Abstract:
The present invention describes peptides and recombinant proteins containing Hepatitis C virus core protein sequence in which one or more of the amino acids have been modified or deleted to remove the ability of these proteins to bind to specific anti-HCV murine monoclonal antibodies. The deletions and modifications are designed as to maintain the ability of this protein to be used in immunoassays used for the detection of anti-HCV antibodies in individuals infected with HCV.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    An estimated 170 million people worldwide have been infected by hepatitis C virus (HCV). In the next few years, the number of U.S. deaths for HCV-caused liver disease and cancer may overtake deaths caused by Acquired Immune Deficiency Syndrome (AIDS).  
           [0002]    The transmission of HCV seems to require blood-to-blood contact. Carrying a single strand of ribonucleic acid (RNA), HCV contains just one gene, coding for a polyprotein that is subsequently cleaved into at least 10 functional proteins. Clearly, the ability to test the blood supply for HCV is of great importance. A sensitive assay that can detect infection at an early stage would be helpful.  
           [0003]    HCV detection assays typically detect antibodies against HCV virus. These antibodies are detected in immunoassays using recombinant proteins and peptides containing HCV protein sequences. Most commercial anti-HCV assays use proteins from the following regions, core protein, NS3, NS4, and NS5 protein sequences.  
           [0004]    Anti-HCV core antibodies are one of the most prevalent anti-HCV antibodies detected in chronic HCV infected individuals. HCV core protein contains multiple epitopes. Using synthetic peptides in the HCV core region, it has been shown that most of these epitopes are in the amino terminal end of this protein. For example, using overlapping peptides, each approximately 15 amino acids in length, an ELISA was developed to screen for anti-HCV antibodies in chronic HCV infected individuals. Table 1 below shows the peptide sequences. As shown in Table 2 below, HCV infected individuals have antibodies to two or more of these core peptides. Thus, it has been shown that the complete core peptide is not required to detect anti-core antibodies.  
                                                 TABLE 1                               HCV           Peptide   Amino Acid   Polyprotein       ID #   Sequence   AA Location                                0   MSTNPKPQKKNKRNT    1-15   SEQ ID NO.:1                   1   KNKRNTNRRPQDVKF   10-24   SEQ ID NO.:2               2   QDVKFPGGGQIVGGV   20-34   SEQ ID NO.:3               3   QIVGGVYLLPRRGPR   29-43   SEQ ID NO.:4               4   RRGPRLGVPATRKTS   39-53   SEQ ID NO.:5               5   ATRKTSERSQPRGRR   48-62   SEQ ID NO.:6               6   PRGRRQPIPKARRPE   58-72   SEQ ID NO.:7               7   KARRPEGRTWAQPGY   67-81   SEQ ID NO.:8               8   AQPGYPWPLYGNEGC   77-91   SEQ ID NO.:9               9   YGNEGCGWAGWLLSP    86-100   SEQ ID NO.:10               10   WLLSPRGSRPSWGPT    96-110   SEQ ID NO.:11               11   SWGPTDPRRRSRLNG   106-120   SEQ ID NO.:12               12   SRLNGKVIDTLTCGF   116-130   SEQ ID NO.:13               13   LTCGFADLMGYIPLV   126-140   SEQ ID NO.:14               14   YIPLVGAPLGGAARA   136-150   SEQ ID NO.:15               15   GAARALAHGVRVLED   146-160   SEQ ID NO.:16               16   RVLEDGVNYATGNLP   156-170   SEQ ID NO.:17               17   TGNLPGCSFSIFLLA   166-180   SEQ ID NO.:18                  
 
           [0005]    [0005]                                                                                                                                                                     TABLE 2                       Peptide   0   1   2   3   4   5   6   7   8   9   10   11   12   13   14   15   16   17                                Neg   0.044   0.004   0.021   0.075   0.059   0.069   0.076   0.103   0.020   0.034   0.002   0.088   0.051   0.028   0.023   0.034   0.041   0.042       Neg   0.045   0.065   0.017   0.081   0.051   0.081   0.103   0.111   0.033   0.035   0.002   0.085   0.038   0.030   0.023   0.030   0.038   0.052       Neg   0.050   0.003   0.014   0.075   0.087   0.079   0.094   0.102   0.025   0.034   0.002   0.071   0.038   0.033   0.021   0.031   0.039   0.047       Neg   0.014   0.043   0.134   0.023   0.034   0.039   0.034   0.039   0.012   0.018   −0.001   0.025   0.009   0.009   0.008   0.013   0.010   0.020       Neg   0.015   0.039   0.119   0.021   0.033   0.030   0.030   0.034   0.013   0.019   0.000   0.026   0.011   0.010   0.005   0.012   0.010   0.016       Neg   0.013   0.036   0.126   0.027   0.053   0.032   0.037   0.042   0.013   0.016   0.001   0.037   0.013   0.009   0.009   0.012   0.009   0.017       Neg   0.016   0.004   0.104   0.020   0.039   0.034   0.003   0.041   0.019   0.019   0.000   0.041   0.011   0.017   0.006   0.010   0.008   0.021        9   0.256   2.500   2.500   0.425   1.443   0.016   0.218   0.248   0.062   0.015   0.025   0.049   0.030   0.013   0.012   0.021   0.012   0.028       11   0.413   2.500   2.500   0.917   0.229   0.647   0.114   0.262   0.166   0.159   0.002   0.055   0.022   0.027   0.021   0.042   0.247   0.088       15   0.044   0.661   2.500   0.067   0.577   0.057   0.110   0.072   0.061   0.032   −0.003   0.045   0.018   0.031   0.020   0.039   0.035   0.080       18   1.643   0.887   2.500   0.641   0.100   0.052   0.032   0.576   0.037   0.022   −0.001   0.040   0.006   0.010   0.009   0.025   0.036   0.039       20   0.021   0.111   2.500   2.500   0.482   0.032   0.029   0.045   0.028   0.015   0.011   0.035   0.009   0.014   0.009   0.017   0.019   0.059       21   2.500   2.500   2.500   2.262   2.500   0.516   2.259   0.080   0.378   0.018   0.022   0.064   0.013   0.024   0.017   0.024   0.135   0.035       27   0.032   2.500   2.500   0.089   2.500   1.652   1.349   0.043   0.039   0.034   0.033   0.026   0.012   0.018   0.013   0.028   0.287   0.008       30   2.500   2.500   2.500   2.500   0.183   0.044   0.075   0.993   0.078   0.035   0.028   0.190   0.023   0.025   0.022   0.028   0.042   0.043       35   0.022   0.278   0.912   0.031   0.036   0.116   0.079   0.049   0.062   0.010   0.005   0.026   0.005   0.006   0.009   0.020   0.007   0.020       36   0.059   0.129   2.500   0.090   0.131   0.060   0.093   0.148   0.090   0.037   0.003   0.050   0.018   0.060   0.077   0.062   0.129   0.079       37   2.500   2.500   2.500   0.219   0.130   0.090   0.169   0.223   0.147   0.040   0.018   0.065   0.016   0.051   0.051   0.025   0.080   0.110       39   0.165   2.500   2.500   2.500   0.124   1.217   2.351   2.500   0.397   0.056   0.015   1.319   0.017   0.050   0.056   0.041   0.200   0.190       40   0.058   2.500   2.500   2.500   0.098   0.066   0.060   0.755   0.093   0.024   0.000   0.059   0.013   0.028   0.017   0.032   0.068   0.101       42   0.022   0.043   2.500   0.031   0.023   0.150   0.018   0.036   0.023   0.011   −0.001   0.032   0.005   0.014   0.006   0.010   0.012   0.031       43   0.158   0.612   0.368   0.033   0.065   0.103   0.069   0.075   0.053   0.051   0.001   0.055   0.025   0.022   0.015   0.023   0.029   0.035       44   0.042   2.240   2.500   0.511   0.070   0.060   0.086   0.301   0.108   0.066   −0.002   0.143   0.028   0.041   0.028   0.047   0.061   0.076       45   1.002   2.500   2.500   0.174   0.713   0.055   0.129   1.722   1.896   0.029   −0.003   0.069   0.038   0.038   0.055   0.022   0.136   0.039       46   0.016   0.031   2.500   0.022   0.021   0.021   0.038   0.035   0.031   0.027   −0.002   0.027   0.016   0.015   0.013   0.026   0.019   0.037       47   2.484   2.500   2.500   0.449   0.750   0.593   0.102   2.006   0.331   0.017   −0.004   0.034   0.012   0.014   0.007   0.026   0.014   0.032       48   0.427   0.201   2.500   0.364   0.356   0.038   0.049   0.070   0.027   0.029   −0.001   0.040   0.018   0.011   0.013   0.025   0.014   0.095       49   0.019   0.483   2.500   0.044   0.045   0.047   0.050   0.246   0.039   0.025   −0.005   0.033   0.019   0.014   0.013   0.030   0.016   0.085       50   0.668   2.500   2.500   0.568   1.233   0.277   0.154   2.500   0.882   0.024   −0.001   0.035   0.014   0.012   0.007   0.017   0.044   0.055       52   2.500   2.500   2.500   0.948   1.116   1.619   0.026   0.267   0.112   0.019   0.026   0.042   0.012   0.007   0.009   0.019   0.008   0.044       53   0.823   2.500   2.500   0.428   2.500   0.780   0.086   2.500   1.566   0.025   0.001   0.050   0.014   0.020   0.011   0.022   0.019   0.028       54   1.577   2.500   2.500   2.500   2.500   2.500   2.500   2.500   2.500   0.020   0.032   0.053   0.021   0.012   0.012   0.020   0.017   0.038       55   0.017   0.083   2.500   0.047   0.018   0.023   0.071   0.042   0.019   0.017   −0.002   0.026   0.015   0.015   0.012   0.026   0.014   0.030       58   2.500   2.500   2.500   2.283   2.500   2.500   2.500   2.500   0.810   0.037   0.073   0.067   0.018   0.019   0.021   0.025   0.159   0.059       59   0.015   1.805   2.500   0.023   2.500   0.019   0.180   0.038   0.070   0.039   0.027   0.017   0.012   0.015   0.018   0.015   0.023   0.049       60   0.053   2.500   2.500   0.255   2.500   0.102   1.266   0.666   2.500   0.034   −0.003   0.153   0.026   0.019   0.022   0.039   0.097   0.063       62   0.018   0.053   2.500   1.178   0.025   0.031   0.041   0.060   0.028   0.017   −0.002   0.039   0.013   0.012   0.010   0.017   0.010   0.048       63   0.009   0.016   0.027   0.012   0.017   0.013   1.225   0.028   0.012   0.017   −0.004   0.026   0.007   0.007   0.006   0.010   0.057   0.020       65   1.440   0.041   0.026   0.040   0.032   0.056   0.052   0.062   0.020   0.040   0.023   0.051   0.018   0.023   0.023   0.019   0.020   0.035       66   2.060   2.500   2.500   1.030   2.500   0.508   0.144   2.500   2.500   0.013   0.009   0.060   0.007   0.009   0.009   0.014   0.025   0.018       67   2.500   2.500   2.500   2.500   2.500   1.338   0.406   2.500   0.135   0.031   0.003   0.091   0.021   0.027   0.028   0.032   0.042   0.077       68   2.500   2.500   2.500   1.105   0.415   0.358   1.025   2.500   0.263   0.028   0.006   0.039   0.023   0.025   0.022   0.024   0.034   0.037       70   0.745   2.500   2.500   0.342   2.500   0.200   0.049   0.075   1.730   0.030   0.009   0.119   0.015   0.025   0.022   0.027   0.056   0.050       73   0.031   2.392   2.500   0.049   0.629   0.935   0.596   1.119   0.081   0.040   0.009   0.052   0.028   0.036   0.031   0.038   0.043   0.059       75   0.037   2.500   2.500   1.435   1.342   0.107   0.287   0.094   0.080   0.049   0.012   0.031   0.013   0.030   0.017   0.021   0.058   0.050       78   2.500   2.500   2.500   0.269   2.500   0.298   0.051   2.500   0.040   0.057   0.006   0.084   0.013   0.022   0.014   0.021   0.022   0.030       80   0.280   2.500   2.500   0.674   0.318   0.972   0.048   2.118   0.040   0.021   0.022   0.024   0.015   0.010   0.008   0.014   0.066   0.031       84   0.012   0.075   2.500   0.025   0.190   0.145   0.106   0.099   0.019   0.028   0.050   0.018   0.009   0.010   0.009   0.013   0.009   0.026       90   0.019   0.306   2.500   0.039   2.500   0.689   0.033   0.301   0.195   0.021   0.005   0.033   0.011   0.017   0.010   0.013   0.014   0.018                    
           [0006]    A recent report indicates that the HCV core protein can be detected in HCV infected individuals before the appearance of anti-HCV antibodies. (S. Lee et al., Vox Sanguinis, 2001; 80: 19-23). Therefore, we suggest that a more efficient way of early detection of HCV infection would be a combination assay able to detect HCV core protein and anti-HCV antibodies including anti-core antibodies.  
         SUMMARY OF THE INVENTION  
         [0007]    The present invention describes peptides and recombinant proteins containing Hepatitis C virus core protein sequence in which one or more of the amino acids have been modified or deleted to remove the ability of these proteins to bind to specific anti-HCV murine monoclonal antibodies. These modifications were made in the in the underlined regions shown in Table 3 below. The deletions and modifications are designed as to maintain the ability of this protein to be used in immunoassays used for the detection of anti-HCV antibodies in individuals infected with HCV. These modified core proteins can be used in a combination assay for the simultaneous detection of HCV core protein and anti-HCV antibodies. The combination assay will be able to detect HCV assay earlier than the currently used antibody assays.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0008]    One object of the invention is to develop peptide sequences that can detect anti-HCV antibodies in the presence of anti-core monoclonal antibodies to detect HCV core antigen. One use therefore of these modified core antigens will be to use them in an anti-HCV/HCV core assay, or “combination assay”.  
           [0009]    For this purpose, two or more monoclonal antibodies from Table 4 can be co-coated with one or more modified HCV core proteins on a solid phase thereby enabling the solid phase to capture anti-HCV core antibodies and core antigen. The modification is accomplished by removing the epitope for the antibody being used for detection or capture of HCV core. The removal of epitopes can be achieved by means known in the art such as deleting parts of the core sequence or altering amino acids in the epitope sequence. This can be achieved, for example, by synthesizing these peptides by chemical synthesis using commercially available peptide synthesizers or by modifying recombinant clones expressing HCV core protein. The recombinant sequences can be modified by primer dependent single or multiple site mutagenesis or primer dependent deletions. (B. Tao and K. C. Lee, PCR Technology Current Innovations,1994 by CRC Press, Inc., Chapter 10, Mutagenesis by PCR).  
           [0010]    Another object of the invention is to identify immunodominant regions of HCV core protein.  
           [0011]    Another object of the present invention is to determine the pattern of reactivity to core peptides among HCV infected individuals presenting anti-core antibodies.  
           [0012]    The peptides of the invention were generated by maintaining the highly reactive portions of the core protein and making modifications to the remaining parts of the sequence such that the peptide would not be detected by an antibody used to detect core protein in an assay.  
           [0013]    In a preferred embodiment, the peptide would be modified by either substitution of amino acids or deletion of amino acids in the regions underlined in Table 1. The remainder of the sequence shown in Table 1 should not be altered.  
           [0014]    In another preferred embodiment the peptides would be used in a combination assay. That is one that is capable of detecting both HCV antigens and antibodies simultaneously.  
                                       TABLE 3                       HCV core protein sequence:                                SEQ ID NO.:19                   MSTNPKPQRK TKRNTNRRPQDVKFPGGGQ IVGGVYLL PRRGP RLGVPATR                     KTSERSQPRGRRQPIPKARRPEGRSW AQPGYPWPLYGNEGCGWAGWLLSP                   RGSRPSWGPTDPRRRSRNLGKVIDTLTCGF            
 
           [0015]    The effectiveness and advantages of the invention are further illustrated by the following examples. 
       
    
    
     EXAMPLE 1  
       [0016]    Synthetic Peptides  
         [0017]    Synthetic peptides covering the entire sequence of HCV core protein sequence were used. Eighteen overlapping peptides shown in Table 1 were chemically synthesized by solid phase peptide synthesis using a peptide synthesizer. All of the peptides were synthesized with a C-terminal cysteine amide residue. Peptides were cleaved form the resin and purified by reverse phase liquid chromatography. Purity of each of these peptides, based on reverse phase HPLC analysis was greater that ninety-five (95%). The sequence of each peptide was confirmed by amino acid analysis of the acid hydrolysate of the peptide. The structural identities of the peptides were confirmed mass spectrometry. All of the peptides had the molecular weight expected.  
       EXAMPLE 2  
       [0018]    Synthetic peptides were coated onto Immulon 2 microwells (made by Dyanatech) in 50 mM borate buffer at a concentration of 1 ug/ml. To each microwell, 200 ul of peptide solution was added and the microwells were incubated at 25 degrees Celsius for 16-20 hours. The microwells were aspirated and washed once with phosphate buffer saline (PBS) containing TWEEN 20 to remove any unbound peptide. The microwells were then post coated with 300 ul of PBS containing one percent (1%) bovine serum albumin (BSA) and three percent (3%) sucrose to block all of the available protein binding sites. After 2-4 hours, the plates were aspirated, turbo dried and stored in sealed pouches at 2-8 degrees Celsius.  
       EXAMPLE 3  
       [0019]    Synthetic Peptide ELISA  
         [0020]    The sample, 10 ul in 200 ul of diluent, suspected of being infected with HCV was added into the peptide coated microwell. After incubation for approximately one hour the microwells were washed. To the washed microwells anti-human IgG were labeled with horse radish peroxidase was added. After an incubation for thirty minutes the microwells were washed and a solution of ORTHO phenylenedaamine buffer and hydrogen peroxide added to each well. After approximately 30 minutes sulfuric acid were added to each well to stop the reaction. An orange or yellow color indicated the presence of anti-HCV antibodies in the sample.  
         [0021]    Table 4  
         [0022]    Monoclonal Antibodies  
         [0023]    The table below identifies 15 antibodies. The antibodies were screened at every stage of antibody development with microtitrewell plates coated with immunogen. The immunogen used to immunize the mice that produced each strain of monoclonal antibody is identified as one of the following: peptide ODS 243, a large peptide further defined in Example 1; “FLC” means full length core antigen, defined in Example 1; or KLH conjugated core peptide #8, a short peptide, further defined herein. The specificity of each to a numbered peptide is shown and the amino acid sequences of each numbered peptide is identified herein. Furthermore, the epitope to which the antibody specifically binds is included in the last column, defined by the amino acids that encode for the epitope.  
                                                   ATCC   AG-FUSION #,                       #   clone   IMMUNOGEN   ISOTYPE   SPECIFICITY   AA                   PTA-   ODS243, 7B4F11   Peptide   IgG 2b   HCV core   77-91       3811       ODS 243       peptide #8       PTA-   ODS243, 1E3D12   Peptide   IgG2a   HCV core    86-100       3803       ODS 243       peptide #9       PTA-   ODS243, 7C12C4   Peptide   IgG2b   HCV core   77-91       3802       ODS 243       peptide #8       PTA-   core#3,   FLC   IgG1   HCV core   106-120       3813   2A11C6           peptide# 11       PTA-   Core#12,   FLC   IgG1   HCV core   29-43       3809   1B7A1           peptide# 3       PTA-   CORE#13,   FLC   IgG1   HCV core   39-53       3805   5A12G12           peptide# 4       PTA-   Core#13,   FLC   IgG1   HCV core   48-62       3812   4H7E7           peptide# 5       PTA-   Core#13, 12F4A   FLC   IgG1   HCV core   58-72       3806   11           peptide# 6       PTA-   Core#13,   FLC   IgG1   HCV core   67-81       3804   14D12A12           peptide# 7       PTA-   c22-8#4,   KLH   IgG1   HCV core   77-91       3807   6D8E8   Conjugated       peptide# 8               core               peptide               #8       PTA-   Core#12,   FLC   IgG2b   HCV core    96-110       3800   4G10G6           peptide# 10       PTA-   Core#13,   FLC   IgG2a   HCV core   106-120       3801   6E7E1           peptide# 11       PTA-   Core#13,   FLC   IgG2b   HCV core   106-120       3810   11D12A6           peptide# 11       PTA-   Core#13,   FLC   IgG3   HCV core   106-120       3808   14B7C3           peptide# 11       PTA-   Core#12,   FLC   IgG1   HCV core   156-170       3799   4A6H3           peptide# 16                  
 
       EXAMPLE 4  
       [0024]    Use of Modified Core Peptides in an Anti-HCV ELISA  
         [0025]    A peptide consisting of HCV core immunodominant region amino acids 1-43 was chemically synthesized. Another peptide with certain deletions in this region, namely, amino acids 1-8 and 31-33 were deleted, was also synthesized. These peptides were used to test 40 chronic HCV patients&#39; serum samples for anti-HCV antibody status. The results indicated that 39 of the 40 patients were not affected by deletion of these sequences. The deletion peptides can be used in combination with anti-HCV monoclonal antibodies, such as the ones shown in Table 4, in a combination assay. Peptides with deletions of amino acids 31-33 can be used with any of the monoclonal antibodies shown in Table 4.  
                                                     TABLE 5                       Peptide/Specimen ID   265-2   266-2   271-2   272-2                                11   2.500   2.500   2.500   2.500       15   2.500   2.500   2.500   2.500       18   2.500   2.500   2.500   2.500       20   2.500   2.500   2.500   2.500       21   2.500   2.500   2.500   2.500       27   2.500   2.500   2.500   2.500       30   2.500   2.500   2.500   2.500       35   0.762   0.911   0.507   0.611       36   2.500   2.500   2.500   2.500       37   2.500   2.500   2.500   2.500       39   2.500   2.500   2.500   2.500       40   2.500   2.500   2.500   2.500       42   2.500   2.500   2.500   2.500       43   1.621   1.255   1.382   1.443       44   2.500   2.500   2.500   2.500       45   2.500   2.500   2.500   2.500       46   2.003   1.863   1.058   1.829       47   2.500   2.500   2.500   2.500       48   2.500   2.500   2.500   2.500       49   2.500   2.500   2.500   2.500       50   2.500   2.500   2.500   2.500       52   2.500   2.500   2.500   2.500       53   2.500   2.500   2.500   2.500       54   2.500   2.500   2.500   2.500       55   2.500   2.500   2.500   2.500       58   2.500   2.500   2.500   2.500       59   2.500   2.500   2.500   2.500       60   2.500   2.500   2.500   2.500       62   2.500   2.500   2.500   2.500       65   0.056   0.045   2.237   1.704       66   2.500   2.500   2.500   2.500       67   2.500   2.500   2.500   2.500       68   2.500   2.500   2.500   2.500       70   2.500   2.500   2.500   2.500       73   2.500   2.500   2.500   2.500       75   2.500   2.500   2.500   2.500       78   2.500   2.500   2.500   2.500       80   2.500   2.500   2.500   2.500       84   2.500   2.500   2.170   2.500       90   2.500   2.500   2.500   2.500                                                  
 
         [0026]    [0026] 
     
       
       
         1 
         
           
             19  
           
           
             1  
             15  
             PRT  
             Hepatitis C virus  
           
            1 

Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr 
1               5                   10                  15 

 
           
             2  
             15  
             PRT  
             Hepatitis C virus  
           
            2 

Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe 
1               5                   10                  15 

 
           
             3  
             15  
             PRT  
             Hepatitis C virus  
           
            3 

Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val 
1               5                   10                  15 

 
           
             4  
             15  
             PRT  
             Hepatitis C virus  
           
            4 

Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg 
1               5                   10                  15 

 
           
             5  
             15  
             PRT  
             Hepatitis C virus  
           
            5 

Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser 
1               5                   10                  15 

 
           
             6  
             15  
             PRT  
             Hepatitis C virus  
           
            6 

Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg 
1               5                   10                  15 

 
           
             7  
             15  
             PRT  
             Hepatitis C virus  
           
            7 

Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu 
1               5                   10                  15 

 
           
             8  
             15  
             PRT  
             Hepatitis C virus  
           
            8 

Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr 
1               5                   10                  15 

 
           
             9  
             15  
             PRT  
             Hepatitis C virus  
           
            9 

Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys 
1               5                   10                  15 

 
           
             10  
             15  
             PRT  
             Hepatitis C virus  
           
            10 

Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro 
1               5                   10                  15 

 
           
             11  
             15  
             PRT  
             Hepatitis C virus  
           
            11 

Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 
1               5                   10                  15 

 
           
             12  
             15  
             PRT  
             Hepatitis C virus  
           
            12 

Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Leu Asn Gly 
1               5                   10                  15 

 
           
             13  
             15  
             PRT  
             Hepatitis C virus  
           
            13 

Ser Arg Leu Asn Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe 
1               5                   10                  15 

 
           
             14  
             15  
             PRT  
             Hepatitis C virus  
           
            14 

Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val 
1               5                   10                  15 

 
           
             15  
             15  
             PRT  
             Hepatitis C virus  
           
            15 

Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala 
1               5                   10                  15 

 
           
             16  
             15  
             PRT  
             Hepatitis C virus  
           
            16 

Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 
1               5                   10                  15 

 
           
             17  
             15  
             PRT  
             Hepatitis C virus  
           
            17 

Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro 
1               5                   10                  15 

 
           
             18  
             15  
             PRT  
             Hepatitis C virus  
           
            18 

Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala 
1               5                   10                  15 

 
           
             19  
             130  
             PRT  
             Hepatitis C virus  
           
            19 

Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 
1               5                   10                  15 

Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 
            20                  25                  30 

Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 
        35                  40                  45 

Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 
    50                  55                  60 

Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ser Trp Ala Gln Pro Gly 
65                  70                  75                  80 

Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp 
                85                  90                  95 

Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 
            100                 105                 110 

Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 
        115                 120                 125 

Gly Phe 
    130