt?ggctgagcct?gctggtgccc????360

tttgtgatcc?ctatccctag?ctcctgggct?ttcaccccag?ccagggtgac?cggaggagtg????420

tttaaggtgg?gaaacttcac?cggcctgtat?ctgcccagcg?atttctttcc?tagcgtgacc????480

ctgtggaagg?ccgggatcct?gtacaagaat?gtgtccatcc?cttggaccca?caagctggtg????540

gtggactttt?cccagttcag?cagatccgct?atctgctccg?tggtgaggag?agctctgatg????600

ccactgtatg?cctgtatctg?a??????????????????????????????????????????????621

<210>210

<211>206

<212>PRT

＜213〉hepatitis type B virus

<400>210

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His

50??????????????????55??????????????????60

Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr?Val

65??????????????????70??????????????????75??????????????????80

Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Ser?Thr?Leu?Pro

85??????????????????90??????????????????95

Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr

100?????????????????105?????????????????110

Lys?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Ile?Pro?Ile?Pro?Ser?Ser

115?????????????????120?????????????????125

Trp?Ala?Phe?Thr?Pro?Ala?Arg?Val?Thr?Gly?Gly?Val?Phe?Lys?Val?Gly

130?????????????????135?????????????????140

Asn?Phe?Thr?Gly?Leu?Tyr?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Thr

145?????????????????150?????????????????155?????????????????160

Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Asn?Val?Ser?Ile?Pro?Trp?Thr

165?????????????????170?????????????????175

His?Lys?Leu?Val?Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Ser?Ala?Ile?Cys

180?????????????????185?????????????????190

Ser?Val?Val?Arg?Arg?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

195?????????????????200?????????????????205

<210>211

<211>660

<212>DNA

＜213〉hepatitis type B virus

<400>211

atgggaatgc?agttgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgaa?cttcctgctg????180

tccctgggaa?tccacctgta?tatggatgac?gtggtgctgg?gagtgggact?gtccaggtac????240

gtggctaggc?tgttcctgct?gaccagaatc?ctgaccatct?ccaccctgcc?agagaccacc????300

gtggtgagga?ggcaggcctt?cacctttagc?cctacctata?agggagccgc?tgcctggctg????360

agcctgctgg?tgccctttgt?gaatatccct?atccctagct?cctgggcttt?caagacccca????420

gccagggtga?ccggaggagt?gtttaaggtg?ggaaacttca?ccggcctgta?taacctgccc????480

agcgatttct?ttcctagcgt?gaagaccctg?tggaaggccg?gaatcctgta?caagaatgtg????540

tccatccctt?ggacccacaa?gggagccgct?ctggtggtgg?acttttccca?gttcagcaga????600

aattccgcta?tctgctccgt?ggtgaggaga?gctctgatgc?cactgtatgc?ctgtatctga????660

<210>212

<211>219

<212>PRT

＜213〉hepatitis type B virus

<400>212

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Asn?Phe?Leu?Leu?Ser?Leu?Gly?Ile

50??????????????????55??????????????????60

His?Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr

65??????????????????70??????????????????75??????????????????80

Val?Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Ser?Thr?Leu

85??????????????????90??????????????????95

Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

100?????????????????105?????????????????110

Tyr?Lys?Gly?Ala?Ala?Ala?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Asn

115?????????????????120?????????????????125

Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Thr?Pro?Ala?Arg?Val?Thr

130?????????????????135?????????????????140

Gly?Gly?Val?Phe?Lys?Val?Gly?Asn?Phe?Thr?Gly?Leu?Tyr?Asn?Leu?Pro

145?????????????????150?????????????????155?????????????????160

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu

165?????????????????170?????????????????175

Tyr?Lys?Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys?Gly?Ala?Ala?Leu?Val

180?????????????????185?????????????????190

Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Asn?Ser?Ala?Ile?Cys?Ser?Val?Val

195?????????????????200?????????????????205

Arg?Arg?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

210?????????????????215

<210>213

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>213

Thr?Leu?Asn?Phe?Pro?Ile?Ser?Pro?Ile

1???????????????5

<210>214

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>214

Ser?Leu?Leu?Asn?Ala?Thr?Asp?Ile?Ala?Val

1???????????????5???????????????????10

<210>215

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>215

Gln?Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys

1???????????????5???????????????????10

<210>216

<211>11

<212>PRT

＜213〉hepatitis type B virus

<400>216

Val?Thr?Val?Tyr?Tyr?Gly?Val?Pro?Val?Trp?Lys

1???????????????5???????????????????10

<210>217

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>217

Phe?Pro?Val?Arg?Pro?Gln?Val?Pro?Leu

1???????????????5

<210>218

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>218

Tyr?Pro?Leu?Ala?Ser?Leu?Arg?Ser?Leu?Phe

1???????????????5???????????????????10

<210>219

<211>10

<212>PRT

＜213〉hepatitis type B virus

<400>219

Val?Ile?Tyr?Gln?Tyr?Met?Asp?Asp?Leu?Tyr

1???????????????5???????????????????10

<210>220

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>220

Ile?Tyr?Gln?Glu?Pro?Phe?Lys?Asn?Leu

1???????????????5

<210>221

<211>9

<212>PRT

＜213〉hepatitis type B virus

<400>221

Ile?Trp?Gly?Cys?Ser?Gly?Lys?Leu?Ile

1???????????????5

<210>222

<211>4

<212>PRT

＜213〉the unknown

<220>

＜223〉peptide connector

<400>222

Gly?Ala?Ala?Ala

1

<210>223

<211>4

<212>PRT

＜213〉the unknown

<220>

＜223〉peptide connector

<400>223

Asn?Ala?Ala?Ala

1

<210>224

<211>4

<212>PRT

＜213〉the unknown

<220>

＜223〉peptide connector

<400>224

Lys?Ala?Ala?Ala

1

<210>225

<211>277

<212>PRT

＜213〉human immunodeficiency virus

<400>225

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Lys?Leu?Val?Gly?Lys?Leu?Asn?Trp?Ala?Gly

20??????????????????25??????????????????30

Ala?Ala?Ile?Leu?Lys?Glu?Pro?Val?His?Gly?Val?Asn?Ala?Ala?Cys?Pro

35??????????????????40??????????????????45

Lys?Val?Ser?Phe?Glu?Pro?Ile?Lys?Ile?Pro?Ile?His?Tyr?Cys?Ala?Pro

50??????????????????55??????????????????60

Ala?Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys

65??????????????????70??????????????????75??????????????????80

Ala?Phe?Pro?Val?Arg?Pro?Gln?Val?Pro?Leu?Gly?Ala?Ala?Lys?Leu?Thr

85??????????????????90??????????????????95

Pro?Leu?Cys?Val?Thr?Leu?Gly?Ala?Ala?Ala?Val?Leu?Ala?Glu?Ala?Met

100?????????????????105?????????????????110

Ser?Gln?Val?Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys?Gly?Ala

115?????????????????120?????????????????125

Ala?Ala?Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys?Lys?Thr?Thr?Leu?Phe

130?????????????????135?????????????????140

Cys?Ala?Ser?Asp?Ala?Lys?Asn?Ile?Pro?Tyr?Asn?Pro?Gln?Ser?Gln?Gly

145?????????????????150?????????????????155?????????????????160

Val?Val?Lys?His?Pro?Val?His?Ala?Gly?Pro?Ile?Ala?Asn?Val?Thr?Val

165?????????????????170?????????????????175

Tyr?Tyr?Gly?Val?Pro?Val?Trp?Lys?Lys?Ala?Ala?Ala?Gln?Met?Ala?Val

180?????????????????185?????????????????190

Phe?Ile?His?Asn?Phe?Lys?Asn?Ala?Ala?Ala?Tyr?Pro?Leu?Ala?Ser?Leu

195?????????????????200?????????????????205

Arg?Ser?Leu?Phe?Asn?Leu?Thr?Phe?Gly?Trp?Cys?Phe?Lys?Leu?Asn?Arg

210?????????????????215?????????????????220

Ile?Leu?Gln?Gln?Leu?Leu?Phe?Ile?Asn?Ala?Lys?Ile?Gln?Asn?Phe?Arg

225?????????????????230?????????????????235?????????????????240

Val?Tyr?Tyr?Arg?Lys?Ala?Ala?Val?Thr?Ile?Lys?Ile?Gly?Gly?Gln?Leu

245?????????????????250?????????????????255

Lys?Lys?Val?Pro?Leu?Gln?Leu?Pro?Pro?Leu?Lys?Ala?Met?Thr?Asn?Asn

260?????????????????265?????????????????270

Pro?Pro?Ile?Pro?Val

275

<210>226

<211>834

<212>DNA

＜213〉human immunodeficiency virus

<400>226

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatcc?????60

agaggaaagc?tggtgggcaa?actcaactgg?gccggagctg?caatcctgaa?ggagcccgtc????120

cacggggtga?atgccgcttg?ccctaaagtc?agcttcgaac?caattaagat?ccccattcat????180

tactgtgcac?ctgccaaagc?taagtttgtg?gccgcttgga?ccctcaaggc?cgctgcaaaa????240

gccttcccag?tgaggcccca?ggtgcctctg?ggcgccgcta?aactcacacc?actgtgcgtc????300

actctgggag?ccgctgcagt?gctggcagag?gccatgtccc?aagtgaaggt?gtatctggct????360

tgggtgcccg?cccacaaggg?ggccgctgca?gccatctttc?agtctagcat?gaccaagaaa????420

acaactctgt?tctgtgcctc?cgacgctaag?aacatccctt?ataatccaca?gtctcagggc????480

gtggtcaagc?atcccgtgca?cgccggacct?attgctaacg?tgaccgtgta?ctatggggtc????540

ccagtgtgga?agaaagccgc?tgcacagatg?gccgtgttta?ttcacaattt?caaaaacgcc????600

gctgcatacc?ccctcgccag?cctgagatcc?ctcttcaacc?tgacattcgg?ctggtgcttt????660

aagctgaacc?ggatcctgca?gcaactgctc?tttatcaatg?ctaaaatcca?gaacttccgc????720

gtctactata?ggaaggctgc?agtgactatc?aaaattggcg?gacaactgaa?gaaagtgcct????780

ctccagctgc?cccctctcaa?ggcaatgacc?aacaatcccc?ctatcccagt?ctga??????????834

<210>227

<211>280

<212>PRT

＜213〉human immunodeficiency virus

<400>227

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Ile?Pro?Ile?His?Tyr?Cys?Ala?Pro?Ala?Lys

20??????????????????25??????????????????30

Ala?Ala?Lys?Ile?Gln?Asn?Phe?Arg?Val?Tyr?Tyr?Arg?Lys?Ala?Ala?Val

35??????????????????40??????????????????45

Thr?Ile?Lys?Ile?Gly?Gly?Gln?Leu?Lys?Lys?Ala?Lys?Phe?Val?Ala?Ala

50??????????????????55??????????????????60

Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Val?Pro?Leu?Gln?Leu?Pro?Pro?Leu

65??????????????????70??????????????????75??????????????????80

Lys?Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys?Lys?Leu?Thr?Pro?Leu?Cys

85??????????????????90??????????????????95

Val?Thr?Leu?Gly?Ala?Gln?Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys?Gly

100?????????????????105?????????????????110

Ala?Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys?Asn?Ala?Ile?Pro

115?????????????????120?????????????????125

Tyr?Asn?Pro?Gln?Ser?Gln?Gly?Val?Val?Lys?Ala?Ile?Leu?Lys?Glu?Pro

130?????????????????135?????????????????140

Val?His?Gly?Val?Gly?Ala?Ala?Ala?Leu?Thr?Phe?Gly?Trp?Cys?Phe?Lys

145?????????????????150?????????????????155?????????????????160

Leu?Asn?Ala?Val?Leu?Ala?G1u?Ala?Met?Ser?Gln?Val?Asn?Arg?Ile?Leu

165?????????????????170?????????????????175

Gln?Gln?Leu?Leu?Phe?Ile?Asn?Ala?Ala?Ala?Cys?Pro?Lys?Val?Ser?Phe

180?????????????????185?????????????????190

Glu?Pro?Ile?Lys?Val?Thr?Val?Tyr?Tyr?Gly?Val?Pro?Val?Trp?Lys?Lys

195?????????????????200?????????????????205

Ala?Ala?His?Pro?Val?His?Ala?Gly?Pro?Ile?Ala?Asn?Ala?Ala?Ala?Tyr

210?????????????????215?????????????????220

Pro?Leu?Ala?Ser?Leu?Arg?Ser?Leu?Phe?Asn?Ala?Ala?Ala?Thr?Thr?Leu

225?????????????????230?????????????????235?????????????????240

Phe?Cys?Ala?Ser?Asp?Ala?Lys?Asn?Lys?Leu?Val?Gly?Lys?Leu?Asn?Trp

245?????????????????250?????????????????255

Ala?Asn?Ala?Ala?Ala?Phe?Pro?Val?Arg?Pro?Gln?Val?Pro?Leu?Asn?Met

260?????????????????265?????????????????270

Thr?Asn?Asn?Pro?Pro?Ile?Pro?Val

275?????????????????280

<210>228

<211>843

<212>DNA

＜213〉human immunodeficiency virus

<400>228

atggggatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatcc?????60

agaggaatcc?ccattcacta?ctgcgcccct?gctaaggcag?ccaaaatcca?gaacttcagg????120

gtgtattaca?gaaaggctgc?agtcaccatt?aaaatcggcg?gacaactgaa?gaaagccaag????180

tttgtggccg?cttggacact?caaggccgct?gcaaaggtcc?cactgcagct?cccccctctg????240

aaggccatct?tccagagctc?catgactaag?aaactgaccc?cactgtgtgt?gacactcggg????300

gcccagatgg?ctgtgttcat?ccataatttt?aaaggcgcca?aggtctacct?ggcttgggtg????360

cccgcacaca?agaacgccat?tccttacaat?ccacagtctc?aaggagtggt?caaagctatt????420

ctgaaggagc?ccgtgcacgg?ggtgggcgcc?gctgcactca?ctttcggatg?gtgctttaaa????480

ctgaacgccg?tgctggctga?agccatgagc?caggtcaatc?ggatcctgca?gcaactgctc????540

ttcattaacg?ccgctgcatg?tcctaaggtg?tccttcgagc?caatcaaagt?gaccgtgtat????600

tacggggtcc?ccgtgtggaa?gaaagccgct?catcctgtcc?acgcaggccc?aatcgccaac????660

gccgctgcat?atcccctcgc?ctctctgcgc?agcctgttta?acgccgctgc?aacaaccctc????720

ttttgcgcct?ccgacgctaa?gaataaactg?gtgggaaagc?tgaactgggc?caacgcagct????780

gcattccctg?tgaggccaca?ggtccccctc?aatatgacta?acaatccccc?tatcccagtg????840

tga??????????????????????????????????????????????????????????????????843

<210>229

<211>211

<212>PRT

＜213〉human immunodeficiency virus

<400>229

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Lys?Leu?Val?Gly?Lys?Leu?Asn?Trp?Ala?Met?Ala?Ser

20??????????????????25??????????????????30

Asp?Phe?Asn?Leu?Pro?Pro?Val?Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys

35??????????????????40??????????????????45

Val?Thr?Ile?Lys?Ile?Gly?Gly?Gln?Leu?Lys?Arg?Ile?Leu?Gln?Gln?Leu

50??????????????????55??????????????????60

Leu?Phe?Ile?Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys?Ile?Pro?Tyr?Asn

65??????????????????70??????????????????75??????????????????80

Pro?Gln?Ser?Gln?Gly?Val?Val?Thr?Thr?Leu?Phe?Cys?Ala?Ser?Asp?Ala

85??????????????????90??????????????????95

Lys?Ile?Leu?Lys?Glu?Pro?Val?His?Gly?Val?Gln?Met?Ala?Val?Phe?Ile

100?????????????????105?????????????????110

His?Asn?Phe?Lys?Gly?Ala?Ala?Val?Phe?Ile?His?Asn?Phe?Lys?Arg?Cys

115?????????????????120?????????????????125

Pro?Lys?Val?Ser?Phe?Glu?Pro?Ile?Lys?Ile?Gln?Asn?Phe?Arg?Val?Tyr

130?????????????????135?????????????????140

Tyr?Arg?Leu?Thr?Phe?Gly?Trp?Cys?Phe?Lys?Leu?Gln?Val?Pro?Leu?Arg

145?????????????????150?????????????????155?????????????????160

Pro?Met?Thr?Tyr?Lys?Met?Thr?Asn?Asn?Pro?Pro?Ile?Pro?Val?Thr?Val

165?????????????????170?????????????????175

Tyr?Tyr?Gly?Val?Pro?Val?Trp?Lys?Val?Leu?Ala?Glu?Ala?Met?Ser?Gln

180?????????????????185?????????????????190

Val?Ile?Pro?Ile?His?Tyr?Cys?Ala?Pro?Ala?Lys?Leu?Thr?Pro?Leu?Cys

195?????????????????200?????????????????205

Val?Thr?Leu

210

<210>230

<211>633

<212>DNA

＜213〉human immunodeficiency virus

<400>230

atgcaggtgc?agatccagag?cctgtttctg?ctcctcctgt?gggtgcccgg?atccagagga?????60

aagctggtgg?ggaagctgaa?ctgggccatg?gccagcgatt?tcaacctgcc?ccccgtggcc????120

atcttccaga?gcagcatgac?caaggtgacc?atcaagatcg?gggggcagct?gaagaggatc????180

ctgcagcagc?tgctgttcat?catggccgtg?ttcatccaca?acttcaagat?cccctacaac????240

ccccagagcc?agggggtggt?gaccaccctg?ttctgcgcca?gcgatgccaa?gatcctgaag????300

gagcccgtgc?acggggtgca?gatggccgtg?ttcatccaca?acttcaaggg?cgccgccgtg????360

ttcatccaca?acttcaagag?gtgccccaag?gtgagcttcg?agcccatcaa?gatccagaac????420

ttcagggtgt?actacaggct?gaccttcggg?tggtgcttca?agctgcaggt?gcccctgagg????480

cccatgacct?acaagatgac?caacaacccc?cccatccccg?tgaccgtgta?ctacggggtg????540

cccgtgtgga?aggtgctggc?cgaggccatg?agccaggtga?tccccatcca?ctactgcgcc????600

cccgccaagc?tgacccccct?gtgcgtgacc?ctg?????????????????????????????????633

<210>231

<211>585

<212>PRT

＜213〉human immunodeficiency virus

<400>231

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Tyr?Trp?Gln?Ala?Thr?Trp?Ile?Pro?Glu?Trp

20??????????????????25??????????????????30

Lys?Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys?Lys?Val?Tyr?Leu?Ala?Trp

35??????????????????40??????????????????45

Val?Pro?Ala?His?Lys?Asn?Ala?Ala?Cys?Pro?Lys?Val?Ser?Phe?Glu?Pro

50??????????????????55??????????????????60

Ile?Lys?His?Pro?Val?His?Ala?Gly?Pro?Ile?Ala?Asn?Leu?Thr?Phe?Gly

65??????????????????70??????????????????75??????????????????80

Trp?Cys?Phe?Lys?Leu?Asn?Lys?Met?Ile?Gly?GIy?Ile?Gly?Gly?Phe?Ile

85??????????????????90??????????????????95

Lys?Phe?Arg?Asp?Tyr?Val?Asp?Arg?Phe?Tyr?Lys?Ala?Ala?Ala?Arg?Ile

100?????????????????105?????????????????110

Leu?Gln?Gln?Leu?Leu?Phe?Ile?Asn?Thr?Thr?Leu?Phe?Cys?Ala?Ser?Asp

115?????????????????120?????????????????125

Ala?Lys?Asn?Gln?Met?Val?His?Gln?Ala?Ile?Ser?Pro?Arg?Gly?Ala?Lys

130?????????????????135?????????????????140

Leu?Val?Gly?Lys?Leu?Asn?Trp?Ala?Gly?Ala?Ala?Ala?Ile?Tyr?Glu?Thr

145?????????????????150?????????????????155?????????????????160

Tyr?Gly?Asp?Thr?Trp?Lys?Ala?Ala?Gln?Val?Pro?Leu?Arg?Pro?Met?Thr

165?????????????????170?????????????????175

Tyr?Lys?Gly?Ala?Ala?Ala?Val?Thr?Val?Leu?Asp?Val?Gly?Asp?Ala?Tyr

180?????????????????185?????????????????190

Asn?Ala?Ala?Ala?Arg?Tyr?Leu?Lys?Asp?Gln?Gln?Leu?Leu?Asn?Thr?Leu

195?????????????????200?????????????????205

Ash?Phe?Pro?Ile?Ser?Pro?Ile?Asn?Met?Thr?Asn?Asn?Pro?Pro?Ile?Pro

210?????????????????215?????????????????220

Val?Asn?Ala?Pro?Tyr?Asn?Thr?Pro?Val?Phe?Ala?Ile?Lys?Ala?Ala?Ala

225?????????????????230?????????????????235?????????????????240

Val?Pro?Leu?Gln?Leu?Pro?Pro?Leu?Lys?Ala?Ala?Ile?Pro?Tyr?Asn?Pro

245?????????????????250?????????????????255

Gln?Ser?Gln?Gly?Val?Val?Lys?Ala?Leu?Leu?Gln?Leu?Thr?Val?Trp?Gly

260?????????????????265?????????????????270

Ile?Gly?Ala?Ala?Ile?Leu?Lys?Glu?Pro?Val?His?Gly?Val?Asn?Ala?Ala

275?????????????????280?????????????????285

Ala?Phe?Pro?Ile?Ser?Pro?Ile?Glu?Thr?Val?Lys?Val?Trp?Lys?Glu?Ala

290?????????????????295?????????????????300

Thr?Thr?Thr?Leu?Phe?Lys?Ala?Ala?Ala?Val?Thr?Ile?Lys?Ile?Gly?Gly

305?????????????????310?????????????????315?????????????????320

Gln?Leu?Lys?Lys?Ile?Tyr?Gln?Glu?Pro?Phe?Lys?Asn?Leu?Lys?Ala?Ala

325?????????????????330?????????????????335

Ala?Val?Leu?Ala?Glu?Ala?Met?Ser?Gln?Val?Asn?Leu?Val?Gly?Pro?Thr

340?????????????????345?????????????????350

Pro?Val?Asn?Ile?Gly?Ala?Ala?Ala?Glu?Val?Asn?Ile?Val?Thr?Asp?Ser

355?????????????????360?????????????????365

Gln?Tyr?Lys?Ala?Ala?Ala?Ile?Pro?Ile?His?Tyr?Cys?Ala?Pro?Ala?Lys

370?????????????????375?????????????????380

Ala?Val?Ile?Tyr?Gln?Tyr?Met?Asp?Asp?Leu?Tyr?Lys?Ala?Ala?Ala?Gln

385?????????????????390?????????????????395?????????????????400

Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys?Asn?Ala?Ala?Thr?Tyr?Gln?Ile

405?????????????????410?????????????????415

Tyr?Gln?Glu?Pro?Phe?Lys?Pro?Tyr?Asn?Glu?Trp?Thr?Leu?Glu?Leu?Lys

420?????????????????425?????????????????430

Ala?Lys?Ile?Gln?Asn?Phe?Arg?Val?Tyr?Tyr?Arg?Lys?Ala?Phe?Pro?Val

435?????????????????440?????????????????445

Arg?Pro?Gln?Val?Pro?Leu?Gly?Ala?Ala?Ala?Ile?Trp?Gly?Cys?Ser?Gly

450?????????????????455?????????????????460

Lys?Leu?Ile?Lys?Val?Met?Ile?Val?Trp?Gln?Val?Asp?Arg?Asn?Ala?Ala

465?????????????????470?????????????????475?????????????????480

Lys?Ala?Ala?Cys?Trp?Trp?Ala?Gly?Ile?Lys?Ala?Lys?Phe?Val?Ala?Ala

485?????????????????490?????????????????495

Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Leu?Thr?Pro?Leu?Cys?Val?Thr?Leu

500?????????????????505?????????????????510

Asn?Ala?Ala?Met?Ala?Ser?Asp?Phe?Asn?Leu?Pro?Pro?Val?Lys?Ser?Leu

515?????????????????520?????????????????525

Leu?Asn?Ala?Thr?Asp?Ile?Ala?Val?Asn?Val?Thr?Val?Tyr?Tyr?Gly?Val

530?????????????????535?????????????????540

Pro?Val?Trp?Lys?Lys?Ala?Ala?Ala?Ala?Ile?Ile?Arg?Ile?Leu?Gln?Gln

545?????????????????550?????????????????555?????????????????560

Leu?Lys?Arg?Ala?Met?Ala?Ser?Asp?Phe?Asn?Leu?Asn?Ala?Ala?Ala?Tyr

565?????????????????570?????????????????575

Pro?Leu?Ala?Ser?Leu?Arg?Ser?Leu?Phe

580?????????????????585

<210>232

<211>1758

<212>DNA

＜213〉human immunodeficiency virus

<400>232

atggggatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatct?????60

agaggatact?ggcaagctac?ttggattcca?gaatggaaag?ctatctttca?atcctcaatg????120

acgaagaagg?tatacctggc?atgggtccca?gcacacaaga?acgccgcttg?cccaaaggtg????180

tcctttgaac?ccattaaaca?cccagtgcac?gcagggccaa?tagcgaattt?gacattcggg????240

tggtgcttca?aactaaacaa?aatgatcggc?ggcattggag?gctttatcaa?gtttagagat????300

tacgtggacc?gattctataa?agccgctgcc?cgtatactcc?agcagctact?attcatcaac????360

accactctct?tctgcgcttc?agacgctaag?aaccaaatgg?tacaccaagc?cataagccct????420

agaggagcca?agctcgtagg?gaaattaaat?tgggcgggtg?cagcagcaat?ctacgagact????480

tacggcgata?cctggaaagc?agcccaggtt?ccgttacgcc?caatgaccta?taaaggcgca????540

gcagcagtaa?cagttctaga?tgtaggagac?gcttacaacg?ctgccgcaag?atacctaaaa?????600

gatcagcagt?tactcaacac?actaaatttc?ccaattagcc?cgataaacat?gacaaataac?????660

ccaccaattc?ccgtcaatgc?tccctacaac?actccagtat?tcgcaatcaa?agccgctgct?????720

gtccccctgc?agctccctcc?tctgaaagct?gcgatacctt?acaacccaca?gagccaaggt?????780

gttgtcaaag?cactgcttca?gctaacagtt?tggggaattg?gtgctgcaat?tctaaaagag?????840

ccagttcatg?gggttaacgc?cgccgccttc?ccaatcagtc?ctattgagac?tgtgaaagta?????900

tggaaagaag?ccacaaccac?actttttaag?gcagccgcag?ttacaattaa?aatagggggc?????960

caacttaaga?aaatatacca?ggaacctttc?aagaatctca?aagccgctgc?agtgctcgcc????1020

gaggctatgt?cacaggtgaa?tttggtcgga?ccaacacccg?taaacatcgg?agccgcagcc????1080

gaagtgaaca?tagtcaccga?ctcacagtac?aaagccgctg?caatacccat?acattattgt????1140

gctcccgcaa?aggccgtgat?ctatcaatat?atggacgacc?tgtataaggc?cgccgcgcag????1200

atggcagtct?ttatccacaa?ctttaaaaac?gcagctactt?atcagatcta?ccaggaacca????1260

ttcaaaccgt?acaatgagtg?gaccttggaa?ctaaaggcca?aaattcagaa?cttcagggta????1320

tattatagaa?aagcatttcc?agtgaggccc?caggtgcctc?tgggtgccgc?agcaatatgg????1380

ggatgttctg?gaaaactgat?caaggtgatg?attgtatggc?aagtggacag?aaatgcagct????1440

aaggcagcct?gttggtgggc?aggtataaaa?gcaaagttcg?tggcagcatg?gacgcttaaa????1500

gcagccgcaa?aactcactcc?tctctgcgtg?acacttaatg?cagccatggc?ctctgatttc????1560

aaccttcccc?ctgtaaaatc?cctgcttaat?gcgacagata?tcgcagtcaa?cgtaacagta????1620

tattatggcg?tgccagtctg?gaaaaaagcc?gccgcggcca?taattcggat?actgcagcag????1680

ctgaaaagag?ctatggcgag?tgacttcaac?ctgaatgcgg?ccgcctaccc?cttggcatcg????1740

ttaaggtcac?tattttga??????????????????????????????????????????????????1758

<210>233

<211>255

<212>PRT

＜213〉hepatitis C virus

<400>233

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Leu?Leu?Phe?Asn?Ile?Leu?Gly?Gly?Trp?Val

20??????????????????25??????????????????30

Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val?Tyr?Leu?Val?Ala?Tyr?Gln?Ala

35??????????????????40??????????????????45

Thr?Val?Ile?Leu?Ala?Gly?Tyr?Gly?Ala?Gly?Val?Arg?Leu?Ile?Val?Phe

50??????????????????55??????????????????60

Pro?Asp?Leu?Gly?Val?His?Met?Trp?Asn?Phe?Ile?Ser?Gly?Ile?Tyr?Leu

65??????????????????70??????????????????75??????????????????80

Leu?Pro?Arg?Arg?Gly?Pro?Arg?Leu?Tyr?Leu?Val?Thr?Arg?His?Ala?Asp

85??????????????????90??????????????????95

Val?Val?Leu?Val?Gly?Gly?Val?Leu?Ala?Ala?Leu?Leu?Phe?Leu?Leu?Leu

100?????????????????105?????????????????110

Ala?Asp?Ala?Phe?Leu?Leu?Leu?Ala?Asp?Ala?Arg?Val?Trp?Met?Asn?Arg

115?????????????????120?????????????????125

Leu?Ile?Ala?Phe?Ala?Cys?Thr?Cys?Gly?Ser?Ser?Asp?Leu?Tyr?Leu?Ser

130?????????????????135?????????????????140

Ala?Phe?Ser?Leu?His?Ser?Tyr?Gly?Val?Ala?Gly?Ala?Leu?Val?Ala?Phe

145?????????????????150?????????????????155?????????????????160

Lys?Leu?Pro?Gly?Cys?Ser?Phe?Ser?Ile?Phe?Lys?Thr?Ser?Glu?Arg?Ser

165?????????????????170?????????????????175

Gln?Pro?Arg?Leu?Ile?Phe?Cys?His?Ser?Lys?Lys?Lys?Phe?Trp?Ala?Lys

180?????????????????185?????????????????190

His?Met?Trp?Asn?Phe?Ile?Pro?Phe?Tyr?Gly?Lys?Ala?Ile?Arg?Met?Tyr

195?????????????????200?????????????????205

Val?Gly?Gly?Val?Glu?His?Arg?Gln?Leu?Phe?Thr?Phe?Ser?Pro?Arg?Arg

210?????????????????215?????????????????220

Arg?Leu?Gly?Val?Arg?Ala?Thr?Arg?Lys?Val?Gly?Ile?Tyr?Leu?Leu?Pro

225?????????????????230?????????????????235?????????????????240

Asn?Arg?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala

245?????????????????250?????????????????255

<210>234

<211>747

<212>DNA

＜213〉hepatitis C virus

<400>234

gaattcgccg?ccaccatgca?ggtgcagatc?cagagcctgt?ttctgctcct?cctgtgggtg?????60

cccggatcca?gaggactgct?gttcaacatc?ctgggggggt?gggtggatct?gatggggtac????120

atccccctgg?tgtacctggt?ggcctaccag?gccaccgtga?tcctggccgg?gtacggggcc????180

ggggtgaggc?tgatcgtgtt?ccccgatctg?ggggtgcaca?tgtggaactt?catcagcggg????240

atctacctgc?tgcccaggag?aggacctaga?ctgtacctgg?tgactagaca?cgctgatgtg????300

gtgctggtgg?gaggagtgct?ggctgctctg?ctgtttctgc?tgctggctga?tgctttcctg????360

ctgctggctg?atgctagagt?gtggatgaac?agactgatcg?ctttcgcttg?tacatgtgga????420

agctccgatc?tgtatctgag?cgctttcagc?ctgcacagct?acggagtggc?tggagctctg????480

gtggctttta?agctgcctgg?atgtagcttt?agcatcttta?agaccagcga?aagaagccag????540

cctagactga?tcttttgtca?cagcaagaag?aagttttggg?ctaagcacat?gtggaatttt????600

atccctttct?atggaaaggc?tatcagaatg?tatgtgggag?gagtggaaca?cagacagctg????660

tttacattta?gccctagaag?gagactggga?gtgagagcta?caagaaaggt?gggaatctat????720

ctgctgccta?atagatgaaa?gcttggg????????????????????????????????????????747

<210>235

<211>281

<212>PRT

＜213〉hepatitis C virus

<400>235

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val?Ala

20??????????????????25??????????????????30

Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Leu?Leu?Phe?Leu

35??????????????????40??????????????????45

Leu?Leu?Ala?Asp?Ala?Leu?Ile?Phe?Cys?His?Ser?Lys?Lys?Lys?Gln?Leu

50??????????????????55??????????????????60

Phe?Thr?Phe?Ser?Pro?Arg?Arg?Tyr?Leu?Val?Thr?Arg?His?Ala?Asp?Val

65??????????????????70??????????????????75??????????????????80

Tyr?Leu?Leu?Pro?Arg?Arg?Gly?Pro?Arg?Leu?Cys?Thr?Cys?Gly?Ser?Ser

85??????????????????90??????????????????95

Asp?Leu?Tyr?His?Met?Trp?Asn?Phe?Ile?Ser?Gly?Ile?Phe?Trp?Ala?Lys

100?????????????????105?????????????????110

His?Met?Trp?Asn?Phe?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala

115?????????????????120?????????????????125

Ala?Ala?Ile?Leu?Ala?Gly?Tyr?Gly?Ala?Gly?Val?Tyr?Leu?Val?Ala?Tyr

130?????????????????135?????????????????140

Gln?Ala?Thr?Val?Gly?Val?Ala?Gly?Ala?Leu?Val?Ala?Phe?Lys?Ile?Pro

145?????????????????150?????????????????155?????????????????160

Phe?Tyr?Gly?Lys?Ala?Ile?Arg?Met?Tyr?Val?Gly?Gly?Val?Glu?His?Arg

165?????????????????170?????????????????175

Val?Leu?Val?Gly?Gly?Val?Leu?Ala?Ala?Phe?Leu?Leu?Leu?Ala?Asp?Ala

180?????????????????185?????????????????190

Arg?Val?Leu?Pro?Gly?Cys?Ser?Phe?Ser?Ile?Phe?Ala?Lys?Phe?Val?Ala

195?????????????????200?????????????????205

Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Thr?Ser?Glu?Arg?Ser?Gln?Pro

210?????????????????215?????????????????220

Arg?Arg?Leu?Gly?Val?Arg?Ala?Thr?Arg?Lys?Arg?Leu?Ile?Val?Phe?Pro

225?????????????????230?????????????????235?????????????????240

Asp?Leu?Gly?Val?Trp?Met?Asn?Arg?Leu?Ile?Ala?Phe?Ala?Leu?Ser?Ala

245?????????????????250?????????????????255

Phe?Ser?Leu?His?Ser?Tyr?Leu?Leu?Phe?Asn?Ile?Leu?Gly?Gly?Trp?Val

260?????????????????265?????????????????270

Val?Gly?Ile?Tyr?Leu?Leu?Pro?Asn?Arg

275?????????????????280

<210>236

<211>789

<212>DNA

＜213〉hepatitis C virus

<400>236

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaga?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gatccagagg?agatctgatg?ggatatatcc?ctctggtggc?taagtttgtg????120

gctgcttgga?cactgaaggc?tgctgctctg?ctgtttctgc?tgctggctga?tgctctgatc????180

ttctgtcaca?gcaagaagaa?gcagctgttt?acatttagcc?caagaagata?tctggtgaca????240

agacacgctg?atgtgtatct?gctgcctaga?cgcggaccta?gactgtgtac?atgtggaagc????300

tccgatctgt?atcacatgtg?gaactttatc?agcggaatct?tttgggctaa?gcacatgtgg????360

aatttcatcc?tggctggata?tggagctgga?gtgtatctgg?tggcttatca?ggctacagtg????420

ggagtggctg?gagctctggt?ggctttcaag?atcccattct?atggaaaggc?tatcagaatg????480

tatgtgggag?gagtggaaca?cagagtgctg?gtgggaggag?tgctggctgc?tttcctgctg????540

ctggctgatg?ctagagtgct?gccaggatgt?agctttagca?tcttcaagac?ttccgaacgc????600

tcccagccta?gaagactggg?agtgagagct?acaaggaaga?gactgatcgt?gtttccagat????660

ctgggagtgt?ggatgaatag?actgatcgct?ttcgctctga?gcgctttcag?cctgcacagc????720

tatctgctgt?tcaacatcct?gggaggatgg?gtggtgggaa?tctatctgct?gccaaacaga????780

tgaaagctt????????????????????????????????????????????????????????????789

<210>237

<211>107

<212>PRT

＜213〉hepatitis C virus

<400>237

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Tyr?Leu?Val?Ala?Tyr?Gln?Ala?Thr?Val?Ala

20??????????????????25??????????????????30

Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Leu?Leu?Phe?Leu

35??????????????????40??????????????????45

Leu?Leu?Ala?Asp?Ala?Leu?Ile?Phe?Cys?His?Ser?Lys?Lys?Lys?Tyr?Leu

50??????????????????55??????????????????60

Val?Thr?Arg?His?Ala?Asp?Val?Leu?Gly?Phe?Gly?Ala?Tyr?Met?Ser?Lys

65??????????????????70??????????????????75??????????????????80

Cys?Thr?Cys?Gly?Ser?Ser?Asp?Leu?Tyr?His?Met?Trp?Asn?Phe?Ile?Ser

85??????????????????90??????????????????95

Gly?Ile?Phe?Trp?Ala?Lys?His?Met?Trp?Asn?Phe

100?????????????????105

<210>238

<211>345

<212>DNA

＜213〉hepatitis C virus

<400>238

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gatccagagg?atacctcgtc?gcctaccagg?ccactgtggc?taaattcgtg????120

gcagcctgga?cactgaaagc?tgcagctctg?ctcttcctgc?tcctggccga?tgcactcatc????180

ttctgccatt?ccaagaaaaa?gtatctggtc?accagacatg?ctgacgtgct?ggggtttggc????240

gcctacatga?gcaagtgcac?ctgtggcagc?tccgacctgt?atcacatgtg?gaactttatt????300

tctggaatct?tttgggccaa?gcacatgtgg?aatttctgaa?agctt????????????????????345

<210>239

<211>106

<212>PRT

＜213〉hepatitis C virus

<400>239

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Val?Leu?Val?Gly?Gly?Val?Leu?Ala?Ala?Ala

20??????????????????25???????????????????30

Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu?Leu?Leu

35??????????????????40??????????????????45

Ala?Asp?Ala?Arg?Val?Leu?Ser?Ala?Phe?Ser?Leu?His?Ser?Tyr?Ile?Leu

50??????????????????55??????????????????60

Ala?Gly?Tyr?Gly?Ala?Gly?Val?Trp?Met?Asn?Arg?Leu?Ile?Ala?Phe?Ala

65??????????????????70??????????????????75??????????????????80

Ile?Pro?Phe?Tyr?Gly?Lys?Ala?Ile?Val?Ala?Gly?Ala?Leu?Val?Ala?Phe

85??????????????????90??????????????????95

Lys?Val?Gly?Ile?Tyr?Leu?Leu?Pro?Asn?Arg

100?????????????????105

<210>240

<211>342

<212>DNA

＜213〉hepatitis C virus

<400>240

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gatccagagg?agtcctggtg?ggcggcgtcc?tggccgctgc?taagtttgtc????120

gctgcttgga?cactgaaggc?agccgctttc?ctgctcctgg?cagacgccag?ggtgctgtct????180

gccttcagcc?tccactccta?catcctcgca?gggtatggcg?caggcgtgtg?gatgaatcgg????240

ctgatcgcct?ttgccattcc?attctatggg?aaagccattg?tggctggcgc?cctggtggca????300

ttcaaggtcg?ggatctacct?cctgcctaac?cgctgaaagc?tt???????????????????????342

<210>241

<211>80

<212>PRT

＜213〉hepatitis C virus

<400>241

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Val?Leu?Val?Gly?Gly?Val?Leu?Ala?Ala?Ala

20??????????????????25??????????????????30

Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu?Leu?Leu

35??????????????????40??????????????????45

Ala?Asp?Ala?Arg?Val?Leu?Ser?Ala?Phe?Ser?Leu?His?Ser?Tyr?Ile?Leu

50??????????????????55??????????????????60

Ala?Gly?Tyr?Gly?Ala?Gly?Val?Trp?Met?Asn?Arg?Leu?Ile?Ala?Phe?Ala

65??????????????????70??????????????????75??????????????????80

<210>242

<211>264

<212>DNA

＜213〉hepatitis C virus

<400>242

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gatccagagg?agtcctggtg?ggcggcgtcc?tggccgctgc?taagtttgtc????120

gctgcttgga?cactgaaggc?agccgctttc?ctgctcctgg?cagacgccag?ggtgctgtct????180

gccttcagcc?tccactccta?catcctcgca?gggtatggcg?caggcgtgtg?gatgaatcgg????240

ctgatcgcct?ttgcctgagg?atcc???????????????????????????????????????????264

<210>243

<211>130

<212>PRT

＜213〉hepatitis C virus

<400>243

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val?Ala

20??????????????????25??????????????????30

Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Arg?Leu?Gly?Val

35??????????????????40??????????????????45

Arg?Ala?Thr?Arg?Lys?Leu?Leu?Phe?Asn?Ile?Leu?Gly?Gly?Trp?Val?Arg

50??????????????????55??????????????????60

Met?Tyr?Val?Gly?Gly?Val?Glu?His?Arg?Arg?Leu?Ile?Val?Phe?Pro?Asp

65?????????????????70???????????????????75??????????????????80

Leu?Gly?Val?Gly?Val?Ala?Gly?Ala?Leu?Val?Ala?Phe?Lys?Leu?Pro?Gly

85??????????????????90??????????????????95

Cys?Ser?Phe?Ser?Ile?Phe?Lys?Thr?Ser?Glu?Arg?Ser?Gln?Pro?Arg?Gln

100?????????????????105?????????????????110

Leu?Phe?Thr?Phe?Ser?Pro?Arg?Arg?Tyr?Leu?Leu?Pro?Arg?Arg?Gly?Pro

115?????????????????120?????????????????125

Arg?Leu

130

<210>244

<211>414

<212>DNA

＜213〉hepatitis C virus

<400>244

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gatccagagg?agacctgatg?ggctacatcc?ctctcgtggc?caagtttgtg????120

gcagcttgga?ccctgaaggc?cgctgccaga?ctgggagtgc?gcgctacacg?gaaactcctg????180

tttaacatcc?tgggagggtg?ggtgcggatg?tacgtcggag?gcgtcgagca?cagaaggctc????240

attgtctttc?cagatctcgg?cgtgggcgtc?gcaggcgcac?tcgtggcctt?caaactgcca????300

gggtgcagct?tcagcatttt?caagacctcc?gaacgctccc?aacccagaca?gctgttcact????360

ttctctcctc?ggaggtatct?gctgcccaga?cgcggaccca?ggctgtgaaa?gctt??????????414

<210>245

<211>98

<212>PRT

＜213〉hepatitis C virus

<400>245

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Leu?Leu?Phe?Asn?Ile?Leu?Gly?Gly?Trp?Val

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Leu?Ala

35??????????????????40??????????????????45

Asp?Gly?Gly?Cys?Ser?Gly?Gly?Ala?Tyr?Arg?Leu?lle?Val?Phe?Pro?Asp

50??????????????????55??????????????????60

Leu?Gly?Val?Lys?Phe?Trp?Ala?Lys?His?Met?Trp?Asn?Phe?Ile?Gly?Val

65??????????????????70??????????????????75??????????????????80

Ala?Gly?Ala?Leu?Val?Ala?Phe?Lys?Lys?Gln?Leu?Phe?Thr?Phe?Ser?Pro

85??????????????????90??????????????????95

Arg?Arg

<210>246

<211>318

<212>DNA

＜213〉hepatitis C virus

<400>246

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gatccagagg?actgctcttc?aacatcctgg?gcggatgggt?gaaggccaag????120

ttcgtggctg?cctggaccct?gaaggctgcc?gctctggccg?acgggggatg?cagcggcgga????180

gcttacaggc?tcattgtctt?tcccgatctc?ggagtcaaat?tttgggcaaa?gcacatgtgg????240

aatttcatcg?gggtggccgg?agccctggtc?gcttttaaaa?agcagctctt?caccttctcc????300

ccaagacggt?gaggtacc??????????????????????????????????????????????????318

<210>247

<211>107

<212>PRT

＜213〉hepatitis C virus

<400>247

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Arg?Leu?Gly?Val?Arg?Ala?Thr?Arg?Lys?Lys

20??????????????????25??????????????????30

Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Thr?Ser

35??????????????????40??????????????????45

Glu?Arg?Ser?Gln?Pro?Arg?Asn?Leu?Pro?Gly?Cys?Ser?Phe?Ser?Ile?Phe

50??????????????????55??????????????????60

Asn?Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val?Lys?Tyr?Leu?Leu?Pro?Arg

65??????????????????70??????????????????75??????????????????80

Arg?Gly?Pro?Arg?Leu?Asn?Thr?Leu?Cys?Gly?Phe?Ala?Asp?Leu?Met?Gly

85??????????????????90??????????????????95

Tyr?Arg?Met?Tyr?Val?Gly?Gly?Val?Glu?His?Arg

100?????????????????105

<210>248

<211>345

<212>DNA

＜213〉hepatitis C virus

<400>248

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gatccagagg?aaggctgggc?gtgagagcca?cccggaagaa?ggccaagttc????120

gtggctgcct?ggaccctgaa?ggctgccgct?aaaacaagcg?agcgctccca?gcccaggaac????180

ctgcctggat?gctctttcag?catctttaat?gacctcatgg?ggtacattcc?actggtgaag????240

tatctgctcc?ccagacgggg?ccctcgcctg?aacactctct?gtggatttgc?tgatctgatg????300

gggtacagga?tgtatgtcgg?cggagtcgaa?cacagatgag?gtacc????????????????????345

<210>249

<211>308

<212>PRT

＜213〉hepatitis C virus

<400>249

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Val?Leu?Val?Gly?Gly?Val?Leu?Ala?Ala?Ala

20??????????????????25??????????????????30

Phe?Leu?Leu?Leu?Ala?Asp?Ala?Arg?Val?Leu?Ser?Ala?Phe?Ser?Leu?His

35??????????????????40??????????????????45

Ser?Tyr?Ile?Leu?Ala?Gly?Tyr?Gly?Ala?Gly?Val?Trp?Met?Asn?Arg?Leu

50??????????????????55??????????????????60

Ile?Ala?Phe?Ala?Gly?Ala?Ala?Ala?Arg?Leu?Gly?Val?Arg?Ala?Thr?Arg

65??????????????????70??????????????????75??????????????????80

Lys?Lys?Ala?Ala?Ala?Lys?Thr?Ser?Glu?Arg?Ser?Gln?Pro?Arg?Asn?Leu

85??????????????????90??????????????????95

Pro?Gly?Cys?Ser?Phe?Ser?Ile?Phe?Asn?Asp?Leu?Met?Gly?Tyr?Ile?Pro

100?????????????????105?????????????????110

Leu?Val?Lys?Tyr?Leu?Leu?Pro?Arg?Arg?Gly?Pro?Arg?Leu?Asn?Thr?Leu

115?????????????????120?????????????????125

Cys?Gly?Phe?Ala?Asp?Leu?Met?Gly?Tyr?Arg?Met?Tyr?Val?Gly?Gly?Val

130?????????????????135?????????????????140

Glu?His?Arg?Lys?Leu?Leu?Phe?Asn?Ile?Leu?Gly?Gly?Trp?Val?Lys?Ala

145?????????????????150?????????????????155?????????????????160

Ala?Ala?Leu?Ala?Asp?Gly?Gly?Cys?Ser?Gly?Gly?Ala?Tyr?Arg?Leu?Ile

165?????????????????170?????????????????175

Val?Phe?Pro?Asp?Leu?Gly?Val?Lys?Phe?Trp?Ala?Lys?His?Met?Trp?Asn

180?????????????????185?????????????????190

Phe?Ile?Gly?Val?Ala?Gly?Ala?Leu?Val?Ala?Phe?Lys?Lys?Gln?Leu?Phe

195?????????????????200?????????????????205

Thr?Phe?Ser?Pro?Arg?Arg?Asn?Gly?Tyr?Leu?Val?Ala?Tyr?Gln?Ala?Thr

210?????????????????215?????????????????220

Val?Ala?Ala?Ala?Leu?Leu?Phe?Leu?Leu?Leu?Ala?Asp?Ala?Leu?Ile?Phe

225?????????????????230?????????????????235?????????????????240

Cys?His?Ser?Lys?Lys?Lys?Tyr?Leu?Val?Thr?Arg?His?Ala?Asp?Val?Leu

245?????????????????250?????????????????255

Gly?Phe?Gly?Ala?Tyr?Met?Ser?Lys?Cys?Thr?Cys?Gly?Ser?Ser?Asp?Leu

260?????????????????265?????????????????270

Tyr?His?Met?Trp?Asn?Phe?Ile?Ser?Gly?Ile?Phe?Trp?Ala?Lys?His?Met

275?????????????????280?????????????????285

Trp?Asn?Phe?Lys?Ala?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu

290?????????????????295?????????????????300

Lys?Ala?Ala?Ala

305

<210>250

<211>948

<212>DNA

＜213〉hepatitis C virus

<400>250

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gctccagagg?agtcctggtg?ggcggcgtcc?tggcagccgc?tttcctgctc????120

ctggcagacg?ccagggtgct?gtctgccttc?agcctccact?cctacatcct?cgcagggtat????180

ggcgcaggcg?tgtggatgaa?tcggctgatc?gcctttgccg?gcgctgccgc?aaggctgggc????240

gtgagagcca?cccggaagaa?ggctgccgct?aaaacaagcg?agcgctccca?gcccaggaac????300

ctgcctggat?gctctttcag?catctttaat?gacctcatgg?ggtacattcc?actggtgaag????360

tatctgctcc?ccagacgggg?ccctcgcctg?aacactctct?gtggatttgc?tgatctgatg????420

gggtacagga?tgtatgtcgg?cggagtcgaa?cacagaaaac?tgctcttcaa?catcctgggc????480

ggatgggtga?aggctgccgc?tctggccgac?gggggatgca?gcggcggagc?ttacaggctc????540

attgtctttc?ccgatctcgg?agtcaaattt?tgggcaaagc?acatgtggaa?tttcatcggg????600

gtggccggag?ccctggtcgc?ttttaaaaag?cagctcttca?ccttctcccc?aagacggaac????660

ggatacctcg?tcgcctacca?ggccactgtg?gctgcagctc?tgctcttcct?gctcctggcc????720

gatgcactca?tcttctgcca?ttccaagaaa?aagtatctgg?tcaccagaca?tgctgacgtg????780

ctggggtttg?gcgcctacat?gagcaagtgc?acctgtggca?gctccgacct?gtatcacatg????840

tggaacttta?tttctggaat?cttttgggcc?aagcacatgt?ggaattttaa?ggccgcagca????900

gctaaattcg?tggcagcctg?gacactgaaa?gcagctgcat?gaggatcc?????????????????948

<210>251

<211>308

<212>PRT

＜213〉hepatitis C virus

<400>251

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Arg?Leu?Gly?Val?Arg?Ala?Thr?Arg?Lys?Lys

20??????????????????25??????????????????30

Ala?Ala?Ala?Lys?Thr?Ser?Glu?Arg?Ser?Gln?Pro?Arg?Asn?Leu?Pro?Gly

35??????????????????40??????????????????45

Cys?Ser?Phe?Ser?Ile?Phe?Asn?Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val

50??????????????????55??????????????????60

Lys?Tyr?Leu?Leu?Pro?Arg?Arg?Gly?Pro?Arg?Leu?Asn?Thr?Leu?Cys?Gly

65??????????????????70??????????????????75??????????????????80

Phe?Ala?Asp?Leu?Met?Gly?Tyr?Arg?Met?Tyr?Val?Gly?Gly?Val?Glu?His

85??????????????????90??????????????????95

Arg?Lys?Leu?Leu?Phe?Asn?Ile?Leu?Gly?Gly?Trp?Val?Lys?Ala?Ala?Ala

100?????????????????105?????????????????110

Leu?Ala?Asp?Gly?Gly?Cys?Ser?Gly?Gly?Ala?Tyr?Arg?Leu?Ile?Val?Phe

115?????????????????120?????????????????125

Pro?Asp?Leu?Gly?Val?Lys?Phe?Trp?Ala?Lys?His?Met?Trp?Asn?Phe?Ile

130?????????????????135?????????????????140

Gly?Val?Ala?Gly?Ala?Leu?Val?Ala?Phe?Lys?Lys?Gln?Leu?Phe?Thr?Phe

145?????????????????150?????????????????155?????????????????160

Ser?Pro?Arg?Arg?Asn?Gly?Tyr?Leu?Val?Ala?Tyr?Gln?Ala?Thr?Val?Ala

165?????????????????170?????????????????175

Ala?Ala?Leu?Leu?Phe?Leu?Leu?Leu?Ala?Asp?Ala?Leu?Ile?Phe?Cys?His

180?????????????????185?????????????????190

Ser?Lys?Lys?Lys?Tyr?Leu?Val?Thr?Arg?His?Ala?Asp?Val?Leu?Gly?Phe

195?????????????????200?????????????????205

Gly?Ala?Tyr?Met?Ser?Lys?Cys?Thr?Cys?Gly?Ser?Ser?Asp?Leu?Tyr?His

210?????????????????215?????????????????220

Met?Trp?Asn?Phe?Ile?Ser?Gly?Ile?Phe?Trp?Ala?Lys?His?Met?Trp?Asn

225?????????????????230?????????????????235?????????????????240

Phe?Lys?Lys?Ala?Ala?Ala?Val?Leu?Val?Gly?Gly?Val?Leu?Ala?Ala?Ala

245?????????????????250?????????????????255

Phe?Leu?Leu?Leu?Ala?Asp?Ala?Arg?Val?Leu?Ser?Ala?Phe?Ser?Leu?His

260?????????????????265?????????????????270

Ser?Tyr?Ile?Leu?Ala?Gly?Tyr?Gly?Ala?GIy?Val?Trp?Met?Asn?Arg?Leu

275?????????????????280?????????????????285

Ile?Ala?Phe?Ala?Asn?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu

290?????????????????295?????????????????300

Lys?Ala?Ala?Ala

305

<210>252

<211>948

<212>DNA

＜213〉hepatitis C virus

<400>252

gaattcgccg?ccaccatggg?aatgcaggtg?cagatccaaa?gcctgtttct?gctcctcctg?????60

tgggtgcccg?gctccagagg?aaggctgggc?gtgagagcca?cccggaagaa?ggctgccgct????120

aaaacaagcg?agcgctccca?gcccaggaac?ctgcctggat?gctctttcag?catctttaat????180

gacctcatgg?ggtacattcc?actggtgaag?tatctgctcc?ccagacgggg?ccctcgcctg????240

aacactctct?gtggatttgc?tgatctgatg?gggtacagga?tgtatgtcgg?cggagtcgaa????300

cacagaaaac?tgctcttcaa?catcctgggc?ggatgggtga?aggctgccgc?tctggccgac????360

gggggatgca?gcggcggagc?ttacaggctc?attgtctttc?ccgatctcgg?agtcaaattt????420

tgggcaaagc?acatgtggaa?tttcatcggg?gtggccggag?ccctggtcgc?ttttaaaaag????480

cagctcttca?ccttctcccc?aagacggaac?ggatacctcg?tcgcctacca?ggccactgtg????540

gctgcagctc?tgctcttcct?gctcctggcc?gatgcactca?tcttctgcca?ttccaagaaa????600

aagtatctgg?tcaccagaca?tgctgacgtg?ctggggtttg?gcgcctacat?gagcaagtgc????660

acctgtggca?gctccgacct?gtatcacatg?tggaacttta?tttctggaat?cttttgggcc????720

aagcacatgt?ggaattttaa?gaaagccgct?gcagtcctgg?tgggcggcgt?cctggcagcc????780

gctttcctgc?tcctggcaga?cgccagggtg?ctgtctgcct?tcagcctcca?ctcctacatc????840

ctcgcagggt?atggcgcagg?cgtgtggatg?aatcggctga?tcgcctttgc?caatgctgca????900

gctaaattcg?tggcagcctg?gacactgaaa?gcagctgcat?gaggatcc??????????????????948

<210>253

<211>123

<212>PRT

＜213〉the unknown

<220>

<223>AOSI.K

<400>253

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Phe?Leu?Leu?Ser?Leu?Gly?Ile

50??????????????????55??????????????????60

His?Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr

65??????????????????70??????????????????75??????????????????80

Val?Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Ser?Thr?Leu

85??????????????????90??????????????????95

Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

100?????????????????105?????????????????110

Tyr?Lys?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val

115?????????????????120

<210>254

<211>372

<212>DNA

＜213〉the unknown

<220>

<223>AOSI.K

<400>254

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgaa?gttcctgctg????180

tccctgggaa?tccacctgta?tatggatgac?gtggtgctgg?gagtgggact?gtccaggtac????240

gtggctaggc?tgttcctgct?gaccagaatc?ctgaccatct?ccaccctgcc?agagaccacc????300

gtggtgagga?ggcaggcctt?cacctttagc?cctacctata?agtggctgag?cctgctggtg????360

ccctttgtgt?ga????????????????????????????????????????????????????????372

<210>255

<211>206

<212>PRT

＜213〉hepatitis type B virus

<400>255

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His

50??????????????????55?????????????????60

Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr?Val

65??????????????????70??????????????????75??????????????????80

Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?lle?Leu?Thr?Ile?Ser?Thr?Leu?Pro

85??????????????????90??????????????????95

Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr

100?????????????????105?????????????????110

Lys?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Ile?Pro?Ile?Pro?Ser?Ser

115?????????????????120?????????????????125

Trp?Ala?Phe?Thr?Pro?Ala?Arg?Val?Thr?Gly?Gly?Val?Phe?Lys?Val?Gly

130?????????????????135?????????????????140

Asn?Phe?Thr?Gly?Leu?Tyr?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Thr

145?????????????????150?????????????????155?????????????????160

Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Asn?Val?Ser?Ile?Pro?Trp?Thr

165?????????????????170?????????????????175

His?Lys?Leu?Val?Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Ser?Ala?Ile?Cys

180?????????????????185?????????????????190

Ser?Val?Val?Arg?Arg?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

195?????????????????200?????????????????205

<210>256

<211>621

<212>DNA

＜213〉hepatitis type B virus

<400>256

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgtt?cctgctgtcc????180

ctgggaatcc?acctgtatat?ggatgacgtg?gtgctgggag?tgggactgtc?caggtacgtg????240

gctaggctgt?tcctgctgac?cagaatcctg?accatctcca?ccctgccaga?gaccaccgtg????300

gtgaggaggc?aggccttcac?ctttagccct?acctataagt?ggctgagcct?gctggtgccc????360

tttgtgatcc?ctatccctag?ctcctgggct?ttcaccccag?ccagggtgac?cggaggagtg????420

tttaaggtgg?gaaacttcac?cggcctgtat?ctgcccagcg?atttctttcc?tagcgtgacc????480

ctgtggaagg?ccgggatcct?gtacaagaat?gtgtccatcc?cttggaccca?caagctggtg????540

gtggactttt?cccagttcag?cagatccgct?atctgctccg?tggtgaggag?agctctgatg????600

ccactgtatg?cctgtatctg?a??????????????????????????????????????????????621

<210>257

<211>219

<212>PRT

＜213〉hepatitis type B virus

<400>257

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Asn?Phe?Leu?Leu?Ser?Leu?Gly?Ile

50??????????????????55??????????????????60

His?Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr

65??????????????????70??????????????????75??????????????????80

Val?Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Ser?Thr?Leu

85??????????????????90??????????????????95

Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

100?????????????????105?????????????????110

Tyr?Lys?Gly?Ala?Ala?Ala?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Asn

115?????????????????120?????????????????125

Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Thr?Pro?Ala?Arg?Val?Thr

130?????????????????135?????????????????140

Gly?GIy?Val?Phe?Lys?Val?Gly?Asn?Phe?Thr?Gly?Leu?Tyr?Asn?Leu?Pro

145?????????????????150?????????????????155?????????????????160

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu

165?????????????????170?????????????????175

Tyr?Lys?Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys?Gly?Ala?Ala?Leu?Val

180?????????????????185?????????????????190

Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Asn?Ser?Ala?Ile?Cys?Ser?Val?Val

195?????????????????200?????????????????205

Arg?Arg?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

210?????????????????215

<210>258

<211>660

<212>DNA

＜213〉hepatitis type B virus

<400>258

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgaa?cttcctgctg????180

tccctgggaa?tccacctgta?tatggatgac?gtggtgctgg?gagtgggact?gtccaggtac????240

gtggctaggc?tgttcctgct?gaccagaatc?ctgaccatct?ccaccctgcc?agagaccacc????300

gtggtgagga?ggcaggcctt?cacctttagc?cctacctata?agggagccgc?tgcctggctg????360

agcctgctgg?tgccctttgt?gaatatccct?atccctagct?cctgggcttt?caagacccca????420

gccagggtga?ccggaggagt?gtttaaggtg?ggaaacttca?ccggcctgta?taacctgccc????480

agcgatttct?ttcctagcgt?gaagaccctg?tggaaggccg?gaatcctgta?caagaatgtg????540

tccatccctt?ggacccacaa?gggagccgct?ctggtggtgg?acttttccca?gttcagcaga????600

aattccgcta?tctgctccgt?ggtgaggaga?gctctgatgc?cactgtatgc?ctgtatctga????660

<210>259

<211>168

<212>PRT

＜213〉the unknown

<220>

<223>PfCTL.1

<400>259

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Ile?Leu?Ser?Val?Ser?Ser?Phe?Leu?Phe?Val?Asn?Ala

20??????????????????25??????????????????30

Ala?Ala?Gln?Thr?Asn?Phe?Lys?Ser?Leu?Leu?Arg?Asn?Leu?Pro?Ser?Glu

35??????????????????40??????????????????45

Asn?Glu?Arg?Gly?Tyr?Lys?Ala?Ala?Ala?Leu?Leu?Ala?Cys?Ala?Gly?Leu

50??????????????????55??????????????????60

Ala?Tyr?Lys?Lys?Ala?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu

65??????????????????70??????????????????75?????????????????80

Lys?Ala?Ala?Ala?Lys?Ala?Phe?Met?Lys?Ala?Val?Cys?Val?Glu?Val?Asn

85??????????????????90??????????????????95

Ala?Ala?Ala?Ser?Phe?Leu?Phe?Val?Glu?Ala?Leu?Phe?Asn?Ala?Thr?Pro

100?????????????????105?????????????????110

Tyr?Ala?Gly?Glu?Pro?Ala?Pro?Phe?Lys?Ala?Ala?Ala?Lys?Tyr?Lys?Leu

115?????????????????120?????????????????125

Ala?Thr?Ser?Val?Leu?Lys?Ala?Gly?Val?Ser?Glu?Asn?Ile?Phe?Leu?Lys

130????????????????135??????????????????140

Asn?Ala?Ala?Ala?Tyr?Phe?Ile?Leu?Val?Asn?Leu?Leu?Ile?Lys?Ala?Gly

145?????????????????150?????????????????155?????????????????160

Leu?Leu?Gly?Val?Val?Ser?Thr?Val

165

<210>260

<211>513

<212>DNA

＜213〉the unknown

<220>

<223>?PfCTL.1

<400>260

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatcc?????60

agaggaatcc?tgagcgtgtc?ctctttcctg?tttgtcaacg?ccgctgcaca?gaccaatttc????120

aagagcctcc?tgaggaacct?cccctccgag?aacgaaagag?gctacaaagc?cgctgcactg????180

ctcgcctgcg?ctggactggc?ctataagaaa?gccgctgcag?ccaagttcgt?ggccgcttgg????240

acactgaagg?ccgctgcaaa?agcctttatg?aaggctgtct?gtgtggaggt?caatgccgct????300

gcatctttcc?tgtttgtgga?ggccctcttt?aacgctactc?cttacgcagg?ggaaccagcc????360

cccttcaagg?ccgctgcaaa?atataagctg?gcaaccagcg?tgctgaaggc?tggcgtgtcc????420

gagaatattt?ttctgaaaaa?cgccgctgca?tacttcatcc?tggtgaatct?gctcattaag????480

gccggactcc?tgggggtggt?ctctacagtg?tga?????????????????????????????????513

<210>261

<211>157

<212>PRT

＜213〉the unknown

<220>

<223>PfCTL.2

<400>261

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Phe?Val?Glu?Ala?Leu?Phe?Gln?Glu?Tyr?Asn?Ala?Ala

20??????????????????25??????????????????30

Ala?Lys?Tyr?Leu?Val?Ile?Val?Phe?Leu?Ile?Asn?Ala?Leu?Ala?Cys?Ala

35??????????????????40??????????????????45

Gly?Leu?Ala?Tyr?Lys?Lys?Phe?Tyr?Phe?Ile?Leu?Val?Asn?Leu?Leu?Lys

50??????????????????55??????????????????60

Ala?Ala?Leu?Phe?Phe?Ile?Ile?Phe?Asn?Lys?Asn?Ala?Ala?Ala?Lys?Phe

65??????????????????70??????????????????75??????????????????80

Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Phe?Ile?Leu?Val?Asn

85??????????????????90??????????????????95

Leu?Leu?Ile?Phe?His?Asn?Phe?Gln?Asp?Glu?Glu?Asn?Ile?Gly?Ile?Tyr

100?????????????????105?????????????????110

Lys?Leu?Pro?Tyr?Gly?Arg?Thr?Asn?Leu?Lys?Ala?Ala?Ala?Val?Leu?Leu

115?????????????????120?????????????????125

Gly?Gly?Val?Gly?Leu?Val?Leu?Asn?Phe?Leu?Ile?Phe?Phe?Asp?Leu?Phe

130?????????????????135?????????????????140

Leu?Val?Lys?Ala?Val?Leu?Ala?Gly?Leu?Leu?Gly?Val?Val

145?????????????????150?????????????????155

<210>262

<211>480

<212>DNA

＜213〉the unknown

<220>

<223>PfCTL.2

<400>262

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatcc?????60

agaggattcg?tggaggccct?gtttcaggaa?tacaacgccg?ctgcaaagta?tctcgtcatc????120

gtgttcctga?tcaatgctct?ggcatgcgcc?ggcctcgctt?acaaaaagtt?ttacttcatt????180

ctggtcaacc?tgctcaaggc?cgctctgttc?tttatcattt?tcaataaaaa?cgccgcagct????240

aagtttgtgg?ccgcatggac?cctgaaggcc?gctgcaaaat?tcatcctcgt?gaatctgctc????300

atttttcaca?acttccaaga?cgaggaaaat?atcggaattt?ataagctgcc?ctacgggagg????360

acaaacctga?aagccgctgc?agtcctgctc?ggcggagtgg?ggctggtgct?caattttctg????420

atcttctttg?atctgttcct?ggtgaaggcc?gtcctggccg?gcctgctcgg?agtcgtgtga????480

<210>263

<211>169

<212>PRT

＜213〉the unknown

<220>

<223>PfCTL.3

<400>263

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Val?Phe?Leu?Ile?Phe?Phe?Asp?Leu?Phe?Leu?Asn?Ala

20??????????????????25??????????????????30

Ala?Ala?Pro?Ser?Asp?Gly?Lys?Cys?Asn?Leu?Tyr?Lys?Ala?Ala?Ala?Val

35??????????????????40??????????????????45

Thr?Cys?Gly?Asn?Gly?Ile?Gln?Val?Arg?Lys?Leu?Phe?His?Ile?Phe?Asp

50??????????????????55??????????????????60

Gly?Asp?Asn?Glu?Ile?Lys?Ala?His?Val?Leu?Ser?His?Asn?Ser?Tyr?Glu

65??????????????????70??????????????????75??????????????????80

Lys?Asn?Tyr?Tyr?Gly?Lys?Gln?Glu?Asn?Trp?Tyr?Ser?Leu?Lys?Lys?Ile

85??????????????????90??????????????????95

Leu?Ser?Val?Phe?Phe?Leu?Ala?Asn?Ala?Ala?Ala?Lys?Phe?Ile?Lys?Ser

100?????????????????105?????????????????110

Leu?Phe?His?Ile?Phe?Lys?Ala?Ala?Ala?Leu?Tyr?Ile?Ser?Phe?Tyr?Phe

115?????????????????120?????????????????125

Ile?Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys

130?????????????????135?????????????????140

Ala?Ala?Ala?Tyr?Tyr?Ile?Pro?His?Gln?Ser?Ser?Leu?Lys?Ala?Ala?Ala

145?????????????????150?????????????????155?????????????????160

Gly?Leu?Ile?Met?Val?Leu?Ser?Phe?Leu

165

<210>264

<211>516

<212>DNA

＜213〉the unknown

<220>

<223>PfCTL.3

<400>264

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatcc?????60

agaggagtgt?tcctgatctt?ctttgacctg?ttcctgaacg?ccgctgcacc?cagcgatggc????120

aagtgcaatc?tctacaaggc?cgctgcagtg?acctgtggaa?acgggattca?ggtcaggaaa????180

ctctttcaca?tcttcgacgg?cgataacgag?atcaaggccc?atgtgctgtc?ccacaattct????240

tatgaaaaaa?actactatgg?aaagcaagag?aattggtaca?gcctgaagaa?aattctgtcc????300

gtgttctttc?tcgccaacgc?cgctgcaaag?tttatcaagt?ctctgttcca?tattttcaag????360

gccgctgcac?tctacatcag?cttctatttt?attaaagcca?aatttgtggc?cgcttggaca????420

ctgaaggccg?ctgcaaaagc?cgctgcatac?tatatccctc?accagagctc?cctgaaggcc????480

gctgcagggc?tgatcatggt?gctctctttc?ctgtga??????????????????????????????516

<210>265

<211>456

<212>PRT

＜213〉the unknown

<220>

<223>PfCTL/HTL/(N)

<400>265

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Ser?Ser?Val?Phe?Asn?Val?Val?Asn?Ser?Ser?Ile?Gly

20??????????????????25??????????????????30

Leu?Ile?Met?Val?Leu?Ser?Phe?Leu?Gly?Pro?Gly?Pro?Gly?Leu?Tyr?Ile

35??????????????????40??????????????????45

Ser?Phe?Tyr?Phe?Ile?Leu?Val?Asn?Leu?Leu?Ile?Phe?His?Ile?Asn?Gly

50??????????????????55??????????????????60

Lys?Ile?Ile?Lys?Asn?Ser?Glu?Gly?Pro?Gly?Pro?Gly?Pro?Asp?Ser?Ile

65??????????????????70??????????????????75??????????????????80

Gln?Asp?Ser?Leu?Lys?Glu?Ser?Arg?Lys?Leu?Ser?Gly?Pro?Gly?Pro?Gly

85??????????????????90??????????????????95

Val?Leu?Ala?Gly?Leu?Leu?Gly?Val?Val?Ser?Thr?Val?Leu?Leu?Gly?Gly

100?????????????????105?????????????????110

Val?Gly?Leu?Val?Leu?Gly?Pro?Gly?Pro?Gly?Leu?Pro?Ser?Glu?Asn?Glu

115?????????????????120?????????????????125

Arg?Gly?Tyr?Tyr?Ile?Pro?His?Gln?Ser?Ser?Leu?Gly?Pro?Gly?Pro?Gly

130?????????????????135?????????????????140

Gln?Thr?Asn?Phe?Lys?Ser?Leu?Leu?Arg?Asn?Leu?Gly?Val?Ser?Glu?Asn

145?????????????????150?????????????????155?????????????????160

Ile?Phe?Leu?Lys?Gly?Pro?Gly?Pro?Gly?Phe?Gln?Asp?Glu?Glu?Asn?Ile

165?????????????????170?????????????????175

Gly?Ile?Tyr?Gly?Pro?Gly?Pro?Gly?Lys?Tyr?Leu?Val?Ile?Val?Phe?Leu

180?????????????????185?????????????????190

Ile?Phe?Phe?Asp?Leu?Phe?Leu?Val?Gly?Pro?Gly?Pro?Gly?Lys?Phe?Ile

195?????????????????200?????????????????205

Lys?Ser?Leu?Phe?His?Ile?Phe?Asp?Gly?Asp?Asn?Glu?Ile?Gly?Pro?Gly

210?????????????????215?????????????????220

Pro?Gly?Lys?Ser?Lys?Tyr?Lys?Leu?Ala?Thr?Ser?Val?Leu?Ala?Gly?Leu

225?????????????????230?????????????????235?????????????????240

Leu?Gly?Pro?Gly?Pro?Gly?Leu?Pro?Tyr?Gly?Lys?Thr?Asn?Leu?Gly?Pro

245?????????????????250?????????????????255

Gly?Pro?Gly?Arg?His?Asn?Trp?Val?Asn?His?Ala?Val?Pro?Leu?Ala?Met

260?????????????????265?????????????????270

Lys?Leu?Ile?Gly?Pro?Gly?Pro?Gly?Met?Arg?Lys?Leu?Ala?Ile?Leu?Ser

275?????????????????280?????????????????285

Val?Ser?Ser?Phe?Leu?Phe?Val?Glu?Ala?Leu?Phe?Gln?Glu?Tyr?Gly?Pro

290?????????????????295?????????????????300

Gly?Pro?Gly?Val?Thr?Cys?Gly?Asn?Gly?Ile?Gln?Val?Arg?Gly?Pro?Gly

305?????????????????310?????????????????315?????????????????320

Pro?Gly?Met?Asn?Tyr?Tyr?Gly?Lys?Gln?Glu?Asn?Trp?Tyr?Ser?Leu?Lys

325?????????????????330?????????????????335

Lys?Gly?Pro?Gly?Pro?Gly?Pro?Ser?Asp?Gly?Lys?Cys?Asn?Leu?Tyr?Ala

340?????????????????345?????????????????350

Asp?Ser?Ala?Trp?Glu?Asn?Val?Lys?Asn?Val?Ile?Gly?Pro?Phe?Met?Lys

355?????????????????360?????????????????365

Ala?Val?Cys?Val?Glu?Val?Gly?Pro?Gly?Pro?Gly?Lys?Ile?Leu?Ser?Val

370?????????????????375?????????????????380

Phe?Phe?Leu?Ala?Leu?Phe?Phe?Ile?Ile?Phe?Asn?Lys?Gly?Pro?Gly?Pro

385?????????????????390?????????????????395?????????????????400

Gly?His?Val?Leu?Ser?His?Asn?Ser?Tyr?Glu?Lys?Gly?Pro?Gly?Pro?Gly

405?????????????????410?????????????????415

Lys?Tyr?Lys?Ile?Ala?Gly?Gly?Ile?Ala?Gly?Gly?Leu?Ala?Leu?Leu?Ala

420?????????????????425?????????????????430

Cys?Ala?Gly?Leu?Ala?Tyr?Lys?Phe?Val?Val?Pro?Gly?Ala?Ala?Thr?Pro

435?????????????????440?????????????????445

Tyr?Ala?Gly?Glu?Pro?Ala?Pro?Phe

450?????????????????455

<210>266

<211>1385

<212>DNA

＜213〉the unknown

<220>

<223>PfCTL/HTL/(N)

<400>266

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatcc?????60

agaggaagta?gtgtgttcaa?tgttgtgaac?tcatcaattg?gtctgatcat?ggtgctgagc????120

tttctcgggc?cagggccagg?attatatatt?tctttctact?tcatccttgt?caacctgtta????180

atattccaca?ttaacggcaa?aataataaag?aacagtgaag?gccctgggcc?tgggcctgac????240

tcgatccagg?attctctaaa?agaatcgagg?aagctctccg?gaccaggccc?tggtgtactc????300

gccgggttgc?tgggagtagt?tagcacagtg?ctgttaggag?gcgtcggcct?cgtcttagga????360

cctggaccag?gtctgccgtc?cgaaaacgaa?agaggatact?acatacctca?ccagagcagc?????420

ctcggcccag?gccccggaca?aaccaatttc?aaatccctct?tgcgaaatct?aggagtgagc?????480

gagaacatat?ttcttaaagg?acccggtccc?ggctttcagg?acgaggagaa?tataggtatt?????540

tacggtccag?gacctggaaa?atacctagtg?atcgtattcc?taattttttt?tgacctattt?????600

ctggtgggcc?caggtcccgg?aaagttcatt?aaatcactct?tccacatttt?tgacggagat?????660

aacgagatag?gacccggtcc?cgggaaatca?aagtacaaac?tagccacttc?agtgctggcc?????720

ggccttctag?ggccgggccc?agggctcccc?tatggaaaga?caaatcttgg?ccccggtcca?????780

ggacggcaca?actgggtgaa?tcatgcggtt?ccattggcca?tgaaactaat?cgggcccggt?????840

ccaggcatgc?gcaaacttgc?aattctaagc?gtaagttcat?ttctgttcgt?agaggcactg?????900

tttcaagaat?atggcccagg?acctggcgtc?acatgtggga?atgggatcca?ggtgagagga?????960

ccgggacctg?gtatgaacta?ttacggtaaa?caggaaaatt?ggtactccct?gaaaaagggt????1020

ccaggccccg?gcccctcaga?tggtaagtgc?aacctgtatg?ctgactcagc?atgggagaac????1080

gtaaaaaatg?taataggccc?attcatgaag?gcagtttgtg?tcgaagtcgg?accaggccca????1140

ggaaaaatac?tttctgtctt?cttcctagct?ctcttcttca?tcatcttcaa?caagggacca????1200

gggccaggtc?acgtgttatc?ccataactct?tatgaaaaag?ggccaggacc?tgggaaatac????1260

aaaatcgcag?gagggatcgc?cggcgggcta?gcgctccttg?cctgcgcagg?cttggcttac????1320

aaattcgttg?taccaggagc?tgcaacaccc?tatgcaggag?aacctgcccc?attttgaaga????1380

tctgc????????????????????????????????????????????????????????????????1385

<210>267

<211>419

<212>PRT

＜213〉the unknown

<220>

<223>Pf33

<400>267

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Phe?Met?Lys?Ala?ValCys?Val?Glu?Val?Asn

20??????????????????25?????????????????30

Val?Thr?Cys?Gly?Asn?Gly?Ile?Gln?Val?Arg?Lys?Gly?Leu?Ile?Met?Val

35??????????????????40??????????????????45

Leu?Ser?Phe?Leu?Asn?Ala?Ala?Leu?Phe?His?Ile?Phe?Asp?Gly?Asp?Asn

50??????????????????55??????????????????60

Glu?Ile?Lys?Ala?Ala?Leu?Leu?Ala?Cys?Ala?Gly?Leu?Ala?Tyr?Lys?Lys

65??????????????????70??????????????????75??????????????????80

Ser?Phe?Leu?Phe?Val?Glu?Ala?Leu?Phe?Asn?Ala?Ala?Pro?Ser?Asp?Gly

85??????????????????90??????????????????95

Lys?Cys?Asn?Leu?Tyr?Lys?Ala?Ala?Gln?Thr?Asn?Phe?Lys?Ser?Leu?Leu

100?????????????????105?????????????????110

Arg?Asn?Leu?Pro?Ser?Glu?Asn?Glu?Arg?Gly?Tyr?Lys?Ala?Ala?Gly?Val

115?????????????????120?????????????????125

Ser?Glu?Asn?Ile?Phe?Leu?Lys?Asn?Ala?Ala?Ala?Tyr?Phe?Ile?Leu?Val

130?????????????????135?????????????????140

Asn?Leu?Leu?Ile?Lys?Ala?Ala?Ala?Ile?Leu?Ser?Val?Ser?Ser?Phe?Leu

145?????????????????150?????????????????155?????????????????160

Phe?Val?Asn?Thr?Pro?Tyr?Ala?Gly?Glu?Pro?Ala?Pro?Phe?Lys?Ala?Ala

165?????????????????170?????????????????175

Ala?Lys?Tyr?Lys?Leu?Ala?Thr?Ser?Val?Leu?Lys?Ala?Ala?Val?Phe?Leu

180?????????????????185?????????????????190

Ile?Phe?Phe?Asp?Leu?Phe?Leu?Asn?Tyr?Tyr?Ile?Pro?His?Gln?Ser?Ser

195?????????????????200?????????????????205

Leu?Lys?Ala?Ala?Gly?Leu?Leu?Gly?Asn?Val?Ser?Thr?Val?Gly?Ala?Val

210?????????????????215?????????????????220

Leu?Leu?Gly?Gly?Val?Gly?Leu?Val?Leu?Asn?Leu?Ala?Cys?Ala?Gly?Leu

225?????????????????230?????????????????235?????????????????240

Ala?Tyr?Lys?Lys?Ala?Lys?Phe?Ile?Lys?Ser?Leu?Phe?His?Ile?Phe?Lys

245?????????????????250?????????????????255

Ala?Ala?Phe?Tyr?Phe?Ile?Leu?Val?Asn?Leu?Leu?Lys?Ala?Phe?Leu?Ile

260?????????????????265?????????????????270

Phe?Phe?Asp?Leu?Phe?Leu?Val?Lys?Ala?Leu?Phe?Phe?Ile?Ile?Phe?Asn

275?????????????????280?????????????????285

Lys?Asn?Tyr?Tyr?Gly?Lys?Gln?Glu?Asn?Trp?Tyr?Ser?Leu?Lys?Phe?Val

290?????????????????295?????????????????300

Glu?Ala?Leu?Phe?Gln?Glu?Tyr?Asn?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala

305?????????????????310?????????????????315?????????????????320

Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Ile?Leu?Ser?Val?Phe?Phe?Leu?Ala

325?????????????????330?????????????????335

Asn?Ala?Val?Leu?Ala?Gly?Leu?Leu?Gly?Asn?Val?Asn?Phe?Gln?Asp?Glu

340?????????????????345?????????????????350

Glu?Asn?Ile?Gly?Ile?Tyr?Lys?Ala?Ala?Ala?Leu?Tyr?Ile?Ser?Phe?Tyr

355?????????????????360?????????????????365

Phe?Ile?Lys?Ala?Phe?Ile?Leu?Val?Asn?Leu?Leu?Ile?Phe?His?Asn?Ala

370?????????????????375?????????????????380

Ala?Leu?Pro?Tyr?Gly?Arg?Thr?Asn?Leu?Lys?Ala?Ala?His?Val?Leu?Ser

385?????????????????390?????????????????395?????????????????400

His?Asn?Ser?Tyr?Glu?Lys?Asn?Ala?Ala?Ala?Lys?Tyr?Leu?Val?Ile?Val

405?????????????????410?????????????????415

Phe?Leu?Ile

<210>268

<211>1269

<212>DNA

＜213〉the unknown

<220>

<223>Pf33

<400>268

gccgccacca?tgggaatgca?ggtgcagatc?cagagcctgt?ttctgctcct?cctgtgggtg?????60

cccggatcca?gaggatttat?gaaagctgtc?tgtgtagagg?tgaatgtaac?atgcggtaac????120

ggaattcagg?tgagaaaggg?actcatcatg?gtactcagct?ttctgaacgc?agccctgttc????180

cacatctttg?acggagacaa?tgaaatcaaa?gccgcattgc?tcgcctgtgc?cggactagcc????240

tataaaaaga?gtttcctttt?cgttgaagca?ctatttaacg?cagcacccag?tgacggtaaa????300

tgcaacctat?ataaagcagc?tcagactaat?ttcaaaagcc?tgttaagaaa?tctgccctca????360

gagaatgaaa?ggggttacaa?agccgccggc?gtgtccgaga?atattttcct?gaagaacgcc????420

gctgcttatt?ttatactcgt?gaatctactc?ataaaggcag?ccgcaatcct?ttcagtgtcc????480

agctttctgt?ttgttaacac?accatatgcg?ggcgagccgg?ctcctttcaa?ggctgcagca????540

aaatacaagc?ttgccacatc?agtattgaaa?gcagctgtgt?ttttgatatt?ctttgatctt????600

tttttaaact?actacatacc?tcatcagtct?agtcttaaag?cagccgggct?actggggaac????660

gtctctactg?tgggggccgt?cttacttgga?ggagttggcc?tcgtgttgaa?cctcgcgtgc????720

gcaggtctgg?cctacaaaaa?agcgaaattc?atcaagtctc?tgttccacat?ttttaaagcc????780

gcattctatt?tcatactagt?gaaccttctc?aaagctttcc?tgatcttctt?cgatctattc????840

ctcgtaaaag?cgctattctt?cattatcttt?aacaaaaatt?attacggcaa?gcaagaaaat????900

tggtactcac?tcaagtttgt?agaagctctg?ttccaggaat?acaacgccgc?tgctaaattc????960

gttgcagctt?ggaccctgaa?agcagctgca?aagatcctat?cggtcttctt?tctcgctaat????1020

gccgtattag?caggacttct?aggcaacgtg?aactttcaag?acgaagagaa?tataggcatc????1080

tacaaagccg?cagcactgta?catttcattc?tacttcatca?aggccttcat?actggtcaac????1140

cttctgatat?ttcataatgc?agcactgcca?tatgggagaa?ccaacttgaa?agcggcccac????1200

gtgttgagcc?acaactccta?cgagaagaac?gccgccgcga?aatatctcgt?cattgtcttc????1260

ctgatttga????????????????????????????????????????????????????????????1269

<210>269

<211>180

<212>PRT

＜213〉the unknown

<220>

<223>TB.1

<400>269

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Arg?Met?Ser?Arg?Val?Thr?Thr?Phe?Thr?Val?Lys?Ala

20??????????????????25??????????????????30

Leu?Val?Leu?Leu?Met?Leu?Pro?Val?Val?Asn?Leu?Met?Ile?Gly?Thr?Ala

35??????????????????40??????????????????45

Ala?Ala?Val?Val?Lys?Ala?Leu?Val?Leu?Leu?Met?Leu?Pro?Val?Gly?Ala

50??????????????????55??????????????????60

Gly?Leu?Met?Thr?Ala?Val?Tyr?Leu?Val?Gly?Ala?Ala?Ala?Met?Ala?Leu

65??????????????????70??????????????????75??????????????????80

Leu?Arg?Leu?Pro?Val?Lys?Arg?Met?Phe?Ala?Ala?Asn?Leu?Gly?Val?Asn

85??????????????????90??????????????????95

Ser?Leu?Tyr?Phe?Gly?Gly?Ile?Cys?Val?Gly?Arg?Leu?Pro?Leu?Val?Leu

100?????????????????105?????????????????110

Pro?Ala?Val?Asn?Ala?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu

115?????????????????120?????????????????125

Lys?Ala?Ala?Ala?Lys?Ala?Ala?Ala?Arg?Leu?Met?Ile?Gly?Thr?Ala?Ala

130????????????????135?????????????????140

Ala?Gly?Phe?Val?Val?Ala?Leu?Ile?Pro?Leu?Val?Asn?Ala?Met?Thr?Tyr

145?????????????????150?????????????????155?????????????????160

Ala?Ala?Pro?Leu?Phe?Val?Gly?Ala?Ala?Ala?Ala?Met?Ala?Leu?Leu?Arg

165?????????????????170?????????????????175

Leu?Pro?Leu?Val

180

<210>270

<211>543

<212>DNA

＜213〉the unknown

<220>

<223>TB.1

<400>270

atgcaggtgc?agatccagag?cctgtttctg?ctcctcctgt?gggtgcccgg?atccagagga?????60

aggatgagca?gagtgaccac?attcactgtc?aaggccctgg?tgctcctgat?gctccccgtc????120

gtgaacctga?tgatcggcac?cgctgcagcc?gtcgtgaaag?ctctcgtcct?gctcatgctc????180

cctgtgggag?cagggctgat?gacagccgtg?tacctggtcg?gcgctgcagc?catggccctc????240

ctgcggctgc?cagtgaagcg?catgtttgct?gcaaatctgg?gagtcaactc?cctctatttc????300

gggggcattt?gcgtgggaag?gctgcccctc?gtgctgcctg?ctgtgaatgc?agccgctgcc????360

aaatttgtcg?ccgcttggac?tctgaaggca?gccgctaagg?ccgctgcaag?actgatgatc????420

gggaccgccg?ctgccggctt?cgtggtcgcc?ctgattcccc?tggtgaacgc?catgacatac????480

gcagctcctc?tgtttgtggg?agccgctgca?gccatggctc?tcctgcggct?gccactggtg????540

tga??????????????????????????????????????????????????????????????543

<210>271

<211>148

<212>PRT

＜213〉the unknown

<220>

<223>BCL?A2?#90

<400>271

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Ile?Met?Ile?Gly?His?Leu?Val?Gly?Val?Asn?Arg?Leu

20??????????????????25??????????????????30

Leu?Gln?Glu?Thr?Glu?Leu?Val?Asn?Ala?Lys?Val?Ala?Glu?Ile?Val?His

35??????????????????40??????????????????45

Phe?Leu?Asn?Ala?Lys?Val?Phe?Gly?Ser?Leu?Ala?Phe?Val?Asn?Ala?Tyr

50??????????????????55??????????????????60

Leu?Ser?Gly?Ala?Asn?Leu?Asn?Val?Gly?Ala?Ala?Tyr?Leu?Gln?Leu?Val

65??????????????????70??????????????????75??????????????????80

Phe?Gly?Ile?Glu?Val?Asn?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr

85??????????????????90?????????????????95

Leu?Lys?Ala?Ala?Ala?Lys?Ala?Ala?Ala?Val?Val?Leu?Gly?Val?Val?Phe

100????????????????105?????????????????110

Gly?Ile?Asn?Ser?Met?Pro?Pro?Pro?Gly?Thr?Arg?Val?Asn?Ala?Ala?Ala

115?????????????????120?????????????????125

Ala?Thr?Val?Gly?Ile?Met?Ile?Gly?Val?Asn?Ala?Lys?Leu?Cys?Pro?Val

130?????????????????135?????????????????140

Gln?Leu?Trp?Val

145

<210>272

<211>447

<212>DNA

＜213〉the unknown

<220>

<223>BCL?A2?#90

<400>272

atgcaggtgc?agatccagag?cctgtttctg?ctcctcctgt?gggtgcccgg?gtccagagga?????60

attatgatcg?gccatctggt?gggcgtcaac?agactgctgc?aggaaaccga?gctggtgaat????120

gccaaggtgg?ccgaaattgt?gcactttctc?aacgcaaagg?tgtttggttc?cctggctttt????180

gtcaatgcct?atctgagcgg?cgctaacctc?aacgtcggag?ccgcctacct?ccagctggtc????240

ttcggcatcg?aggtcaacgc?tgctgcaaaa?ttcgtggcag?cttggaccct?caaggctgca????300

gcaaaggctg?ccgccgtcgt?gctcggagtg?gtgttcggga?tcaactctat?gccacctccc????360

gggactaggg?tcaatgctgc?cgccgcaaca?gtgggaatca?tgattggggt?gaatgccaaa????420

ctgtgcccag?tgcaactgtg?ggtgtga????????????????????????????????????????447

<210>273

<211>144

<212>PRT

＜213〉the unknown

<220>

<223>BCL?A2?#88

<400>273

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Val?Val?Leu?Gly?Val?Val?Phe?Gly?Ile?Asn?Ala?Ala

20??????????????????25??????????????????30

Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Val

35??????????????????40??????????????????45

Ala?Glu?Ile?Val?His?Phe?Leu?Asn?Ala?Tyr?Leu?Ser?Gly?Ala?Asn?Leu

50??????????????????55??????????????????60

Asn?Val?Gly?Ala?Ala?Tyr?Leu?Gln?Leu?Val?Phe?Gly?Ile?Glu?Val?Asn

65??????????????????70??????????????????75??????????????????80

Ile?Met?Ile?Gly?His?Leu?Val?Gly?Val?Asn?Arg?Leu?Leu?Gln?Glu?Thr

85??????????????????90??????????????????95

Glu?Leu?Val?Asn?Ala?Lys?Val?Phe?Gly?Ser?Leu?Ala?Phe?Val?Asn?Ala

100?????????????????105?????????????????110

Lys?Leu?Cys?Pro?Val?Gln?Leu?Trp?Val?Asn?Ala?Ala?Ala?Ala?Thr?Val

115?????????????????120?????????????????125

Gly?Ile?Met?Ile?Gly?Val?Asn?Ser?Met?Pro?Pro?Pro?Gly?Thr?Arg?Val

130????????????????135??????????????????140

<210>274

<211>435

<212>DNA

＜213〉the unknown

<220>

<223>BCL?A2?#88

<400>274

atgcaggtgc?agatccagag?cctgtttctg?ctcctcctgt?gggtgcccgg?gtccagagga?????60

gtcgtgctgg?gagtcgtctt?cggcattaat?gccgccgctg?caaagttcgt?ggctgcctgg????120

accctgaagg?ccgcagctaa?agtggcagag?atcgtgcact?ttctgaacgc?ctacctgagc????180

ggagcaaatc?tgaacgtcgg?cgctgcctat?ctgcagctcg?tgtttggaat?tgaagtgaac????240

atcatgattg?gacatctggt?gggcgtgaac?aggctgctcc?aggaaactga?gctggtcaac????300

gctaaagtgt?tcgggtctct?cgcctttgtg?aacgctaagc?tctgccccgt?ccaactctgg????360

gtcaatgccg?cagccgctac?agtggggatc?atgatcggcg?tgaactccat?gcctccacca????420

gggaccagag?tgtga???????????????????????????????????????????????????????435

<210>275

<211>147

<212>PRT

＜213〉the unknown

<220>

<223>BCL?A2?#63

<400>275

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Lys?Leu?Cys?Pro?Val?Gln?Leu?Trp?Val?Asn?Ala?Ala

20??????????????????25??????????????????30

Ala?Ala?Thr?Val?Gly?Ile?Met?Ile?Gly?Val?Asn?Ile?Met?Ile?Gly?His

35??????????????????40??????????????????45

Leu?Val?Gly?Val?Asn?Arg?Leu?Leu?Gln?Glu?Thr?Glu?Leu?Val?Asn?Ala

50??????????????????55??????????????????60

Lys?Val?Ala?Glu?Ile?Val?His?Phe?Leu?Asn?Ala?Lys?Val?Phe?Gly?Ser

65??????????????????70??????????????????75??????????????????80

Leu?Ala?Phe?Val?Asn?Ala?Tyr?Leu?Ser?Gly?Ala?Asn?Leu?Asn?Val?Gly

85?????????????????90??????????????????95

Ala?Ala?Tyr?Leu?Gln?Leu?Val?Phe?Gly?Ile?Glu?Val?Asn?Ala?Ala?Ala

100?????????????????105?????????????????110

Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Ala?Ala?Ala

115????????????????120?????????????????125

Val?Val?Leu?Gly?Val?Val?Phe?Gly?Ile?Asn?Ser?Met?Pro?Pro?Pro?Gly

130?????????????????135?????????????????140

Thr?Arg?Val

145

<210>276

<211>450

<212>DNA

＜213〉the unknown

<220>

<223>BCL?A2?#63

<400>276

atgcaggtgc?agatccagag?cctgtttctg?ctcctcctgt?gggtgcccgg?gtccagagga?????60

aagctctgcc?ccgtgcaact?gtgggtcaac?gccgccgccg?caaccgtcgg?cattatgatc????120

ggggtgaaca?tcatgatcgg?acacctggtc?ggcgtgaaca?ggctgctgca?ggagacagaa????180

ctggtcaatg?ccaaggtggc?tgaaattgtc?catttcctga?atgccaaagt?gttcggctct????240

ctcgctttcg?tgaacgctta?tctgagcgga?gctaacctca?acgtgggggc?cgcatacctc????300

cagctcgtct?ttgggattga?ggtgaatgcc?gcagctaaat?ttgtcgctgc?ctggaccctg????360

aaggcagcag?ccaaggctgc?cgcagtggtg?ctgggagtgg?tgtttggaat?caattccatg????420

cctccaccag?gcactagagt?gtgaggatcc?????????????????????????????????????450

<210>277

<211>183

<212>PRT

＜213〉the unknown

<220>

＜223〉prostate 1

<400>277

Leu?Thr?Phe?Phe?Trp?Leu?Asp?Arg?Ser?Val?Lys?Ala?Ala?Ala?Val?Leu

1???????????????5???????????????????10??????????????????15

Val?His?Pro?Gln?Trp?Val?Leu?Thr?Val?Lys?Ala?Ala?Ala?Leu?Leu?Gln

20??????????????????25??????????????????30

Glu?Arg?Gly?Val?Ala?Tyr?Ile?Lys?Ala?Ala?Leu?Leu?Leu?Ser?Ile?Ala

35??????????????????40??????????????????45

Leu?Ser?Val?Asn?Pro?Leu?Val?Cys?Asn?Gly?Val?Leu?Gln?Gly?Val?Lys

50??????????????????55??????????????????60

Ala?Ala?Ile?Met?Tyr?Ser?Ala?His?Asp?Thr?Thr?Val?Lys?Ala?Ala?Ala

65??????????????????70??????????????????75??????????????????80

Phe?Leu?Thr?Pro?Lys?Lys?Leu?Gln?Cys?Val?Asn?Ala?Met?Met?Asn?Asp

85??????????????????90??????????????????95

Gln?Leu?Met?Phe?Leu?Asn?Ala?Gly?Leu?Pro?Ser?Ile?Pro?Val?His?Pro

100?????????????????105?????????????????110

Val?Lys?Ala?Ala?Ala?Leu?Gly?Thr?Thr?Cys?Tyr?Val?Gly?Ala?Ala?Ile

115?????????????????120?????????????????125

Leu?Leu?Trp?Gln?Pro?Ile?Pro?Val?Asn?Phe?Leu?Arg?Pro?Arg?Ser?Leu

130?????????????????135?????????????????140

Gln?Cys?Val?Lys?Ala?Phe?Leu?Thr?Leu?Ser?Val?Thr?Trp?Ile?Gly?Val

145?????????????????150?????????????????155?????????????????160

Asn?Ala?Leu?Leu?Tyr?Ser?Leu?Val?His?Asn?Leu?Gly?Ala?Ala?Thr?Leu

165?????????????????170?????????????????175

Met?Ser?Ala?Met?Thr?Asn?Leu

180

<210>278

<211>648

<212>DNA

＜213〉the unknown

<220>

＜223〉prostate 1

<400>278

atgcaggtgc?agatccagag?cctgtttctg?ctcctcctgt?gggtgcccgg?gtccagagga?????60

ttgacatttt?tttggctgga?tagatcggtt?aaggctgcag?ccgtgcttgt?tcatccccag????120

tgggtcttga?ccgtaaaggc?tgccgcgctg?ctacaagaaa?gaggggtcgc?atacatcaaa????180

gctgctctcc?tcttgagtat?tgcgctaagt?gtaaacccgc?tagtttgtaa?tggggtgtta????240

caaggtgtga?aagcggcgat?tatgtacagt?gcccacgaca?ctaccgtaaa?agcagccgct????300

ttcctgaccc?caaaaaaact?ccaatgcgtg?aacgcaatga?tgaatgatca?gctgatgttt????360

ttaaacgctg?gcttaccttc?tataccggtt?catccagtca?aggccgcggc?attgggtacg????420

acgtgttatg?ttggagcagc?gatacttctt?tggcagccca?taccagtaaa?ttttttaaga????480

cctagatcct?tacaatgcgt?caaagcattc?cttacactct?cagtaacttg?gatcggagtc????540

aatgctctgc?tatatagcct?cgtacacaac?ttgggcgcgg?ccacacttat?gagtgcaatg????600

acgaatttag?ctaagttcgt?ggcggcctgg?actctaaagg?ccgcagca?????????????????648

<210>279

<211>322

<212>PRT

＜213〉human immunodeficiency virus

<400>279

Met?Glu?Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys?Gly?Ile?Gly

1????????????????5??????????????????10??????????????????15

Gly?Gly?Pro?Gly?Pro?Gly?Gln?Lys?Gln?Ile?Thr?Lys?Ile?Gln?Asn?Phe

20??????????????????25??????????????????30

Arg?Val?Tyr?Tyr?Arg?Gly?Pro?Gly?Pro?Gly?Trp?Glu?Phe?Val?Asn?Thr

35??????????????????40??????????????????45

Pro?Pro?Leu?Val?Lys?Leu?Trp?Tyr?Gln?Gly?Pro?Gly?Pro?Gly?Tyr?Arg

50??????????????????55??????????????????60

Lys?Ile?Leu?Arg?Gln?Arg?Lys?Ile?Asp?Arg?Leu?Ile?Asp?Gly?Pro?Gly

65??????????????????70??????????????????75??????????????????80

Pro?Gly?Gln?His?Leu?Leu?Gln?Leu?Thr?Val?Trp?Gly?Ile?Lys?Gln?Leu

85??????????????????90??????????????????95

Gln?Gly?Pro?Gly?Pro?Gly?Gly?Glu?Ile?Tyr?Lys?Arg?Trp?Ile?Ile?Leu

100?????????????????105?????????????????110

Gly?Leu?Asn?Lys?Ile?Val?Arg?Met?Tyr?Gly?Pro?Gly?Pro?Gly?Gln?Gly

115?????????????????120?????????????????125

Gln?Met?Val?His?Gln?Ala?Ile?Ser?Pro?Arg?Thr?Leu?Asn?Gly?Pro?Gly

130?????????????????135?????????????????140

Pro?Gly?Ile?Lys?Gln?Phe?Ile?Asn?Met?Trp?Gln?Glu?Val?Gly?Lys?Ala

145?????????????????150?????????????????155?????????????????160

Met?Tyr?Gly?Pro?Gly?Pro?Gly?Trp?Ala?Gly?Ile?Lys?Gln?Glu?Phe?Gly

165?????????????????170?????????????????175

Ile?Pro?Tyr?Asn?Pro?Gln?Gly?Pro?Gly?Pro?Gly?Lys?Thr?Ala?Val?Gln

180?????????????????185?????????????????190

Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys?Arg?Gly?Pro?Gly?Pro?Gly?Ser

195?????????????????200?????????????????205

Pro?Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys?Ile?Leu?Glu?Pro?Gly?Pro

210?????????????????215?????????????????220

Gly?Pro?Gly?Glu?Val?Asn?Ile?Val?Thr?Asp?Ser?Gln?Tyr?Ala?Leu?Gly

225?????????????????230?????????????????235?????????????????240

Ile?Ile?Gly?Pro?Gly?Pro?Gly?His?Ser?Asn?Trp?Arg?Ala?Met?Ala?Ser

245?????????????????250?????????????????255

Asp?Phe?Asn?Leu?Pro?Pro?GIy?Pro?Gly?Pro?Gly?Ala?Glu?Thr?Phe?Tyr

260?????????????????265?????????????????270

Val?Asp?Gly?Ala?Ala?Asn?Arg?Glu?Thr?Lys?Gly?Pro?Gly?Pro?Gly?Gly

275?????????????????280?????????????????285

Ala?Val?Val?Ile?Gln?Asp?Asn?Ser?Asp?Ile?Lys?Val?Val?Pro?Gly?Pro

290?????????????????295?????????????????300

Gly?Pro?Gly?Phe?Arg?Lys?Tyr?Thr?Ala?Phe?Thr?Ile?Pro?Ser?Ile?Asn

305?????????????????310?????????????????315?????????????????320

Asn?Glu

<210>280

<211>969

<212>DNA

＜213〉human immunodeficiency virus

<400>280

atggagaagg?tgtacctggc?ctgggttcca?gcccacaaag?gcatcggggg?agggcccgga?????60

cctgggcaga?aacagatcac?caagatccag?aacttccggg?tatactaccg?gggacctggt????120

ccaggttggg?agtttgtgaa?cacaccaccc?ttagtaaagc?tctggtacca?gggccccggt????180

cccggatacc?gtaaaatcct?gaggcaaaga?aagatagatc?gcctcattga?tggcccgggc????240

ccaggccagc?accttctgca?gcttacagtg?tggggaatta?aacagctgca?ggggccgggc????300

cccggggggg?aaatttataa?aaggtggatc?attctgggtc?tgaacaagat?cgtccgcatg????360

tatggccctg?gacccggaca?ggggcagatg?gtccaccaag?caatcagccc?tcgaaccttg????420

aatggaccgg?gcccaggaat?caagcaattc?attaacatgt?ggcaagaagt?tggtaaggct????480

atgtacggtc?ccggccctgg?atgggcaggg?ataaaacagg?agtttggaat?cccttacaat????540

ccccagggtc?ctgggccagg?taaaacggca?gtgcagatgg?ccgtgttcat?tcataatttt????600

aagcggggcc?ctggacctgg?cagcccagct?atatttcaaa?gttcgatgac?caaaatcttg????660

gagcccggcc?cagggccggg?cgaagtgaac?attgtcacag?attctcagta?tgccctcggc????720

atcatagggc?ccggaccagg?gcattccaat?tggcgcgcca?tggcgtctga?ctttaatcta????780

cctcctgggc?caggccctgg?cgcggaaact?ttctatgtgg?acggcgctgc?aaacagggag????840

actaagggac?ccggacccgg?cggcgctgta?gtcattcagg?acaactcaga?catcaaggtg????900

gttcccggtc?caggccccgg?gttcagaaag?tataccgcct?tcactattcc?gtccatcaac????960

aatgagtga????????????????????????????????????????????????????????????969

<210>281

<211>340

<212>PRT

＜213〉human immunodeficiency virus

<400>281

Met?Glu?Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys?Gly?Ile?Gly

1????????????????5??????????????????10??????????????????15

Gly?Gly?Pro?Gly?Pro?Gly?Gln?Lys?Gln?Ile?Thr?Lys?Ile?Gln?Asn?Phe

20??????????????????25??????????????????30

Arg?Val?Tyr?Tyr?Arg?Gly?Pro?Gly?Pro?Gly?Trp?Glu?Phe?Val?Asn?Thr

35??????????????????40??????????????????45

Pro?Pro?Leu?Val?Lys?Leu?Trp?Tyr?Gln?Gly?Pro?Gly?Pro?Gly?Tyr?Arg

50??????????????????55??????????????????60

Lys?Ile?Leu?Arg?Gln?Arg?Lys?Ile?Asp?Arg?Leu?Ile?Asp?Gly?Pro?Gly

65??????????????????70??????????????????75??????????????????80

Pro?Gly?Gln?His?Leu?Leu?Gln?Leu?Thr?Val?Trp?Gly?Ile?Lys?Gln?Leu

85??????????????????90??????????????????95

Gln?Gly?Pro?Gly?Pro?Gly?Gly?Glu?Ile?Tyr?Lys?Arg?Trp?Ile?Ile?Leu

100?????????????????105?????????????????110

Gly?Leu?Asn?Lys?Ile?Val?Arg?Met?Tyr?Gly?Pro?Gly?Pro?Gly?Gln?Gly

115?????????????????120?????????????????125

Gln?Met?Val?His?Gln?Ala?Ile?Ser?Pro?Arg?Thr?Leu?Asn?Gly?Pro?Gly

130?????????????????135?????????????????140

Pro?Gly?Ile?Lys?G1n?Phe?Ile?Asn?Met?Trp?G1n?G1u?Val?G1y?Lys?Ala

145?????????????????150?????????????????155?????????????????160

Met?Tyr?Gly?Pro?Gly?Pro?Gly?Trp?Ala?Gly?Ile?Lys?Gln?Glu?Phe?Gly

165?????????????????170?????????????????175

Ile?Pro?Tyr?Asn?Pro?Gln?Gly?Pro?Gly?Pro?Gly?Lys?Thr?Ala?Val?Gln

180?????????????????185?????????????????190

Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys?Arg?Gly?Pro?Gly?Pro?Gly?Ser

195?????????????????200?????????????????205

Pro?Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys?Ile?Leu?Glu?Pro?Gly?Pro

210?????????????????215?????????????????220

Gly?Pro?Gly?Glu?Val?Asn?Ile?Val?Thr?Asp?Ser?Gln?Tyr?Ala?Leu?Gly

225?????????????????230?????????????????235?????????????????240

Ile?Ile?Gly?Pro?Gly?Pro?Gly?His?Ser?Asn?Trp?Arg?Ala?Met?Ala?Ser

245?????????????????250?????????????????255

Asp?Phe?Asn?Leu?Pro?Pro?Gly?Pro?Gly?Pro?Gly?Ala?Glu?Thr?Phe?Tyr

260?????????????????265?????????????????270

Val?Asp?Gly?Ala?Ala?Asn?Arg?Glu?Thr?Lys?Gly?Pro?Gly?Pro?Gly?Gly

275?????????????????280?????????????????285

Ala?Val?Val?Ile?Gln?Asp?Asn?Ser?Asp?Ile?Lys?Val?Val?Pro?Gly?Pro

290?????????????????295?????????????????300

Gly?Pro?Gly?Phe?Arg?Lys?Tyr?Thr?Ala?Phe?Thr?Ile?Pro?Ser?Ile?Asn

305?????????????????310?????????????????315?????????????????320

Asn?Glu?Gly?Pro?Gly?Pro?Gly?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu

325?????????????????330?????????????????335

Lys?Ala?Ala?Ala

340

<210>282

<211>1023

<212>DNA

＜213〉human immunodeficiency virus

<400>282

atggagaagg?tgtacctggc?ctgggttcca?gcccacaaag?gcatcggggg?agggcccgga?????60

cctgggcaga?aacagatcac?caagatccag?aacttccggg?tatactaccg?gggacctggt????120

ccaggttggg?agtttgtgaa?cacaccaccc?ttagtaaagc?tctggtacca?gggccccggt????180

cccggatacc?gtaaaatcct?gaggcaaaga?aagatagatc?gcctcattga?tggcccgggc????240

ccaggccagc?accttctgca?gcttacagtg?tggggaatta?aacagctgca?ggggccgggc????300

cccggggggg?aaatttataa?aaggtggatc?attctgggtc?tgaacaagat?cgtccgcatg????360

tatggccctg?gacccggaca?ggggcagatg?gtccaccaag?caatcagccc?tcgaaccttg????420

aatggaccgg?gcccaggaat?caagcaattc?attaacatgt?ggcaagaagt?tggtaaggct????480

atgtacggtc?ccggccctgg?atgggcaggg?ataaaacagg?agtttggaat?cccttacaat????540

ccccagggtc?ctgggccagg?taaaacggca?gtgcagttgg?ccgtgttcat?tcataatttt????600

aagcggggcc?ctggacctgg?cagcccagct?atatttcaaa?gttcgatgac?caaaatcttg????660

gagcccggcc?cagggccggg?cgaagtgaac?attgtcacag?attctcagta?tgccctcggc????720

atcatagggc?ccggaccagg?gcattccaat?tggcgcgcca?tggcgtctga?ctttaatcta????780

cctcctgggc?caggccctgg?cgcggaaact?ttctatgtgg?acggcgctgc?aaacagggag????840

actaagggac?ccggacccgg?cggcgctgta?gtcattcagg?acaactcaga?catcaaggtg????900

gttcccggtc?caggccccgg?gttcagaaag?tataccgcct?tcactattcc?gtccatcaac????960

aatgagggcc?ccggcccagg?tgccaagttc?gtggctgcct?ggaccctgaa?ggctgccgct???1020

tga?????????????????????????????????????????????????????????????????1023

<210>283

<211>75

<212>PRT

＜213〉human immunodeficiency virus

<400>283

Glu?Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys?Gly?Ile?Gly?Gly

1???????????????5???????????????????10??????????????????15

Pro?Gly?Pro?Gly?Gln?Gly?Gln?Met?Val?His?Gln?Ala?Ile?Ser?Pro?Arg

20??????????????????25??????????????????30

Thr?Leu?Asn?Gly?Pro?Gly?Pro?Gly?Ser?Pro?Ala?Ile?Phe?Gln?Ser?Ser

35??????????????????40??????????????????45

Met?Thr?Lys?Ile?Leu?Glu?Pro?Gly?Pro?Gly?Pro?Gly?Phe?Arg?Lys?Tyr

50??????????????????55??????????????????60

Thr?Ala?Phe?Thr?Ile?Pro?Ser?Ile?Asn?Asn?Glu

65??????????????????70??????????????????75

<210>284

<211>228

<212>DNA

＜213〉human immunodeficiency virus

<400>284

gagaaggtgt?acctggcctg?ggtgcctgcc?cacaagggaa?tcggaggacc?tggccctgga?????60

cagggacaga?tggtgcacca?ggccatcagc?cctaggaccc?tgaacggacc?tggacctgga????120

agccctgcca?tcttccagag?cagcatgacc?aagatcctgg?agcccggacc?tggacctgga????180

ttcaggaagt?acaccgcctt?caccatcccc?agcatcaaca?acgagtga?????????????????228

<210>285

<211>276

<212>PRT

＜213〉the unknown

<220>

<223>PfHTL

<400>285

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Arg?His?Asn?Trp?Val?Asn?His?Ala?Val?Pro?Leu?Ala

20??????????????????25??????????????????30

Met?Lys?Leu?Ile?Gly?Pro?Gly?Pro?Gly?Lys?Cys?Asn?Leu?Tyr?Ala?Asp

35??????????????????40??????????????????45

Ser?Ala?Trp?Glu?Asn?Val?Lys?Asn?Gly?Pro?Gly?Pro?Gly?Lys?Ser?Lys

50??????????????????55??????????????????60

Tyr?Lys?Leu?Ala?Thr?Ser?Val?Leu?Ala?Gly?Leu?Leu?Gly?Pro?Gly?Pro

65??????????????????70??????????????????75??????????????????80

Gly?Gln?Thr?Asn?Phe?Lys?Ser?Leu?Leu?Arg?Asn?Leu?Gly?Val?Ser?Glu

85??????????????????90??????????????????95

Gly?Pro?Gly?Pro?Gly?Ser?Ser?Val?Phe?Asn?Val?Val?Asn?Ser?Ser?Ile

100?????????????????105?????????????????110

Gly?Leu?Ile?Met?Gly?Pro?Gly?Pro?Gly?Val?Lys?Asn?Val?Ile?Gly?Pro

115????????????????120?????????????????125

Phe?Met?Lys?Ala?Val?Cys?Val?Glu?Gly?Pro?Gly?Pro?Gly?Met?Asn?Tyr

130?????????????????135?????????????????140

Tyr?Gly?Lys?Gln?Glu?Asn?Trp?Tyr?Ser?Leu?Lys?Lys?Gly?Pro?Gly?Pro

145?????????????????150?????????????????155?????????????????160

Gly?Gly?Leu?Ala?Tyr?Lys?Phe?Val?Val?Pro?Gly?Ala?Ala?Thr?Pro?Tyr

165?????????????????170?????????????????175

Gly?Pro?Gly?Pro?Gly?Pro?Asp?Ser?Ile?Gln?Asp?Ser?Leu?Lys?Glu?Ser

180?????????????????185?????????????????190

Arg?Lys?Leu?Asn?Gly?Pro?Gly?Pro?Gly?Leu?Leu?Ile?Phe?His?Ile?Asn

195?????????????????200?????????????????205

Gly?Lys?Ile?Ile?Lys?Asn?Ser?Glu?Gly?Pro?Gly?Pro?Gly?Ala?Gly?Leu

210?????????????????215?????????????????220

Leu?Gly?Asn?Val?Ser?Thr?Val?Leu?Leu?Gly?Gly?Val?Gly?Pro?Gly?Pro

225?????????????????230?????????????????235?????????????????240

Gly?Lys?Tyr?Lys?Ile?Ala?Gly?Gly?Ile?Ala?Gly?Gly?Leu?Ala?Leu?Leu

245?????????????????250?????????????????255

Gly?Pro?Gly?Pro?Gly?Met?Arg?Lys?Leu?Ala?Ile?Leu?Ser?Val?Ser?Ser

260?????????????????265?????????????????270

Phe?Leu?Phe?Val

275

<210>286

<211>837

<212>DNA

＜213〉the unknown

<220>

<223>PfHTL

<400>286

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccggatcc?????60

agaggaaggc?acaactgggt?gaatcatgct?gtgcccctgg?ctatgaagct?gatcggccct????120

ggaccaggga?aatgcaacct?ctacgcagac?agcgcctggg?agaacgtcaa?gaatggcccc????180

ggacctggga?aatccaagta?taagctcgct?acctctgtgc?tggcaggcct?gctcggacca????240

ggccccggac?agacaaattt?caaaagcctg?ctcagaaacc?tgggagtgtc?cgaggggcct????300

ggcccaggat?ctagcgtctt?taatgtggtc?aactcctcta?ttgggctcat?catgggaccc????360

ggacctgggg?tgaaaaatgt?cattggccca?ttcatgaagg?ccgtgtgtgt?cgaaggaccc????420

gggcctggca?tgaactacta?tggaaagcaa?gaaaattggt?acagcctgaa?gaaaggccct????480

gggccaggcg?gactggctta?caagtttgtg?gtcccagggg?cagccactcc?ctatgggcct????540

gggccaggcc?ccgattccat?ccaggactct?ctcaaagaga?gccggaaact?gaacggaccc????600

gggcctggac?tgctcatttt?ccacatcaat?ggcaaaatta?tcaagaacag?cgagggacct????660

gggccaggcg?ccggactgct?ggggaacgtg?tccaccgtcc?tgctcggcgg?agtggggccc????720

ggccctggga?agtacaagat?cgctggaggg?atcgcaggcg?gactggccct?cctgggccca????780

ggaccaggga?tgcgcaaact?ggctattctc?tctgtctcca?gctttctgtt?tgtgtga???????837

<210>287

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>287

Val?Leu?Ala?Glu?Ala?Met?Ser?Gln?Val

1???????????????5

<210>288

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>288

Met?Thr?Asn?Asn?Pro?Pro?Ile?Pro?Val

1???????????????5

<210>289

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>289

Met?Ala?Ser?Asp?Phe?Asn?Leu?Pro?Pro?Val

1???????????????5???????????????????10

<210>290

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>290

Lys?Leu?Val?Gly?Lys?Leu?Asn?Trp?Ala

1???????????????5

<210>291

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>291

Leu?Val?Gly?Pro?Thr?Pro?Val?Asn?Ile

1???????????????5

<210>292

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>292

Ile?Leu?Lys?Glu?Pro?Val?His?Gly?Val

1???????????????5

<210>293

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>?293

Lys?Ala?Ala?Cys?Trp?Trp?Ala?Gly?Ile

1???????????????5

<210>294

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>294

Lys?Met?Ile?Gly?Gly?Ile?Gly?Gly?Phe?Ile

1???????????????5???????????????????10

<210>295

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>295

Arg?Ala?Met?Ala?Ser?Asp?Phe?Asn?Leu

1???????????????5

<210>296

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>296

Thr?Leu?Asn?Phe?Pro?Ile?Ser?Pro?Ile

1???????????????5

<210>297

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>297

Lys?Leu?Thr?Pro?Leu?Cys?Val?Thr?Leu

1???????????????5

<210>298

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>298

Leu?Leu?Gln?Leu?Thr?Val?Trp?Gly?Ile

1???????????????5

<210>299

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>299

Ser?Leu?Leu?Asn?Ala?Thr?Asp?Ile?Ala?Val

1???????????????5???????????????????10

<210>300

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>300

Leu?Thr?Phe?Gly?Trp?Cys?Phe?Lys?Leu

1???????????????5

<210>301

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>301

Ala?Ile?Ile?Arg?Ile?Leu?Gln?Gln?Leu

1???????????????5

<210>302

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>302

Arg?Ile?Leu?Gln?Gln?Leu?Leu?Phe?Ile

1???????????????5

<210>303

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>303

Gln?Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys

1???????????????5???????????????????10

<210>304

<211>11

<212>PRT

＜213〉human immunodeficiency virus

<400>304

Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys

1???????????????5???????????????????10

<210>305

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>305

Lys?Ile?Gln?Asn?Phe?Arg?Val?Tyr?Tyr?Arg

1???????????????5???????????????????10

<210>306

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>306

Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys

1???????????????5

<210>307

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>307

Val?Thr?Ile?Lys?Ile?Gly?Gly?Gln?Leu?Lys

1???????????????5???????????????????10

<210>308

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>308

Thr?Thr?Leu?Phe?Cys?Ala?Ser?Asp?Ala?Lys

1???????????????5???????????????????10

<210>309

<211>11

<212>PRT

＜213〉human immunodeficiency virus

<400>309

Val?Thr?Val?Tyr?Tyr?Gly?Val?Pro?Val?Trp?Lys

1???????????????5???????????????????10

<210>310

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>310

Gln?Val?Pro?Leu?Arg?Pro?Met?Thr?Tyr?Lys

1???????????????5???????????????????10

<210>311

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>311

Val?Met?Ile?Val?Trp?Gln?Val?Asp?Arg

1???????????????5

<210>312

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>312

Gln?Met?Val?His?Gln?Ala?Ile?Ser?Pro?Arg

1???????????????5???????????????????10

<210>313

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>313

Tyr?Pro?Leu?Ala?Ser?Leu?Arg?Ser?Leu?Phe

1???????????????5???????????????????10

<210>314

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>314

His?Pro?Val?His?Ala?Gly?Pro?Ile?Ala

1???????????????5

<210>315

<21l>9

<212>PRT

＜213〉human immunodeficiency virus

<400>315

Phe?Pro?Ile?Ser?Pro?Ile?Glu?Thr?Val

l???????????????5

<210>316

<211>11

<212>PRT

＜213〉human immunodeficiency virus

<400>316

Ile?Pro?Tyr?Asn?Pro?Gln?Ser?Gln?Gly?Val?Va1

1???????????????5???????????????????10

<210>317

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>317

Ile?Pro?Ile?His?Tyr?Cys?Ala?Pro?Ala

1???????????????5

<210>318

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>318

Cys?Pro?Lys?Val?Ser?Phe?Glu?Pro?Ile

1???????????????5

<210>319

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>319

Phe?Pro?Val?Arg?Pro?Gln?Val?Pro?Leu

1???????????????5

<210>320

<211>8

<212>PRT

＜213〉human immunodeficiency virus

<400>320

Val?Pro?Leu?Gln?Leu?Pro?Pro?Leu

1???????????????5

<210>321

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>321

Glu?Val?Asn?Ile?Val?Thr?Asp?Ser?G1n?Tyr

1???????????????5???????????????????10

<210>322

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>322

Phe?Arg?Asp?Tyr?Val?Asp?Arg?Phe?Tyr

1???????????????5

<210>323

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>323

Val?Ile?Tyr?Gln?Tyr?Met?Asp?Asp?Leu?Tyr

1???????????????5???????????????????10

<210>324

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>324

Val?Thr?Val?Leu?Asp?Val?Gly?Asp?Ala?Tyr

1???????????????5???????????????????10

<210>325

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>325

Ile?Tyr?Gln?Glu?Pro?Phe?Lys?Asn?Leu

1???????????????5

<210>326

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>326

Pro?Tyr?Asn?Thr?Pro?Val?Phe?Ala?Ile

1???????????????5

<210>327

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>327

Thr?Tyr?Gln?Ile?Tyr?Gln?Glu?Pro?Phe

1???????????????5

<210>328

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>328

Tyr?Trp?Gln?Ala?Thr?Trp?Ile?Pro?Glu?Trp

1???????????????5???????????????????10

<210>329

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>329

Ile?Trp?Gly?Cys?Ser?Gly?Lys?Leu?Ile

1???????????????5

<210>330

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>330

Arg?Tyr?Leu?Lys?Asp?Gln?Gln?Leu?Leu

1???????????????5

<210>331

<211>10

<212>PRT

＜213〉human immunodeficiency virus

<400>331

Val?Trp?Lys?Glu?Ala?Thr?Thr?Thr?Leu?Phe

1???????????????5???????????????????10

<210>332

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>332

Ile?Tyr?Glu?Thr?Tyr?Gly?Asp?Thr?Trp

1???????????????5

<210>333

<211>9

<212>PRT

＜213〉human immunodeficiency virus

<400>333

Pro?Tyr?Asn?Glu?Trp?Thr?Leu?Glu?Leu

1???????????????5

<210>334

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>334

Lys?Arg?Trp?Ile?Ile?Leu?Gly?Leu?Asn?Lys?Ile?Val?Arg?Met?Tyr

1???????????????5???????????????????10??????????????????15

<210>335

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>335

Trp?Glu?Phe?Val?Asn?Thr?Pro?Pro?Leu?Val?Lys?Leu?Trp?Tyr?Gln

1???????????????5???????????????????10??????????????????15

<210>336

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>336

Gln?Lys?Gln?Ile?Thr?Lys?Ile?Gln?Asn?Phe?Arg?Val?Tyr?Tyr?Arg

1???????????????5???????????????????10??????????????????15

<210>337

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>337

Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys?Gly?Ile?Gly?Gly

1???????????????5???????????????????10??????????????????15

<210>338

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>338

Gly?Glu?Ile?Tyr?Lys?Arg?Trp?Ile?Ile?Leu?Gly?Leu?Asn?Lys?Ile

1???????????????5???????????????????10??????????????????15

<210>339

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>339

Glu?Lys?Val?Tyr?Leu?Ala?Trp?Val?Pro?Ala?His?Lys?Gly?Ile?Gly

1???????????????5???????????????????10??????????????????15

<210>340

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>340

Gln?His?Leu?Leu?Gln?Leu?Thr?Val?Trp?Gly?Ile?Lys?Gln?Leu?Gln

1???????????????5???????????????????10??????????????????15

<210>341

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>341

Gln?Gly?Gln?Met?Val?His?Gln?Ala?Ile?Ser?Pro?Arg?Thr?Leu?Asn

1???????????????5???????????????????10??????????????????15

<210>342

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>342

Ser?Pro?Ala?Ile?Phe?Gln?Ser?Ser?Met?Thr?Lys?Ile?Leu?Glu?Pro

1???????????????5???????????????????10??????????????????15

<210>343

<211>16

<212>PRT

＜213〉human immunodeficiency virus

<400>343

Ile?Lys?Gln?Phe?Ile?Asn?Met?Trp?Gln?Glu?Val?Gly?Lys?Ala?Met?Tyr

1???????????????5???????????????????10??????????????????15

<210>344

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>344

Phe?Arg?Lys?Tyr?Thr?Ala?Phe?Thr?Ile?Pro?Ser?Ile?Asn?Asn?Glu

1???????????????5???????????????????10??????????????????15

<210>345

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>345

His?Ser?Asn?Trp?Arg?Ala?Met?Ala?Ser?Asp?Phe?Asn?Leu?Pro?Pro

1???????????????5???????????????????10??????????????????15

<210>346

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>346

Lys?Thr?Ala?Val?Gln?Met?Ala?Val?Phe?Ile?His?Asn?Phe?Lys?Arg

1???????????????5???????????????????10??????????????????15

<210>347

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>347

Tyr?Arg?Lys?Ile?Leu?Arg?Gln?Arg?Lys?Ile?Asp?Arg?Leu?Ile?Asp

1???????????????5???????????????????10??????????????????15

<210>348

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>348

Trp?Ala?Gly?Ile?Lys?Gln?Glu?Phe?Gly?Ile?Pro?Tyr?Asn?Pro?Gln

1???????????????5???????????????????10??????????????????15

<210>349

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>349

Glu?Val?Asn?Ile?Val?Thr?Asp?Ser?Gln?Tyr?Ala?Leu?Gly?Ile?Ile

1???????????????5???????????????????10??????????????????15

<210>350

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>350

Ala?Glu?Thr?Phe?Tyr?Val?Asp?Gly?Ala?Ala?Asn?Arg?Glu?Thr?Lys

1???????????????5???????????????????10??????????????????15

<210>351

<211>15

<212>PRT

＜213〉human immunodeficiency virus

<400>351

Gly?Ala?Val?Val?Ile?Gln?Asp?Asn?Ser?Asp?Ile?Lys?Val?Val?Pro

1???????????????5???????????????????10??????????????????15

<210>352

<211>10

<212>PRT

＜213〉hepatitis C virus

<400>352

Leu?Leu?Phe?Asn?Ile?Leu?Gly?Gly?Trp?Val

1???????????????5???????????????????10

<210>353

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>353

Phe?Leu?Leu?Leu?Ala?Asp?Ala?Arg?Val

1???????????????5

<210>354

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>354

Tyr?Leu?Val?Ala?Tyr?Gln?Ala?Thr?Val

1???????????????5

<210>355

<211>10

<212>PRT

＜213〉hepatitis C virus

<400>355

Arg?Leu?Ile?Val?Phe?Pro?Asp?Leu?Gly?Val

1???????????????5???????????????????10

<210>356

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>356

Asp?Leu?Met?Gly?Tyr?Ile?Pro?Leu?Val

1???????????????5

<210>357

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>357

Trp?Met?Asn?Arg?Leu?Ile?Ala?Phe?Ala

1???????????????5

<210>358

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>358

Val?Leu?Val?Gly?Gly?Val?Leu?Ala?Ala

1???????????????5

<210>359

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>359

His?Met?Trp?Asn?Phe?Ile?Ser?Gly?Ile

1???????????????5

<210>360

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>360

Ile?Leu?Ala?Gly?Tyr?Gly?Ala?Gly?Val

1???????????????5

<210>361

<211>10

<212>PRT

＜213〉hepatitis C virus

<400>361

Tyr?Leu?Leu?Pro?Arg?Arg?Gly?Pro?Arg?Leu

1???????????????5???????????????????10

<210>362

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>362

Leu?Leu?Phe?Leu?Leu?Leu?Ala?Asp?Ala

1???????????????5

<210>363

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>363

Tyr?Leu?Val?Thr?Arg?His?Ala?Asp?Val

1???????????????5

<210>364

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>364

Lys?Thr?Ser?Glu?Arg?Ser?Gln?Pro?Arg

1???????????????5

<210>365

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>365

Arg?Leu?Gly?Val?Arg?Ala?Thr?Arg?Lys

1???????????????5

<210>366

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>366

Gln?Leu?Phe?Thr?Phe?Ser?Pro?Arg?Arg

1???????????????5

<210>367

<211>10

<212>PRT

＜213〉hepatitis C virus

<400>367

Arg?Met?Tyr?Val?Gly?Gly?Val?Glu?His?Arg

1???????????????5???????????????????10

<210>368

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>368

Leu?Ile?Phe?Cys?His?Ser?Lys?Lys?Lys

1???????????????5

<210>369

<211>10

<212>PRT

＜213〉hepatitis C virus

<400>369

Gly?Val?Ala?Gly?Ala?Leu?Val?Ala?Phe?Lys

1???????????????5???????????????????10

<210>370

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>370

Val?Ala?Gly?Ala?Leu?Val?Ala?Phe?Lys

1???????????????5

<210>371

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>371

Leu?Gly?Phe?Gly?Ala?Tyr?Met?Ser?Lys

1???????????????5

<210>372

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>372

Leu?Pro?Gly?Cys?Ser?Phe?Ser?Ile?Phe

1???????????????5

<210>373

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>373

Leu?Ser?Ala?Phe?Ser?Leu?His?Ser?Tyr

1???????????????5

<210>374

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>374

Cys?Thr?Cys?Gly?Ser?Ser?Asp?Leu?Tyr

l???????????????5

<210>375

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>375

Leu?Thr?Asp?Pro?Ser?His?Ile?Thr?Ala

1???????????????5

<210>376

<211>11

<212>PRT

＜213〉hepatitis C virus

<400>376

Leu?Thr?Cys?Gly?Phe?Ala?Asp?Leu?Met?Gly?Tyr

1???????????????5???????????????????10

<210>377

<211>11

<212>PRT

＜213〉hepatitis C virus

<400>377

Leu?Ala?Asp?Gly?Gly?Cys?Ser?Gly?Gly?Ala?Tyr

1???????????????5???????????????????10

<210>378

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>378

Phe?Trp?Ala?Lys?His?Met?Trp?Asn?Phe

1???????????????5

<210>379

<211>9

<212>PRT

＜213〉hepatitis C virus

<400>379

Arg?Met?Ile?Leu?Met?Thr?His?Phe?Phe

1???????????????5

<210>380

<211>8

<212>PRT

＜213〉hepatitis C virus

<400>380

Val?Met?Gly?Ser?Ser?Tyr?Gly?Phe

1???????????????5

<210>381

<211>10

<212>PRT

＜213〉hepatitis C virus

<400>381

Phe?Trp?Ala?Lys?His?Met?Trp?Asn?Phe?Ile

1???????????????5???????????????????10

<210>382

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>382

Phe?Met?Lys?Ala?Val?Cys?Val?Glu?Val

1???????????????5

<210>383

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>383

Gly?Leu?Leu?Gly?Val?Val?Ser?Thr?Val

1???????????????5

<210>384

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>384

Ile?Leu?Ser?Val?Ser?Ser?Phe?Leu?Phe?Val

1???????????????5???????????????????10

<210>385

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>385

Gln?Thr?Asn?Phe?Lys?Ser?Leu?Leu?Arg

1???????????????5

<210>386

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>386

Gly?Val?Ser?Glu?Asn?Ile?Phe?Leu?Lys

1???????????????5

<210>387

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>387

Leu?Leu?Ala?Cys?Ala?Gly?Leu?Ala?Tyr?Lys

1???????????????5???????????????????10

<210>388

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>388

Thr?Pro?Tyr?Ala?Gly?Glu?Pro?Ala?Pro?Phe

1???????????????5???????????????????10

<210>389

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>389

Leu?Pro?Ser?Glu?Asn?Glu?Arg?Gly?Tyr

1???????????????5

<210>390

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>390

Lys?Tyr?Lys?Leu?Ala?Thr?Ser?Val?Leu

1???????????????5

<210>391

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>391

Ser?Phe?Leu?Phe?Val?Glu?Ala?Leu?Phe

1???????????????5

<210>392

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>392

Tyr?Phe?Ile?Leu?Val?Asn?Leu?Leu?Ile

1???????????????5

<210>393

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>393

Phe?Leu?Ile?Phe?Phe?Asp?Leu?Phe?Leu?Val

1???????????????5???????????????????10

<210>394

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>394

Val?Leu?Ala?Gly?Leu?Leu?Gly?Val?Val

1???????????????5

<210>395

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>395

Val?Leu?Leu?Gly?Gly?Val?Gly?Leu?Val?Leu

1???????????????5???????????????????10

<210>396

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>396

Leu?Ala?Cys?Ala?Gly?Leu?Ala?Tyr?Lys

1???????????????5

<210>397

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>397

Ala?Leu?Phe?Phe?Ile?Ile?Phe?Asn?Lys

1???????????????5

<210>398

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>398

Phe?Ile?Leu?Val?Asn?Leu?Leu?Ile?Phe?His

1???????????????5???????????????????10

<210>399

<211>8

<212>PRT

＜213〉plasmodium falciparum

<400>399

Leu?Pro?Tyr?Gly?Arg?Thr?Asn?Leu

1???????????????5

<210>400

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>400

Phe?Val?Glu?Ala?Leu?Phe?Gln?Glu?Tyr

1???????????????5

<210>401

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>401

Phe?Gln?Asp?Glu?Glu?Asn?Ile?Gly?Ile?Tyr

1???????????????5???????????????????10

<210>402

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>402

Phe?Tyr?Phe?Ile?Leu?Val?Asn?Leu?Leu

1???????????????5

<210>403

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>403

Lys?Tyr?Leu?Val?Ile?Val?Phe?Leu?Ile

1???????????????5

<210>404

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>404

Gly?Leu?Ile?Met?Val?Leu?Ser?Phe?Leu

1???????????????5

<210>405

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>405

Lys?Ile?Leu?Ser?Val?Phe?Phe?Leu?Ala

1???????????????5

<210>406

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>406

Val?Thr?Cys?Gly?Asn?Gly?Ile?Gln?Val?Arg

1???????????????5??????????????????10

<210>407

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>407

His?Val?Leu?Ser?His?Asn?Ser?Tyr?Glu?Lys

1???????????????5???????????????????10

<210>408

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>408

Pro?Ser?Asp?Gly?Lys?Cys?Asn?Leu?Tyr

1???????????????5

<210>409

<211>9

<212>PRT

＜213〉plasmodium falciparum

<400>409

Tyr?Tyr?Ile?Pro?His?Gln?Ser?Ser?Leu

1???????????????5

<210>410

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>410

Lys?Phe?Ile?Lys?Ser?Leu?Phe?His?Ile?Phe

1???????????????5???????????????????10

<210>411

<211>10

<212>PRT

＜213〉plasmodium falciparum

<400>411

Val?Phe?Leu?Ile?Phe?Phe?Asp?Leu?Phe?Leu

1???????????????5???????????????????10

<210>412

<211>11

<212>PRT

＜213〉plasmodium falciparum

<400>412

Leu?Phe?His?Ile?Phe?Asp?Gly?Asp?Asn?Glu?Ile

1???????????????5???????????????????10

<210>413

<211>11

<212>PRT

＜213〉plasmodium falciparum

<400>413

Tyr?Tyr?Gly?Lys?Gln?Glu?Asn?Trp?Tyr?Ser?Leu

1???????????????5???????????????????10

<210>414

<211>8

<212>PRT

＜213〉plasmodium falciparum

<400>414

Leu?Tyr?Ile?Ser?Phe?Tyr?Phe?Ile

1???????????????5

<210>415

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>415

Met?Arg?Lys?Leu?Ala?Ile?Leu?Ser?Val?Ser?Ser?Phe?Leu?Phe?Val

1???????????????5???????????????????10??????????????????15

<210>416

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>416

Met?Asn?Tyr?Tyr?Gly?Lys?Gln?Glu?Asn?Trp?Tyr?Ser?Leu?Lys?Lys

1???????????????5???????????????????10??????????????????15

<210>417

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>417

Ser?Ser?Val?Phe?Asn?Val?Val?Asn?Ser?Ser?Ile?Gly?Leu?Ile?Met

1???????????????5???????????????????10??????????????????15

<210>418

<211>16

<212>PRT

＜213〉plasmodium falciparum

<400>418

Arg?His?Asn?Trp?Val?Asn?His?Ala?Val?Pro?Leu?Ala?Met?Lys?Leu?Ile

1???????????????5???????????????????10??????????????????15

<210>419

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>419

Pro?Asp?Ser?Ile?Gln?Asp?Ser?Leu?Lys?Glu?Ser?Arg?Lys?Leu?Asn

1???????????????5???????????????????10??????????????????15

<210>420

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>420

Lys?Cys?Asn?Leu?Tyr?Ala?Asp?Ser?Ala?Trp?Glu?Asn?Val?Lys?Asn

1???????????????5???????????????????10??????????????????15

<210>421

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>421

Val?Lys?Asn?Val?Ile?Gly?Pro?Phe?Met?Lys?Ala?Val?Cys?Val?Glu

1???????????????5???????????????????10??????????????????15

<210>422

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>422

Lys?Tyr?Lys?Ile?Ala?Gly?Gly?Ile?Ala?Gly?Gly?Leu?Ala?Leu?Leu

1???????????????5???????????????????10??????????????????15

<210>423

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>423

Gly?Leu?Ala?Tyr?Lys?Phe?Val?Val?Pro?Gly?Ala?Ala?Thr?Pro?Tyr

1???????????????5???????????????????10??????????????????15

<210>424

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>424

Lys?Ser?Lys?Tyr?Lys?Leu?Ala?Thr?Ser?Val?Leu?Ala?Gly?Leu?Leu

1???????????????5???????????????????10??????????????????15

<210>425

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>425

Ala?Gly?Leu?Leu?Gly?Asn?Val?Ser?Thr?Val?Leu?Leu?Gly?Gly?Val

1???????????????5???????????????????10??????????????????15

<210>426

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>426

Leu?Leu?Ile?Phe?His?Ile?Asn?Gly?Lys?Ile?Ile?Lys?Asn?Ser?Glu

1???????????????5???????????????????10??????????????????15

<210>427

<211>15

<212>PRT

＜213〉plasmodium falciparum

<400>427

Gln?Thr?Asn?Phe?Lys?Ser?Leu?Leu?Arg?Asn?Leu?Gly?Val?Ser?Glu

1???????????????5???????????????????10??????????????????15

<210>428

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>428

Arg?Met?Ser?Arg?Val?Thr?Thr?Phe?Thr?Val

1???????????????5???????????????????10

<210>?429

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>429

Ala?Leu?Val?Leu?Leu?Met?Leu?Pro?Val?Val

1???????????????5???????????????????10

<210>430

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>430

Leu?Met?Ile?Gly?Thr?Ala?Ala?Ala?Val?Val

1???????????????5???????????????????10

<210>431

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>431

Ala?Leu?Val?Leu?Leu?Met?Leu?Pro?Val

1???????????????5

<210>432

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>432

Gly?Leu?Met?Thr?Ala?Val?Tyr?Leu?Val

1???????????????5

<210>433

<211>8

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>433

Met?Ala?Leu?Leu?Arg?Leu?Pro?Val

1???????????????5

<210>434

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>434

Arg?Met?Phe?Ala?Ala?Asn?Leu?Gly?Val

1???????????????5

<210>435

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>435

Ser?Leu?Tyr?Phe?Gly?Gly?Ile?Cys?Val

1???????????????5

<210>436

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>436

Arg?Leu?Pro?Leu?Val?Leu?Pro?Ala?Val

1???????????????5

<210>437

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>437

Arg?Leu?Met?Ile?Gly?Thr?Ala?Ala?Ala

1???????????????5

<210>438

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>438

Phe?Val?Val?Ala?Leu?Ile?Pro?Leu?Val

1???????????????5

<210>439

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>439

Met?Thr?Tyr?Ala?Ala?Pro?Leu?Phe?Val

1???????????????5

<210>440

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>TB

<400>440

Ala?Met?Ala?Leu?Leu?Arg?Leu?Pro?Leu?Val

1???????????????5???????????????????10

<210>441

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>p53?139

<400>441

Lys?Leu?Cys?Pro?Val?Gln?Leu?Trp?Val

1???????????????5

<210>442

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>CEA?687

<400>442

Ala?Thr?Val?Gly?Ile?Met?Ile?Gly?Val

1???????????????5

<210>443

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>CEA?691

<400>443

Ile?Met?Ile?Gly?His?Leu?Val?Gly?Val

1???????????????5

<210>444

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>Her2?neu?689

<400>444

Arg?Leu?Leu?Gln?Glu?Thr?Glu?Leu?Val

1???????????????5

<210>445

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>MAGE3?112

<400>445

Lys?Val?Ala?Glu?Ile?Val?His?Phe?Leu

1???????????????5

<210>446

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>Her2/neu?665

<400>446

Val?Val?Leu?Gly?Val?Val?Phe?Gly?Ile

1???????????????5

<210>447

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>p53?149

<400>447

Ser?Met?Pro?Pro?Pro?Gly?Thr?Arg?Val

l???????????????5

<210>448

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>PAP.21.T2

<400>448

Leu?Thr?Phe?Phe?Trp?Leu?Asp?Arg?Ser?Val

1???????????????5???????????????????10

<210>449

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>PAP.112

<400>449

Thr?Leu?Met?Ser?Ala?Met?Thr?Asn?Leu

1???????????????5

<210>450

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>PAP.284

<400>450

Ile?Met?Tyr?Ser?Ala?His?Asp?Thr?Thr?Val

1???????????????5???????????????????10

<210>451

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>PSM.288.V10

<400>451

Gly?Leu?Pro?Ser?Ile?Pro?Val?Hi?s?Pro?Val

1???????????????5????????????????????10

<210>452

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>PSM.441

<400>452

Leu?Leu?Gln?Glu?Arg?Gly?Val?Ala?Tyr?Ile

1???????????????5???????????????????10

<210>453

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>PSM.469L2

<400>453

Leu?Leu?Tyr?Ser?Leu?Val?His?Asn?Leu

1???????????????5

<210>454

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>PSM.663

<400>454

Met?Met?Asn?Asp?Gln?Leu?Met?Phe?Leu

1???????????????5

<210>455

<211>11

<212>PRT

＜213〉the unknown

<220>

<223>PSA.3.V11

<400>455

Phe?Leu?Thr?Leu?Ser?Val?Thr?Trp?Ile?Gly?Val

1???????????????5???????????????????10

<210>456

<211>8

<212>PRT

＜213〉the unknown

<220>

<223>PSA.143.V8

<400>456

Ala?Leu?Gly?Thr?Thr?Cys?Tyr?Val

1???????????????5

<210>457

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>PSA.161

<400>457

Phe?Leu?Thr?Pro?Lys?Lys?Leu?Gln?Cys?Val

1???????????????5???????????????????10

<210>458

<211>9

<212>PRT

＜213〉the unknown

<220>

<223>HuK2.4.L2

<400>458

Leu?Leu?Leu?Ser?Ile?Ala?Leu?Ser?Val

1???????????????5

<210>459

<211>11

<212>PRT

＜213〉the unknown

<220>

<223>HuK2.53.V11

<400>459

Val?Leu?Val?His?Pro?Gln?Trp?Val?Leu?Thr?Val

1???????????????5???????????????????10

<210>460

<211>10

<212>PRT

＜213〉the unknown

<220>

<223>HuK2.165

<400>460

Phe?Leu?Arg?Pro?Arg?Ser?Leu?Gln?Cys?Val

1???????????????5???????????????????10

<210>461

<211>11

<212>PRT

＜213〉the unknown

<220>

<223>HuK2.216.Vll

<400>461

Pro?Leu?Val?Cys?Asn?Gly?Val?Leu?Gln?Gly?Val

1???????????????5???????????????????10

<210>462

<211>219

<212>PRT

＜213〉hepatitis type B virus

<400>462

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Asn?Phe?Leu?Leu?Ser?Leu?Gly?Ile

50??????????????????55??????????????????60

His?Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr

65??????????????????70??????????????????75??????????????????80

Val?Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Ser?Thr?Leu

85??????????????????90??????????????????95

Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

100?????????????????105?????????????????110

Tyr?Lys?Gly?Ala?Ala?Ala?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Asn

115?????????????????120?????????????????125

Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Thr?Pro?Ala?Arg?Val?Thr

130?????????????????135????????????????140

Gly?Gly?Val?Phe?Lys?Val?Gly?Asn?Phe?Thr?Gly?Leu?Tyr?Asn?Leu?Pro

145?????????????????150?????????????????155?????????????????160

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu

165?????????????????170?????????????????175

Tyr?Lys?Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys?Gly?Ala?Ala?Leu?Val

180?????????????????185?????????????????190

Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Asn?Ser?Ala?Ile?Cys?Ser?Val?Val

195?????????????????200?????????????????205

Arg?Arg?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

210?????????????????215

<210>463

<211>660

<212>DNA

＜213〉hepatitis type B virus

<400>463

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgaa?cttcctgctg????180

tccctgggaa?tccacctgta?tatggatgac?gtggtgctgg?gagtgggact?gtccaggtac????240

gtggctaggc?tgttcctgct?gaccagaatc?ctgaccatct?ccaccctgcc?agagaccacc????300

gtggtgagga?ggcaggcctt?cacctttagc?cctacctata?agggagccgc?tgcctggctg????360

agcctgctgg?tgccctttgt?gaatatccct?atccctagct?cctgggcttt?caagacccca????420

gccagggtga?ccggaggagt?gtttaaggtg?ggaaacttca?ccggcctgta?taacctgccc????480

agcgatttct?ttcctagcgt?gaagaccctg?tggaaggccg?gaatcctgta?caagaatgtg????540

tccatccctt?ggacccacaa?gggagccgct?ctggtggtgg?acttttccca?gttcagcaga????600

aattccgcta?tctgctccgt?ggtgaggaga?gctctgatgc?cactgtatgc?ctgtatctga????660

<210>464

<211>333

<212>PRT

＜213〉hepatitis type B virus

<400>464

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Asn?Phe?Leu?Leu?Ser?Leu?Gly?Ile

50??????????????????55??????????????????60

His?Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr

65??????????????????70??????????????????75??????????????????80

Val?Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Ser?Thr?Leu

85??????????????????90??????????????????95

Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

100?????????????????105?????????????????110

Tyr?Lys?Gly?Ala?Ala?Ala?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Asn

115?????????????????120?????????????????125

Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Thr?Pro?Ala?Arg?Val?Thr

130????????????????135??????????????????140

Gly?Gly?Val?Phe?Lys?Val?Gly?Asn?Phe?Thr?Gly?Leu?Tyr?Asn?Leu?Pro

145?????????????????150?????????????????155?????????????????160

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu

165?????????????????170?????????????????175

Tyr?Lys?Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys?Gly?Ala?Ala?Leu?Val

180?????????????????185?????????????????190

Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Asn?Ser?Ala?Ile?Cys?Ser?Val?Val

195?????????????????200?????????????????205

Arg?Arg?Lys?Ala?Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys

210?????????????????215?????????????????220

Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Ala?His?Pro?Ala?Ala?Met

225?????????????????230?????????????????235?????????????????240

Pro?His?Leu?Leu?Lys?Ala?Ala?Ala?Asp?Leu?Leu?Asp?Thr?Ala?Ser?Ala

245?????????????????250?????????????????255

Leu?Tyr?Asn?Ala?Ala?Ala?Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro

260?????????????????265?????????????????270

Phe?Asn?Ala?Ala?Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu?Lys?Leu

275?????????????????280?????????????????285

Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Lys?Ala?Leu?Ser?Leu?Asp

290?????????????????295?????????????????300

Val?Ser?Ala?Ala?Phe?Tyr?Gly?Ala?Ala?Glu?Tyr?Leu?Val?Ser?Phe?Gly

305?????????????????310?????????????????315?????????????????320

Val?Trp?Gly?Ala?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

325?????????????????330

<210>465

<211>1002

<212>DNA

＜213〉hepatitis type B virus

<400>465

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgaa?cttcctgctg????180

tccctgggaa?tccacctgta?tatggatgac?gtggtgctgg?gagtgggact?gtccaggtac????240

gtggctaggc?tgttcctgct?gaccagaatc?ctgaccatct?ccaccctgcc?agagaccacc????300

gtggtgagga?ggcaggcctt?cacctttagc?cctacctata?agggagccgc?tgcctggctg????360

agcctgctgg?tgccctttgt?gaatatccct?atccctagct?cctgggcttt?caagacccca????420

gccagggtga?ccggaggagt?gtttaaggtg?ggaaacttca?ccggcctgta?taacctgccc????480

agcgatttct?ttcctagcgt?gaagaccctg?tggaaggccg?gaatcctgta?caagaatgtg????540

tccatccctt?ggacccacaa?gggagccgct?ctggtggtgg?acttttccca?gttcagcaga????600

aatagcgcca?tctgttcggt?cgtgagaagg?aaagcctgga?tgatgtggta?ctggggtcct????660

agtctgtata?agaagtacac?ctcattccca?tggctcttga?atgcccatcc?cgctgcaatg?????720

ccacacctgc?ttaaagctgc?ggcggatctg?ctggacacag?cctcagcttt?atataatgct?????780

gcagcaagat?tctcctggtt?gtctctctta?gtgcccttca?acgcagcttc?ctggccaaaa?????840

tttgccgttc?cgaacctgaa?gctcactttt?ggaagagaga?cagtacttga?atacaaagca?????900

ctaagccttg?acgtgtcagc?agccttctac?ggagcagcag?aatatctagt?atcttttggg?????960

gtctggggcg?cagccctcat?gcctctatac?gcctgcattt?ga???????????????????????1002

<210>466

<211>333

<212>PRT

＜213〉hepatitis type B virus

<400>466

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr

20??????????????????25??????????????????30

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Phe?Leu

35??????????????????40??????????????????45

Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Asn?Phe?Leu?Leu?Ser?Leu?Gly?Ile

50??????????????????55??????????????????60

His?Leu?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Gly?Leu?Ser?Arg?Tyr

65??????????????????70??????????????????75??????????????????80

Val?Ala?Arg?Leu?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Ser?Thr?Leu

85??????????????????90??????????????????95

Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

100?????????????????105?????????????????110

Tyr?Lys?Gly?Ala?Ala?Ala?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Asn

115?????????????????120?????????????????125

Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Thr?Pro?Ala?Arg?Val?Thr

130?????????????????135?????????????????140

Gly?Gly?Val?Phe?Lys?Val?Gly?Asn?Phe?Thr?Gly?Leu?Tyr?Asn?Leu?Pro

145?????????????????150?????????????????155?????????????????160

Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu

165?????????????????170?????????????????175

Tyr?Lys?Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys?Gly?Ala?Ala?Leu?Val

180?????????????????185?????????????????190

Val?Asp?Phe?Ser?Gln?Phe?Ser?Arg?Asn?Ser?Ala?Ile?Cys?Ser?Val?Val

195?????????????????200?????????????????205

Arg?Arg?Lys?Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Gly?Leu?Ser?Leu

210?????????????????215?????????????????220

Asp?Val?Ser?Ala?Ala?Phe?Tyr?Asn?Ala?Ala?Ala?Lys?Tyr?Thr?Ser?Phe

225?????????????????230?????????????????235?????????????????240

Pro?Trp?Leu?Leu?Asn?Ala?His?Pro?Ala?Ala?Met?Pro?His?Leu?Leu?Lys

245?????????????????250?????????????????255

Ala?Ala?Ala?Asp?Leu?Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr?Asn?Ser?Trp

260?????????????????265?????????????????270

Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu?Lys?Leu?Thr?Phe?Gly?Arg?Glu?Thr

275?????????????????280?????????????????285

Val?Leu?Glu?Tyr?Lys?Ala?Ala?Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser

290?????????????????295?????????????????300

Leu?Tyr?Lys?Ala?Ala?Ala?Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro

305?????????????????310?????????????????315?????????????????320

Phe?Gly?Ala?Ala?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile

325?????????????????330

<210>467

<211>1002

<212>DNA

＜213〉hepatitis type B virus

<400>467

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggacaca?ccctgtggaa?ggccggaatc?ctgtataagg?ccaagttcgt?ggctgcctgg????120

accctgaagg?ctgccgcttt?cctgcctagc?gatttctttc?ctagcgtgaa?cttcctgctg????180

tccctgggaa?tccacctgta?tatggatgac?gtggtgctgg?gagtgggact?gtccaggtac????240

gtggctaggc?tgttcctgct?gaccagaatc?ctgaccatct?ccaccctgcc?agagaccacc????300

gtggtgagga?ggcaggcctt?cacctttagc?cctacctata?agggagccgc?tgcctggctg????360

agcctgctgg?tgccctttgt?gaatatccct?atccctagct?cctgggcttt?caagacccca????420

gccagggtga?ccggaggagt?gtttaaggtg?ggaaacttca?ccggcctgta?taacctgccc????480

agcgatttct?ttcctagcgt?gaagaccctg?tggaaggccg?gaatcctgta?caagaatgtg????540

tccatccctt?ggacccacaa?gggagccgct?ctggtggtgg?acttttccca?gttcagcaga????600

aattcagcaa?tttgttcggt?ggtgagaaga?aaggaatatc?ttgtttcatt?tggcgtctgg????660

gggctgtcac?tggatgtaag?tgcggcattt?tacaatgccg?ccgcaaaata?tacaagcttc????720

ccatggctcc?taaacgcaca?cccagctgca?atgccgcatc?tactgaaagc?agccgctgac????780

ctcttagaca?ctgcctccgc?tctgtacaac?tcttggccca?agtttgccgt?gcctaatctc????840

aagttgacct?tcggtagaga?gacagtctta?gaatacaaag?cggcctggat?gatgtggtac????900

tggggaccct?ctctgtataa?agccgctgca?aggttctcct?ggcttagcct?tctcgtacca????960

ttcggagcag?ctgccctaat?gcctttgtac?gcatgcatct?ga??????????????????????1002

<210>468

<211>295

<212>PRT

＜213〉hepatitis type B virus

<400>468

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu

20??????????????????25??????????????????30

Lys?Ala?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala

35??????????????????40??????????????????45

Ala?Lys?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Lys?His?Pro

50??????????????????55??????????????????60

Ala?Ala?Met?Pro?His?Leu?Leu?Lys?Ala?Ala?Ala?His?Thr?Leu?Trp?Lys

65??????????????????70??????????????????75??????????????????80

Ala?Gly?Ile?Leu?Tyr?Lys?Lys?Ala?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr

85??????????????????90??????????????????95

Ile?Gly?Ala?Leu?Ser?Leu?Asp?Val?Ser?Ala?Ala?Phe?Tyr?Asn?Ala?Ala

100?????????????????105?????????????????110

Ala?Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Ala?Ala?Ala?Arg?Phe

115?????????????????120?????????????????125

Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala?Thr?Pro?Ala?Arg

130?????????????????135?????????????????140

Val?Thr?Gly?Gly?Val?Phe?Lys?Ala?Ala?Glu?Tyr?Leu?Val?Ser?Phe?Gly

145?????????????????150?????????????????155?????????????????160

Val?Trp?Gly?Ala?Ala?Ala?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Asn

165?????????????????170?????????????????175

Asp?Leu?Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr?Asn?Ala?Ala?Ala?Phe?Pro

180?????????????????185?????????????????190

His?Cys?Leu?Ala?Phe?Ser?Tyr?Met?Lys?Ala?Ala?Ala?Trp?Met?Met?Trp

195?????????????????200?????????????????205

Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Ala?Ser?Ala?Ile?Cys?Ser?Val

210?????????????????215?????????????????220

Val?Arg?Arg?Lys?Asn?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu?Asn?Ile

225?????????????????230?????????????????235?????????????????240

Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Ala?Ala?Trp?Leu?Ser?Leu?Leu

245?????????????????250?????????????????255

Val?Pro?Phe?Val?Asn?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val

260?????????????????265?????????????????270

Lys?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Lys?Gln?Ala?Phe

275?????????????????280?????????????????285

Thr?Phe?Ser?Pro?Thr?Tyr?Lys

290?????????????????295

<210>469

<211>888

<212>DNA

＜213〉hepatitis type B virus

<400>469

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggatctt?ggcctaaatt?cgcagtgcca?aaccttaaag?ccgcggctgc?taagttcgta????120

gctgcctgga?cactaaaggc?cgccgctaag?agcacactgc?cagagaccac?cgtggtccgg????180

cgaaagcatc?cagccgcaat?gccccacttg?ctcaaagcag?ccgcccacac?tctttggaag????240

gctgggatat?tgtacaagaa?agccttcctt?ctgaccagga?tattaactat?cggagctctg????300

tcactcgacg?tttctgctgc?cttctacaac?gcggcggcaa?aatacactag?ctttccatgg????360

ctactcaacg?cagccgccag?attttcttgg?ctatcactac?tggtgccatt?taatgcagca????420

acacctgcta?gagtgactgg?cggcgtcttt?aaagcagccg?agtacttggt?gagctttggc????480

gtctggggtg?cagcggcata?tatggatgat?gtagtgttag?gggtgaacga?cctcctggac????540

acagccagtg?cgctgtacaa?tgcagctgca?ttcccgcatt?gcctagcctt?cagttatatg????600

aaagcagcag?cctggatgat?gtggtactgg?ggaccgtccc?tttataaagc?agcttcagca????660

atctgttccg?ttgtgaggag?aaaaaacttt?ttactctccc?tcggtattca?cctgaacatt????720

cccatccctt?cctcatgggc?attcaaagcc?gcttggctga?gtctactcgt?acctttcgtt????780

aatgcatttc?tgcccagcga?ctttttcccc?tcggtaaaac?tgacattcgg?acgcgaaaca????840

gtccttgaat?ataagcaggc?cttcacgttc?tcaccaacct?ataaatga?????????????????888

<210>470

<211>296

<212>PRT

＜213〉hepatitis type B virus

<400>470

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Asn

20??????????????????25??????????????????30

Ala?Ala?Ala?Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Asn?Asp?Leu?Leu

35??????????????????40??????????????????45

Asp?Thr?Ala?Ser?Ala?Leu?Tyr?Gly?Ala?Ala?His?Thr?Leu?Trp?Lys?Ala

50??????????????????55??????????????????60

Gly?Ile?Leu?Tyr?Lys?Lys?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser

65??????????????????70??????????????????75??????????????????80

Val?Lys?Ala?Phe?Pro?His?Cys?Leu?Ala?Phe?Ser?Tyr?Met?Lys?Ala?Ala

85??????????????????90??????????????????95

Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala?Ser?Trp

100?????????????????105?????????????????110

Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu?Lys?Ala?Ala?Ala?Gln?Ala?Phe?Thr

115?????????????????120?????????????????125

Phe?Ser?Pro?Thr?Tyr?Lys?Asn?Ala?Ala?Ala?Ser?Ala?Ile?Cys?Ser?Val

130?????????????????135?????????????????140

Val?Arg?Arg?Lys?Ala?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Asn?Ile

145?????????????????150?????????????????155?????????????????160

Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Ala?Ala?Trp?Met?Met?Trp?Tyr

165?????????????????170?????????????????175

Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Ala?Ala?Thr?Pro?Ala?Arg?Val?Thr

180?????????????????185?????????????????190

Gly?Gly?Val?Phe?Lys?Ala?Ala?Asn?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His

195?????????????????200?????????????????205

Leu?Asn?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Lys?His?Pro

210?????????????????215?????????????????220

Ala?Ala?Met?Pro?His?Leu?Leu?Lys?Ala?Ala?Ser?Thr?Leu?Pro?Glu?Thr

225?????????????????230?????????????????235?????????????????240

Thr?Val?Val?Arg?Arg?Lys?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val?Asn

245?????????????????250?????????????????255

Ala?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala

260?????????????????265?????????????????270

Lys?Leu?Ser?Leu?Asp?Val?Ser?Ala?Ala?Phe?Tyr?Asn?Ala?Ala?Ala?Lys

275?????????????????280?????????????????285

Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu

290?????????????????295

<210>471

<211>891

<212>DNA

＜213〉hepatitis type B virus

<400>471

atgggaatgc?aggtgcagat?ccagagcctg?tttctgctcc?tcctgtgggt?gcccgggtcc?????60

agaggataca?tggatgacgt?tgtgttaggc?gttaatgcag?ccgcagaata?tctcgtgtca????120

ttcggcgtct?ggaacgacct?gttggacact?gcatctgctc?tgtacggtgc?agcccatacc????180

ctgtggaagg?ccggaatcct?ctacaaaaag?gcattcctac?ctagcgactt?ttttccttca????240

gtgaaagcct?tcccacattg?cctagcattc?tcgtatatga?aagcggctag?gttctcatgg????300

cttagtcttc?tagtaccttt?caatgccgcc?tcctggccca?aattcgccgt?accaaatcta????360

aaagcggccg?cgcaggcctt?tacattctct?ccgacttata?aaaatgcagc?agcctccgct????420

atttgtagcg?tcgtgcgccg?aaaggccttc?ctgctaaccc?ggattttgac?gataaacatc????480

cccatccctt?ctagctgggc?tttcaaagca?gcatggatga?tgtggtactg?gggtcccagc????540

ttatacaaag?ctgcggcaac?cccagcaaga?gtgacagggg?gcgtgtttaa?ggccgccaac????600

ttcctcctga?gtctcggaat?acacctgaac?ttaacctttg?ggagagagac?agtactggag????660

tataaacacc?cagcagctat?gccgcaccta?ctcaaagccg?cttcaacact?cccagaaaca????720

actgtagtga?ggagaaaatg?gctctccctg?cttgtcccat?ttgtcaacgc?cgccgccgct????780

aagtttgtgg?ccgcttggac?acttaaggct?gcagcaaagt?tgtcacttga?tgttagtgca????840

gcgttctata?acgcagctgc?aaaatacact?tcctttccct?ggctgctgtg?a?????????????891

<210>472

<211>403

<212>PRT

＜213〉hepatitis type B virus

<400>472

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Asn

20??????????????????25??????????????????30

Ala?Ala?Ala?Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu?Lys?Ala?Ala

35??????????????????40??????????????????45

Ala?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Lys?Ala?Asp?Leu

50??????????????????55??????????????????60

Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr?Asn?Gln?Ala?Phe?Thr?Phe?Ser?Pro

65??????????????????70??????????????????75??????????????????80

Thr?Tyr?Lys?Gly?Ala?Ala?Ala?Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys

85??????????????????90??????????????????95

Gly?Ala?Ala?Ala?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu?Asn?Ile?Pro

100?????????????????105?????????????????110

Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Ala?Ala?Ala?Leu?Trp?Phe?His?Ile

115?????????????????120?????????????????125

Ser?Cys?Leu?Thr?Phe?Lys?Ala?Ala?Ala?Ile?Leu?Leu?Leu?Cys?Leu?Ile

130?????????????????135?????????????????140

Phe?Leu?Leu?Asn?Ala?Ala?Ala?Tyr?Pro?Ala?Leu?Met?Pro?Leu?Tyr?Ala

145?????????????????150?????????????????155?????????????????160

Cys?Ile?Asn?Ala?His?Pro?Ala?Ala?Met?Pro?His?Leu?Leu?Lys?Ala?Ala

165?????????????????170?????????????????175

Ala?Ser?Phe?Cys?Gly?Ser?Pro?Tyr?Lys?Ala?Ala?Gly?Leu?Ser?Arg?Tyr

180?????????????????185?????????????????190

Val?Ala?Arg?Leu?Asn?Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Phe

195?????????????????200?????????????????205

Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Phe?Pro?His?Cys?Leu

210?????????????????215?????????????????220

Ala?Phe?Ser?Tyr?Met?Lys?Ala?Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp

225?????????????????230?????????????????235?????????????????240

Asn?Ala?Ala?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Lys?Ala

245?????????????????250?????????????????255

Ala?Ala?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Ala?Tyr?Met?Asp

260?????????????????265?????????????????270

Asp?Val?Val?Leu?Gly?Val?Asn?Leu?Val?Val?Asp?Phe?Ser?Gln?Phe?Ser

275?????????????????280?????????????????285

Arg?Asn?Ala?Ala?Ala?Arg?Trp?Met?Cys?Leu?Arg?Arg?Phe?Ile?Ile?Asn

290?????????????????295?????????????????300

Ala?Ala?Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala

305?????????????????310?????????????????315?????????????????320

Thr?Pro?Ala?Arg?Val?Thr?Gly?Gly?Val?Phe?Lys?Ala?Ala?Trp?Leu?Ser

325?????????????????330?????????????????335

Leu?Leu?Val?Pro?Phe?Val?Asn?Ser?Ala?Ile?Cys?Ser?Val?Val?Arg?Arg

340?????????????????345?????????????????350

Lys?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Trp

355?????????????????360?????????????????365

Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Ala?Ser?Thr?Leu

370?????????????????375?????????????????380

Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Lys?Leu?Ser?Leu?Asp?Val?Ser?Ala

385?????????????????390?????????????????395?????????????????400

Ala?Phe?Tyr

<210>473

<211>1215

<212>DNA

＜213〉hepatitis type B virus

<400>473

atgggaatgc?aggtccagat?acagagcttg?ttcctcctcc?tgctttgggt?ccccggatca??????60

aggggtttcc?tcctaacccg?catcctgaca?attaacgccg?cagcctcctg?gccaaaattt?????120

gccgtgccaa?atctcaaggc?agctgcacac?acactatgga?aagcagggat?actgtacaag?????180

aaagccgatc?tgctagacac?agcgtctgcg?ttgtacaacc?aggcttttac?tttctctcct?????240

acatataaag?gcgcagctgc?aaacgtgagt?atcccttgga?cgcacaaagg?agccgctgcc?????300

aacttcttac?tgtccctggg?catccatcta?aatatcccta?ttccttcatc?ctgggcattt?????360

aaagcagccg?ccttatggtt?ccacataagt?tgtctgacct?tcaaagccgc?agcaatcctg?????420

ctcctttgcc?tcattttctt?actaaacgcc?gctgcctatc?cagctcttat?gccattgtac?????480

gcatgtatca?acgcccaccc?cgcagcaatg?ccccacctcc?ttaaagctgc?cgccagtttc?????540

tgcggttctc?cttataaagc?agcagggctg?tccagatacg?tagctaggct?aaacaagtat?????600

accagcttcc?cctggttact?taatttcctg?ccgtcagatt?tctttccatc?agttaaggcc?????660

ttccctcatt?gtctggcctt?tagctacatg?aaggctgaat?atttggtatc?cttcggcgtg?????720

tggaatgcgg?cactgacatt?tggaagggag?acagtgctcg?agtacaaagc?cgccgcacta?????780

ccctcggact?tcttcccatc?ggtcaaagct?tacatggacg?atgtagtcct?cggcgttaac?????840

ttagtagtgg?acttttctca?attttccaga?aacgcagcgg?ccagatggat?gtgccttcgg?????900

cgttttataa?taaacgccgc?tcgattcagc?tggctatcac?tcctagttcc?atttaatgca?????960

gctacacccg?cacgggtgac?aggtggagtt?ttcaaggcag?cgtggctttc?actgcttgtg????1020

ccatttgtga?actcagctat?ttgctcagta?gtgagaagga?aggcaaaatt?cgtcgctgcc????1080

tggactctca?aagctgccgc?aaagtggatg?atgtggtatt?ggggaccgag?cttgtacaaa????1140

gcggcctcta?ctctgccaga?aactaccgta?gtgagaagaa?aactgagcct?ggacgtcagc????1200

gcggcattct?actga?????????????????????????????????????????????????????1215

<210>474

<211>403

<212>PRT

＜213〉hepatitis type B virus

<400>474

Met?Gly?Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp

1???????????????5???????????????????10??????????????????15

Val?Pro?Gly?Ser?Arg?Gly?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu?Asn

20??????????????????25??????????????????30

Ala?Ala?Ala?Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Ala?Ala?Ala

35??????????????????40??????????????????45

Arg?Phe?Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala?Phe?Pro

50??????????????????55??????????????????60

His?Cys?Leu?Ala?Phe?Ser?Tyr?Met?Lys?Ala?Ala?Leu?Val?Val?Asp?Phe

65??????????????????70??????????????????75??????????????????80

Ser?Gln?Phe?Ser?Arg?Gly?Ala?Ile?Leu?Leu?Leu?Cys?Leu?Ile?Phe?Leu

85??????????????????90??????????????????95

Leu?Asn?Ala?Ala?Ala?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys

100?????????????????105?????????????????110

Lys?Ala?Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Tyr

115?????????????????120?????????????????125

Pro?Ala?Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile?Gly?Ala?Ala?Ala?Trp?Leu

130?????????????????135?????????????????140

Ser?Leu?Leu?Val?Pro?Phe?Val?Asn?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr

145?????????????????150?????????????????155?????????????????160

Ile?Asn?Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Ala?Ala?Ala?G1u

165?????????????????170?????????????????175

Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Asn?Leu?Pro?Ser?Asp?Phe?Phe?Pro

180?????????????????185?????????????????190

Ser?Val?Lys?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Lys?Asp?Leu

195?????????????????200?????????????????205

Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr?Asn?Ser?Trp?Pro?Lys?Phe?Ala?Val

210?????????????????215?????????????????220

Pro?Asn?Leu?Lys?Ala?Ala?Ala?Ser?Ala?Ile?Cys?Ser?Val?Val?Arg?Arg

225?????????????????230?????????????????235?????????????????240

Lys?Leu?Ser?Leu?Asp?Val?Ser?Ala?Ala?Phe?Tyr?Asn?Ala?Ala?Ala?Lys

245?????????????????250?????????????????255

Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala?Lys?Ala?Ala?Asn?Val

260?????????????????265?????????????????270

Ser?Ile?Pro?Trp?Thr?His?Lys?Gly?Ala?Ala?Gly?Leu?Ser?Arg?Tyr?Val

275?????????????????280?????????????????285

Ala?Arg?Leu?Asn?Ala?Ala?Ala?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val

290?????????????????295?????????????????300

Arg?Arg?Lys?His?Pro?Ala?Ala?Met?Pro?His?Leu?Leu?Lys?Ala?Ala?Ala

305?????????????????310?????????????????315?????????????????320

Arg?Trp?Met?Cys?Leu?Arg?Arg?Phe?Ile?Ile?Asn?Ala?Ser?Phe?Cys?Gly

325?????????????????330?????????????????335

Ser?Pro?Tyr?Lys?Ala?Ala?Tyr?Met?Asp?Asp?Val?Val?Leu?Gly?Val?Asn

340?????????????????345?????????????????350

Ala?Leu?Trp?Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Lys?Ala?Ala?Ala?Thr

355?????????????????360?????????????????365

Pro?Ala?Arg?Val?Thr?Gly?Gly?Val?Phe?Lys?Ala?Ala?Ala?Leu?Thr?Phe

370?????????????????375?????????????????380

Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro

385?????????????????390?????????????????395?????????????????400

Thr?Tyr?Lys

<210>475

<211>1212

<212>DNA

＜213〉hepatitis type B virus

<400>475

atgggaatgc?aggtgcaaat?acagtctctc?ttccttttgc?ttctctgggt?tccaggatca?????60

cggggcttct?tgcttagctt?gggcatccac?ctaaatgctg?ctgcaaaata?cacatctttt????120

ccttggctcc?ttaatgccgc?cgctaggttt?tcatggctga?gtctgctagt?acctttcaat????180

gcggctttcc?cacattgcct?agcttttagc?tatatgaaag?ctgctttagt?cgtggacttt????240

tcacagttta?gcagaggagc?aatcctgctg?ctatgtctga?tattccttct?aaacgcagca????300

gcccacacac?tctggaaagc?tggtatcctt?tacaagaaag?cctggatgat?gtggtattgg????360

ggacccagcc?tctacaaagc?ataccctgcc?ctgatgccac?tatacgcatg?cattggcgcg????420

gcagcctggt?tatccctttt?agtaccgttt?gtcaactttc?tattaaccag?aatcctgacg????480

attaatattc?cgatcccaag?ttcctgggca?ttcaaagcag?ccgcggagta?tctggtttca????540

tttggcgtat?ggaacctgcc?aagcgacttc?tttccttctg?ttaagttcct?cccctccgat????600

ttctttccat?cggtgaaaga?cctccttgat?accgcgagcg?ctctgtacaa?ctcgtggcca????660

aaattcgcag?ttccaaacct?aaaagccgcc?gccagtgcca?tttgttccgt?ggtaaggaga????720

aaattatcac?tcgacgtgtc?cgcagcattt?tataacgctg?ctgcaaagtt?tgtcgcagca?????780

tggacattga?aggctgcagc?gaaagcagca?aatgtatcaa?taccctggac?ccacaagggt?????840

gcagccgggc?tgtctaggta?tgtggcgagg?ctaaacgccg?ccgcctcaac?actgcctgag?????900

actactgtcg?tgagacgcaa?acaccctgcc?gcaatgcccc?acctgctgaa?agcagccgca?????960

cgatggatgt?gcctcagaag?attcataata?aacgcttctt?tctgtgggtc?accctacaaa????1020

gccgcttaca?tggacgatgt?ggtcctcgga?gtgaatgccc?tctggttcca?tatcagctgc????1080

ctgacattca?aggcagccgc?cacccccgct?cgtgtgacag?gaggtgtctt?caaagccgcg????1140

gcactgactt?tcggtcggga?aactgtattg?gaatataagc?aggccttcac?attctcccca????1200

acatacaagt?ga????????????????????????????????????????????????????????1212

<210>476

<211>410

<212>PRT

＜213〉hepatitis type B virus

<400>476

Met?Gln?Val?Gln?Ile?Gln?Ser?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Val?Pro

1???????????????5???????????????????10??????????????????15

Gly?Ser?Arg?Gly?Phe?Leu?Leu?Ser?Leu?Gly?Ile?His?Leu?Asn?Ala?Ala

20??????????????????25??????????????????30

Ala?Lys?Tyr?Thr?Ser?Phe?Pro?Trp?Leu?Leu?Asn?Ala?Ala?Ala?Arg?Phe

35??????????????????40??????????????????45

Ser?Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Asn?Ala?Ala?Phe?Pro?His?Cys

50??????????????????55??????????????????60

Leu?Ala?Phe?Ser?Tyr?Met?Lys?Ala?Ala?Leu?Val?Val?Asp?Phe?Ser?Gln

65??????????????????70??????????????????75??????????????????80

Phe?Ser?Arg?Gly?Ala?Ile?Leu?Leu?Leu?Cys?Leu?Ile?Phe?Leu?Leu?Asn

85??????????????????90???????????????????95

Ala?Ala?Ala?His?Thr?Leu?Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Lys?Ala

100?????????????????105?????????????????110

Trp?Met?Met?Trp?Tyr?Trp?Gly?Pro?Ser?Leu?Tyr?Lys?Ala?Tyr?Pro?Ala

115?????????????????120?????????????????125

Leu?Met?Pro?Leu?Tyr?Ala?Cys?Ile?Gly?Ala?Ala?Ala?Trp?Leu?Ser?Leu

130?????????????????135?????????????????140

Leu?Val?Pro?Phe?Val?Asn?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Asn

145?????????????????150?????????????????155?????????????????160

Ala?Ala?Ala?Ile?Pro?Ile?Pro?Ser?Ser?Trp?Ala?Phe?Lys?Ala?Ala?Ala

165?????????????????170?????????????????175

Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp?Asn?Leu?Pro?Ser?Asp?Phe?Phe

180????????????????185?????????????????190

Pro?Ser?Val?Lys?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser

195?????????????????200?????????????????205

Val?Lys?Ala?Ala?Ala?Asp?Leu?Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr?Asn

210?????????????????215?????????????????220

Ser?Trp?Pro?Lys?Phe?Ala?Val?Pro?Asn?Leu?Lys?Ala?Ala?Ala?Ser?Ala

225?????????????????230?????????????????235?????????????????240

Ile?Cys?Ser?Val?Val?Arg?Arg?Lys?Leu?Ser?Leu?Asp?Val?Ser?Ala?Ala

245?????????????????250?????????????????255

Phe?Tyr?Asn?Ala?Ala?Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala

260?????????????????265?????????????????270

Ala?Ala?Lys?Ala?Ala?Asn?Val?Ser?Ile?Pro?Trp?Thr?His?Lys?Gly?Ala

275?????????????????280?????????????????285

Ala?Gly?Leu?Ser?Arg?Tyr?Val?Ala?Arg?Leu?Asn?Ala?Ala?Ala?Ser?Thr

290?????????????????295?????????????????300

Leu?Pro?Glu?Thr?Thr?Val?Val?Arg?Arg?Lys?His?Pro?Ala?Ala?Met?Pro

305?????????????????310?????????????????315?????????????????320

His?Leu?Leu?Lys?Ala?Ala?Ala?Arg?Trp?Met?Cys?Leu?Arg?Arg?Phe?Ile

325?????????????????330?????????????????335

Ile?Asn?Ala?Ser?Phe?Cys?Gly?Ser?Pro?Tyr?Lys?Ala?Ala?Tyr?Met?Asp

340?????????????????345?????????????????350

Asp?Val?Val?Leu?Gly?Val?Asn?Ala?Leu?Trp?Phe?His?Ile?Ser?Cys?Leu

355?????????????????360?????????????????365

Thr?Phe?Lys?Ala?Ala?Ala?Thr?Pro?Ala?Arg?Val?Thr?Gly?Gly?Val?Phe

370?????????????????375?????????????????380

Lys?Ala?Ala?Ala?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Leu?Glu?Tyr?Lys

385?????????????????390?????????????????395?????????????????400

Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr?Tyr?Lys

405?????????????????410

<210>477

<211>1239

<212>DNA

＜213〉hepatitis type B virus

<400>477

atgggaatgc?aggtgcaaat?acagtctctc?ttccttttgc?ttctctgggt?tccaggatca?????60

cggggcttct?tgcttagctt?gggcatccac?ctaaatgctg?ctgcaaaata?cacatctttt????120

ccttggctcc?ttaatgccgc?cgctaggttt?tcatggctga?gtctgctagt?acctttcaat????180

gcggctttcc?cacattgcct?agcttttagc?tatatgaaag?ctgctttagt?cgtggacttt????240

tcacagttta?gcagaggagc?aatcctgctg?ctatgtctga?tattccttct?aaacgcagca????300

gcccacacac?tctggaaagc?tggtatcctt?tacaagaaag?cctggatgat?gtggtattgg????360

ggacccagcc?tctacaaagc?ataccctgcc?ctgatgccac?tatacgcatg?cattggcgcg?????420

gcagcctggt?tatccctttt?agtaccgttt?gtcaactttc?tattaaccag?aatcctgacg?????480

attaatgctg?ccgccattcc?gatcccaagt?tcctgggcat?tcaaagcagc?cgcggagtat?????540

ctggtttcat?ttggcgtatg?gaacctgcca?agcgacttct?ttccttctgt?taaggccgct?????600

gctttcctcc?cctccgattt?ctttccatcg?gtgaaagccg?ctgccgacct?ccttgatacc?????660

gcgagcgctc?tgtacaactc?gtggccaaaa?ttcgcagttc?caaacctaaa?agccgccgcc?????720

agtgccattt?gttccgtggt?aaggagaaaa?ttatcactcg?acgtgtccgc?agcattttat?????780

aacgctgctg?caaagtttgt?cgcagcatgg?acattgaagg?ctgcagcgaa?agcagcaaat?????840

gtatcaatac?cctggaccca?caagggtgca?gccgggctgt?ctaggtatgt?ggcgaggcta?????900

aacgccgccg?cctcaacact?gcctgagact?actgtcgtga?gacgcaaaca?ccctgccgca?????960

atgccccacc?tgctgaaagc?agccgcacga?tggatgtgcc?tcagaagatt?cataataaac????1020

gcttctttct?gtgggtcacc?ctacaaagcc?gcttacatgg?acgatgtggt?cctcggagtg????1080

aatgccctct?ggttccatat?cagctgcctg?acattcaagg?cagccgccac?ccccgctcgt????1140

gtgacaggag?gtgtcttcaa?agccgcggca?ctgactttcg?gtcgggaaac?tgtattggaa????1200

tataagcagg?ccttcacatt?ctccccaaca?tacaagtga???????????????????????????1239

<210>478

<211>344

<212>PRT

＜213〉hepatitis type B virus

<400>478

Met?Gly?Thr?Ser?Phe?Val?Tyr?Val?Pro?Ser?Ala?Leu?Asn?Pro?Ala?Asp

1???????????????5???????????????????10??????????????????15

Gly?Pro?Gly?Pro?Gly?Leu?Cys?Gln?Val?Phe?Ala?Asp?Ala?Thr?Pro?Thr

20??????????????????25??????????????????30

Gly?Trp?Gly?Leu?Gly?Pro?Gly?Pro?Gly?Arg?His?Tyr?Leu?His?Thr?Leu

35??????????????????40??????????????????45

Trp?Lys?Ala?Gly?Ile?Leu?Tyr?Lys?Gly?Pro?Gly?Pro?Gly?Pro?His?His

50??????????????????55??????????????????60

Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu?Leu?Met?Thr?Leu

65??????????????????70??????????????????75??????????????????80

Ala?Gly?Pro?Gly?Pro?Gly?Glu?Ser?Arg?Leu?Val?Val?Asp?Phe?Ser?Gln

85??????????????????90??????????????????95

Phe?Ser?Arg?Gly?Asn?Gly?Pro?Gly?Pro?Gly?Pro?Phe?Leu?Leu?Ala?Gln

100?????????????????105?????????????????110

Phe?Thr?Ser?Ala?Ile?Cys?Ser?Val?Val?Gly?Pro?Gly?Pro?Gly?Leu?Val

115?????????????????120?????????????????125

Pro?Phe?Val?Gln?Trp?Phe?Val?Gly?Leu?Ser?Pro?Thr?Val?Gly?Pro?Gly

130?????????????????135?????????????????140

Pro?Gly?Leu?His?Leu?Tyr?Ser?His?Pro?Ile?Ile?Leu?Gly?Phe?Arg?Lys

145?????????????????150?????????????????155?????????????????160

Ile?Gly?Pro?Gly?Pro?Gly?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Ser?Leu?Asp

165?????????????????170?????????????????175

Val?Ser?Ala?Ala?Phe?Gly?Pro?Gly?Pro?Gly?Leu?Gln?Ser?Leu?Thr?Asn

180?????????????????185?????????????????190

Leu?Leu?Ser?Ser?Asn?Leu?Ser?Trp?Leu?Gly?Pro?Gly?Pro?Gly?Ala?Gly

195?????????????????200?????????????????205

Phe?Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile?Pro?Gln?Ser?Gly?Pro?Gly

210?????????????????215?????????????????220

Pro?Gly?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg

225?????????????????230?????????????????235?????????????????240

Pro?Pro?Asn?Ala?Pro?Ile?Gly?Pro?Gly?Pro?Gly?Val?Gly?Pro?Leu?Thr

245?????????????????250?????????????????255

Val?Asn?Glu?Lys?Arg?Arg?Leu?Lys?Leu?Ile?Gly?Pro?Gly?Pro?Gly?Lys

260?????????????????265?????????????????270

Gln?Cys?Phe?Arg?Lys?Leu?Pro?Val?Asn?Arg?Pro?Ile?Asp?Trp?Gly?Pro

275?????????????????280?????????????????285

Gly?Pro?Gly?Ala?Ala?Asn?Trp?Ile?Leu?Arg?Gly?Thr?Ser?Phe?Val?Tyr

290?????????????????295?????????????????300

Val?Pro?Gly?Pro?Gly?Pro?Gly?Lys?Gln?Ala?Phe?Thr?Phe?Ser?Pro?Thr

305?????????????????310?????????????????315?????????????????320

Tyr?Lys?Ala?Phe?Leu?Cys?Gly?Pro?Gly?Pro?Gly?Ala?Lys?Phe?Val?Ala

325?????????????????330?????????????????335

Ala?Trp?Thr?Leu?Lys?Ala?Ala?Ala

340

<210>479

<211>1035

<212>DNA

＜213〉hepatitis type B virus

<400>479

atgggaactt?cttttgtgta?tgtcccttcc?gctctgaacc?cagcagacgg?acccgggcct?????60

ggcctgtgcc?aggtcttcgc?cgacgcaact?cccacagggt?gggggctggg?gccaggacca????120

ggcaggcact?acctgcatac?tctgtggaag?gcaggaatcc?tctataaagg?gcccggccca????180

ggccctcacc?acaccgccct?gaggcaggcc?atcctgtgct?ggggggagct?catgaccctg????240

gccggacctg?gacccgggga?gagcagactg?gtggtggatt?tcagccaatt?cagcagagga????300

aacggacccg?gccctgggcc?ttttctgctg?gctcagttta?catctgctat?ttgttctgtg????360

gtcggccccg?ggcccggact?cgtgcctttc?gtgcagtggt?tcgtgggact?gtcccctaca????420

gtcgggcccg?gcccagggct?gcatctgtac?tcccacccaa?tcatcctcgg?cttccgcaag?????480

attggacccg?gcccaggctc?cagcaatctc?tcctggctct?ctctggacgt?gtctgccgcc?????540

tttggccctg?gaccaggcct?gcaaagcctg?actaatctgc?tcagcagcaa?cctgtcctgg?????600

ctgggacctg?gcccaggggc?tggcttcttt?ctgctcaccc?ggattctcac?aattccccag?????660

tccggaccag?gaccaggagt?cagtttcggg?gtgtggatca?ggacccctcc?tgcttataga?????720

ccacccaatg?ctccaatcgg?ccccggccct?ggcgtcgggc?cactgaccgt?gaatgagaag?????780

cgccggctga?agctgatcgg?ccctggccct?ggcaagcagt?gctttcgcaa?actgcccgtg?????840

aacagaccta?ttgattgggg?ccccggccct?ggagcagcca?actggattct?caggggaaca?????900

agcttcgtct?acgtgcccgg?gcccggacca?gggaagcagg?cttttacctt?ctctcccact?????960

tacaaggcct?tcctctgtgg?gccaggcccc?ggcgccaagt?ttgtggcagc?atggaccctc????1020

aaagccgctg?cctga?????????????????????????????????????????????????????1035

Claims (17)

1. polynucleotides, it contains:

(a) multi-epitope constructs, it contains coding hepatitis type B virus (HBV) cytotoxic T lymphocyte (CTL) epi-position pol 562, and pol 745, env 332, and pol 530, and pol 388, env249, env 359, and pol 640, env 335, and env 183, and env 313, core 117, core 19, core 18, core 419, pol 392, and pol 531, pol 415, and pol 47, pol455, core 141, pol 429, and env 236, pol 166, and pol 538, core 101, the nucleic acid of pol354 and core 137 (being that each HBV CTL epi-position is made up of the correlated series in the table 7), wherein this nucleic acid is connected to each other in identical reading frame directly or indirectly;

(b) multi-epitope constructs in (a), it also contains the nucleic acid of coding HBV CTL epi-position pol 665 (i.e. 665 epi-positions of pol in the table 7), and this nucleic acid is connected to the CTL epi-position nucleic acid of (a) directly or indirectly in identical reading frame;

(c) multi-epitope constructs, it contains coding hepatitis type B virus (HBV) cytotoxic T lymphocyte (CTL) epi-position pol 149, core 18, pol 562, pol 538, and pol 455, env183, core 141, pol 665, and env 335, env 313, and pol 354, and pol 629, core 19, pol 150, and pol 47, pol 388, the nucleic acid of pol 531 and pol 642, and wherein this nucleic acid is connected to each other in identical reading frame directly or indirectly;

(d) (a) or (b) or multi-epitope constructs (c), it also contains one or more nucleic acid at interval, and this interval nucleic acid is connected to CTL epi-position nucleic acid directly or indirectly in identical reading frame;

(e) multi-epitope constructs (d), wherein these one or more at interval nucleic acid be positioned between the CTL epi-position nucleic acid of (a), between (a) and the CTL epi-position nucleic acid (b), (a) and (b) and (a), between the CTL epi-position nucleic acid of (c) or between the CTL epi-position nucleic acid (c);

(f) (d) or multi-epitope constructs (e), wherein this interval nucleic acid coding length is the amino acid sequence of 1～8 residue;

(g) (d) multi-epitope constructs of each in (f), wherein 2 or different (the being inequality) amino acid sequence of a plurality of intervals nucleic acid coding;

(h) (d) multi-epitope constructs of each in (g), wherein 2 or a plurality of intervals nucleic acid coding and other different amino acid sequence of amino acid sequence of nucleic acid coding at interval;

(i) (d) multi-epitope constructs of each in (h), wherein 2 or the identical amino acid sequence of a plurality of intervals nucleic acid coding;

(j) (d) multi-epitope constructs of each in (i), wherein one or more nucleic acid codings amino acid sequences of containing 3 continuous alanine (Ala) residues or forming at interval by 3 continuous alanine residues;

(k) (a) multi-epitope constructs of each in (j), it also contains the nucleic acid of one or more codings HTL epi-positions, and this nucleic acid is connected to CTL epi-position nucleic acid and/or nucleic acid at interval directly or indirectly in identical reading frame;

(l) multi-epitope constructs (k), wherein this HTL epi-position is PADRE ^_Epi-position;

(m) multi-epitope constructs (k), wherein this HTL epi-position is a HBV HTL epi-position;

(n) multi-epitope constructs (m), wherein this HBV HTL epi-position is selected from pol 774, pol694, pol 145, core 50, and pol 385, pol 523, and env 339, and pol 501, pol420, pol 412, and env 180, core 120, pol 96, and pol 618, pol 767 and pol 664 (being that each HBV HTL epi-position is made up of the correlated series in the table 11);

(o) (k) multi-epitope constructs of each in (n), it also contains one or more interval nucleic acid between CTL epi-position and HTL epi-position or between the HTL epi-position;

(p) (a) multi-epitope constructs of each in (o), it also contains one or more I class MHC and/or II class MHC target nucleic acid;

(q) multi-epitope constructs (p), wherein this target nucleic acid coding is selected from following target sequence: Ig κ burst, tissue plasminogen activator's burst, the insulin signaling sequence, the endoplasmic reticulum burst, LAMP-1 lysosome target sequence, LAMP-2 lysosome target sequence, HLA-DM lysosome target sequence, the HLA-DM-relating sequence of HLA-DO, Ig-α cytoplasm domain, Ig-β cytoplasm domain, Ii albumen, influenza stromatin, the HBV surface antigen, HBV cAg and yeast Ty albumen;

(r) (a) multi-epitope constructs of each in (q), it was optimized CTL and/or the processing of HTL epi-position;

(s) (a) multi-epitope constructs of each in (r), wherein this CTL nucleic acid minimizes so that CTL and/or HTL connect the quantity of epi-position through screening;

(t) (k) multi-epitope constructs of each in (s), wherein this HTL nucleic acid minimizes so that CTL and/or HTL connect the quantity of epi-position through screening;

(u) (a) multi-epitope constructs of each in (t), it contains the nucleic acid of one or more one or more flanking amino acid residues of encoding;

(the v) multi-epitope constructs of (u), wherein these one or more flanking amino acid residues are selected from: be positioned at the K of the C+1 position of CTL epi-position nucleic acid, R, N, Q, G, A, S, C and T;

(w) (a) to (multi-epitope constructs of each v), wherein this HBV CTL nucleic acid connects with the order shown in Figure 27 A;

(x) (n) multi-epitope constructs of each in (w), wherein this HBV HTL nucleic acid connects with the order shown in Figure 28 A;

(y) (c) to (multi-epitope constructs of each v) or (x), wherein this HBV CTL nucleic acid connects with order shown in Figure 34;

(z) (a) multi-epitope constructs of each in (x), its coding contain the amino acid sequence shown in Figure 24 B or the table 13,14,18 or 19 or by its peptide of forming;

(aa) multi-epitope constructs (z), it contains and is selected from following nucleotide sequence: the nucleotide of the nucleotide sequence in the table 13+1 is to 1248, the nucleotide of the nucleotide sequence in the table 14+1 is to 1032, nucleotide sequence among Figure 24 C, the nucleotide of the nucleotide sequence in the table 18+1 to 2292 and table 19 in nucleotide+1 of nucleotide sequence to 2232;

(bb) (c) to (multi-epitope constructs of each v) or (x) or (y) or (z), its coding contain the amino acid sequence shown in table 23 or 24 or by its peptide of forming;

(cc) multi-epitope constructs (bb), it contains and is selected from following nucleotide sequence: the nucleotide of the nucleotide sequence in the table 23+1 is to 618, or the nucleotide of the nucleotide sequence in the table 24+1 is to 657;

(dd) (a) multi-epitope constructs and one or more regulating and controlling sequences of each in (cc);

(ee) (a) multi-epitope constructs and one or more IRES of each in (dd);

(ff) (a) multi-epitope constructs and one or more promotors of each in (ee);

(gg) (a) multi-epitope constructs and one or more CMV promotors of each in (ff);

(hh) (a) multi-epitope constructs and 2 or a plurality of CMV promotor of each in (gg);

The (ii) multi-epitope constructs of each in (a) to (hh), and carrier;

(jj) multi-epitope constructs (ii), wherein this carrier is an expression vector;

(kk) (a) multi-epitope constructs of each in (jj), it has Figure 29 A (i), (ii) or the structure of the multi-epitope constructs (iii).

2. the polynucleotides that contain 2 multi-epitope constructs, the 1st contain above each HBV multi-epitope constructs in (a) to (kk), the 2nd contains HBV HTL epi-position, those in (n) for example, wherein the 1st is not directly to be connected with the 2nd multi-epitope constructs, and/or is not to be connected in the identical frame.

3. polynucleotides as claimed in claim 4, wherein the 1st and the 2nd multi-epitope constructs are operably connected to regulating and controlling sequence.

4. polynucleotides as claimed in claim 5, wherein the 1st and the 2nd multi-epitope constructs are operably connected at least 1 and are selected from following regulating and controlling sequence: at least 1 promotor and at least 1 IRES, 1 promotor and 1 IRES, 2 promotors, or 2 or a plurality of promotor and/or IRES.

5. polynucleotides as claimed in claim 5, wherein this promotor is the CMV promotor.

6. polynucleotides as claimed in claim 4, wherein these 2 multi-epitope constructs have Figure 29 A (i) or (ii) shown in structure.

7. polynucleotides as claimed in claim 4, wherein the 2nd multi-epitope constructs coding contains the amino acid sequence shown in Figure 24 C or the table 14 or by its peptide of forming.

8. polynucleotides as claimed in claim 4, the wherein nucleotide sequence of the 2nd multi-epitope constructs nucleotide+1 of containing the nucleotide sequence that is selected from Figure 24 C and the nucleotide sequence in the table 14 to 1032.

9. polynucleotides, it contains and is selected from following nucleotide sequence: the nucleotide of the nucleotide sequence in the table 13+1 is to 1248, the nucleotide of the nucleotide sequence in the table 18+1 is to 2292, the nucleotide of the nucleotide sequence in the table 19+1 is to 2232, the nucleotide of table 23+1 to 618 and nucleotide+1 of table 24 to 657.

10. carrier, it contains each the polynucleotides among the claim 1-9.

11. as the carrier of claim 10, it has Figure 29 A (i), (ii) or the carrier structure (iii).

12. polypeptide, it contains each the amino acid sequence of polynucleotide encoding among the claim 1-11.

13. as the polypeptide of claim 12, it contains and is selected from following amino acid sequence: the amino acid sequence shown in Figure 24 B or the table 13,14,18,19,23 or 24.

14. composition, it contains each polynucleotides or the carrier of claim 10 or 11 or polypeptide or its any combination of claim 12 or 13 among the claim 1-9.

15. cell, it contains each polynucleotides or the carrier of claim 10 or 11 or the polypeptide of claim 12 or 13 among the claim 1-9.

16. in the individuality of this method of needs, induce immunoreactive method at hepatitis type B virus (HBV), it comprise to described individuality use among the claim 1-9 each polynucleotides, claim 10 or 11 carrier, claim 12 or 13 polypeptide, the composition of claim 14 or the cell of claim 15.

17. the method for the cell of the polypeptide of the carrier of the polynucleotides of each among the preparation claim 1-9, claim 10 or 11, claim 12 or 13, the composition of claim 14 or claim 15.

Images