Polypeptides And Antibodies For Treating HBV Infection And Related Diseases

Abstract

The present invention relates to epitope peptides (or mutants thereof) for treating hepatitis B virus infection, recombinant proteins comprising such epitope peptides (or mutants thereof) and carrier proteins, and uses of such epitope peptides (or mutants thereof) and recombinant proteins. The present invention also relates to antibodies against such epitope peptides, cell lines producing said antibodies, and uses thereof. Furthermore, the present invention relates to vaccines or pharmaceutical compositions for treating or alleviating one or more symptoms associated with hepatitis B virus infection, which comprise the recombinant proteins or antibodies according to the invention, respectively.

Claims

1-26. (canceled)

27. A monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody is derived from one of the following monoclonal antibodies or is selected from the following antibodies: 1) the monoclonal antibody produced by the hybridoma cell line HBs-E6F6, wherein the hybridoma cell line HBs-E6F6 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201270; 2) the monoclonal antibody produced by the hybridoma cell line HBs-E7G11, wherein the hybridoma cell line HBs-E7G11 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201271; 3) the monoclonal antibody produced by the hybridoma cell line HBs-G12F5, wherein the hybridoma cell line HBs-G12F5 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201272; and 4) the monoclonal antibody produced by the monoclonal antibody produced by hybridoma cell line HBs-E13C5, wherein the hybridoma cell line HBs-E13C5 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201273.

28. The monoclonal antibody or antigen binding fragment of claim 27, wherein the monoclonal antibody has one or more of the following features: (1) the monoclonal antibody can specifically bind to positions 119 to 125 of HBsAg protein; (2) the monoclonal antibody or antigen binding fragment thereof is selected from a group consisting of Fab, Fab′, F(ab′)2, Fd, Fv, dAb, complementary determining region fragment, single chain antibody, mouse antibody, humanized antibody, chimeric antibody, or bispecific or poly-specific antibody; (3) the monoclonal antibody binds to HBsAg protein with a K.sub.D of less than about 10 M, or less than about 10.sup.−6 M, 10.sup.−7 M, 10.sup.−8 M, 10.sup.−9 M, 10.sup.−10 M or less; (4) the monoclonal antibody comprises non-CDR region, and the non-CDR region is from species other than murine species; and (5) the monoclonal antibody is capable of reducing serum level of HBV DNA and/or HBsAg in a subject.

29. The monoclonal antibody or antigen binding fragment of claim 28, wherein the single chain antibody is scFv, and/or, the chimeric antibody is a human-mouse chimeric antibody.

30. The monoclonal antibody or antigen binding fragment of claim 28, wherein the non-CDR region is from human antibody.

31. An isolated nucleic acid molecule, encoding the monoclonal antibody or antigen binding fragment of claim 27.

32. A vector, comprising the isolated nucleic acid molecule of claim 31.

33. A host cell, comprising the isolated nucleic acid molecule according to claim 31 or a vector comprising the isolated nucleic acid molecule.

34. A method for producing the monoclonal antibody or antigen binding fragment of claim 27, comprising 1) expressing an isolated nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment thereof in a host cell, to produce the monoclonal antibody or antigen binding fragment thereof; and 2) isolating the monoclonal antibody or antigen binding fragment thereof.

35. A hybridoma cell line, selected from: 1) hybridoma cell line HBs-E6F6, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201270; 2) hybridoma cell line HBs-E7G11, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201271; 3) hybridoma cell line HBs-G12F5, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201272; and 4) hybridoma cell line HBs-E13C5, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO.C201273.

36. A kit for detecting the presence or level of HBsAg protein in a sample or for diagnosing whether a subject is infected by HBV, comprising the monoclonal antibody or antigen binding fragment of claim 27.

37. The kit according to claim 36, wherein the monoclonal antibody or antigen binding fragment thereof further comprises a detectable marker.

38. The kit according to claim 37, wherein the detectable marker comprised in the monoclonal antibody or antigen binding fragment thereof is selected from a radioisotope, a fluorescent substance, a luminescent substance, a chromophoric substance and an enzyme.

39. The kit according to claim 36, wherein the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen binding fragment thereof; optionally, the second antibody further comprises a detectable marker.

40. The kit according to claim 39, wherein the detectable marker comprised in the second antibody is selected from a radioisotope, a fluorescent substance, a luminescent substance, a chromophoric substance and an enzyme.

41. A method for detecting the presence or level of HBsAg protein in a sample, comprising contacting the monoclonal antibody or antigen binding fragment of claim 27 with the sample, and detecting any binding between the monoclonal antibody or antigen binding fragment thereof and HBsAg protein.

42. A method for diagnosing whether a subject is infected by HBV, comprising (1) contacting the monoclonal antibody or antigen binding fragment of claim 27 with a sample from a subject that is suspected to be infected by HBV, and (2) detecting the presence of HBV in the sample by detecting any binding between the monoclonal antibody or antigen binding fragment thereof and HBsAg protein.

43. A pharmaceutical composition comprising the monoclonal antibody or antigen binding fragment of claim 27, and a pharmaceutically acceptable carrier and/or excipient.

44. A method for preventing or treating HBV infection or a disease associated with HBV infection in a subject, comprising administering a prophylactically or therapeutically effective amount of the monoclonal antibody or antigen binding fragment of claim 27 to a subject in need thereof.

45. The method according to claim 44, wherein the disease associated with HBV infection is hepatitis B.

46. A method for reducing serum level of HBV DNA and/or HBsAg in a subject, comprising administering to a subject infected with HBV and in need of reducing serum level of HBV DNA and/or HBsAg an effective amount of one of the following: (1) the monoclonal antibody or antigen binding fragment of claim 27; and (2) a monoclonal antibody or an antigen binding fragment thereof, capable of blocking the binding of HBsAg protein to the monoclonal antibody or antigen binding fragment thereof as defined in (1) by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

Description

DESCRIPTION OF THE DRAWINGS

[0125] FIGS. 1A-1H: Evaluation of efficacy of different mouse monoclonal antibodies in the treatment of HBV transgenic mice.

[0126] FIGS. 2A-2C: Evaluation of efficacy of HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5, 0.9% NS and entecavir (ETV) in the treatment of HBV transgenic mice. The values shown therein are the average values of 4 mice in each experimental group. FIG. 2A: Decrease in HBsAg level in serum after treating HBV transgenic mice with mouse monoclonal antibodies and entecavir (ETV), respectively; FIG. 2B: Decrease in HBV DNA level in serum after treating HBV transgenic mice with mouse monoclonal antibodies and entecavir (ETV), respectively; FIG. 2C: Changes in alanine aminotransferase (ALT) level in serum after treating HBV transgenic mice with mouse monoclonal antibodies and entecavir (ETV), respectively.

[0127] FIGS. 3A-3B: Dynamic changes in HBV DNA and HBsAg after the injection of HBs-E6F6. FIG. 3A: Decrease in HBsAg level in serum after injecting HBV transgenic mice with HBs-E6F6; FIG. 3B: Decrease in HBV DNA level in serum after injecting HBV transgenic mice with HBs-E6F6.

[0128] FIGS. 4A-4B: Evaluation of efficacy of chimeric antibodies in the treatment of HBV transgenic mice.

[0129] FIGS. 5A-5B: Construction, expression, purification and electron microscopic observation of 9 recombinant proteins. FIG. 5A: Scheme of construction of pC149-SEQ clone; FIG. 5B: Results of SDS-PAGE detection and electron microscopic observation of 9 recombinant proteins.

[0130] FIG. 6: Analysis on epitopes of HBs-E6F6, HBs-E7G11, HBs-G12F5, and HBs-E13C5.

[0131] FIGS. 7A-7B: Analysis on sensitivity of HBs-E6F6/HBs-E7G11 to the amino acid mutations of the epitope peptide SEQ1, wherein “Ref.” means that HBsAg is used as a reference antigen indicating antibody reactivity, “−” means that the reactivity is identical to that of HBsAg, “++” means that the reactivity is lower than that of HBsAg by 2 orders of magnitude (log.sub.10), “++++” means that the reactivity is lower than that of HBsAg by 4 orders of magnitude (log.sub.10).

[0132] FIGS. 8A-8B: The preparation of 5 recombinant proteins comprising epitope peptides and evaluation of their immunogenicity. FIG. 8A: Results of SDS-PAGE detection and electron microscopic observation of said 5 recombinant proteins. FIG. 8B: Changes in antibody titer in serum after immunizing BALB/C mice with said 5 recombinant proteins.

[0133] FIGS. 9A-9B: Evaluation of therapeutic effects of mouse blood-derived polyclonal antibodies.

[0134] FIGS. 10A-10D: Evaluation of effects of said 5 recombinant proteins in the treatment of HBV transgenic mice.

[0135] FIGS. 11A-11E: The preparation of 3 recombinant proteins comprising epitope peptides and evaluation of their therapeutic effects. FIG. 11A: Results of SDS-PAGE detection and electron microscopic observation of said 3 recombinant proteins. FIGS. 11B-11E: Changes in serum HBsAg level, serum HBV DNA level, Anti-HBsAg antibody level, and anti-carrier protein antibody level in mice after immunizing HBV transgenic mice with said 3 recombinant proteins.

[0136] FIG. 12: Illustration of CRM197-SEQ6, CRM389-SEQ6, CRMA-SEQ6 recombinant protein.

SEQUENCE INFORMATION

[0137] The information on sequences involved in the invention is provided in the following Table 1.

TABLE-US-00001 SEQ ID NO Name Sequence information  1 SEQ1 GPCKTCT  2 SEQ2 GPCRTCT  3 SEQ3 STTTSTGPCKTCTTP  4 SEQ4 TTSTGPCKTCT  5 SEQ5 CKTCTTPAQ  6 SEQ6 SSTTSTGPCKTCTTPAQGTSMFP  7 SEQ7 PGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCT  8 SEQ8 STTTSTGPC  9 SEQ9 STGPCKT 10 SEQ10 CKTC 11 SEQ11 TCTTPAQGNSMFPAQ 12 S1 MENIASGLLGPLLVL 13 S2 LGPLLVLQAGFFLLT 14 S3 AGFFLLTKILTIPQS 15 S4 ILTIPQSLDSWWTSL 16 S5 DSWWTSLNFLGGTPV 17 S6 FLGGTPVCLGQNSQS 18 S7 LGQNSQSQISSHSPT 19 S8 ISSHSPTCCPPICPG 20 S9 CPPICPGYRWMCLRR 21 S10 RWMCLRRFIIFLCIL 22 S11 IIFLCILLLCLIFLL 23 S12 LCLIFLLVLLDYQGM 24 S13 LLDYQGMLPVCPLIP 25 S14 PVCPLIPGSSTTSTG 26 S15 SSTTSTGPCKTCTTP 27 S16 CKTCTTPAQGTSMFP 28 S17 QGTSMFPSCCCTKPT 29 S18 CCCTKPTDGNCTCIP 30 S19 GNCTCIPIPSSWAFA 31 S20 PSSWAFAKYLWEWAS 32 S21 YLWEWASVRFSWLSL 33 S22 RFSWLSLLVPFVQWF 34 S23 VPFVQWFVGLSPTVW 35 S24 GLSPTVWLSVIWMMW 36 S25 SVIWMMWFWGPSLYN 37 S26 WGPSLYNILSPFMPL 38 S27 LSPFMPLLPIFFCLWVYI 39 HBsAg MENIASGLLGPLLVLQAGFFLLTKILTIPQSLDSWWTSLNFLGGTPVCLGQ NSQSQISSHSPTCCPPICPGYRWMCLRRFIIFLCILLLCLIFLLVLLDYQGM LPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCTCIPIPS SWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWFW GPSLYNILSPFMPLLPIFFCLWVYI 40 HBcAg MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH HTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQL LWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRR RGRSPRRRTPSPRRRRSQSPHRRRSQSRESQC 41 WHcAg MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP HHTTIRQALVCWDELTKLIAWMSSNITSEQVRTIIVNYVNDTWGLKVRQSL WFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVIRRR GGARASRSPRRRTPSPRRRRSQSPRRRRSQSPSANC 42 CRM197 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS 43 C149/mut MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSFEFGGGGS GGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWI RTPPAYRPPNAPILSTLPETTVV 44 C183/mut MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSFEFGGGGS GGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWI RTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPHRRR SQSRESQC 45 WHC149/ MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP mut HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSFEFGGGGS GGGGSRTIIVNYVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVW IRTPAPYRPPNAPILSTLPEHTVI 46 Linker GGGGSGGGGSGGGGS 47 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ1 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSGPCKTCTEF GGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVV 48 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ2 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSGPCRTCTEF GGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVV 49 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ3 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSTTTSTGPC KTCTTPEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGR ETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV 50 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ4 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSTTSTGPCKT CTEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVL EYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV 51 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ5 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSCKTCTTPAQ EFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEY LVSFGVWIRTPPAYRPPNAPILSTLPETTVV 52 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ6 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSSTTSTGPC KTCTTPAQGTSMFPEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWF HISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV 53 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ7 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSPGSSTTSTG PCKTCTTPAQGTSMFPSCCCTKPTDGNCTEFGGGGSGGGGSRELVVSY VNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPI LSTLPETTVV 54 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ8 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSTTTSTGPC EFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEY LVSFGVWIRTPPAYRPPNAPILSTLPETTVV 55 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ9 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSTGPCKTEF GGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVV 56 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ10 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSCKTCEFGG GGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSF GVWIRTPPAYRPPNAPILSTLPETTVV 57 C149- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ11 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSTCTTPAQG NSMFPAQEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFG RETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV 58 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ1 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSGPCKTCTEF GGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQS PHRRRSQSRESQC 59 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ2 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSGPCRTCTEF GGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQS PHRRRSQSRESQC 60 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ3 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSTTTSTGPC KTCTTPEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGR ETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPS PRRRRSQSPHRRRSQSRESQC 61 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ4 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSTTSTGPCKT CTEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVL EYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRR RSQSPHRRRSQSRESQC 62 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ5 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSCKTCTTPAQ EFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEY LVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRS QSPHRRRSQSRESQC 63 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ6 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSSSTTSTGPC KTCTTPAQGTSMFPEFGGGGSGGGGSRELVVSYVNVNMGLKIRQLLWF HISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGR SPRRRTPSPRRRRSQSPHRRRSQSRESQC 64 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ7 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSPGSSTTSTG PCKTCTTPAQGTSMFPSCCCTKPTDGNCTEFGGGGSGGGGSRELVVSY VNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPI LSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPHRRRSQSRESQC 65 C183- MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH SEQ10 HTALRQAILCWGELMNLATWVGSNLEDGGGGSGGGGTGSCKTCEFGG GGSGGGGSRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSF GVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSP HRRRSQSRESQC 66 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ1 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSGPCKTCTEF GGGGSGGGGSRTIIVNYVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLV SFGVWIRTPAPYRPPNAPILSTLPEHTVI 67 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ2 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSGPCRTCTEF GGGGSGGGGSRTIIVNYVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLV SFGVWIRTPAPYRPPNAPILSTLPEHTVI 68 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ3 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSSTTTSTGPC KTCTTPEFGGGGSGGGGSRTIIVNYVNDTWGLKVRQSLWFHLSCLTFGQ HTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVI 69 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ4 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSTTSTGPCKT CTEFGGGGSGGGGSRTIIVNYVNDTWGLKVRQSLWFHLSCLTFGQHTV QEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVI 70 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ5 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSCKTCTTPAQ EFGGGGSGGGGSRTIIVNYVNDTWGLKVRQSLWFHLSCLTFGQHTVQE FLVSFGVWIRTPAPYRPPNAPILSTLPEHTVI 71 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ6 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSSSTTSTGPC KTCTTPAQGTSMFPEFGGGGSGGGGSRTIIVNYVNDTWGLKVRQSLWF HLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAPILSTLPEHTVI 72 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ7 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSPGSSTTSTG PCKTCTTPAQGTSMFPSCCCTKPTDGNCTEFGGGGSGGGGSRTIIVNYV NDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFGVWIRTPAPYRPPNAP ILSTLPEHTVI 73 WHC149- MDIDPYKEFGSSYQLLNFLPLDFFPDLNALVDTATALYEEELTGREHCSP SEQ10 HHTTIRQALVCWDELTKLIAWMSSNITSGGGGSGGGGTGSCKTCEFGGG GSGGGGSRTIIVNYVNDTWGLKVRQSLWFHLSCLTFGQHTVQEFLVSFG VWIRTPAPYRPPNAPILSTLPEHTVI 74 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ1 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKWDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGSG PCKTCT 75 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ2 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGSG PCRTCT 76 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ3 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGSS TTTSTGPCKTCTTP 77 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ4 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKIKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGST TSTGPCKTCT 78 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ5 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGSC KTCTTPAQ 79 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ6 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGSS STTSTGPCKTCTTPAQGTSMFP 80 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ7 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGSP GSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCT 81 CRM197- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ10 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSN EISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSGGGGSGGGGSGGGGSC KTC 82 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ1 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSGPCKTCT 83 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ2 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSGPCRTCT 84 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ3 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSSTTTSTGPCKTCTTP 85 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ4 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSTTSTGPCKTCT 86 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ5 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSCKTCTTPAQ 87 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ6 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSSSTTSTGPCKTCTTPAQGTSMFP 88 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ7 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCT 89 CRM389- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ10 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGS SLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLE EFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLE KTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGEL VDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFGGGGSGGGGS GGGGSCKTC 90 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ1 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSGPCKTCT 91 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ2 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSGPCRTCT 92 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ3 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSSTTTSTGPCKTCTTP 93 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ4 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSTTSTGPCKTCT 94 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ5 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSCKTCTTPAQ 95 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ6 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSSSTTSTGPCKTCTTPAQGTSMFP 96 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ7 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCT 97 CRMA- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDD SEQ10 WKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDN AETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEY INNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRGGGGSGG GGSGGGGSCKTC

Description of Deposition of Biological Materials

[0138] The invention relates to the following biological materials deposited in China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China): [0139] 1) hybridoma cell line HBs-E6F6, with an deposition number of CCTCC NO.C201270, deposited on Jun. 7, 2012; [0140] 2) hybridoma cell line HBs-E7G11, with an deposition number of CCTCC NO.C201271, deposited on Jun. 7, 2012; [0141] 3) hybridoma cell line HBs-G12F5, with an deposition number of CCTCC NO.C201272, deposited on Jun. 7, 2012; and [0142] 4) hybridoma cell line HBs-E13C5, with an deposition number of CCTCC NO.C201273, deposited on Jun. 7, 2012.

Specific Modes for Carrying Out the Invention

[0143] The present invention is illustrated by reference to the following examples (which are used only for the purpose of illustration and are not intended to limit the protection scope of the present invention).

[0144] Unless indicated otherwise, the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes are used under the conditions recommended by manufacturers of the products. The reagents used in the present invention, whose manufacturers are not indicated, are conventional products in the art or commercially available. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the present invention.

Example 1: Preparation and Property Analysis of Mouse Monoclonal Antibodies

[0145] Purpose: obtainment of mouse monoclonal antibodies specific for HBsAg

[0146] 1.1 Preparation of Anti-HBsAg Mouse Monoclonal Antibodies

[0147] 1.1.1 Immunization of Mice

[0148] 1.1.1.1 Preparation of immunogen: immunogen was a recombinant HBV surface antigen protein expressed by CHO (HBsAg, purchased from BEIJING WANTAI BIOLOGY PHARMACY CO., LTD.). The recombinant protein was diluted to a concentration of 0.4 mg/mL, and was mixed with an equivalent volume of Freund's adjuvant, to form a water-in-oil emulsion (a method for determining whether the mixed solution was completely emulsified: a drop of the mixed solution was dropped on the liquid surface of clean water, if the mixed solution aggregated and was not dispersed, it can be believed that the solution was substantially mixed homogeneously). Freund's complete adjuvant was used for primary immunization, and Freund's incomplete adjuvant was used for the subsequent boost immunization, no adjuvant was added for the last boost immunization which was conducted 72 h before fusion.

[0149] 1.1.1.2 Fundamental immunization of mice: 6-8 week old BALB/c female mice were immunized by subcutaneous multi-point injection of said immunogen at an amount of 400 μL per mouse per time, and 200 μL venous blood of eyeball was collected before each immunization, for titer assay. A boost immunization was performed every two weeks. Indirect ELISA was used to determine serum titer, and when the serum titer of mice was in plateau phase, immunization of mice was stopped and the fusion was performed after resting for 2 months.

[0150] 1.1.1.3 Final boost 72 h before fusion: final boost of spleen was performed 72 h before fusion of mouse spleen cell and mouse myeloma cell, the immunogen for this boost comprised no adjuvants, injection of 100 μl 0.5 mg/mL recombinant protein was performed. Before immunization of spleen, mice were anaesthetized with ether, abdominal cavity was opened by cutting skin of abdominal wall to take spleen, the spleen was injected with 100 μL antigen vertically, and the cut on skin of abdominal wall was rapidly sutured surgically.

[0151] 1.1.2 Preparation and Screening of Fused Hybridomas

[0152] After final boost which was conducted 72 h before fusion, mouse spleen was taken and was prepared into cell suspension and was subjected to cell fusion with mouse myeloma cells Sp2/0 to obtain hybridoma cells. Previous to this, feeder cells were prepared. During the culture of hybridoma cells, a large number of myeloma cells and splenocytes died one after another in 1640-HAT culture medium after fusion, a single cell or a few scattered cells were not easy to survive, and other cells had to be added to make them survive. The living cells added were known as feeder cells. The laboratory used mouse peritoneal macrophages or thymocytes of 13-day old mice as feeder cells.

[0153] 1.1.2.1 Preparation of mouse macrophages comprised the following steps. (i) A 6-week old BALB/c mouse was killed by cervical dislocation. The mouse was washed with running water, and bathed in 75% ethanol solution for 5 min; the mouse was placed on a superclean bench, with abdomen upward; skin of the mouse abdomen was lifted with a pair of tweezers; a small hole was cut; the skin was tore upward and downward with a bigger pair of tweezers to ensure sufficient explosure of abdomen. (ii) A pair of aseptic ophthalmic tweezers were used to lift the peritoneum, a small hole was cut in the middle of the peritoneum with another pair of scissors, 1 mL pipette was used to inject a suitable amount of culture medium into the abdominal cavity via the hole, the solution was stirred carefully with the pipette in the abdominal cavity, and the culture medium was sucked out and put in a centrifugation tube. (iii) The cell solution from the abdominal cavity was dissolved in HAT culture medium or HT culture medium, to get macrophagous feeder cells at a concentration of 2×105 cells/mL. (iv) 0.1 mL the feeder cells was added to each well of a 96-well cell culture plate, and was cultured in an incubator; or was added to a 96-well cell culture plate after mixing with fusion cells.

[0154] 1.1.2.2 Preparation of mouse thymocytes comprised the following steps. (i) A 13-week old BALB/c mouse was killed by cervical dislocation. The mouse was washed with running water and bathed in 75% ethanol solution for 5 min; the mouse was placed on a superclean bench, with abdomen upward. (ii) Skin of the mouse abdomen was lifted with a pair of tweezers, and the outer skin of abdomen and chest was cut. (iii) Another pair of clean scissors was used to cut the thoracic cavity, ivory-white thymus gland was taken out with a pair of tweezers, after grinding, the resultant mixture passed through a 200-mesh cell sieve to get a thymic feeder cell solution.

[0155] 1.1.2.3 Preparation of mouse myeloma cells comprising the following steps. (i) Mouse myeloma cell line Sp2/0-Ag14 (Sp2/0) was the most ideal fusion cell now as the cell line was easy to culture and has a high fusion rate; however, Sp2/0 myeloma cell line was more sensitive to the culture conditions as compared to NS-1, and did not grow well when it was over-diluted (at a density of less than 3×105/mL) and at basic pH (pH higher than 7.3). (ii) Cells in logarithmic growth phase were chosen for fusion. (iii) Before fusion, myeloma cells were removed from culture flask to a centrifugation tube, and were washed with RPMI-1640 culture medium for three times (1000 rpm×5 min); the cells were re-suspended in RPMI-1640 culture medium, and the cells were counted. (iv) Generally, mouse myeloma cells were thawn 5 days before fusion, and about 6 bottles of 35 cm2 Sp2/0 cells were needed for each fusion.

[0156] 1.1.2.4 Preparation of immunological splenocyte comprised the following steps. (i) BALB/C mice to be fused were used, the eyeballs were removed and the mice bled to death, the collected blood was used to prepare antiserum, which was used as positive control for antibody detection. The mice were washed with running water and bathed in 75% ethanol solution for 5 min; and the mice was placed on a superclean bench, with right arm recumbent. (ii) Abdominal cavity was opened and spleen was taken out by aseptic operation, the spleen was cut into small pieces, and the small pieces were placed on a 200-mesh cell sieve and were squeezed and ground by a grinding rod (plunger) whilst adding RPMI-1640 culture medium dropwise with a blowpipe. (iii) A suitable amount of RPMI-1640 culture medium was added, and the mixture was kept standing for 3-5 min, the upper ⅔ of the suspension was removed to a 50 mL plastic centrifugation tube; the operation was repeated for 2-3 times. (iv) The cells were washed with RPMI-1640 culture medium for three times (1000 rpm×10 min). (v) The cells were re-suspended in RPMI-1640 culture medium, and the number of cells was counted.

[0157] 1.1.2.5 The preparation of hybridomas by fusion using PEG fusogen comprised the following steps. (i) Before fusion, 1 mL PEG-1500 and 10 mL RPMI-1640 serum-free culture medium and 200 mL complete medium were pre-heated to 37° C. (ii) The prepared myeloma cells and splenocytes were mixed in a 50 mL centrifuge tube (1×108 splenocytes+1×107 myeloma cells, about 10:1), and were centrifugated at 1500 rpm×8 min; after centrifugation, the tube was flicked at the bottom to make the cells loose and be paste. (iii) 1 mL suction pipet was used to remove 0.8 mL (1×108 splenocytes+0.8 mL PEG) to a centrifugation tube under slight stirring, and the addition of PEG was finished within 60 s, followed by the addition of 10 mL RPMI-1640 complete medium that was preheated to 37° C., under mild stirring. Finally, RPMI-1640 culture medium was added to 40 mL, and centrifugation at 1000 rpm×5 min was performed. (iv) The supernatant was discarded, and a few amount of HT culture medium was used to blow off the cells carefully, and the cells were removed to a prepared HT culture medium and were added to a 96-well cell culture plate, at 0.1 mL per well; and were cultured in a CO2 incubator. (v) After 12 h, a suitable amount of HAT complete medium was prepared, and 0.1 mL of the medium was added to each well; 5 days later, HT complete medium was used to replace 50-100% of the cell supernatant in wells; about 9-14 days later, the supernatant was taken for detection.

[0158] 1.1.2.6 Screening of hybridomas: by indirect ELISA screening, the plate was coated with the recombinant antigen HBsAg at 100 ng/well, 50 uL cell supernant was added, and positive clone wells were picked.

[0159] 1.1.2.7 Cloning of hybridoma cells: limiting dilution assay was used, cells were firstly diluted to a given concentration gradient, and then were seeded to each well of a 96-well cell culture plate, with one cell grew in each well as far as possible. Hybridoma monoclonal positive cell line generally had to be cloned repeatedly for 2-3 times, and was regarded as stable clone line until 100% positive was reached.

[0160] 1.1.3 Production of MAb Ascites

[0161] 2-3 BALB/c mice were used, and 0.5 mL saxol was injected to abdominal cavity. After 1 week, hybridoma cells in logarithmic growth phase were centrifugated at 1000 rpm for 5 min, and the supernatant was discarded. The hybridoma cells were suspended in serum-free culture medium, and the number of cells was adjusted to (1-2)×106/mL, and 0.5 mL of the suspension was injected to abdominal cavity of each mouse. 7-10 d later, mice were killed by cervical dislocation when the abdominal cavity was inflated obviously. The mice were washed with running water, embathed, and bathed in 75% ethanol for 5 min. The abdomen of the mouse was upward, and the four limbs were fixed onto a dissecting table with syringe needles. Skin of the mouse abdomen was lifted with a pair of tweezers, a small hole was cut, and then the skin was cut from both sides to dorsum of the mouse. The skin was tore upward and downward with a bigger pair of tweezers to ensure sufficient explosure of abdomen. A pair of aspectic ophthalmic tweezers was used to lift the peritoneum, a small hole was cut in the middle of the peritoneum, and then 1 mL pipette was used to take all the ascites from the abdominal cavity. The ascites collected was mixed and centrifugated in a centrifuge tube at 3000 rpm for 20 min. The supernatant was collected after centrifugation.

[0162] 1.1.4 Purification of MAb Ascites

[0163] After ammonium sulfate precipitation and Protein A affinity chromatography (purchased from US GE Co.), purified monoclonal antibodies were obtained.

[0164] 1.2 Analysis on Properties of Anti-HBsAg Mouse Monoclonal Antibodies

[0165] 1.2.1 Synthesis of Polypeptides

[0166] HBV sequence (GenBank ID: AAF24729.1) was used as reference sequence, and 27 polypeptides were synthesized (synthesized by XiaMen Jingju Biology Science Co., Ltd.). Said 27 polypeptides (S1-S27) together covered full-length 226 amino acids of HBsAg. Information on polypeptides S1-S27 was shown in Table 2. The full-length amino acid sequence of HBsAg was set forth in SEQ ID NO: 42.

TABLE-US-00002 TABLE 2 Information on polypeptides S1-S27 Name Amino acid position Amino acid sequence S1 HBsAg-aa1-aa15 MENIASGLLGPLLVL S2 HBsAg-aa9-aa23 LGPLLVLQAGFFLLT S3 HBsAg-aa17-aa31 AGFFLLTKILTIPQS S4 HBsAg-aa25-aa39 ILTIPQSLDSWWTSL S5 HBsAg-aa33-aa47 DSWWTSLNFLGGTPV S6 HBsAg-aa41-aa55 FLGGTPVCLGQNSQS S7 HBsAg-aa49-aa63 LGQNSQSQISSHSPT S8 HBsAg-aa57-aa71 ISSHSPTCCPPICPG S9 HBsAg-aa65-aa79 CPPICPGYRWMCLRR S10 HBsAg-aa73-aa87 RWMCLRRFIIFLCIL S11 HBsAg-aa81-aa95 IIFLCILLLCLIFLL S12 HBsAg-aa89-aa103 LCLIFLLVLLDYQGM S13 HBsAg-aa97-aa111 LLDYQGMLPVCPLIP S14 HBsAg-aa105-aa119 PVCPLIPGSSTTSTG S15 HBsAg-aa113-aa127 SSTTSTGPCKTCTTP S16 HBsAg-aa121-aa135 CKTCTTPAQGTSMFP S17 HBsAg-aa129-aa143 QGTSMFPSCCCTKPT S18 HBsAg-aa137-aa151 CCCTKPTDGNCTCIP S19 HBsAg-aa145-aa159 GNCTCIPIPSSWAFA S20 HBsAg-aa153-aa167 PSSWAFAKYLWEWAS S21 HBsAg-aa161-aa175 YLWEWASVRFSWLSL S22 HBsAg-aa169-aa183 RFSWLSLLVPFVQWF S23 HBsAg-aa177-aa191 VPFVQWFVGLSPTVW S24 HBsAg-aa185-aa199 GLSPTVWLSVIWMMW S25 HBsAg-aa193-aa207 SVIWMMWFWGPSLYN S26 HBsAg-aa201-aa215 WGPSLYNILSPFMPL S27 HBsAg-aa209-aa226 LSPFMPLLPIFFCLWVYI

[0167] 1.2.2 Assay on Reactivity of Anti-HBsAg Mouse Monoclonal Antibodies with Polypeptides S1-S27

[0168] (1.2.2.1) Preparation of Reaction Plates

[0169] The polypeptides were diluted with pH9.6 50 mM CB buffer (NaHCO3/Na2CO3 buffer, a final concentration of 50 mM, pH 9.6) to a final concentration of 1 μg/mL; to each well of a 96-well ELISA plate, 100 μL coating solution was added, the coating was performed at 2˜8° C. for 16˜24 h, and then was performed at 37° C. for 2 h. The plate was washed with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) once; 200 μL blocking solution (pH 7.4 20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer containing 20% fetal bovine serum and 1% casein) was then added to each well, and the blocking was performed at 37° C. for 2 h; the blocking solution was discarded. After drying, the plate was packaged in an aluminum foil bag and was stored at 2-8° C. for further use.

[0170] (1.2.2.2) ELISA of Anti-HBsAg Mouse Monoclonal Antibodies

[0171] 25 Anti-HBsAg mouse monoclonal antibodies obtained in 1.1 were diluted with PBS solution containing 20% newborn calf serum to a concentration of 1 μg/mL, for qualitative ELISA.

[0172] Sample reaction: 100 μL diluted sample was added to each well of 27 ELISA plates coated with polypeptides, and the plates were placed in an incubator at 37° C. for 30 min.

[0173] Enzyme labelling reaction: after sample reaction step was finished, the ELISA plate was washed with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for 5 times, 100 μL HRP-labelled goat anti-mouse IgG (GAM) (purchased from BEIJING WANTAI BIOLOGY PHARMACY CO., LTD) was added to each well, and the plate was placed in an incubator at 37° C. for 30 min.

[0174] Color development reaction: After the enzyme labelling reaction, the ELISA plate was washed with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for 5 times, 50 μL TMB colour developing reagent (purchased from BEIJING WANTAI BIOLOGY PHARMACY CO., LTD) was added to each well, and the plate was placed in an incubator at 37° C. for 15 min.

[0175] Stopping reaction and value readout: After the color development reaction step was finished, 50 μL stopping buffer (purchased from BEIJING WANTAI BIOLOGY PHARMACY CO., LTD) was added to each well of the ELISA plate, and OD450/630 value was readout with ELIASA for each well.

[0176] Determination of reactivity of Anti-HBsAg mouse monoclonal antibodies with 27 polypeptides: the reactivity was determined by the read values. If test value/background value was above 5, the sample was regarded as positive.

[0177] (1.2.2.3) Analysis of Recognization Properties of Anti-HBsAg Mouse Monoclonal Antibodies

[0178] The results were shown in Table 3. The types recognized by 25 Anti-HBsAg mouse monoclonal antibodies may be divided into 5 groups (depending on the recognization properties), i.e. sA, sB, sC, sD, sE, wherein antibodies of sA group recognized polypeptides S15 and S16; the antibodies of sB group recognized polypeptide S16; antibodies of group sC showed negative in the reaction with said 27 polypeptides; the antibodies of sD group recognized the polypeptide S18; and the antibodies of sE group recognized the polypeptide S8.

TABLE-US-00003 TABLE 3 Analysis of properities of Anti- HBsAg mouse monoclonal antibodies Recognized Antibody Antibody Group polypeptides name subtype sA S15, S16 HBs-E7G11 IgG1 sA S15, S16 HBs-G12F5 IgG1 sA S15, S16 HBs-E6F6 IgG1 sA S15, S16 HBs-E13C5 IgG1 sA S15, S16 HBs-3E9 IgG1 sA S15, S16 HBs-77D1 IgG2a sA S15, S16 HBs-86H6 IgG2b sA S15, S16 HBs-4D12 IgG2b sA S15, S16 HBs-32H10 IgG1 sA S15, S16 HBs-70A6 IgG1 sA S15, S16 HBs-6C10 IgM sA S15, S16 HBs-61B1 IgG1 sA S15, S16 HBs-37E12 IgG2b sA S15, S16 HBs-85D12 IgG1 sA S15, S16 HBs-H8D9 IgG1 sA S15, S16 HBs-E11E4 IgG2a sA S15, S16 HBs-83H12 IgG1 sB S16 HBs-127D7 IgG1 sC no HBs-2C1 IgG1 sC no HBs-S1A IgG2a sC no HBs-5F11 IgG2a sC no HBs-20A2 IgG2b sD S18 HBs-42B6 IgG1 sD S18 HBs-A13A2 IgG2b sE S8 HBs-45E9 IgG3

Example 2: Evaluation of Efficacy of Anti-HBsAg Mouse Monoclonal Antibodies in the Treatment of HBV Transgenic Mice

[0179] Purpose:

[0180] Evaluation of efficacy of Anti-HBsAg mouse monoclonal antibodies in the treatment of HBV transgenic mice

[0181] 2.1 Establishment of Denaturation-Chemiluminescence Quantitative Assay of HBsAg

[0182] After treatment, a large number of antibodies were present in serum, and therefore the determination of HBsAg might be disturbed by antigen-antibody complexes. Thus, it needs to establish a method for quantitative determination of HBsAg without interference from antibodies. The inventors had the antigen-antibody complexes lyzed in samples by denaturation method so as to exclude interference from antibodies and to carry out accurate quantitative assay of HBsAg.

[0183] 2.1.1 Preparation of Reaction Plates

[0184] The mouse monoclonal antibody HBs-45E9 was diluted with pH7.4 20 mM PB buffer (Na2HPO4/NaH2PO4 buffer, a final concentration of 50 mM, pH 7.4) to a final concentration of 2 μg/mL; to each well of a 96-well ELISA plate, 100 μL coating solution was added, the coating was performed at 2˜8° C. for 16˜24 h, and then at 37° C. for 2 h. The plate was washed with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) once; 200 μL blocking solution (pH 7.4 20 mM Na2HPO4/NaH2PO4 buffer containing 20% fetal bovine serum and 1% casein) was then added to each well, the blocking was performed at 37° C. for 2 h; and the blocking solution was discarded. After drying, the plate was packaged in an aluminum foil bag and was stored at 2-8° C. for further use.

[0185] 2.1.2 Denaturation-Chemiluminescence Quantitative Assay of HBsAg

[0186] Sample dilution: mouse serum was diluted with PBS solution containing 20% new-born calf serum to 2 gradient concentrations, i.e. 1:30 and 1:150.

[0187] Sample denaturation: 15 μL said diluted sample was mixed with 7.5 μL denaturation buffer (15% SDS, dissolved in 20 mM PB7.4), the mixed solution was incubated at 37° C. for 1 h, and 90 μL neutralization buffer (4% CHAPS, dissolved in 20 mM PB7.4) was then added to the mixed solution, and the resultant solution was mixed homogeneously.

[0188] Sample reaction: 100 μL said mixed solution sample obtained by the denaturation treatment was added to the reaction plate. The plate was placed in an incubator at 37° C. for 60 min.

[0189] Enzyme labelling reaction: after sample reaction step was finished, the chemiluminescent reaction plate was washed with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for 5 times, 100 μL HBs-A6A7-HRP solution (provided by BEIJING WANTAI BIOLOGY PHARMACY CO., LTD) was added to each well, and the plate was placed in an incubator at 37° C. for 60 min.

[0190] Luminous reaction and determination: after enzyme labelling reaction step was finished, the chemiluminescent reaction plate was washed with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for 5 times, Luminous solution (provided by BEIJING WANTAI BIOLOGY PHARMACY CO., LTD) was added for determining light intensity.

[0191] The obtainment of HBsAg concentration in a sample to be tested: standard substances were used for the same experiment, and standard curve was plotted with the results of the standard substances (linear regression of the light intensity values and concentration values); according to the standard curve, the HBsAg concentration in a sample to be tested was obtained by calculation.

[0192] 2.2 Real-Time Fluorescent Quantitative Assay of HBV DNA

[0193] Real-time fluorescent quantitative assay kit of HBV DNA was purchased from SHANGHAI KEHUA BIO-ENGINEERING CO., LTD., and real-time fluorescent quantitative assay of HBV DNA was conducted according to the instruction of the kit.

[0194] 2.3 Efficacy of Anti-HBsAg Mouse Monoclonal Antibodies in the Treatment of HBV Transgenic Mice

[0195] 25 antibodies obtained in Example 1 were injected to caudal vein of HBV transgenic mice at a single dose of 20 mg/kg. HBV transgenic mice injected with normal saline (0.9% NS) were used as negative control group. Each group included 4 HBV transgenic mice, two were male and the other two were female. Mouse blood was taken from periorbital venous plexus, and HBsAg and HBV DNA level in mouse serum were monitored.

[0196] The results are shown in FIGS. 1A-1H. The results indicated that after HBV transgenic mice were treated with five groups of Anti-HBsAg mouse monoclonal antibodies against different epitopes, antibodies of sA and sD groups showed the effect of significant viral clearance; HBsAg and HBV DNA level were significantly decreased in serum of mice from treatment group using the two groups of antibodies; After treatment with the other three groups of antibodies, HBsAg and HBV DNA level were not significantly decreased in mouse serum. Among the antibodies of sA and sD groups, HBsAg and HBV DNA level in serum were decreased to a larger extent after treatment with antibodies of sA group relative to the treatment with antibodies of sD group, and the four antibodies with the longest duration of inhibition belonged to antibodies of sA group, which were HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5, respectively.

Example 3: Efficacy and Side-Effect of Mouse Monoclonal Antibodies of sA Group in the Treatment of HBV Transgenic Mice

[0197] Purpose: evaluation of efficacy and side-effect of mouse monoclonal antibodies of sA group in the treatment of HBV transgenic mice, monitoring the duration of effective inhibition of viruses after treatment with a single dose of an antibody, and monitoring ALT.

[0198] Four antibodies HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5 having the best therapeutic effect, as screened in Example 2, were chosen for the experiment, and were injected to caudal vein of HBV transgenic mice at a single dose of 20 mg/kg. HBV transgenic mice treated with normal saline (0.9% NS) were used as negative control group, and HBV transgenic mice treated with 3.2 mg/kg/d entecavir (ETV) administrated by intragastric route were used as effective drug control group. Each group included 4 HBV transgenic mice, two were male and the other two were female. Mouse blood was taken from periorbital venous plexus, and HBsAg, HBV DNA, ALT level in mouse serum were monitored.

[0199] According to the methods described in Example 2, HBsAg and HBV DNA level were determined, and ALT was determined by alanine aminotransferase (ALT) assay kit provided by BEIJING WANTAI BIOLOGY PHARMACY CO., LTD.

[0200] The results of treating HBV transgenic mice with HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5, 0.9% NS or entecavir (ETV) are shown in FIGS. 2A-2C (the values showed were the average values of four mice of each experimental group). The results indicated that after treatment with single dose of monoclonal antibody HBs-E6F6, HBs-E7G11, HBs-G12F5 or HBs-E13C5, HBsAg and HBV DNA level were significantly decreased in serum of HBV transgenic mice, wherein the antibody treatment group was comparable to ETV treatment group with respect to the decrease in HBV DNA level. By contrast, HBsAg level did not significantly decrease in serum of mice of ETV treatment group, while HBsAg level decreased significantly in serum in antibody treatment group. In addition, during the treatment with any of the antibodies, no increase in ALT was observed.

Example 4: Dynamic Changes in HBV DNA and HBsAg after the Injection of HBs-E6F6

[0201] Purpose: study on the shortest time that monoclonal antibodies take to exert the maximal efficacy

[0202] The mouse monoclonal antibody HBs-E6F6 having the best effect was selected from four antibodies used in Example 3, and was injected to caudal vein of HBV transgenic mice at a single dose of 20 mg/kg. In the experiment, 4 male mice were used, and dynamic changes in HBV DNA and HBsAg in mouse serum were monitored.

[0203] According to the methods described in Example 2, HBsAg and HBV DNA level in serum were determined.

[0204] The results are shown in FIGS. 3A-3B. The results indicated that HBsAg and HBV DNA level in mouse serum decreased to the maximal inhibition level within 1 to 24 h after the injection.

Example 5: Evaluation of Therapeutic Effect of Human-Mouse Chimeric Antibody HBs-E6F6 and HBs-E7G11

[0205] Purpose: evaluation of therapeutic effect of chimeric antibodies

[0206] Igv gene of HBs-E6F6 and HBs-E7G11 antibody was linked to Igc gene encoding human antibody constant region, and the chimeric antibody HBs-E6F6 and the chimeric antibody HBs-E7G11 were obtained through recombinant expression in CHO cells and purification. The chimeric antibodies were injected to caudal vein of HBV transgenic mice at a single dose of 10 mg/kg. Dynamic changes in HBV DNA and HBsAg in mouse serum were monitored. The results are shown in FIGS. 4A-4B. The results indicated that both the chimeric antibody HBs-E6F6 and the chimeric antibody HBs-E7G11 can effectively eliminate HBsAg and HBV DNA in mice.

Example 6: Identification of Epitopes Recognized by Antibodies of sA Group

[0207] Purpose: Identification of epitopes recognized by antibodies of sA group and determination of the recognized core amino acid sequence

[0208] 6.1 Construction of pC149-SEQ Clone

[0209] When HBcAg was used as a carrier protein, full-length HBcAg protein or a fragment thereof (e.g., N-terminal aa 1-149 of HBcAg protein) might be used (see, Yang Haijie et al., Construction of Peptide Display Vector Based on HBV Core Protein, JOURNAL OF XIAMEN UNIVERSITY(NATURAL SCIENCE), 2004.05, Vol. 43, No. 4). In this experiment, a fragment of HBcAg protein (aa 1-149) was used as a carrier protein to construct a series of clones.

[0210] The sequence encoding HBcAg aa79-81 was deleted from the nucleotide sequence encoding a fragment of HBcAg protein (aa 1-149) by site-directed mutagenesis, two linkers were separately introduced to the two ends of the deletion, BamH I/EcoR I digestion site was designed between the two linkers, and thus the sequence encoding the carrier protein C149/mut (the amino acid sequence of C149/mut was set forth in SEQ ID NO: 43, with a structure of HBc (1-78)-G4SG4T-GS-G4SG4-HBc (82-149); in C149/mut, 3 amino acids (aa 79-81) of HBcAg were replaced with the flexible linker rich in Gly, G4SG4T-GS-G4SG4) was obtained. The sequence encoding C149/mut was cloned into pTO-T7 prokaryotic expression vector (Luo Wenxin, et al., Chinese Journal of Biotechnology, 2000, 16:53-57), to get the recombinant plasmid pC149/mut, which encoded the protein C149/mut. Later, by using BamH I/EcoR I digestion site, the sequence encoding the polypeptide of interest (represented by SEQ in FIG. 5A) was cloned between the flexible linkers to obtain the recombinant vector pC149-SEQ, which encoded the recombinant protein C149-SEQ (comprising the carrier protein C149/mut and the polypeptide of interest SEQ). The clone design and structure of the recombinant vector pC149-SEQ are shown in FIG. 5A.

[0211] The gene sequences of the 9 polypeptides shown in Table 4 were separately ligated to the recombinant plasmid pC149/mut, to obtain 9 pC149-SEQ recombinant vectors (pC149-SEQ1, 3, 4, 5, 6, 8, 9, 10, 11), which encoded the recombinant protein C149-SEQ1, 3, 4, 5, 6, 8, 9, 10, 11, respectively.

TABLE-US-00004 TABLE 4 Polypeptides of interest presented by C149/mut Poly- peptide Polypeptide name position Amino acid sequence SEQ1 HBsAg-aa119-aa125 GPCKTCT SEQ3 HBsAg-aa113-aa127 STTTSTGPCKTCTTP SEQ4 HBsAg-aa115-aa125 TTSTGPCKTCT SEQ5 HBsAg-aa121-aa129 CKTCTTPAQ SEQ6 HBsAg-aa113-aa135 STTTSTGPCKTCTTPAQGNSMFP SEQ8 HBsAg-aa113-aa121 STTTSTGPC SEQ9 HBsAg-aa117-aa123 STGPCKT SEQ10 HBsAg-aa121-aa124 CKTC SEQ11 HBsAg-aa123-aa137 TCTTPAQGNSMFPAQ

[0212] 6.2 Expression and Purification of C149-SEQ Proteins

[0213] Expression and purification of a recombinant protein were described by using C149-SEQ6 as an example.

[0214] (6.2.1) Obtainment of high-efficiency expression strains: according to the method described in 6.1, the vector of interest pC149-SEQ6 was constructed, after identification with DNA sequencing, the vector of interest was transformed into E. coli ER2566 strain (E. coli, ER2566), to get an expression strain.

[0215] (6.2.2) Expression of C149-SEQ6 protein: the expression strain was seeded to a triangular flask (500 mL), and was cultured in a shaking table at 37° C. until OD was about 1.0. Isopropyl β-D-Thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, the expression was induced under shaking at 25° C. for 6 h.

[0216] (6.2.3) Purification of C149-SEQ6 protein:

[0217] (6.2.3.1) Ultrasonication of bacteria: bacteria in 6.2.2 were collected by centrifugation; bacteria were subjected to ultrasonication, the ultrasonication buffer comprising the components: 20 mM phosphate buffer (PH6.0) +300 mM NaCl.

[0218] (6.2.3.2) Primary purification of proteins of interest: since proteins of interest were thermotolerant, the ultrasonated mixture was put in water bath at 65° C. for 30 min, and the supernatant was collected after centrifugation. The supernant was added to a saturated ammonium sulfate solution at a volume ratio of 1:1, and the precipitate was collected after centrifugation. An appropriate volume of buffer was added to resuspend the precipitate to get primarily purified proteins of interest, wherein the buffer comprised 20 mM phosphate buffer (pH=7.4)+150 mM NaCl.

[0219] (6.2.3.3) Chromatographic purification of proteins of interest: the proteins obtained in 6.2.3.2 were further purified by Sepharose 4FF (GE) molecular sieve column chromatography to obtain the purified proteins of interest. The purified proteins of interest were subjected to SDS-PAGE, and the assembly state of the particles of the proteins of interest was observed by transmission electron microscope (TEM).

[0220] FIG. 5B showed the SDS-PAGE and TEM results of said 9 recombinant proteins. The results indicated that said 9 recombinant proteins had a purity of above 95%, and could be assembled into protein particles of a uniform size.

[0221] 6.2 Evaluation of Reactivity of Said 9 Recombinant Proteins with Antibodies of sA Group

[0222] 6.2.1 Preparation of Reaction Plates

[0223] According to the method described in Example 1-1.2, reaction plates were prepared, and the coating antigens are said 9 recombinant proteins presenting polypeptides of interest.

[0224] 6.2.2 Determination of reactivity of HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5 with said recombinant proteins by ELISA

[0225] According to the method described in Example 1-1.2, the reactivity of HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5 with said recombinant proteins was determined.

[0226] 6.2.3 Analysis on epitopes recognized by HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5

[0227] The ELISA results in 6.2.2 are shown in FIG. 6. The results indicated that HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5 had good reactivity with the recombinant proteins presenting polypeptides SEQ1, SEQ3, SEQ4, SEQ5, SEQ6, SEQ10, but had no reactivity with the recombinant proteins presenting polypeptides SEQ8, SEQ9, SEQ11. The sequence analysis of these polypeptides showed that the common feature of the polypeptides SEQ1, SEQ3, SEQ4, SEQ5, SEQ6, SEQ10 lies in comprising HBsAg aa121-aa124, and the common feature of SEQ8, SEQ9, SEQ11 resided in not comprising an intact HBsAg aa121-124. Therefore, it could be concluded that HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5 recognized the same epitope, and the amino acid sequence of the shortest epitope recognized by them was HBsAg aa121-124, i.e. CKTC. ELISA results showed that the recombinant proteins C149-SEQ1 and 3-6 had comparable reactivity with antibodies, and had reactivity higher than C149-SEQ10. Therefore, the polypeptides SEQ1 and 3-6 were the preferred epitope peptides recognized by antibodies HBs-E6F6, HBs-E7G11, HBs-G12F5, HBs-E13C5. In addition, since the sequence of SEQ1 was shorter than SEQ3-6, SEQ1 was regarded as the preferred core epitope.

Example 7: Analysis on Sensitivity of HBs-E6F6 and HBs-E7G11 to the Amino Acid Mutations of the Epitope Peptide SEQ1

[0228] SEQ1 (GPCKTCT) was subjected to amino acid point-mutation, and 7 mutants were prepared. The amino acid sequences of said 7 mutant polypeptides were shown in Table 5. According to the method described in Example 6, recombinant proteins comprising the mutant polypeptides and C149/mut were prepared, and HBs-E6F6 and HBs-E7G11 were evaluated for their reactivity with said 7 mutant polypeptides.

TABLE-US-00005 TABLE 5 Amino acid sequences of mutant polypeptides Name Mutated amino acid Amino acid sequence M1 P120S GSCKTCT M2 P120T GTCKTCT M3 C121S GPSKTCT M4 K122R GPCRTCT M5 T123I GPCKICT M6 C124S GPCKTST M7 C121S/C124S GPSKTST SEQ1 HBsAg aa119-aa125 GPCKTCT

[0229] The results are shown in FIGS. 7A-7B. The results indicated that mutant M1 (P120S), mutant M2 (P120T), mutant M4 (K122R) were comparable to the epitope peptide SEQ1 with respect to the binding to antibody HBs-E6F6 and HBs-E7G11, while the binding of the other mutants to the antibodies was significantly decreased. It indicated that P120S, P120T, K122R mutation had no effect on the reactivity of HBs-E6F6 and HBs-E7G11 with the epitope SEQ1, while C121S, C124S, C121S/C124S, T123I mutation significantly decreased the reactivity of HBs-E6F6 and HBs-E7G11 with the epitope SEQ1.

Example 8: Preparation of Recombinant Proteins Comprising Epitope Peptides and Evaluation of their Immunogenicity

[0230] 8.1 Preparation of Recombinant Proteins Comprising Epitope Peptides

[0231] According to the method described in Example 6, C149/mut was used as carrier protein to present epitope peptides SEQ1, SEQ3, SEQ4, SEQ6, SEQ7, wherein SEQ3, SEQ4, SEQ6, SEQ7 were polypeptides obtained by extension of N and/or C-terminus of the preferred core epitope SEQ1 (i.e., comprising the core epitope SEQ1), from which 5 recombinant proteins (used as antigens for immunization) capable of forming nucleocapsid-like particles (CLP) were prepared.

[0232] As described in Example 6, 5 recombinant proteins C149-SEQ1, SEQ3, SEQ4, SEQ6, SEQ7 were prepared, and said 5 recombinant proteins were subjected to SDS-PAGE and were observed by transmission electron microscope. The results are shown in FIG. 8A. The results indicated that said 5 recombinant proteins had a purity of above 95%, and could be assembled into protein particles of a uniform size.

[0233] 8.2 Evaluation of Immunogenicity of Recombinant Proteins Comprising Epitope Peptides

[0234] 8.2.1 Immunization of Mice

[0235] BALB/C mice were immunized with said 5 recombinant proteins and the carrier protein C149/mut (as control), respectively. Immunoadjuvant was aluminium hydroxide adjuvant, the immune dose was 3 μg/dose, the immunization route was intramuscular injection of lateral hind thigh, and the immune procedure was as followed: a boost immunization was performed every 2 weeks after primary immunization; immunization was performed for four times.

[0236] 8.2.2 Determination of Anti-HBs Antibody Titer in Serum

[0237] 8.2.2.1 Preparation of Reaction Plate

[0238] According to the method described in Example 1-1.2, the reaction plate was prepared, the coating antigen was hepatitis B surface antigen protein (HBsAg) recombinantly expressed in CHO cells.

[0239] 8.2.2.2 ELISA of Anti-HBs Antibody Titer in Serum

[0240] Sample dilution: mouse serum was diluted with PBS solution containing 20% new-born calf serum to 7 gradient concentrations, i.e. 1:100, 1:500, 1:2500, 1:12500, 1:62500, 1:312500, 1:1562500.

[0241] Sample reaction: 100 μL diluted sample was added to each well of the coated reaction plate, and the plate was placed in an incubator at 37° C. for 30 min.

[0242] Enzyme labelling reaction: after sample reaction was finished, the ELISA plate was washed with PBST (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for 5 times, 100 μL GAM-HRP solution was added to each well, and the plate was placed in an incubator at 37° C. for 30 min.

[0243] Color development reaction: After the Enzyme labelling reaction, the ELISA plate was washed with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20) for 5 times, 50 μL TMB colour developing reagent (provided by BEIJING WANTAI BIOLOGY PHARMACY CO., LTD) was added to each well, and the plate was placed in an incubator at 37° C. for 15 min.

[0244] Stopping reaction and value readout: After the color development reaction step was finished, 50 μL stopping buffer (provided by BEIJING WANTAI BIOLOGY PHARMACY CO., LTD) was added to each well of the ELISA plate, and OD450/630 value was read with ELIASA for each well.

[0245] Calculation of Anti-HBsAg antibody titer in serum: a regression curve was plotted with dilution factors of samples with a readout value between 0.2 and 2.0 and the readout values, the dilution factor of the sample at which the readout value was the double of the background value was calculated, and the dilution factor was used as Anti-HBsAg antibody titer in serum.

[0246] 8.2.3 Determination of Anti-C149/Mut Antibody Titer in Serum

[0247] 8.2.3.1 Preparation of Reaction Plates

[0248] According to the method described in Example 1-1.2, reaction plates were prepared, and the antigen for coating was the fusion carrier protein C149/mut.

[0249] 8.2.3.2 Determination of Anti-C149/Mut Antibody Titer in Serum by ELISA

[0250] According to the method described in Example 8.2.2.2, sample dilution, sample reaction, enzyme labelling reaction, color development reaction, stopping reaction and value readout, were carried out, and Anti-C149/mut antibody titer in serum was calculated.

[0251] 8.2.4 Analysis of Immunogenicity of Recombinant Proteins Comprising Epitope Peptides

[0252] By carrying out said steps, Anti-HBsAg antibody titer and Anti-C149/mut antibody titer in serum were obtained. The results are shown in FIG. 8B. The results indicated that the recombinant proteins comprising the epitope peptides SEQ1, SEQ3, SEQ4, SEQ6, SEQ7 induced a high titer of Anti-HBsAg in BALB/C mice, while C149/mut alone could not induce a high titer of Anti-HBsAg.

Example 9: Evaluation of Therapeutic Effect of Mouse Blood-Derived Anti-SEQ6 Polyclonal Antibodies

[0253] 9.1 Preparation of Mouse Blood-Derived Anti-SEQ6 Polyclonal Antibodies

[0254] 9.1.1 Immunization of Mice

[0255] According to the method described in Example 8-8.2.1, BALB/C mice were immunized with an immunogen that was a recombinant protein comprising SEQ6 (C149-SEQ6).

[0256] 9.1.2 Purification of Mouse Blood-Derived Anti-SEQ6 Polyclonal Antibodies

[0257] After immune procedure was completed, titer of Anti-HBsAg antibody in serum of mice reached a high level, blood was taken from periorbital venous plexus for several times. After purification by ammonium sulfate precipitation and Protein A affinity chromatography, purified polyclonal antibodies were obtained.

[0258] 9.2 Evaluation of Therapeutic Effect of Mouse Blood-Derived Anti-SEQ6 Polyclonal Antibodies

[0259] Mouse blood-derived Anti-SEQ6 polyclonal antibodies were injected to caudal vein of HBV transgenic mice, changes in HBV DNA and HBsAg in serum were monitored. The results are shown in FIGS. 9A-9B. The results indicated that polyclonal antibodies, obtained by immunization of mice with C149-SEQ6, significantly decreased HBV DNA and HBsAg level in HBV transgenic mice, and were effective in clearing up HBV.

Example 10: Effect of Recombinant Proteins in the Treatment of HBV Transgenic Mice

[0260] 10.1 Immunization of Mice

[0261] HBV transgenic mouse model was used to evaluate the therapeutic effect of said 5 recombinant proteins (C149-SEQ1, C149-SEQ3, C149-SEQ4, C149-SEQ6, C149-SEQ7) obtained in Example 8, and the carrier protein C149/mut was used as control. Immunoadjuvant was aluminium hydroxide adjuvant, the immune dose was 12 μg/dose, the immunization route was intramuscular injection of lateral hind thigh, and the immune procedure was as following: a boost immunization was performed 2 weeks after primary immunization, followed by a boost immunization every week, i.e. immunization was performed at week 0, 2, 3, 4, 5, i.e. immunization was performed for five times.

[0262] 10.2 Determination of Antibody Titer in Serum

[0263] According to the methods described in Example 8-8.2.2 and 8.2.3, serum antibody titer of Anti-HBsAg and Anti-C149/mut was determined, and virological indexes, HBV DNA and HBsAg level in serum of mice were monitored.

[0264] 10.3 Analysis on Therapeutic Effect of the Recombinant Proteins

[0265] The results are shown in FIGS. 10A-10D. The results indicated that in groups receiving immunotherapy of recombinant proteins, Anti-HBsAg and Anti-C149/mut were detected in serum of mice, and HBV DNA and HBsAg level in serum of mice were decreased to different extents. By contrast, in control group, no Anti-HBsAg was produced in serum of mice, and HBV DNA and HBsAg level in serum did not decrease. The Example shows that the epitopes and epitope peptides identified by the invention are effective targets for treatment of HBV infection. The recombinant proteins produced based on these epitopes and epitope peptides have potential for treating chronic HBV infection. In particular, the recombinant proteins comprising the epitope peptide SEQ1-SEQ7 may be used as protein vaccines to change the immunotolerant state directed to HBV in HBV transgenic mice and induce effective, specific and therapeutic anti-HBV immune response.

Example 11: Construction and Evaluation of Recombinant Proteins Based on Different Carrier Proteins and SEQ6

[0266] 11.1 Construction of 3 Fusion Expression Vectors

[0267] According to the method described in Example 6, 3 carrier proteins were constructed, which were C149/mut (SEQ ID NO: 43), C183/mut (SEQ ID NO: 44), WHC149/mut (SEQ ID NO: 45), respectively. C149/mut was obtained by engineering 149 amino acid residues at N-terminus of HBV core protein, C183/mut was obtained by engineering 183 amino acid residues of full-length HBV core protein, and WHC149/mut was obtained by engineering 149 amino acid residues at N-terminus of woodchuck hepatitis virus core protein (the engineering method was described in 6.1). SEQ6 was ligated to the three vectors to get recombinant proteins C149-SEQ6, C183-SEQ6, and WHC149-SEQ6, respectively.

[0268] 11.2 Expression and Purification of 3 Different Carrier Recombinant SEQ6 Vaccines

[0269] According to the method described in 6.2, 3 recombinant proteins (C149-SEQ6, C183-SEQ6, WHC149-SEQ6) were expressed and purified. The proteins of interest obtained were subjected to SDS-PAGE, and assembly state of the protein particles was identified by transmission electron microscope. The results are shown in FIG. 11A. The results indicated that the 3 recombinant proteins obtained had a purity above 95%, and could be assembled into protein particles of a uniform size.

[0270] 11.3 Effects of Said 3 Recombinant Proteins in the Treatment of HBV Transgenic Mice

[0271] HBV transgenic mouse model was used to evaluate the therapeutic effect of said 3 recombinant proteins obtained in 11.2 as protein vaccines. Immunoadjuvant was aluminium hydroxide adjuvant, the immune dose was 12 μg/dose, and the immune procedure was as followed: a boost immunization was performed 2 weeks after primary immunization, followed by a boost immunization every week, i.e. immunization was performed at week 0, 2, 3, 4, 5, i.e. immunization was performed for five times.

[0272] According to the methods described in Example 8-8.2.2 and 8.2.3, serum antibody titer of Anti-HBsAg and anti-carrier was determined, and virological indexes, HBV DNA and HBsAg level in serum of mice were monitored.

[0273] The results are shown in FIGS. 10A-10D. The results indicated that in groups receiving immunotherapy of recombinant proteins, Anti-HBsAg and anti-carrier antibody were detected in serum of mice, and HBV DNA and HBsAg level in serum of mice reduced to different extents. By contrast, in control group, no Anti-HBsAg was produced in serum of mice, and HBV DNA and HBsAg level in serum did not reduce. The Example showed that different carrier proteins may be used to present the epitopes and epitope peptides identified by the invention, and the recombinant proteins produced therefrom had potential for treating chronic HBV infection. Such recombinant proteins may be used as protein vaccines to change the immunotolerant state directed to HBV in HBV transgenic mice and induced effective, specific and therapeutic anti-HBV immune response.

[0274] Similarly, based on C149/mut (SEQ ID NO: 43), C183/mut (SEQ ID NO: 44) or WHC149/mut (SEQ ID NO: 45), and SEQ1-5, 7 and 10, the recombinant proteins C149-SEQ1-5, 7, 10; C183-SEQ1-5, 7, 10; and WHC149-SEQ1-5, 7, 10 were also designed and constructed. The amino acid sequences of these recombinant proteins were shown in Table 1.

Example 12: Construction and Expression of Recombinant Proteins Based on CRM197 or Fragments Thereof

[0275] In the Example, a series of recombinant proteins were designed and constructed based on CRM197 or fragments thereof and SEQ6.

[0276] The amino acid sequence of CRM197 is set forth in SEQ ID NO: 42, which consists of 535 amino acids. An exemplary fragment of CRM197 is CRM 389, consisting of 389 amino acids at N-terminus of CRM197. Another exemplary fragment of CRM197 is CRM A, consisting of 190 amino acids at N-terminus of CRM197.

[0277] As shown in FIG. 12, SEQ6 was linked to C-terminus of CRM197, CRM389 or CRMA via a linker, wherein the amino acid sequence of the linker was GGGGSGGGGSGGGGS (SEQ ID NO: 46). The main function of the linker was to promote the relatively independent folding of the two peptides linked thereby to obtain a high biological activity. The recombinant proteins thus obtained were designated as CRM197-SEQ6, CRM389-SEQ6 and CRMA-SEQ6, respectively.

[0278] Genes of interest encoding CRM197-SEQ6, CRM389-SEQ6 and CRMA-SEQ6 were constructed, the genes of interest were separately ligated to pTO-T7 prokaryotic expression vector (Luo Wenxin et al., Chinese Journal of Biotechnology, 2000, 16:53-57), and were transformed into ER2566 bacteria; plasmids were extracted, positive expression clones comprising gene fragments of interest were obtained after identification by NdeI/SalI enzyme digestion.

[0279] Three recombinant proteins CRM197-SEQ6, CRM389-SEQ6 and CRMA-SEQ6 were expressed and purified according to the methods described in Example 6-6.2, and the therapeutic effect of said 3 recombinant proteins were evaluated by the methods described in Example 11.

[0280] Similarly, based on CRM197 or fragments thereof as well as SEQ1-5, 7 and 10, recombinant proteins CRM197-SEQ1-5, 7, 10; CRM389-SEQ1-5, 7, 10; and CRMA-SEQ1-5, 7, 10 were designed and constructed. The amino acid sequences of these recombinant proteins were shown in Table 1.

[0281] Although the specific embodiments of the invention have been described in details, those skilled in the art would understand that, according to all the disclosed teachings, various modifications and changes can be made without departing from the sprit or scope of the invention as generally described, and that such modifications and changes are within the scope of the present invention. The scope of the present invention is given by the appended claims and any equivalents thereof.