CA2419418A1 - Vaccines containing ribavirin and methods of use thereof - Google Patents

Abstract

Compositions and methods for enhancing the effect of vaccines in animals, such as domestic, sport, or pet species, and humans are disclosed. More particularly, vaccine compositions comprising ribavirin and an antigen, preferably an antigen that has an epitope present in Hepatitis C virus (HCV), are disclosed for use in treating and preventing disease, preferably HCV
infection.

Description

VACCINES CONTAINING RIBAVIRIN AND METHODS OF USE THEREOF
FIELD OF THE INVENTION
The present invention relates to compositions and methods for enhancing the effect of vaccines in animals, such as domestic, sport, or pet species, and humans. More particularly, preferred embodiments concern the use of ribavirin as an adjuvant and compositions having ribavirin and an antigen.
BACKGROUND OF THE INVENTION
The use of vaccines to prevent disease in humans, farm livestock, sports animals, and household pets is a common practice. Frequently, however, the antigen used in a vaccine is not sufficiently immunogenic to raise ~he antibody titre to levels that are sufficient to provide protection against subsequent challenge or to maintain the potential for mounting these levels over extended time periods. Further, many vaccines are altogether deficient in inducing cell-mediated immunity, which is a primary immune defense against bacterial and viral infection. A
considerable amount of research is currently focussed on the development of more potent vaccines and ways to enhance the immunogenicity of antigen-containing preparations.
(See e.g., U.S. Pat.
Nos. 6,056,961; 6,060,068; 6,063,380; and Li et al., Scie»ce 288:2219-2222 (2000)).
Notorious among such "weak" vaccines are hepatitis B vaccines. For example, recombinant vaccines against hepatitis B virus such as Genhevacb (Pasteur Merieux Serums et Vaccines, 58, Avenue Leclerc 69007 Lyon, France), Engerixb (Smith, Kline and Symbol French), and Recombivaxhb (Merck, Sharp, and Dhome) are effective only after at least three injections at 0, 30, and 60 or 180 days, followed by an obligatory booster after one year.
(Chedid et al., U.S.
Patent No. 6,063,380). Additionally, many subjects receiving these vaccines respond poorly, if at all. Because many regions of the world are endemic for HBV infection, the poorly immunogenic character of existing HBV vaccines his become an extremely serious problem.
To obtain a stronger, humoral and/or cellular response, it is common to administer a vaccine in a material that enhances the immune response of the patient to the antigen present in the vaccine. The most commonly used adjuvants for vaccine protocols are oil preparations and alum.
(Chedid et al., U.S. Patent No. 6,063,380). A greater repertoire of safe and effective adjuvants is needed.
Nucleoside analogs have been widely used in anti-viral therapies due to their capacity to reduce viral replication. (Hosoya et al., J. I».f. Dis., 168:641-646 (1993)).
ribavirin (1-(3-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a synthetic guanosine analog that has been used to inhibit RNA and DNA virus replication. (Huffman et al., Antimicrob. Agents.
Cher»other., 3:235 (1973); Sidwell et al., Scie»ee, 177:705 (1972)). ribavirin has been shown to be a competitive inhibitor of inositol mono-phosphate (IMP) dehydrogenase (IMPDH), which converts IMP to IMX
(which is then converted to GMP). De Clercq, Anti viral Agents: characteristic activity spectrum depending on the molecular target with which they interact, Academic press, Inc., New York N.Y., pp. 1-55 (1993). Intracellular pools of GTP become depleted as a result of long term ribavirin treatment.
In addition to antiviral activity, investigators have observed that some guanosine analogs have an effect on the immune system. (U.S. Patent Nos. 6,063,772 and 4,950,647). ribavirin has been shown to inhibit functional humoral immune responses (Peavy et al., J.
Irranaunol., 126:861-864 (1981); Powers et al., Antimicrob. Agents. Chernother., 22:108-114 (1982)) and IgE-mediated modulation of mast cell secretion. (Marquardt et al., J. Pharrnacol. Exp.
Therapeutics, 240:145-149 (1987)). Some investigators report that a daily oral therapy of ribavirin has an immune modulating effect on humans and mice. (Hultgren et al., J. Gen. Yirol., 79:2381-2391 (1998) and Cramp et al., Gastron. Enterol., 118:346-355 (2000)). Nevertheless, the current understanding of the effects of ribavirin on the immune system is in its infancy.
I S SUMMARY OF THE INVENTION
It has been discovered that ribavirin can be used as an adjuvant to enhance or facilitate an immune response to an antigen. Embodiments of the invention described herein include "strong"
vaccine preparations that comprise an antigen and ribavirin. Generally, these preparations have an amount of ribavirin that is sufficient to enhance or facilitate an immune response to the antigen.
Other aspects of the invention include methods of enhancing or facilitating an immune response of an animal, including a human, to an antigen. By one approach, for example, an animal in need of a potent immune response to an antigen is identified and then is provided an amount of ribavirin together with the antigen. In some -methods, the ribavirin and the antigen are provided in combination (e.g., in a single composition) and in others, the ribavirin and the antigen are provided separately. Several embodiments also concern the manufacture and use of compositions having ribavirin and an antigen.
Although the embodied compositions include ribavirin and virtually any antigen or epitope, preferred compositions comprise ribavirin and a hepatitis viral antigen or epitope. The antigen or epitope can be peptide or nucleic acid-based (e.g., a RNA encoding a peptide antigen or a construct that expresses a peptide antigen when introduced to a subject).
Compositions having ribavirin and a peptide comprising an antigen or epitope from the hepatitis A
virus (HAV) or a nucleic acid encoding said peptide are embodiments. Compositions having ribavirin and a peptide comprising an antigen or epitope from the hepatitis B virus (HBV) or a nucleic acid encoding said peptide are embodiments. HBV antigens that are suitable include, for example, hepatitis B surface antigen (HBsAg), hepatitis core antigen (HBcAg), hepatitis a antigen (HBeAg), and nucleic acids encoding these molecules. Still further, compositions having ribavirin and a peptide comprising an antigen or epitope from the hepatitis C virus (HCV) or a nucleic acid encoding said peptide are embodiments. Suitable HCV antigens include, but are not limited to, one or more domains of the HCV sequence (e.g., NS3 and/or NS4~'A) and nucleic acids encoding said molecules.
A new HCV sequence was also discovered, A novel NS3/4A fragment of the HCV
genome was cloned and sequenced from a patient infected with HCV (SEQ. ID.
NO.: 16). This sequence was found to be only 93% homologous to the most closely related HCV
sequence. This novel peptide (SEQ. ID. NO.: 17) and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length, nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are also embodiments of the present invention. A
particularly preferred embodiment is a vaccine composition comprising ribavirin and the HCV
peptide of SEQ. ID. NO.: 17 or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NO.: 25) or a nucleic acid encoding said peptide or fragments.
Additionally, it was discovered that truncated mutants and mutants of the NS3/4A peptide, which lack a proteolytic cleavage site, are highly immunogenic. These novel peptides (SEQ. ID.
NOs.: 29- 32 and 43-49) and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 26, 27, and 33-42), nucleic acids encoding these molecules, vectors t having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are also embodiments. A particularly preferred embodiment is a vaccine composition comprising ribavirin and at least one HCV peptide of SEQ. ID. NOs.: 29- 32 and 43-49 or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 26, 27, and 33-42) or a nucleic acid encoding said peptides or fragments.
Furthermore, compositions having a mixture of the antigens above are embodiments of the invention. For example, some compositions comprise a HBV antigen, a HAV
antigen, and ribavirin or a HBV antigen, a HCV antigen, and ribavirin or a HAV antigen, a HCV antigen, and ribavirin or a HBV antigen, a HAV antigen, a HCV antigen, and ribavirin. Other embodiments comprise ribavirin and a nucleic acid encoding a mixture of the antigens described above. Some embodiments also include other adjuvants, binders, emulsifiers, carriers, and fillers, as known in the art, including, but not limited to, alum, oil, and other compounds that enhance an immune response.
Methods of making and usityg the compositions described herein are also aspects of the invention. Some methods are practiced by mixing ribavirin with a peptide or nucleic acid antigen (e.g., an HAV, HBV, HCV antigen) so as to formulate a single composition (e.g., a vaccine composition). Preferred methods involve the mixing of ribavirin with an HCV
antigen that has an epitope present on one or more domains of HCV (e.g., NS3 and/or NS4A).

Preferred methods of using tlRe compositions described herein involve providing an animal in need with a sufficient amount of ribavirin and a hepatitis viral antigen (e.g., HBV antigen, HAV
antigen, HCV antigen a nucleic acid encoding one of these antigens or any combination thereof).
By one approach, for example, an animal in need of potent immune response to a hepatitis viral antigen (e.g., an animal at risk or already infected with a hepatitis infection) is identified and said animal is provided an amount of ribavirin and a hepatitis viral antigen (either in a single composition or separately) that is effective to enhance or facilitate an immune response to the hepatitis viral antigen. Preferably, an animal in need of a potent immune response to HCV is identified and said animal is provided a composition comprising ribavirin and a peptide comprising an antigen or epitope present on SEQ. ID. NO.: 1, 6, 7, or 17 or a nucleic acid encoding said peptide. Particularly preferred methods involve the identification of an animal in need of an potent immune response to HCV and providing said animal a composition comprising ribavirin and an amount of an HCV antigen (e.g., NS3/4A (SEQ. ID. NO.: 17), mutant NS3/4A SEQ.
ID. NOs.:
29- 32 and 43-49, or a fragment thereof at least 3, 4-10, 10-20, 20-30, or 30-50 amino acids in A
length (e.g., SEQ. ID. NOs.: 25-27, and 33-42) or a nucleic acid encoding one or more of these molecules) that is sufficient to enhance or facilitate an immune response to said antigen.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is a graph showing the humoral response to 10 and 100pg recombinant Hepatitis C virus (HCV) non structural 3 protein (NS3), as determined by mean end point titres, when a single dose of lmg of ribavirin was co-administered.
FIGURE 2 is a graph showing the humoral response to 20pg recombinant Hepatitis C virus (HCV) non structural 3 protein (NS3), as determined by mean end point titres, when a single dose of 0.1, 1.0, or l Omg of ribavirin was co-administered.
FIGURE 3 is a graph showing the effects of a single dose of lmg ribavirin on NS3-specific lymph node proliferative responses, as determined by ih vitro recall responses.
FIGURE 4 is a graph showing the antibody titer in H-2d mice against NS3 as a function of time after the first immunization. Diamonds denote antibody titer in mice immunized with NS3/4A-pVAX and squares denote ay~tibody titer in mice immunized with NS3-pVAX.
FIGURE SA is a graph showing the percentage of specific CTL-mediated lysis of target cells as a function of the effector to target ratio. Phosphate Buffered Saline (PBS) was used as a control immunogen.
FIGURE SB Is a graph showing the percentage specific CTL-mediated lysis of target cells as a function of the effector to target ratio. Plasmid NS3/4A-pVAX was used as the immunogen.

DETAILED DESCRIPTION OF THE INVENTION
It has been discovered that compositions comprising ribavirin and an antigen (e.g., a molecule containing an epitope of a pathogen such as a virus, bacteria, mold, yeast, or parasite) enhance and/or facilitate an animal's immune response to the antigen. That is, it was discovered that ribavirin is an effective "adjuvant," which for the purposes of this disclosure, refers to a material that has the ability to enhance or facilitate an immune response to a particular antigen.
The adjuvant activity of ribavirin was manifested by a significant increase in immune-mediated protection against the antigen, an increase in the titer of antibody raised to the antigen, and an increase in proliferative T cell responses.
Several compositions (e.g., vaccines) that comprise ribavirin and an antigen or epitope are described herein. Vaccine formulations containing ribavirin, for example, can vary according to the amount of ribavirin, the form of ribavirin, and the type of antigen. The antigen can be a peptide or a nucleic acid (e.g., a RNA encoding a peptide antigen or a construct that expresses a peptide antigen when introduced into a subject): Preferred compositions comprise ribavirin and a hepatitis viral antigen (e.g., HAV antigen, HBV antigen, HCV antigen, a nucleic acid encoding these molecules, or any combination thereof). In particular, at least one HCV
antigen or an epitope present on SEQ. ID. NO.: 1 or a nucleic acid encoding said HCV antigen are desired for mixing with ribavirin to make said compositions. That is, some embodiments include, but are not limited to, compositions comprising ribavirin and a peptide comprising SEQ. ID. NO.:
1, or a fragment thereof having at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids of SEQ. ID. NO.: 1. Additional embodiments concern compositions comprising ribavirin and a nucleic acid encoding SEQ. ID. NO.: 13 or a fragment thereof having at least 9, 12, 15, 20, 30, 50, 75, 100, 200, 500 consecutive nucleotides of SEQ. ID. NO.: 13.
Other embodiments include a composition (e.g., a vaccine) that comprises ribavirin and a specific fragment of SEQ. ID. NO.>1, wherein said fragment corresponds to a particular domain of HCV. Some embodiments, for example, comprise a fragment of HCV
corresponding to amino acids 1-182, 183-379, 380-729, 730-1044, -1045-1657, 1658-1711, 1712-1971, or 1972-3011 of SEQ. ID. NO.: 1. Compositions comprising ribavirin and a nucleic acid encoding one or more of these fragments are also embodiments of the invention.
Additionally, a novel HCV sequence was discovered. A novel nucleic acid and protein corresponding to the NS3/4A domain of HCV was cloned from a patient infected with HCV (SEQ.
ID. NO.: 16). A Genebank search revealed that the cloned sequence had the greatest homology to HCV sequences but was only 93% homologous to the closest HCV relative (accession no AJ
278830). This novel peptide (SEQ. ID. NO.: 17) and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length, nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are also embodiments of the invention. Further, some of the vaccine embodiments described herein comprise ribavirin and this novel NS3/4A peptide or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NO.: 25) or a nucleic acid encoding one or more of these molecules.
Mutants of the novel NS3/4A peptide were also created. It was discovered that truncated mutants (e.g., SEQ. ID. NO.: 29) and mutants, which lack a proteolytic cleavage site, are highly immunogenic. These novel peptides SEQ. ID. NOs.: 29- 32 and 43-49 and fragments thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.:
26, 27, and 33-42), nucleic acids encoding these molecules, vectors having said nucleic acids, and cells having said vectors, nucleic acids, or peptides are also embodiments. Furthermore, some of the compositions described herein comprise ribavirin and at least one of the mutant HCV
peptides described above or a fragment thereof at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length. Other vaccine embodiments comprise ribavirin and a nucleic acid (e.g., DNA) encoding one or more of the peptides described above.
Methods of making and using the compositions above are also embodiments. For example, the compositions described above can be made by providing ribavirin, providing an antigen (e.g., a peptide comprising an HCV antigen or a nucleic acid encoding said peptide), and mixing said ribavirin and said antigen so as to formulate a composition that can be used to enhance or facilitate an immune response in a subject to said antigen. Preferred methods entail mixing a preferred antigen or epitope (e.g., a peptide comprising SEQ. ID. NO.: 1, 6, 7, or 17 or specific fragments thereof, such as amino aFids 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, 1972-3011 of SEQ. ID. NO.: 1 and nucleic acids encoding these molecules) with ribavirin. Other antigens or epitopes can also be mixed with ribavirin including, but not limited to, fragments of SEQ. ID. NO.: 1 that have at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids and nucleic acids encoding these fragments. Particularly preferred methods concern the making of vaccine compositions comprising the newly discovered NS3/4A fragment or an NS3/4A mutant (e.g., a truncated mutant or a mutant lacking a proteolytic cleavage site), or a fragment thereof of at least four amino acids in length or a nucleic acid encoding one or more of these molecules.
Methods of enhancing or facilitating the immune response of an animal, including humans, to an antigen are embodiments of the invention. Such methods can be practiced, for example, by identifying an animal in need of a potent immune response to an antigen/epitope and providing said animal a composition comprising the antigen/epitope and an amount of ribavirin that is effective to enhance or facilitate an immune response to the antigen/epitope.
In some embodiments, the ribavirin and the antigen are administered separately, instead of in a single mixture. Preferably, in this instance, the ribavirin is administered a short time before or a short time after admininstering the antigen. Preferred methods involve providing the animal in need with ribavirin and a hepatitis antigen (e.g., HAV antigen, HBV antigen, HCV
antigen, a nucleic acid encoding these molecules, or any combination thereof). Some of these methods involve HCV
antigens, such as a peptide comprising SEQ. ID. NO.: 1, or a fragment thereof having at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids of SEQ. ID.
NO.: 1. Additional methods involve compositions comprising ribavirin and a nucleic acid encoding SEQ. ID. NO.: 13 or a nucleic acid encoding one or more of the fragments discussed above.
Some preferred methods, for example, concern the use of a composition (e.g., a vaccine) that comprises ribavirin and a peptide comprising SEQ. ID. NO.: 1 or a specific fragment thereof, which corresponds to an HCV domain including, but not limited to, a peptide comprising amino acids 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, or 1972-3011 of SEQ. ID. NO.: 1. Particularly preferred methods concern the use of a vaccine composition comprising the NS3/4A fragment of SEQ. ID. NO.: 17 or the mutant NS3/4A (e.g., SEQ. ID.
NOs:. 29- 32 and 43-49), which lack a proteolytic cleavage site, or a fragment thereof of at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 26, 27, and 33-42).
Compositions comprising ribavirin and a nucleic acid encoding these fragments can also be used with the methods described herein.
Other embodiments concern methods of treating and preventing HCV infection. By one approach, ribavirin and an HCV antigen or epitope are used to prepare a medicament for the treatment and/or prevention of HCV infection. By another approach, an individual in need of a medicament that prevents and/or treats HCV infection is identified and said individual is provided a medicament comprising ribavirin and an HCV antigen or epitope, preferably an epitope present on SEQ. ID. NO.: 1, more preferably a fragment of SEQ. ID. NO.: 1 having at least 2500, 2000, 1600, 1200, 800, 400, 200, 100, 50, 10, or 3 consecutive amino acids or most preferably a fragment of SEQ. ID. NO.: 1 such as 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, or 1972-3011 or a nucleic acid encoding SEQ. ID. NO.: 1 or said fragments above.
Particularly preferred methods concern the use of a vaccine composition comprising ribavirin and the NS3/4A fragment of SEQ. ID. NO.: 17 or the mutant NS3/4A, which lacks a proteolytic cleavage site (e.g., SEQ. ID. NOs.: 29- 32 and 43-49) or a fragment thereof of at least 3, 4, 6, 8, 10, 12, 15 or 20 amino acids in length (e.g., SEQ. ID. NOs.: 25-27, and 33-42) or a nucleic acid encoding one or more of these molecules. The section below discusses the use of ribavirin as an adjuvant in greater detail.
_7_ Ribaviritt The compositions described b~erein can be manufactured in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to animals, e.g., mammals including humans. Ribavirin can be obtained from commercial suppliers (e.g., Sigma and ICN). Ribavirin and/or the antigen can be formulated into the vaccine with and without modification. For example, the ribavirin and/or antigen can be modified or derivatized to make a more stable molecule and/or a more potent adjuvant. By one approach, the stability of ribavirin and/or an antigen can be enhanced by coupling the molecules to a support such as a hydrophilic polymer (e.g., polyethylene glycol).
Many more ribavirin derivatives can be generated using conventional techniques in rational drug design and combinatorial chemistry. For example, Molecular Simulations Inc.
(MSI), as well as many other suppliers, provide software that allows one of skill to build a combinatorial library of organic molecules. The C2.Analog Builder program, for example, can be integrated with MSI's suite of Cerius2 molecular diversity software to develop a library of ribavirin derivatives that can be used with the embodiments described herein. (See e.g., http://msi.comllife/products/cerius2/index.html).
By one approach, the chemical structure of ribavirin is recorded on a computer readable medium and is accessed by one or more modeling software application programs.
The C2.Analog Builder program in conjunction with C2Diversity program allows the user to generate a very large virtual library based on the diversity of R-groups for each substituent position, for example.
Compounds having the same structure as the modeled ribavirin derivatives created in the virtual library are then made using conventional chemistry or can be obtained from a commercial source.
The newly manufactured ribavirin derivatives are then screened in assays, which determine the extent of adjuvant activity of the molecule and/or the extent of its ability to modulate of an immune response. Some assays may involve virtual drug screening software, such as C2.Ludi.
C2.Ludi is a software program that allows a user to explore databases of molecules (e.g., ribavirin derivatives) for their ability to interact with the active site of a protein of interest (e.g., RAC2 or another GTP binding protein). Based upon predicted interactions discovered with the virtual drug screening software, the ribavirin derivatives can be prioritized for further characterization in conventional assays that determine adjuvant activity and/or the extent of a molecule to modulate an immune response. Example 1 describes several assays that were used to evaluate the adjuvant activity of ribavirin.

This following assays can be used with any ribavirin derivative or combinations of ribavirin derivatives to determine the extent of adjuvant activity of the particular composition. In a _g_ first set of experiments, groups of three to five Balblc mice (BK Universal, Uppsala~ Sweden) were immunized i.p or s. c. (e.g., at the base of the tail) with lOp.g or 100wg of recombinant hepatitis C
virus non-structural 3 (rNS3) protein at weeks zero and four. The rNS3 was dissolved in phosphate buffered saline (PBS) alone or PBS containing lmg ribavirin (obtained from ICN, Costa Mesa, CA). Mice were injected with a total volume of 100p1 per injection.
At two, four, and six weeks following first i.p. immunization, all mice were bled by retro-orbital sampling. Serum samples were collected and analyzed for the presence of antibodies to rNS3. To determine the antibody titer, an enzyme immunoassay (EIA) was performed: (See e.g., Hultgren et al., J Gen Virol. 79:2381-91 (1998) and Hultgren et al., Clin.
Diagn. Lab. Immuraol.
4:630-632 (1997)). The antibody levels were recorded as the highest serum dilution giving an optical density at 405nm more than twice that of non-immunized mice.
Mice that received 10~g oro 100pg rNS3 mixed with lmg ribavirin in PBS
displayed consistently higher levels of NS3 antibodies. The antibody titer that was detected by EIA at two weeks post-immunization is shown in FIGURE 1. The vaccine formulations having lmg of ribavirin and either lOp.g or 100~g of i'NS3 induced a significantly greater antibody titer than the vaccine formulations composed of only rNS3.
In a second set of experiments, groups of eight Balb/c mice were at weeks zero and four immunized intraperitoneally with 10 or 50 pg of rNS3 in 100 p1 phosphate buffered saline containing either 0 mg, 1 mg, 3 mg, or 10 mg ribavirin (Sigma). At four, six and eight weeks the mice were bled and serum was separated and frozen. After completion of the study, sera were tested for the levels of antibodies to recombinant NS3, as described above.
Mean antibody levels to rNS3 were compared between the groups using Student's t-test (parametric analysis) or Mann-Whitney (non-parametric analysis) and the software package StatView 4.5 (Abacus Concepts, Berkely, CA). The adjuvant effect of ribavirin when added in three doses to 10 pg of rNS3 are provided in TABLE 1. The adjuvant effect of ribavirin when added in three doses to 50 p.g of rNS3 are provided in TABLE 2. P"arametrical comparison of the mean rNS3 antibody titres in mice receiving different lOpg or 50 pg of rNS3 and different doses of ribavirin are provided in TABLES 3 and 4, respectively. Non-parametrical comparison of mean NS3 antibody titres in mice receiving different lOpg or 50 pg of rNS3 and different doses of ribavirin are provided in TABLES 5 and 6, respectively. The values given represent end point titres to recombinant rNS3.

Amount Amount Mouse Antibody ribavirinimmunogen ID titre to (mg/dose)(pg/dose) rNS3 at indicated week Week 4 Week 6 Week 8 None 10 5:1 300 1500 1500 None 10 5:2 <60 7500 1500 None 10 5:3 <60 1500 300 None 10 5:4 60 1500 1500 None 10 5:5 <60 1500 nt None 10 :6 60 1500 1500 None 10 5:7 <60 7500 7500 None 10 5:8 300 37500 7500 Group 180 7500 3042 mean X139 X12421 X3076 titre (mean~SD) -1 10 6:1 300 37500 37500 1 10 6:2 <60 1500 1500 1 10 6:3 300 37500 187500 1 10 6:4 300 37500 7500 1 10 6:5 60 nt nt 1 10 6:6 <60 37500 7500 1 10 6:7 <60 37500 7500 1 10 6:8 300 7500 7500 Group 252 28071 36642 mean X107 X16195 X67565 titre (mean~SD) 3 10 7:1 60 37500 7500 3 10 7:2 60 37500 37500 3 10 7:3 300 7500 7500 3 10 7:4 300 37500 7500 3 10 e7:5 300 37500 37500 3 10 7:6 300 37500 37500 3 10 7:7 60 7500 7500 3 10 7:8 60 37500 37500 Group 180 30000 22500 mean 128 13887 34637 titre (mean~SD) -10 8:1 300 37500 37500 10 10 8:2 300 ' 37500 37500 10 10 8:3 <60 300 300 10 10 8:4 60 7500 7500 10 10 8:5 <60 300 300 10 10 8:6 <60 37500 37500 10 10 8:7 <60 7500 7500 10 10 8:8 <60 nt nt Group 220 18300 18300 mean 139 18199 18199 titre (mean~SD) Amount Amount Mouse Antibody ribavirinimmunogen ID titre to (mg/dose)(~.gldose) rNS3 at indicated week Week 4 Week 6 Week 8 None 50 1:1 60 7500 7500 None 50 1:2 60 7500 7500 None 50 1:3 60 7500 7500 None 50 1:4 <60 1500 300 None 50 1:5 300 37500 37500 None 50 1:6 60 7500 7500 None 50 1:7 60 37500 7500 None 50 1:8 Group 100 15214 10757 mean X98 X15380 X12094 titre (mean~SD) 1 50 2:1 60 7500 7500 1 50 2:2 300 37500 7500 1 SO 2:3 60 187500 7500 1 50 2:4 60 37500 187500 1 50 2:5 60 37500 7500 1 50 2:6 60 37500 37500 1 50 2:7 300 37500 7500 1 50 2:8 300 37500 37500 Group 150 52500 37500 mean X124 X55549 X62105 titre (mean~SD) ,, 3 SO 3:1 60 37500 7500 3 50 3:2 300 37500 37500 3 50 3:3 300 37500 7500 3 50 3~4 60 37500 7500 3 50 3:5 300 37500 7500 3 50 3:6 60 37500 7500 3 50 3:7 - 7500 37500 3 50 3:8 1500 7500 37500 Group 387 30000 18750 mean X513 X13887 X15526 titre (mean~SD) 50 4:1 300 7500 7500 10 50 4:2 300 37500 37500 10 50 4:3 60 7500 7500 10 50 4:4 60 7500 7500 10 50 4:5 60 1500 1500 10 50 4:6 60 7500 37500 10 50 4:7 - 7500 7500 10 50 8:8 60 37500 7500 Group 140 10929 15214 mean X124 X11928 X15380 titre (mean~SD) Group Week Mean~SD Group Mean~SD analysisp-value l Opg NS3/no4 180 10 ~.g NS3/252 Students0.4071 ribavirin X139 1 mg ribavirinX107 t-test 6 7500 28071 Students0.0156 ~ 12421 ~ 16195 t-test 8 3042 36642 Students0.2133 X3076 X67565 t-test l Opg NS3/no4 180 10 ~.g NS3/180 Students1.000 ribavirin X139 3 mg ribavirin128 t-test 6 7500 30000 Students0.0042 X12421 13887 t-test 8 3042 22500 Students0.0077 X3076 34637 t-test 10~,g NS3/no4 180 10 ltg NS3/220 Students0.7210 ribavirin X139 lOmg ribavirin139 t-test 6 7500 18300 Students0.1974 X12421 18199 t-test 8 3042 18300 Students0.0493 ~307C~ 18199 t-test Group Week Mean~SD Group Mean~SD analysisp-value 50wg NS3/no4 100 50 ~.g NS3/150 Students0.4326 ribavirin X98 1 mg ribavirinX124 t-test 6 15214 52500 Students0.1106 X15380 X55549 t-test 8 10757 37500 Students0.2847 ~ 12094 X62105 t-test 50pg NS3lno4 100 50 ~g NS3/ 387 Students0.2355 ribavirin X98 3 mg ribavirinX513 t-test 6 15214 30000 Students0.0721 X15380 X13887 t-test 8 10757 18750 Students0.2915 X12094 X15526 t-test 50~g NS3/no4 100 50 ~g NS3/ 140 Students0.5490 ribavirin ~9 lOmg ribavirinX124 t-test 6 15214 10929 Students0.5710 X15380 X11928 t-test 8 10757 15214 Students0.5579 X12094 X15380 t-test _ Significance levels: NS = not significant; * = p<0.05; ** = p<0.01; *** =
p<0.001 Group Week Mean~SD Group Mean~SD analysisp-value l Opg NS3/no4 180 10 pg NS3/252 Mann- 0.4280 ribavirin X139 1 mg ribavirinX107 Whitney 6 7500 28071 Mann- 0.0253 X12421 X16195 Whitney 8 3042 36642 Mann- 0.0245 X3076 X67565 Whitney l Opg NS3/no4 180 10 pg NS3/180 Mann- 0.0736 ribavirin X139 3 mg ribavirin128 Whitney 6 7500 30000 Mann- 0.0050 X12421 13887 Whitney 8 3042 22500 Mann- 0.0034 X3076 34637 Whitney l Opg NS3/no4 180' 10 pg NS3/220 Mann- 0.8986 ribavirin X139 l Omg ribavirin139 Whitney 6 7500 18300 Mann- 0.4346 X12421 18199 Whitney 8 3042 _ 18300 Mann- 0.2102 X3076 18199 Whitney Group Week Mean~SD Group Mean~SD analysisp-value SOpg NS3/no4 100 50 p.g NS3/150 Mann- 0.1128 ribavirin X98 1 mg ribavirinX124 Whitney 6 15214 52500 Mann- 0.0210 X15380 X55549 Whitney 8 10757 37500 Mann- 0.1883 X12094 X62105 Whitney SOpg NS3/no4 100 SO pg NS3/ 387 Mann- 0.1400 ribavirin X98 3 mg ribavirinX513 Whitney 6 1521Qr 30000 Mann- 0.0679 X15380 X13887 Whitney 8 10757 18750 Mann- 0.2091 X12094 X15526 Whitney SOpg NS3/no4 100 SO pg NS3/ 140 Mann- 0.4292 ribavirin X98 10 mg ribavirinX124 Whitney 6 15214 10929 Mann- 0.9473 X15380 X11928 Whitney 8 10757 15214 Mann- 0.6279 ~ 12094 ~ 153 Whitney Significance levels: NS = not significant; * = p<0.05; ** = p<0.01; *** =
p<0.001 The data above demonstrate that ribavirin facilitates or enhances an immune response to an HCV antigen or HCV epitopes. A potent immune response to rNS3 was elicited after immunization with a vaccine composition comprising as little as 1 mg ribavirin and 10 pg of rNS3 antigen. The data above also provide evidence that the amount of ribavirin that is sufficient to facilitate an immune response to an antigen is between 1 and 3 mg per injection for a 25-30g Balb/c mouse. It should be realized, however, that these amounts are intended for guidance only and should not be interpreted to limit the scope of the invention in any way.
Nevertheless, the data shows that vaccine compositions comprising approximately 1 to 3 mg doses of ribavirin induce an immune response that is more than 12 times higher than the immune response elicited in the absence of ribavirin (TABLES 3 and 4). Thus, ribavirin has a significant adjuvant effect on the humoral immune response of an animal and thereby, enhances or facilitates the immune response to the antigen. The example below describes experiments that were performed to better understand the amount of ribavirin needed to enhance or facilitate an immune response to an antigen.

To determine a dose of ribavirin that is sufficient to provide an adjuvant effect, the following experiments were performed. In a first set of experiments, groups of mice (three per group) were immunized with a 20p.g rNS3 alone or a mixture of 20pg rNS3 and O.lmg, lmg, or lOmg ribavirin. The levels of antibody to the antigen were then determined by EIA. The mean endpoint titers at weeks 1 and 3 were plotted and are shown in FIGURE 2. It was discovered that the adjuvant effect provided by ribavirin had different kinetics depending on the dose of ribavirin provided. For example, even low doses (<lmg) of ribavirin were found to enhance antibody levels at week one but not at week three, whereas, higher doses (1-l Omg) were found to enhance antibody levels at week three.
A second set of experiments was also performed. In these experiments, groups of mice were injected with vaccine compositions comprising various amounts of ribavirin and rNS3 and the IgG response in these animals was monitored. The vaccine compositions comprised approximately 100 p,1 phosphate buffered saline and 20 pg rNS3 with or without 0.1 mg, 1.0 mg, or 10 mg ribavirin (Sigma). The mice were bled at week six and rNS3-specific IgG
levels were determined by EIA as described previously. As shown in TABLE 7, the adjuvant effects on the sustained antibody levels were most obvious in the dose range of 1 to 10 mg per injection for a 25-30g mouse.

Immunogen Amount Mouse Endpoint (mg) ID titre ribavirin of rNS3 mixed IgG at with the indicated immunogen week ' Week 1 Week 2 Week 3 20 pg rNS3 None 1 60 360 360 20 pg rNS3 None 2~ 360 360 2160 20 ~.g rNS3None 3 360 2160 2160 Mean 260173 9601039 15601039 , 20 ~g rNS3 0.1 4 2160 12960 2160 20 ~.g rNS30.1 5 60 60 60 20 pg rNS3 0.1 6 <60 2160 2160 20 ~,g rNS31.0 7 <60 60 12960 20 ~.g rNS31.0 8 <60 2160 2160 20 pg rNS3 1.0 9 360 2160 2160 Mean 360 146011212 57606235 20 pg rNS3 10.0 10 360 12960 77760 20 pg rNS3 10.0 11 <60 2160 12960 20 ~,g rNS3I0.0 12 360 2160 2160 Mean 360 576016235 3096040888 i In a third set of experiments, the adjuvant effect of ribavirin after primary and booster injections was investigated. In these experiments, mice were given two intraperitoneal injections of a vaccine composition comprising 10 pg rNS3 with or without ribavirin and the IgG subclass responses to the antigen was monitored, as before. Accordingly, mice were immunized with 100 p.1 phosphate buffered containing 10 p.g recombinant NS3 alone, with or without 0.1 or 1.0 mg ribavirin (Sigma) at weeks 0 and 4. The mice were bled at week six and NS3-specific IgG
subclasses were determined by EIA as described previously. As shown in TABLE
8, the addition of ribavirin to the immunogen prior to the injection does not change the IgG
subclass response in the NS3-specific immune response. Thus, the adjuvant effect of a vaccine composition comprising ribavirin and an antigen can not be explained by a shift in the ThI/Th2-balance. It appears that another mechanism may be responsible for the adjuvant effect of ribavirin.

Immunogen Amount Mouse Endpoint (mg) ID titre ribavirin of indicated mixed NS3 with the IgG
immunogen subclass IgG 1 IgG2a IgG2b IgG3 p.g None 1 360 60 <60 60 rNS3 10 pg rNS3None 2 360 <60 <60 60 10 p.g None 3 2160 60 <60 360 rNS3 Mean 9601039 60 - 160173 10 pg rNS30.1 4 360 <60 <60 60 10 p.g 0.1 5 60 <60 <60 <60 rNS3 10 pg rNS30.1 6 2160 60 60 360 10 p.g 1.0 7 2160 <60 <60 60 rNS3 10 p,g 1.0 8 360 <60 <60 <60 rNS3 10 pg rNS31.0 9 2160 <60 <60 60 Mean 15601039- - 60 5 The data presented in this example further verify that ribavirin can be administered as an adjuvant and establish that that the dose of ribavirin can modulate the kinetics of the adjuvant effect. The example below describes another assay that was performed to evaluate the ability of ribavirin to enhance or facilitate an immune response to an antigen.

10 This assay can be used with any ribavirin derivative or combinations of ribavirin derivatives to determine the extent that a particular vaccine formulation modulates a cellular immune response. To determine CD4+ T cell responses to a ribavirin-containing vaccine, groups of mice were immunized s.c. with either 100p.g rNS3 in PBS or 100pg rNS3 and lmg ribavirin in PBS. The mice were sacrificed ten days post-immunization and their lymph nodes were harvested 1 S and drained. lyz vitro recall assays were then performed. (See e.g., Hultgren et al., J Gezz Yirol.
79:2381-91 (1998) and Hultgren et al., Cli~z. Diagn. Lab. Iznzzaunol. 4:630-632 (1997)). The amount of CD4+ T cell proliferation was determined at 96 h of culture by the incorporation of [3H]
thymidine.
As shown in FIGURE 3, mice that were immunized with 100wg rNS3 mixed with lmg ribavirin had a much greater T cell proliferative response than mice that were immunized with 1001Zg rNS3 in PBS. These data provide additional evidence that ribavirin enhances or facilitates a cellular immune response (e.g., by promoting the effective priming of T
cells). The section below discusses some of the antigens and epitopes that can be used with the embodiments described herein.
Antigens and epitopes Virtually any antigen that can be used to generate an immune response in an animal can be combined with ribavirin so as to prepare the compositions described herein.
That is, antigens that can be incorporated into such compositions (e.g., vaccines) comprise bacterial antigens or epitopes, fungal antigens or epitopes, plant antigens or epitopes, mold antigens or epitopes, viral antigens or epitopes, cancer cell antigens or epitopes, toxin antigens or epitopes, chemical antigens or epitopes, and self antigens or epitopes. Although many of these molecules induce a significant immune response without an adjuvant, ribavirin can be administered in conjunction with or combined with "strong" or "weak" antigens or epitopes to enhance or facilitate the immune response to said antigen or epitope. In addition, the use of ribavirin as an adjuvant may allow for the use of lesser amounts of antigens while retaining immunogenicity.
In addition to peptide antigens, nucleic acid-based antigens can be used in the vaccine compositions described herein. Various nucleic acid-based vaccines are known and it is contemplated that these compositions and approaches to immunotherapy can be augmented by reformulation with ribavirin (See e.g., U.S. Pat. No. 5,589,466 and 6,235,888). By one approach, for example, a gene encoding a polypeptide antigen of interest is cloned into an expression vector capable of expressing the polypeptide when introduced into a subject. The expression construct is introduced into the subject in a mixture of ribavirin or in conjunction with ribavirin (e.g., ribavirin is administered shortly after the expression construct at the same site).
Alternatively, RNA
encoding a polypeptide antigen of interest is provided to the subject in a mixture with ribavirin or in conjunction with ribavirin.
Where the antigen is to be DNA (e.g., preparation of a DNA vaccine composition), suitable promoters include Simian Virus 40~(SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine and human metalothionein can be used. Examples of polyadenylation signals useful with some embodiments, especially in the production of a genetic vaccine for humans, include but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal, which is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for gene expression, other elements may also be included in a gene construct. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human actin, human myosin, human hemoglobin, human muscle creative and viral enhancers such as those from CMV, RSV and EBV.
Gene constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, CA) contain the Epstein Barr virus origin of replication and nuclear antigen EBhIA-1 coding region, which produces high copy episomal replication without integration. All forms of DNA, whether replicating or non-replicating, which do not become integrated into the genome, and which are expressible, can be used. The example below describes the use of a composition comprising a nucleic acid-based antigen and ribavirin.

The following describes the immunization of an animal with a vaccine comprising a nucleic acid-based antigen and ribavirin. Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3m1 of 2.5% Avertin. A l.Scm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. One group of mice are injected with approximately 20:g of an expression construct having the gp-120 gene, driven by a cytomegalovirus (CMV) promotor and second group of mice are injected with approximately S:g of capped in vitro transcribed RNA (e.g., SP6, T7, or T3 (Ambion)) encoding gp-120. These two groups are controls. A third group of mice is injected with approximately 20:g of the expression vector having the gp-120 gene and the CMV promoter mixed with lmg of ribavirin and a fourth group of mice is injected with approximately S:g of capped in vitro transcribed RNA mixed with lrng ribavirin. The vaccines are injected in O.lml of solution (PBS) in a 1 cc syringe through a 27 gauge needle over one minute, approximately O.Scm from the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for future localization, and the skin is then closed with stainless steel clips.
Blood samples are obtained prior to the injection (Day 0) and up to more than 40 days post injection. The serum from each sample is serially diluted and assayed in a standard ELISA
technique assay for the detection of antibody, using recombinant gp-120 protein made in yeast as the antigen. Both IgG and IgM antibodies specific for gp-120 will be detected in all samples, however, groups three and four, which contained the ribavirin, will exhibit a greater immune response to the gp-120 as measured by the amount and/or titer of antibody detected in the sera.
Preferred embodiments of the invention comprise ribavirin and a viral antigen or an epitope present on a virus, preferably a hepatitis virus. Compositions comprise, for example, ribavirin and an HAV antigen, HBV ~ ntigen, HCV antigen or any combination of these antigens or epitopes present on one or more of these viruses. The hepatitis antigens can be peptides or nucleic acids. Compositions that can be used to vaccinate against HAV infection, for example, comprise ribavirin and an HAV peptide with a length of at least 3-10 consecutive amino acids, 10-50 consecutive amino acids, 50-100 consecutive amino acids, 100-200 consecutive amino acids, 200-400 consecutive amino acids, 400-800 consecutive amino acids, 800-1200 consecutive amino acids, 1200-1600 consecutive amino acids, 1600-2000 consecutive amino acids, and 2000-2227 consecutive amino acids of SEQ ID. NO.: 12.
Additionally, compositions comprising ribavirin and a nucleic acid encoding one or more of the HAV peptides, described above, can be used to treat or prevent HAV
infection. Preferred nucleic acid-based antigens include a nucleotide sequence of at least 9 consecutive nucleotides of an HAV sequence (e.g., SEQ. ID. NO.: 15). That is, a nucleic acid based antigen can comprise at least 9-25 consecutive nucleotides, 25-50 consecutive nucleotides, 50-100 consecutive nucleotides, 100-200 consecutive nucleotides, 200-500 consecutive nucleotides, 500-1000 consecutive nucleotides, 1000-2000 consecutive nucleotides, 2000-4000 consecutive nucleotides, 4000-8000 consecutive nucleotides, and 8000-9416 consecutive nucleotides of SEQ. ID.
NO.: 15 or an RNA
that corresponds to these sequences.
Similarly, preferred HBV vaccine embodiments comprise ribavirin and a HBV
peptide of at least 3 consecutive amino acids o~ HBsAg (SEQ. ID. NO.: 10) or HBcAg and HBeAg (SEQ.
ID. NO.: 11). That is, some embodiments have ribavirin and a HBV peptide with a length of at least 3-10 consecutive amino acids, 10-50 consecutive amino acids, 50-100 consecutive amino acids, 100-150 consecutive amino acids, 150-200 consecutive amino acids, and consecutive amino acids of either SEQ. ID. NO.: 10 or SEQ. ID. NO.: 11.
Additionally, compositions comprising ribavirin and a nucleic acid encoding one or more of the HBV peptides, described above, can be used to treat or prevent HBV
infection. Preferred nucleic acid-based antigens include a nucleotide sequence of at least 9 consecutive nucleotides of an HBV (e.g., SEQ. ID. N0.:14). That is, a nucleic acid based antigen can comprise at least 9-25 consecutive nucleotides, 25-50 consecutive nucleotides, 50-100 consecutive nucleotides, 100-200 consecutive nucleotides, 200-500 consecutive nucleotides, 500-1000 consecutive nucleotides, 1000-2000 consecutive nucleotides, 2000-4000 consecutive nucleotides, 4000-8000 consecutive nucleotides, and 8000-9416 consecutive nucleotides of SEQ. ID. NO.: 14 or an RNA that corresponds to these sequences. The example below describes the use of ribavirin in conjunction with a commercial HBV vaccine pre~Saration.

The adjuvant effect of ribavirin was tested when mixed with two doses of a commercially available vaccine containing HBsAg and alum. (Engerix, SKB). Approximately 0.2pg or 2pg of Engerix vaccine was mixed with either PBS or lmg ribavirin in PBS and the mixtures were injected intra peritoneally into groups of mice (three per group). A booster containing the same mixture was given on week four and all mice were bled on week six. The serum samples were diluted from 1:60 to 1:37500 and the dilutions were tested by EIA, as described above, except that purified human HBsAg was used as the solid phase antigen. As shown in TABLE 9, vaccine formulations having ribavirin enhanced the response to 2pg of an existing vaccine despite the fact that the vaccine already contained alum. That is, by adding ribavirin to a suboptimal vaccine dose (i.e., one that does not induce detectable antibodies alone) antibodies became detectable, providing evidence that the addition of ribavirin allows for the use of lower antigen amounts in a vaccine formulation without compromising the immune response.

Endpoint Week antibody titer to HBsAg in EIA

0.02pg 0.2pg Engerix Engerix No lmg No lmg ribavirin ribavirin ribavirin ribavirin #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 6 <60 <60 <60 <60 <60 <60 <60 <60 <60 300 60 <60 Some HCV vaccine compositions comprise ribavirin and a HCV peptide of at least consecutive amino acids of SEQ. ID. NO.: 1 or a nucleic acid encoding said HCV
peptide. That is, a vaccine composition can comprise ribavirin and one or more HCV peptides with a length of at least 3-10 consecutive amino acids, 10-50 consecutive amino acids, 50-100 consecutive amino acids, 100-200 consecutive amino acids, 200-400 consecutive amino acids, 400-80'0 consecutive amino acids, 800-1200 consecutive amino acids, 1200-1600 consecutive amino acids, 1600-2000 consecutive amino acids, 2000-2500 consecutive amino acids, and 2500-3011 consecutive amino acids of SEQ. ID. NO.: 1 or a nucleic acid encoding one or more of said fragments.
Preferred HCV compositions comprise ribavirin and a peptide of at least 3 consecutive amino acids of HCV core protein (SEQ. ID. NO.: 2), HCV E1 protein (SEQ. ID.
NO.; 3), HCV
E2 protein (SEQ. ID. NO.: 4), HCV NS2 (SEQ. ID. NO.: 5), HCV NS3 (SEQ. ID.
NO.: 6), HCV
NS4A (SEQ. ID. NO.: 7), HCV NS4B (SEQ. ID. NO.: 8), or HCV NSSAB (SEQ. ID.
NO.: 9) or peptides consisting of combinations of these domains. That is, preferred HCV
vaccines comprise ribavirin and a peptide with a length o~ at least 3-10 consecutive amino acids, 10-50 consecutive amino acids, 50-100 consecutive amino acids, 100-200 consecutive amino acids, consecutive amino acids, 400-800 consecutive amino acids, and 800-1040 consecutive amino acids of any one or more of (SEQ. ID. NOs.: 2-9). These domains correspond to amino acid residues 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-1711, 1712-1971, or 1972-3011 of SEQ. ID.
NO.: 1. Thus, preferred embodiments also include one or more of 1-182, 183-379, 380-729, 730-1044, 1045-1657, 1658-171 l, 1712-1971, or 1972-3011 of SEQ. ID. NO.: 1 or fragments thereof.
Vaccine compositions comprising ribavirin and a nucleic acid encoding one or more of the peptides described above are also embodiments. Preferred nucleic acid-based antigens include a nucleotide sequence of at least 9 consecutive nucleotides of HCV (SEQ. ID.
NO.: 13). That is, a nucleic acid based antigen can comprise at least 9-25 consecutive nucleotides, 25-50 consecutive nucleotides, 50-100 consecutive nucleotides, 100-200 consecutive nucleotides, consecutive nucleotides, S00-1000 consecutive nucleotides, 1000-2000 consecutive nucleotides, 2000-4000 consecutive nucleotides, 4000-8000 consecutive nucleotides, and 8000-consecutive nucleotides of any one of SEQ. ID. NOs.: 13 or an RNA that corresponds to these sequences. The section below discusses some of the compositions containing ribavirin and an antigen.
Compositions containing ribavi~in a>zd an antigen Compositions (e.g., vaccines) that comprise ribavirin and an antigen or epitope of a pathogen (e.g., virus, bacteria, mold, yeast, and parasite) may contain other ingredients including, but not limited to, adjuvants, binding agents, excipients such as stabilizers (to promote long teen storage), emulsifiers, thickening agents, salts, preservatives, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. These compositions are suitable for treatment of animals either as a preventive measure to avoid a disease or condition or as a therapeutic to treat animals already afflicted with a disease or condition.
Many other ingredients can be present in the vaccine. For example, the ribavirin and antigen can be employed in admixture with conventional excipients (e.g., pharmaceutically acceptable organic or inorganic carriex substances suitable for parenteral, enteral (e.g., oral) or topical application that do not deleteriously react with the ribavirin and/or antigen). Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyetylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Many more suitable carriers are described in Remznizzgtozz's Pharmaceutical Sciences, 15th Edition, Easton:Mack Publishing Company, pages 1405-1412 and 1461-1487(1975) and The National Fonmulary XIV, 14th Edition, Washington, American Pharmaceutical Association (1975).
The gene constructs described herein may be formulated with or administered in conjunction with agents that increase uptake and/or expression of the gene construct by the cells relative to uptake and/or expression of the gene construct by the cells that occurs when the identical genetic vaccine is administered in the absence of such agents. Such agents and the protocols for administering them in conjunction with gene constructs are described in U.S. Ser. No.
08/008,342 filed Jan. 26, 1993, U.S~. Ser. No. 08/029,336 filed Mar. 11, 1993, U.S. Ser. No.
08/125,012 filed Sep. 21, 1993, PCT Patent Application Serial Number PCT/LTS94/00899 filed Jan.
26, 1994, and U.S. Ser. No. 08/221,579 filed Apr. l, 1994. Examples of such agents include:
CaPO4, DEAF dextran, anionic lipids; extracellular matrix-active enzymes;
saponins; lectins;
estrogenic compounds and steroidal hormones; hydroxylated lower alkyls;
dimethyl sulfoxide (DMSO); urea; and benzoic acid esters anilides, amidines, urethanes and the hydrochloride salts thereof such as those of the family of local anesthetics. In addition, the gene constructs are encapsulated within/administered in conjunction with lipids/polycationic complexes.
Vaccines can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with ribavirin or the antigen.
The effective dose and method of administration of a particular vaccine formulation can vary based on the individual patient end the type and stage of the disease, as well as other factors known to those of skill in the art. Therapeutic efficacy and toxicity of the vaccines can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., EDSp (the dose therapeutically effective in 50% of the population). The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for human use. The dosage of the vaccines lies preferably within a range of circulating concentrations that include the EDSp with no toxicity. The dosage varies within this range depending upon the type of ribavirin derivative and antigen, the dosage form employed, the sensitivity of the patient, and the route of administration.
Since ribavirin has been on the market for several years, many dosage forms and routes of administration are known. All known dosage forms and routes of administration can be provided within the context of the embodiments described herein. Preferably, an amount of ribavirin that is effective to enhance an immune response to an antigen in an animal can be considered to be an amount that is sufficient to achieve a blood serum level of antigen approximately 0.25 - l2.Spg/ml in the animal, preferably, about 2.~pg/ml. In some embodiments, the amount of ribavirin is determined according to the body weight of the animal to be given the vaccine.
Accordingly, the amount of ribavirin in a vaccine formulation can be from about 0.1 - 6.Omg/kg body weight. That is, some embodiments have an amount of ribavirin that corresponds to approximately 0.1 -l.Omg/kg, 1.1 - 2.Omg/kg, 2.1 - 3.Om~/kg, 3.1 - 4.Omg/kg, 4.1 - S.Omg/kg, 5.1, and 6.Omglkg body weight of an animal. More conventionally, the vaccines contain approximately 0.25mg - 2000mg of ribavirin. That is, some embodiments have approximately 250pg, SOOpg, lmg, 25mg, SOmg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, SOOmg, SSOmg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, Ig, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, and 2g of ribavirin.
Conventional vaccine preparations can be modified by adding an amount of ribavirin that is sufficient to enhance an immune response to the antigen. That is, existing conventional vaccine formulations can be modified by simply adding ribavirin to the preparation or by administering the conventional vaccine in conjunction with ribavirin (e.g., shortly before or after providing the antigen). As one of skill in the art will appreciate, the amount of antigens in a vaccine can vary depending on the type of antigen and its immunogenicity. The amount of antigens in the vaccines can vary accordingly. Nevertheless, as a general guide, the vaccines can have approximately 0.25mg - Smg, 5-lOmg, 10-100mg, 100-SOOmg, and upwards of 2000mg of an antigen (e.g., a hepatitis viral antigen).
In some approaches described herein, the exact amount of ribavirin and/or antigen is chosen by the individual physician in view of the patient to be treated.
Further, the amounts of ribavirin can be added in combination with or separately from the same or equivalent amount of antigen and these amounts can be adjusted during a particular vaccination protocol so as to provide sufficient levels in light of patient-specific or antigen-specific considerations. In this vein, patient specific and antigen-specific factors that can be taken into account include, but are not limited to, the severity of the disease state of the patient, age, and weight of the patient, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. The next section describes the discovery of a novel HCV gene and the creation of mutant HCV sequences, which can be used with the embodiments described herein.
Novel NS3/4A and mutant NS3/4A sequences A novel nucleic acid and protein corresponding to the NS3/4A domain of HCV was cloned from a patient infected with HCV (SEQ. ID. NOs.: 16 and 17). A Genebank search revealed that the cloned sequence had the greatest homology to HCV sequences but was only 93% homologous to the closest HCV relative (accession no AJ 278830). A truncated mutant of the novel NS3/4A
peptide and NS3/4A mutants, which lack a proteolytic cleavage site, were also created. It was discovered that these novel peptides and nucleic acids encoding said peptides were potent immunogens that can be mixed with ~ibavirin so as to make a composition that provides a recipient with a potent immune response to HCV. The cloning of the novel NS3/4A domain and the creation of the various NS3/4A mutants is described in the following example.

The NS3l4A sequence was amplified from the serum of an HCV-infected patient (HCV
genotype la) using the Polymerase Chain Reaction (PCR). Total RNA was extracted from serum, cDNA synthesis, and PCR was performed according to standard protocols (Chen M
et al., J. Med.
Viol. 43:223-226 (1995)). The cDNA synthesis was initiated using the antisense primer "NS4KR"
(5'-CCG TCT AGA TCA GCA CTC TTC CAT TTC ATC-3' (SEQ. ID. NO.: 18)). From this cDNA, a 2079 base pair DNA fragment of HCV, corresponding to amino acids 1007 to 1711, which encompasses the NS3 and NS4A genes, was amplified. A high fidelity polymerase (Expand High Fidelity PCR, Boehringer-Mannheim, Mannheim, Germany) was used with the "NS3KF"
primer (5'-CCT GAA TTC ATG GCG CCT ATC ACG GCC TAT-3' (SEQ. ID. NO.: 19) and the NS4KR primer. The NS3KF primer contained a EcoRI restriction enzyme cleavage site and a start t codon and the primer NS4KR contained a Xbal restriction enzyme cleavage site and a stop codon.
The amplified fragment was then sequenced SEQ. ID. NO.: 16. Sequence comparison analysis revealed that the gene fragment was indeed amplified from a viral strain of genotype la.
A computerized BLAST search against the Genbank database using the NCBI
website revealed that the closest HCV homologue was 93% identical in nucleotide sequence.
The amplified DNA fragment was then digested with EcoRI and Xbal, and was inserted into a pcDNA3.1/His plasmid (Invitrogen) digested with the same enzymes. The pcDNA3.l plasmid was then digested with EcoRI and Xba I and the insert was purified using the QiaQuick kit (Qiagen, Hamburg, Germany) and was ligated to a EcoRII~'ba I
digested pVAX
vector (Invitrogen) so as to generatethe NS3/4A-pVAX plasmid.
The rNS3 truncated mutant was obtained by deleting NS4A sequence from the DNA. Accordingly, the NS3 gene sequence of NS3/4A-pVAX was PCR amplified using the primers NS3KF and 3'Notl (5'-CCA CGC GGC CGC GAC GAC CTA CAG-3' (SEQ. ID. NO.:
20)) containing EcoRl and Not I restriction sites, respectively. The NS3 fragment (1850 bp) was then ligated to a EcoRl and Not I digested pVAX plasmid to generate the NS3-pVAX vector.
Plasmids were grown in BL21 E.coli cells. The plasmids were sequenced and were verified by restriction cleavage and the results wer~as to be expected based on the original sequence.
To change the proteolytic cleavage site between NS3 and NS4A, the NS3/4A-pVAX
plasmid was mutagenized using the QUICKCHANGETM mutagenesis kit (Stratagene), following the manufacturer's recommendations. To generate the "TPT" mutation, the plasmid was amplified using the primers 5'-CTGGAGGTCGTCACGCCTACCTGGGTGCTCGTT-3' (SEQ. ID. NO.:

21) and S'-ACCGAGCACCCAGGTAGGCGTGACGACCTCCAG-3' (SEQ. TD. NO.: 22) resulting in NS3/4A-TPT-pVAX. To generate the "RGT" mutation, the plasmid was amplified using the primers S'-CTGGAGGTCGTCCGCGGTACCTGGGTGCTCGTT-3' (SEQ. ID. NO.:
23) and S'-ACCGAGCACCCAGGTACC-GCGGACGACCTCCAG-3' (SEQ. ID. NO.: 24) S resulting in NS3/4A-RGT-pVAX.
All mutagenized constructs were sequenced to verify that the mutations had been correctly made. Plasmids were grown in competent BL21 E. coli. The plasmid DNA used for in vivo injection was purified using Qiagen DNA purification columns, according to the manufacturers instructions (Qiagen GmbH, Hilden, FRG). The concentration of the resulting plasmid DNA was determined spectrophotometrically (Dynaquant, Pharmacia Biotech, Uppsala, Sweden) and the purified DNA was dissolved in sterile phosphate buffer saline (PBS) at concentrations of 1 mg/ml.
P
The amino acid sequences of the wild-type and mutated junctions are shown in TABLE 10. The section below describes several nucleic acids that encode HCV peptides.

Plasmid Deduced amino acid sealuence *NS3/4A-pVAX TKYMTCMSADLEVVTSTWVLVGGVL (SEQ. ID. NO.:
25) NS3/4A-TGT-pVAX TKYMTCMSADLEVVTGTWVLVGGVL (SEQ. ID. NO.:
26) NS3/4A-RGT-pVAX TKYMTCMSADLEVVRGTWVLVGGVL (SEQ. ID. NO.:
27) NS3/4A-TPT-pVAX TKYMTCMSADLEVVTPTWVLVGGVL (SEQ. ID. NO.:
33) NS3/4A-RPT-pVAX TKYMTCMSADLEVVRPTWVLVGGVL (SEQ. ID. NO.:
34) NS3/4A-RPA-pVAX TKYMTCMSADLEVVRPAWVLVGGVL (SEQ. ID. NO.:
35) NS3/4A-CST-pVAX TKYMTCMSADLEVVCSTWVLVGGVL (SEQ. ID. NO.:
36) 2S NS3/4A-CCST-pVAX TKYMTCMSADLEVCCSTWVLVGGVL (SEQ. ID. NO.:
37) NS3/4A-SSST-pVAX TKYMTCMSADLEVSSSTWVLVGGVL (SEQ. ID. NO.:
38) NS3/4A-SSSSCST-pVAX TKYMTCMSADSSSSCSTWVLVGGVL (SEQ. ID. NO.:
39) NS3A/4A-VVVVTST-pVAX
TKYMTCMSADVVVVTSTWVLVGGVL
(SEQ. ID. NO.: 40) NSS-pVAX HSEDVVCCSMSYTWTG (SEQ. ID. NO.: 41) NSSA/B-pVAX SSEDVVCCSMWVLVGGVL (SEQ. ID. NO.: 42) *The wild type sequence for the NS3/4A fragment is NS3/4A-pVAX. The NS3/4A
breakpoint is identified by underline, wherein the P1 position corresponds to the first Thr (T) and the P1' position corresponds to the next following amino acid the NS3/4A-pVAX
sequence, In the wild type NS3/4A sequence the NS3 protease cleaves between the P1 and P 1 ' positions.
Nucleic acids encoding HCVpeptides The nucleic acid embodiments include nucleotides encoding the HCV peptides described herein (e.g., SEQ. ID. NO.: 17, 29, 31, 32, and 43-49) or fragments thereof at least 4, 6, 8, 10, 12, 1S, or 20 amino acids in length (e.g., SEQ. TD. NOs.: 25-27, and 33-42). Some embodiments for example, include genomic DNA, RNA, and cDNA encoding these HCV peptides. The HCV
nucleotide embodiments not only include the DNA sequences shown in the sequence listing (e.g., SEQ. ID. NO.: 16) but also include nucleotide sequences encoding the amino acid sequences shown in the sequence listing (e.g., SEQ. ID. NO.: 17) and any nucleotide sequence that hybridizes to the DNA sequences shown in the sequence listing under stringent conditions (e.g., hybridization to filter-bound DNA in 0.5 M NaHPOq, 7.0% sodium dodecyl sulfate (SDS), 1 mM
EDTA at 50°C) and washing in 0.2 X SSC/0.2% SDS at 50°C and any nucleotide sequence that hybridizes to the DNA sequences that encode an amino acid sequence provided in the sequence listing (SEQ. ID. NOs.: 17) under less stringent conditions (e.g., hybridization in 0.5 M NaHPOq., 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 37°C and washing in 0.2X SSC/0.2% SDS at 37°C).
The nucleic acid embodiments also include fragments, modifications, derivatives, and variants of the sequences described above. Desired embodiments, for example, include nucleic acids having at least 12 consecutive bases of one of the novel HCV sequences or a sequence complementary thereto and preferred fragments include at least 12 consecutive bases of a nucleic acid encoding the NS3/4A molecule of SEQ. ID. NO.: 17 or a sequence complementary thereto.
In this regard, the nucleic acid embodiments of the invention can have from 12 to approximately 2079 consecutive nucleotides. Some DNA fragments of the invention, for example, include nucleic acids having at least 12-15, 15-20, 20-30, 30-50, 50-100, 100-200, 200-500, 500-1000, 1000-1500, 1500-2079 consecutive nucleotides of SEQ. ID. NO.: 16 or a complement thereof. The nucleic acid embodiments can also be altered by mutation such as substitutions, additions, or deletions. Due to the degeneracy of nucleotide coding sequences, for example, other DNA sequences that encode substantially the same HCV amino acid sequence as depicted in SEQ.
ID. NOs: 17 can be used in some embodiments. These include, but are not limited to, nucleic acid sequences encoding all or portions of NS3/4A (SEQ. ID. NO.: 16) or nucleic acids that complement all or part of this sequence that have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change, or a functionally non-equivalent amino acid residue within the sequence, thus producing a detectable change.
By using the nucleic acid sequences described above, probes that complement these molecules can be designed and manufactured by oligonucleotide synthesis.
Desirable probes comprise a nucleic acid sequence of (SEQ. ID. NO.: 16) that is unique to this HCV isolate. These probes can be used to screen cDNA from patients so as to isolate natural sources of HCV, some of which may be novel HCV sequences in themselves. Screening can be by filter hybridization or by PCR, for example. By filter hybridization, the labeled probe preferably contains at least I S-30 base pairs of the nucleic acid sequence of (SEQ. ID. NO.: 16) that is unique to to this NS3/4A
peptide. The hybridization washing conditions used are preferably of a medium to high stringency.
The hybridization can be performed in O.SM NaHP04, 7.0% sodium dodecyl sulfate (SDS), 1 mM
EDTA at 42°C overnight and washing can be performed in 0.2X SSC/0.2%
SDS at 42°C. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A
LaboratorYManual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Bioloay, Green Publishing Associates and Wiley Interscience, N.Y.
' HCV nucleic acids can also be isolated from patients infected with HCV using the nucleic acids described herein. (See also Example ~. Accordingly, RNA obtained from a patient infected with HCV is reverse transcribed and the resultant cDNA is amplified using PCR
or another amplification technique. The primers are preferably obtained from the NS3/4A
sequence (SEQ.
ID. NO.: 16).
For a review of PCR technology, see Molecular Cloning to Genetic Engineering, White, B.A.
Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and the publication entitled "PCR Methods and Applications" (1991, Cold Spring Harbor Laboratory Press).
For amplification of mRNAs, it is within the scope of the invention to reverse transcribe mRNA
into cDNA followed by PCR (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Patent No.
5,322,770. Another technique involAes the use of Reverse Transcriptase Asymmetric Gap Ligase Chain Reaction (RT-AGLCR), as described by Marshall R.L. et al. (PCR Methods and Applicatiofis 4:80-84, 1994).
Briefly, RNA is isolated, following standard procedures. A reverse transcription reaction is performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment as a primer of first strand synthesis. The resulting RNA/DNA hybrid is then "tailed" with guanines using a standard terminal transferase reaction. The hybrid a5 then digested with RNAse H, and second strand synthesis is primed with a poly-C primer.
Thus, cDNA
sequences upstream of the amplified fragment are easily isolated. For a review of cloning strategies which can be used, see e.g., Sambrook et al., 1989, supra.
In each of these amplification procedures, primers on either side of the sequence to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerise, such as Taq polymerise, Pfu polymerise, or Vent polymerise. The nucleic acid in the sample is denatured and the primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are then extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR

has further been described in several patents including US Patents 4,683,195, 4,683,202 and 4,965,188.
The primers are selected to be substantially complementary to a portion of the nucleic acid sequence of (SEQ. ID. NO.: 16) that is unique to this NS3/4A molecule, thereby allowing the sequences between the primers to be amplified. Preferably, primers axe at least 16-20, 20-25, or 25-30 nucleotides in length. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The G+C content of the amplification primers described herein preferably range between 10 and 75 %, more preferably between 35 and 60 %, and most preferably between 40 and 55 %.
The appropriate length for primers under a particular set of assay conditions can be empirically determined by one of skill in the art.
The spacing of the primers relates to the length of the segment to be amplified. In the context of the embodiments described herein, amplified segments carrying nucleic acid sequence encoding HCV peptides can range in size from at least about 25 by to the entire length of the HCV
genome. Amplification fragments from 25-1000 by are typical, fragments from 50-1000 by are preferred and fragments from 100-600 by are highly preferred. It will be appreciated that amplification primers can be of any sequence that allows for specific amplification of the NS3/4A
region and can, for example, include modifications such as restriction sites to facilitate cloning.
The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of an HCV peptide. The PCR fragment can then be used to isolate a full length eDNA clone by a variety of methods. For example, the amplified fragment can be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library.
Alternatively, the labeled fragment can be used to isolate genomic clones via the screening of a genomic library.
Additionally, an expression library can be constructed utilizing cDNA
synthesized from, for example, RNA isolated from an infected patient. In this manner, HCV
geneproducts can be isolated using standard antibody screening techniques in conjunction with antibodies raised against i the HCV gene product. (For screening techniques, see, for example, Harlow, E.
and Lane, eds., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor.) Embodiments also include (a) DNA vectors that contain any of the foregoing nucleic acid sequences and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing nucleic acid sequences operatively associated with a regulatory element that directs the expression of the nucleic acid; and (c) genetically engineered host cells that contain any of the foregoing nucleic acid sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. These recombinant constructs are capable of replicating autonomously in a host cell. Alternatively, the recombinant constructs can become integrated into the chromosomal DNA of a host cell. Such recombinant polynucleotides typically comprise an HCV genomic or cDNA polynucleotide of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising these sequences and complements thereof that are not naturally occurring are provided.
Although nucleic acids encoding an HCV peptide or nucleic acids having sequences that complement an HCV gene as they appear in nature can be employed, they will often be altered, e.g., by deletion, substitution, or insertion and can be accompanied by sequence not present in humans. As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include, but are not limited to, the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast -mating factors.
In addition, recombinant HCV peptide-encoding nucleic acid sequences and their complementary sequences can be engineered so as to modify their processing or expression. For example, and not by way of limitation, the HCV nucleic acids described herein can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence can be inserted upstream of HCV peptide-encoding sequences so as to permit secretion of the peptide and thereby facilitate harvesting or bioavailability. Additionally, a given HCV nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, andlor termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitf~o modification. (See Example 6). Any technique for mutagenesis known i in the art can be used, including but not limited to, ira vitro site-directed mutagenesis. (Hutchinson et al., J. Biol. Chem., 253:6551 (1978)).
Further, nucleic acids encoding Bother proteins or domains of other proteins can be joined to nucleic acids encoding an HCV peptide so as to create a fusion protein.
Nucleotides encoding fusion proteins can include, but are not limited to, a full length NS3/4A
sequence (SEQ. ID. NO.:
16), a truncated NS3/4A sequence or a peptide fragment of an NS3/4A sequence fused to an unrelated protein or peptide, such as for example, poly histidine, hemagglutinin, an enzyme, fluorescent protein, or luminescent protein, as discussed below.

Surprisingly, it was discovered that the NS3-pVAX and NS3/4A-pVAX vectors were capable of eliciting a potent immune response when injected into an immunocompetent mammal.
The example below describes these experiments in greater detail.

To determine whether a humoral immune response was elicited by the NS3-pVAX
and NS3/4A-pVAX vectors, the expression constructs described in Example 6 were purified using the Qiagen DNA purification system, according to the manufacturer's instructions and the purified DNA vectors were used to immunize~groups of four to ten Balb/c mice. The plasmids were injected directly into regenerating tibialis anterior (TA) muscles as previously described (Davis et al., Human Gene Therapy 4(6):733 (1993)). In brief, mice were injected intramuscularly with 50 pl/TA of O.OlmM cardiotoxin (Latoxan, Rosans, France) in 0.9% sterile NaCI.
Five days later, each TA muscle was injected with 50 p1 PBS containing either rNS3 or DNA.
Inbred mouse strains C57/BL6 (H-2b) Balb/C (H-2d), and CBA (H-2k) were obtained from the breeding facility at Mollegard Denmark, Charles River Uppsala, Sweden, or B&K Sollentuna Sweden. All mice were female and were used at 4-8 weeks of age. For monitoring of humoral responses, all mice received a booster injection of 50 p1 /TA of plasmid DNA
every fourth week.
In addition, some mice were given recombinant NS3 (rNS3) protein, which was purified as described herein. The mice receiving rNS3 were immunized no more than twice.
All mice were bled twice a month.
Enzyme immunosorbent assays (EIAs) were used to detect the presence of murine antibodies. These assays were performed essentially as described in (Chen et al., Hepatology 28(1): 219 (1998)). Briefly, rNS3 vas passively adsorbed overnight at 4°C to 96-well microtiter plates (Nunc, Copenhagen, Denmark) at 1 pg/ml in 50 mM sodium carbonate buffer (pH 9.6). The plates were then blocked by incubation with dilution buffer containing PBS, 2%
goat serum, and 1% bovine serum albumin for one hour at 37°C. Serial dilutions of mouse sera starting at 1:60 were then incubated on the plates for one hour. Bound murine serum antibodies were detected by an alkaline phosphatase conjugated goat anti-mouse IgG (Sigma Cell Products, Saint Louis, MO) followed by addition of the substrate pNPP (1 tablet/Sml of 1M Diethanol amine buffer with 0.5 mM MgCl2). The reaction was stopped by addition of 1M NaOH and absorbency was read at 405 nm.
After four weeks, four out of five mice immunized with NS3/4A-pVAX had developed NS3 antibodies, whereas one out of five immunized with NS3-pVAX had developed antibodies (FIGURE 4). After six weeks, four out of five mice immunized with NS3/4A-pVAX
had developed high levels (>104) of NS3 antibodies (mean levels 108004830) and one had a titer of 2160. Although all mice immunized with NS3-pVAX developed NS3 antibodies, none of them developed levels as high as that produced by the NS3/4A-pVAX construct (mean levels 1800805). The antibody levels elicited by the NS3/4A fusion construct were significantly higher than those induced by NS3-pVAX at six weeks (mean ranks 7.6 v.s 3.4, p<0.05, Mann-Whitney rank sum test, and p<0.01, Students t-test). Thus, immunization with either NS3-pVAX or NS3/4A-pVAX resulted in the production of anti-NS3 antibodies, but the NS3/4A
fusion gene was a more potent immunogen. The example below describes experiments that were performed to determine if the NS3/4A-TPT-pVAX construct could elicit a potent immune response.

To test if the enhanced imrnunogenicity of NS3l4A could be solely attributed to the presence of NS4A, or if the NS3/4A fusion protein in addition had to be cleaved at the NS3/4A
junction, new experiments were performed. In a first experiment, the immunogenicity of the NS3-pVAX, NS3/4A-pVAX, and NS3/4A-TPT-pVAX vectors were compared in Balb/c mice.
Mice were immunised on week 0 as described above, and, after two weeks, all mice were bled and the presence of antibodies to NS3 at a serum dilution of 1:60 was determined (TABLE 11). Mice were bled again on week 4. Although, the NS3/4A-TPT-pVAX vector was comparable to the NS3-pVAX vector (4/10 vs. 0/10; NS, Fisher's exact test), the NS3/4A pVAX vector continued to be the most potent immunogen. Thus, all of the HCV constructs that were introduced into mice were capable of eliciting an immune response against NS3, however, the NS4A
sequence and a functional proteolytic cleavage site 'between the NS3 and NS4A sequences provided for a more potent immune response.

Weeks from No. of antibody 1st responders to the respective immunogen after one 100~g i.m immunizationimmunization NS3-pVAX NS3l4A-pVAX NS3/4A-TPT-pVAX

0/10 20/20 (390639) 4 (<60) (24153715) 50% >102 55% > 103 10% > 103 10% > 104 During the chronic phase of infection, HCV replicates in hepatocytes, and spreads within the liver. A major factor in combating chronic and persistent viral infections is the cell-mediated immune defense system. CD4+ and CD8+ lymphocytes infiltrate the liver during the chronic phase of HCV infection, but they are incapable of clearing the virus or preventing liver damage. In addition, persistent HCV infection is associated with the onset of hepatocellular carcinoma (HGC).
The examples below describe experiments that were performed to determine whether the NS3 and NS3/4A construct were capable of eliciting a T-cell mediated immune response against NS3.

To study whether the constructs described above were capable of eliciting a cell-mediated response against NS3, an i~a vivo tumor growth assay was perfomed. To this end, an SP2/0 tumor cell tine stabiy transfected with the NS3l4A gene was made. The pcDNA3.1 plasmid containing the NS3/4A gene was linearized by BgIII digestion. A total of 5pg linearized plasmid DNA was mixed with 60pg transfection reagent (Superfect, Qiagen, Germany) and the mixture was added to a 50% confluent layer of SP2/0 cells in a 35 mm dish. The transfected SP2/0 cells (NS3/4A-SP2/0) were grown for 14 days in the presence of 800pg/ml geneticin and individual clones were isolated. A stable NS3/4A-expressing SP2/0 clone was identified using PCR and RTPCR. The cloned cell line was maintained in DMEM containing 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin.
The in vivo growth kinetics of the SP2/0 and the NS3/4A-SP2/0 cell lines were then evaluated in Balb/c mice. Mice were injected subcutaneously with 2 x 106 tumor cells in the right flank. Each day the size of the tumor was determined through the skin. The growth kinetics of the two cell lines was comparable. For example, the mean tumor sizes did not differ between the two cell lines at any time point. (See TABLE 12). The example below describes experiments that were performed to determine whether mice immunized with the NS3/4A constructs had developed a T-cell response against NS3.

Mouse Tumor Maximum ID cell in line vivo tumor size at indicated time point 1 SP2/0 1.6 2.5 4.5 6.0 10.0 10.5 11.0 12.0 12.0 2 SP2/0 I .0 1.0 2.0 3.0 7.5 7.5 8.0 11.5 11.5 ~

3 SP2/0 2.0 5.0 7.5 8.0 11.0 11.5 12.0 12.0 13.0 4 SP2/0 4.0 7.0 8.0 10.0 13.0 15.0 16.5 16.5 17.0 5 SP2/0 I.0 1.0 3.0 4.0 5.0 6.0 6.0 6.0 7.0 Mouse Tumor Maximum ID ita vivo tumor size at indicated time point cell line Group 1,92 3.3 5.0 6.2 9.3 10.1 10.7 11.6 12.1 mean 6 NS3/4A-1.0 2.0 3.0 3.5 4.0 5.5 6.0 7.0 8.0 7 NS3/4A-2.0 2.5 3.0 5.0 7.0 9.0 9.5 9.5 11.0 8 NS3/4A-1.0 2.0 3.5 3.5 9.5 11.0 12.0 14.0 14.0 9 NS3/4A-1.0 I.0 2.0 6.0 11.5 13.0 14.5 16.0 18.0 NS3/4A-3.5 6.0 7.0 10.5 15.0 15.0 15.0 15.5 20.0 Group 1,7 2.7~ 3.7 5.7 9.4 10.7 11.4 12.4 14.2 mean p-value 0,77360.69180.40270.79030.96700.79860.79270.75080.4623 of student's t-test comparison between group means To examine whether a T-cell response is elicited by the NS3/4A immunization, the 5 capacity of an immunized mouse's immune defense system to attack the NS3-expressing tumor cell line was assayed. The protocol for testing for in vivo inhibition of tumor growth of the SP2/0 myeloma cell line in Balb/c mice has been described in detail previously (Encke et al., J. Imnaunol.
161:4917 ( 1998)). Inhibition of tumor growth in this model is dependent on the priming of cytotoxic T lymphocytes (CTLs). Briefly, groups of ten mice were immunized i.m. five times with 10 one month intervals with either 100pg NS3-pVAX or 100 pg NS3/4A-pVAX. Two weeks after the last immunization 2 x 106 SP2/0 or NS3/4A-SP2/0 cells were injected into the right flank of each mouse. Two weeks later the mice were sacrificed and the maximum tumor sizes were measured.
There was no difference between the mean SP2/0 and NS3l4A-SP2/0 tumor sizes in the NS3-pVAX immunized mice (See TABLE )~,3).

Mouse Immunogen Dose Tumor cell Tumor Maximum tumor ID (p,g) line growth size (mm) 1 NS3-pVAX 100 SP210 Yes 5 2 NS3-pVAX 100 SP2/0 Yes 15 3 NS3-pVAX 100 SP2/0 No -4 NS3-pVAX 100 SP2/0 Yes 6 NS3-pVAX 100 SP2/0 Yes 13 Group 4/5 9.754.992 total 6 NS3-pVAX 100 NS3/4A-SP2/0Yes 9 ~

7 NS3-pVAX 100 NS3/4A-SP2/0Yes 8 8 NS3-pVAX 100 NS3/4A-SP2/0Yes 7 9 NS3-pVAX 100 NS3/4A-SP2/0No -NS3-pVAX 100 NS3/4A-SP2/0No -3/5 8.001.00 Note: Statistical analysis (StatView): Student's t-test on maximum tumor size.
Y-values <
0.05 are considered significant.

Unpaired t-test for Max diam Grouping Variable: Column 1 Hypothesized Difference = 0 10 Row exclusion: NS3DNA-Tumor-001213 Mean Diff. DF t-Value P-Value NS3-sp2, NS3-spNS3 ~ 1.750 5~ 0.58 0.584 Group Info for Max diam Grouping Variable: Column 1 Row exclusion: NS3DNA-Tumor-001213 Count Mean - Variance _S_td. Dev. Std. Err NS3-sp2 4 9.750 24.917 4.992 2.496 NS3-spNS3 ~ 8.000 1.000 1.000 0.57 In the next set of experiments, the inhibition of SP2/0 or NS3/4A-SP2/0 tumor growth was evaluated in NS3/4A-pVAX immunized Balb/c mice. In mice immunized with the NS3/4A-pVAX
plasmid the growth of NS3/4A-SP2/0 tumor cells was significantly inhibited as compared to growth of the non-transfected SP2/0 cells. (See TABLE 14). Thus, NS3/4A-pVAX
immunization elicits CTLs that inhibit growth of cells expressing NS3/4A i~ vivo. The example below describes experiments that were performed to analyze the efficiency of various NS3 containing compositions in eliciting a cell-mediated response to NS3.

Mouse Immunogen Dose Tumor cell Tumor Maximum tumor ID (p,g) line growth size (mm) 11 NS3/4A-pVAX 100 SP2/0 No -12 NS3/4A-pVAX 100 SP2/0 Yes 24 13 NS3/4A-pVAX 100 SP2/0 Yes 9 14 NS3/4A-pVAX 100 SP2/0 Yes 11 15 NS3/4A-pVAX 100 SP2/0 Yes 25 - 4/5 17.258.421 16 NS3/4A-pVAX 100 NS3/4A-SP2/0No -17 NS3/4A-pVAX 100 NS3/4A-SP2/0Yes 9 18 NS3/4A-pVAX 100 NS3/4A-SP210Yes 7 19 NS3/4A-pVAX 100 NS3/4A-SP2/0Yes 5 20 NS3/4A-pVAX 100 NS3/4A-SP2/0Yes 4 4/5 6.252.217 Note: Statistical analysis (StatView): Student's t-test on maximum tumor size.
P-values < 0.05 are considered significant.
Unpaired t-test for Max diam Grouping Variable: Column 1 Hypothesized Difference = 0 Row exclusion: NS3DNA-Tumor-001213 Mean Difif. DF t-Value P-Value NS3/4-sp2, NS3/4-spNS3 ~ --11.000 6 2.526 0.044 Group Info for Max diam Grouping Variable: Column 1 Row exclusion: NS3DNA-Tumor-001213 Count Mean _Variance Std. Dev. S_td. Err NS3/4-sp2 17.250 70.917 8.421 _ 4.211 NS3/4-spNS3 ~~ 6.250 4.917 2.217 ~1-10~

To analyze whether administration of different NS3 containing compositions affected the elicitation of a cell-mediated immune response, mice were immunized with PBS, rNS3, irrelevant DNA or the NS3/4A construct, and tumor sizes were determined, as described above. Only the NS3/4A construct was able to elicit ~ T-cell response sufficient to cause a statistically significant reduction in tumor size (See TABLE 15). The example below describes experiments that were performed to determine whether the reduction in tumor size can be attributed to the generation of NS3-specific T-lymphocytes.

Mouse Immunogen Dose Tumor cell Anti-NS3Tumor Maximum ID (fig) line growthtumor size (mm) 1 NS3-pVAX 10 NS3/4A-SP2/0<60 + 12.0 2 NS3-pVAX IO NS3/4A-SP2/0<60 + 20.0 3 NS3-pVAX 10 NS3/4A-SP2/060 + 18.0 4 NS3-pVAX 10 NS3/4A-SP2i0<60 + 13.0 NS3-pVAX 10 NS3/4A-SP2/0<60 + 17.0 Group 60 5/5 16.0+3.391 mean 6 NS3-pVAX 100 NS3/4A-SP2/02160 + 10.0 7 NS3-pVAX I00 NS3/4A-SP2/0<60 -8 NS3-pVAX 100 NS3/4A-SP2/0<60 -9 NS3-pVAX 100 NS3/4A-SP2/0360 -NS3-pVAX 100 NS3/4A-SP2/0<60 + 12.5 Group 1260 2i5 11.251.768 mean 11 NS3/4A-pVAX10 NS3/4A-SP2/0<60 + 10.0 12 NS3/4A-pVAX10 NS3/4A-SP2/0<60 -13 NS3/4A-pVAX10 NS3/4A-SP2/0<60 -14 NS3/4A-pVAX10 NS3/4A-SP2/0<60 + 13.0 NS3/4A-pVAX10 NS3/4A-SP2/0<60 + 13.5 Group <60 3/5 12.167+1.893 mean 16 NS3/4A-pVAX100 NS3/4A-SP2/060 + 10.0 _ 17 NS3/4A-pVAX100 NS3/4A-SP2/0360 -18 NS3/4A-pVAX100 NS3/4A-SP2/02160 + 8.0 19 NS3/4A-pVAX100 NS3/4A-SP2/02160 + 12.0 NS3/4A-pVAX100 NS3/4A-SP2/02160 + 7.0 Group 1380 4/5 9.25+2.217 mean 36 pl7-pcDNA3 100 NS3/4A-SP2/0<60 + 20.0 37 p17-pcDNA3 100 NS3/4A-SP2/0<60 + 7.0 38 pl7-pcDNA3 100 NS3/4A-SP2/0<60 + 11.0 39 p17-pcDNA3 100 NS3/4A-SP2/0<60 + 15.0 40 p17-pcDNA3 100 NS3/4A-SP2/0<60 + 18.0 Group <60 5/5 14.20+5.263 mean 41 rNS3/CFA 20 NS3/4A-SP2/0>466560 + 13.0 42 rNS3/CFA 20 NS3/4A-SP2/0>466560 -43 rNS3/CFA 20 NS3/4A-SP210>466560 + 3.5 44 rNS3/CFA 20 NS3/4A-SP2/0>466560 + 22.0 45 rNS3/CFA 20'' NS3/4A-SP2/0>466560 + 17.0 Group 466560 4/5 17.333+4.509 mean 46 PBS - NS3/4A-SP2/0<60 + 10.0 47 PBS - NS3/4A-SP2/0<60 + 16.5 48 PBS - NS3/4A-SP21060 + 15.0 49 PBS - NS3/4A-SP2/0<60 + 21.0 50 PBS - NS3/4A-SP2/0<60 + 15.0 51 PBS - NS3/4A-SP2/0<60 -Group 60 5/6 15.50+3.937 mean Note: Statistical analysis (StatView): Student's t-test on maxinnum tumor size. P-values <
0.05 are considered as significant.

Unpaired t-test for Largest Tumor size Grouping Variable: group Hypothesized Difference = 0 Mean Diff. DF t-Value P-Value p17-spa-4, NS3-100-spa-4 2.950 5 .739 .4933 p17-spa-4, NS3/4-10-spa-4 2.033 6 .628 .5532 p17-spa-4, NS3-10-spa-4 -1.800 8 -.643 .5383 p17-spa-4, NS3/4-100-spa-44.950 7 1.742 .1250 p17-spa-4, PBS-spa-4 -1.300 8 -.442 .6700 p17-spa-4, rNS3-spa-4 -3.133 6 -.854 .4259 NS3-100-spa-4, NS3/4-10-spa-4-.917 3 -.542 .6254 NS3-100-spa-4, NS3-10-spa-4-4.750 5 -1.811.1299 NS3-100-spa-4, NS3/4-100-spa-42.000 4 1.092 .3360 NS3-100-spa-4, PBS-spa-4 -4.250 5 -1.408.2183 NS3-100-spa-4, rNS3-spa-4 -6.083 3 -1.744.1795 NS3/4-10-spa-4, NS3-10-spa-4-3.833 6 -1.763.1283 NS3/4-10-spa-4, NS314-100-spa-42.917 5 1.824 .1277 NS3/4-10-spa-4, PBS-spa-4 -3.333 6 -1.344.2274 NS3/4-10-spa-4, rNS3-spa-4-5.167 4 -1.830.1412 NS3-10-spa-4, NS3/4-100-spa-46.750 7 3.416 .0112 NS3-10-spa-4, PBS-spa-4 .500 8 .215 .8350 NS3-10-spa-4, rNS3-spa-4 -1.333 6 -.480 .6480 NS3/4-100-spa-4, PBS-spa-4-6.250 7 -2.814.0260 NS3/4-100-spa-4, rNS3-spa-4-8.083 5 -3.179.0246 PBS-spa-4, rNS3-spa-4 -1.833 6 -.607 .5662 I

To determine whether NS3-specific T-cells were elicited by the NS3/4A
immunizations, an in vitf~o T-cell mediated tumor cell lysis assay was employed. The assay has been described in detail previously (Townsend et al., J. Virol. 71:3365 (1997)). Briefly, groups of five Balb/c mice were immunized three times with 100pg NS3/4A-pVAX i.nz Two weeks after the last injection the mice were sacrificed and splenocy~es were harvested. Re-stimulation cultures with 3 x 106 splenocytes and 3 x 106 NS3/4A-SP2/0 cells were set. After five days, a standard Crs 1-release assay was performed using NS3/4A-SP2/0 or SP2/0 cells as targets. Percent specific lysis was calculated as the ratio between lysis of NS3/4A-SP2/0 cells and lysis of SP2/0 cells. Only mice immunized with NS3/4A-pVAX displayed specific lysis over 10% in four out of five tested mice, using an effector to target ratio of 20:1 (See FIGURES SA and B). Accordingly, mice immunized with NS3/4A exhibited a reduction in cancer cell proliferation and/or NS3/4A
caused the lysis of cancer cells. The section below describes several of the embodied HCV
polypeptides in greater detail.

HCV peptides The nucleic acids encoding the HCV peptides, described in the previous section, can be manipulated using conventional techniques in molecular biology so as to create recombinant constructs that express the HCV peptides. The embodied HCV peptides or derivatives thereof, include but are not limited to, those containing as a primary amino acid sequence all of the amino acid sequence substantially as depicted in the Sequence Listing (SEQ. ID.
NOs.: 17, 29- 32 and 43-49) and fragments thereof at least four amino acids in length (e.g., SEQ.
ID. NOs.: 25-27, and 33-42) including altered sequences~in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
Preferred fragments of a sequence of SEQ. ID. NOs.: 17, 29- 32 and 43-49 are at least four amino acids and comprise amino acid sequence unique to the discovered NS3/4A peptide (SEQ. ID. NO.: 17) including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. The HCV peptides can be, for example, at least 12-15, 15-20, 20-25, 25-50, 50-100, 100-150, 150-250, 250-500 or 500-704 amino acids in length.
Other fragments (e.g., SEQ. ID. NOs.: 25-27, and 33-42) are also aspects of the invention.
Embodiments of the invention also include HCV peptides that are substantially identical to those described above. That is, HCV peptides that have one or more amino acid residues within SEQ. ID. NO.: 17 and fragments thereof that are substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The aromatic amino acids include phenylalanine, tryptophan, and tyrosine.
The HCV peptides described herein can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art such as those set forth by Merrifield et al., J. Ana. Chefn. Soc. 85:2149 (1964), Houghten et al., Pr~oc.
Natl. Acad. Sci. USA, 82:51:32 (1985), Stewart and Young (Solid phase peptide s nt~ hesis, Pierce Chem Co., Rockford, IL (1984), and Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman &
Co., N.Y. Such polypeptides can be synthesized with or without a methionine on the amino terminus. Chemically synthesized HCV peptides can be oxidized using methods set forth in these references to form disulfide bridges.

While the HCV peptides described herein can be chemically synthesized, it can be more effective to produce these polypeptides by recombinant DNA technology. Such methods can be used to construct expression vectors containing the HCV nucleotide sequences described above, for example, and appropriate transcriptidnal and translational control signals.
These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and irz vivo genetic recombination. Alternatively, RNA capable of encoding HCV nucleotide sequences can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford. Accordingly, several embodiments concern cell lines that have been engineered to express the embodied HCV
peptides. For example, some cells are made to express the HCV peptides of (SEQ. ID. NOs.: 17, 29- 32 and 43-49) or fragments of these molecules.
A variety of host-expression vector systems can be utilized to express the embodied HCV
peptides. Suitable expression systems include, but are not limited to, microorganisms such as bacteria (e.g., E. coli or B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing HCV nucleotide sequences;
yeast (e.g., Saccharo~rayees, Pichia) transformed with recombinant yeast expression vectors containing the HCV nucleotide sequences; insect cell systems infected with recombinant virus expression vectors i (e.g., baculovirus) containing the HCV sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing HCV
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the HCV gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of HCV peptide or for raising antibodies to the HCV peptide, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J., 2:1791 0983), in which the HCV coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13:3101-3109 (1985); Van Heake &
Schuster, J. Biol. Chern., 264:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autograplza californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The HCV
coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of an HCV gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus, (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.
(See e.g., Smith et al., J. Virol. 46: 584 (1983); and Smith, U.S. Pat. No. 4,215,051).
In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the HCV nucleotide sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the HCV gene product in infected hosts. (See e.g., Logan & Shenk, Proc. Natl.
Acad. Sci. USA
81:3655-3659 (1984)). Specific initiation signals can also be required for efficient translation of inserted HCV nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences.
However, in cases where only a portion of the HCV coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG
initiation codon, can be provided. Furthermore, the initiation codon can be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., Methods in Enzymol., 153:516-544 ( 1987)).
In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products are important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the HCV peptides described above can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are allowed to grow for 1-2 days in an enriched media; and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn are cloned and expanded into cell lines. This method is advantageously used to engineer cell lines which express the HCV gene product.
A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223 (1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA
48:2026 (1962), and adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes:--dhfr, which confers resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA 77:3567 (1980); O'Hare, et al., Proc. Natl. Acad.
Sei. USA 78:1527 (1981); gpt, which confers resistance to mycophenolic acid (Mulligan ~. Berg, Proc. Natl. Acad.
Sci. USA 78:2072 (1981); neo, which confers resistance to the aminoglycoside G-418 (Calberre-Garapin, et al., J. Mol. Biol. 150:1 (1981); and hygro, which confers resistance to hygromycin (Santerre, et al., Gene 30:147 (1984)).
Alternatively, any fusion protein can be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines.
(Janknecht, et. al., Proc. Natl. Acad. Sci. USA 88: 8972-8976 (1991)). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
The example below describes a method that was used to express the HCV peptides encoded by the embodied nucleic acids.

To characterize the NS3/4A fusion protein, and the truncated and mutated versions thereof, the vector constructs, described in Example 6, were transcribed and translated in vitro, and the resulting polypeptides were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro transcription and translation were performed using the T7 coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions. All ira vitro translation reactions of the expression constructs were carried out at 30°C
with 35S-labeled methionine (Amersham International, Plc, Buckinghamshire, UK). The labeled proteins were separated on 12% SDS-PAGE gels and visualized by exposure to X-ray film (Hyper Film-MP, Amersham) for 6-18 hours.
The in vitro analysis revealed that all proteins were expressed to high amounts from their respective expression constructs. The rNS3 construct (NS3-pVAX vector) produced a single peptide of approximately 6lkDa, whereas, the TPT construct (NS3/4A-TPT-pVAX) and the RGT
construct (NS3/4A-RGT-pVAX) produced a single polypeptide of approximately 67 kDa, which is identical to the molecular weight of the uncleaved NS3/4A peptide produced from the NS3/4A-pVAX construct. The cleaved product produced from the expressed NS3/4A peptide was approximately 61 kDa, which was identical in size to the rNS3 produced from the NS3-pVAX
f vector. These results demonstrated that the expression constructs were functional, the NS3/4A
construct was enzymatically active, the rNS3 produced a peptide of the predicted size, and the TPT
and RGT mutations completely abolished cleavage at the NS3 NS4A junction.
The sequences, constructs, vectors, clones, and other materials comprising the embodied HCV nucleic acids and peptides can be in enriched or isolated form. As used herein, "enriched"
means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight. Enriched preparations from about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. The term "isolated" requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated. It is also advantageous that the sequences be iy~ purified form. The term "purified" does not require absolute purity; rather, it is intended as a relative definition. Isolated proteins have been conventionally purified to electrophoretic homogeneity by Coomassie staining, for example.
Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or fiye orders of magnitude is expressly contemplated.
The HCV gene products described herein can also be expressed in plants, insects, and animals so as to create a transgenic organism. Desirable transgenic plant systems having an HCV
peptide include Arabadopsis, maize, and Clalanzydornonas. Desirable insect systems having an HCV peptide include, but are not limited to, D. rnelarzogaster and C.
elegarrs. Animals of any species, including, but not limited to, amphibians, reptiles, birds, mice, hamsters, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, dogs, cats, and non-human primates, e.g., baboons, monkeys, and chimpanzees can be used to generate transgenic animals having an embodied HCV molecule.
These transgenic organisms desirably exhibit germline transfer of HCV peptides described herein.
Any technique known in the art is preferably used to introduce the HCV
transgene into animals to produce the founder lines of transgenic animals or to knock out or replace existing HCV
genes. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., ZISA 82:6148-6152 (1985); gene targeting in embryonic stem cells (Thompson et~al., Cell 56:313-321 (1989); electroporation of embryos (Lo, Mol Cell. Biol. 3:1803-1814 (1983); and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); see also Gordon, Transgenie Arzirnals, Intl. Rev. Cytol.
115:171-229 (1989).
The section below describes the manufacture of antibodies that interact with the HCV peptides described herein, Anti-HCV antibodies Following synthesis or expression and isolation or purification of the HCV
peptides, the isolated or purified peptide can be used to generate antibodies. Depending on the context, the term "antibodies" can encompass polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that recognize the HCV peptides have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, and humans etc. can be immunized by injection with an HCV peptide. Depending on the host species, A
various adjuvants can be used to increase immunological response. Such adjuvants include, but are not limited to, ribavirin, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluro~ic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are also potentially useful adjuvants.

Peptides used to induce specific antibodies can have an amino acid sequence consisting of at least four amino acids, and preferably at least 10 to 15 amino acids. By one approach, short stretches of amino acids encoding fragments of NS3/4A are fused with those of another protein such as keyhole limpet hemocyanin such that an antibody is produced against the chimeric molecule. Additionally, a composition comprising ribavirin and NS3/4A (SEQ.
TD. NO.: 17), a fragment thereof at least 4, 6, 8, 10, 12, 15, or 20 amino acids in length, or a nucleic acid encoding one or more of these moleucles is administered to an animal. While antibodies capable of specifically recognizing HCV can be generated by injecting synthetic 3-mer, 10-mer, and 15-mer peptides that correspond to an HCV peptide into mice, a more diverse set of antibodies can be generated by using recombinant HCV peptides, prepared as decribed above.
To generate antibodies to an HCV peptide, substantially pure peptide is isolated from a transfected or transformed cell. The concentration of the peptide in the final preparation is adjusted, for example, by concentration on any Amicon filter device, to the level of a few micrograms/ml.
Monoclonal or polyclonal antibody to the peptide of interest can then be prepared as follows:
Monoclonal antibodies to an HCV. peptide can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975), the human B-cell hybridoma technique (Kosbor et al.
Inamunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used. (Morrison et al.
Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature 312:604-608(1984); Takeda et al. Nature 314:452-454(1985). Alternatively, techniques described for the production of single chain antibodies (L1.5. Pat. No. 4,946,778) can be adapted to produce HCV-specific single chain antibodies. Antibodies can also be,~produced by inducing irz vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature 349:93-299 (1991).
Antibody fragments that contain specific binding sites for an HCV peptide can also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
By one approach, monoclonal antibodies to an HCV peptide are made as follows.
Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused in the presence of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol.
70:419(1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York.
Section 21-2.
Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and can require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera.
Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective t immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J.
CliiZ. Endocrinol. Metab.
33:988-991 (1971).
Booster injections are given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., Chap.
19 in: Handbook of Experimental Immunolo~y D. Wier (ed) Blackwell (1973).
Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12~M).
Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunoloay, 2d Ed.
(Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibody preparations prepared according to either protocol are useful in quantitative immunoassays that determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively (e.g., in diagnostic embodiments that identify the presence of HCV in biological samples). The section below describes some of the diagnostic embodiments in greater detail.
Diagnostic errzbodiments Generally, the embodied diagnostics are classified according to whether a nucleic acid or protein-based assay is used. Some diagnostic assays detect the presence or absence of an embodied HCV nucleic acid sequence in a sample obtained from a patient, whereas, other assays seek to identify whether an embodied HCV peptide is present in a biological sample obtained from a patient.
Additionally, the manufacture of kits that incorporate the reagents and methods described herein that allow for the rapid detection and identification of HCV are also embodied.
These diagnostic kits can include, for example, an embodied nucleic acid probe or antibody, which speciftcally detects HCV.
The detection component of these kits will typically be supplied in combination with one or more of the following reagents. A support capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied. Available supports include membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents.
One or more restriction enzymes, control reagents, buffers, amplification enzymes, and non-human polynucleotides like calf thymus or salmon-sperm DNA can be supplied in these kits.
Useful nucleic acid-based diagnostics include, but are not limited to, direct DNA
sequencing, Southern Blot analysis, dot blot analysis, nucleic acid amplification, and combinations of these approaches. The starting point for these analysis is isolated or purified nucleic acid from a biological sample obtained from a patient suspected of contracting HCV or a patient at risk of contracting HCV. The nucleic acid is extracted from the sample and can be amplified by RT-PCR
and/or DNA amplification using primers that correspond to regions flanking the embodied HCV
nucleic acid sequences (e.g., NS3/4A (SEQ. ID. NO.: 16)).
In some embodiments, nucleic acid probes that specifically hybridize with HCV
sequences are attached to a support in an ordered array, wherein the nucleic acid probes are attached to distinct regions of the support that do not overlap with each other.
Preferably, such an ordered array is designed to be "addressable" where the distinct locations of the probe are recorded and can be accessed as part of an assay procedure. These probes are joined to a support in different known locations. The knowledge of the precise location of each nucleic acid probe makes these "addressable" arrays particularly useful in binding assays. The nucleic acids from a preparation of several biological samples are then labeled by conventional approaches (e.g., radioactivity or fluorescence) and the labeled samples are applied to the array under conditions that permit hybridization.
If a nucleic acid in the samples hybridizes to a probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the hybrid. Since the -~16-identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence of the polymorphic variant can be rapidly determined. These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic or detection analysis.
Additionally, an opposite approach to that presented above can be employed.
Nucleic acids present in biological samples cart be disposed on a support so as to create an addressable array. Preferably, the samples are disposed on the support at known positions that do not overlap.
The presence of HCV nucleic acids in each sample is determined by applying labeled nucleic acid probes that complement nucleic acids, which encode HCV peptides, at locations on the array that correspond to the positions at which the biological samples were disposed.
Because the identity of the biological sample and its position on the array is known, the identification of a patient that has been infected with HCV can be rapidly determined. These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis.
Any addressable array technology known in the art can be employed. One particular embodiment of polynucleotide arrays is known as GenechipsTM, and has been generally described in US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays are generally produced using mechanical synth~'sis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis.
(Fodor et al., Science, 251:767-777, (1991)). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as "Very Large Scale Immobilized Polymer Synthesis" (VLSPISTM) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSPISTM
technologies are provided in US Patents 5,143,854 and 5,412,087 and in PCT
Publications WO
90/15070, WO 92/10092 and WO 95/11995, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and diagnostic information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO
97/29212, and WO
97/31256.
A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid assays. There are several ways to produce labeled nucleic acids for hybridization or PCR including, but not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, a nucleic acid encoding an HCV peptide can be cloned into a vector for the production of an mRNA
probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway N.J.), Promega (Madison Wis.), and U.S. Biochemical Corp (Cleveland Ohio) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as, substrates, cofactors, inhibitors, magnetic particles and the like.
The presence of an HCV peptide in a protein sample obtained from a patient can also be detected by using conventional assays and the embodiments described herein.
For example, antibodies that are immunoreactive with the disclosed HCV peptides can be used to screen biological samples for the presence of HCV infection. In preferred embodiments, antibodies that are reactive to the embodied HCV peptides are used to immunoprecipitate the disclosed HCV
peptides from biological samples orf are used to react with proteins obtained from a biological sample on Western or Immunoblots. Favored diagnostic embodiments also include enzyme-linked immunosorbant assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA~, including sandwich assays using monoclonal and/or polyclonal antibodies specific for the disclosed HCV peptides. Exemplary sandwich assays are described by David et al., in U.S. Patent Nos.4,376,110 and 4,486,530. Other embodiments employ aspects of the immune-strip technology disclosed in U.S. Patent Nos.
5,290,678;
5,604,105; 5,710,008; 5,744,358; and 5;747,274.
In another preferred protein-based diagnostic, the antibodies described herein are attached to a support in an ordered array, wherein a plurality of antibodies are attached to distinct regions of the support that do not overlap with each other. As with the nucleic acid-based arrays, the protein-based arrays are ordered arrays that are designed to be "addressable" such that the distinct locations are recorded and can be accessed as part of an assay procedure. These probes are joined to a support in different known locations. The knowledge of the precise location of each probe makes these "addressable" arrays particularl''y useful in binding assays. For example, an addressable array can comprise a support having several regions to which are joined a plurality of antibody probes that specifically recognize HCV peptides present in a biological sample and differentiate the isotype of HCV identified herein.
By one approach, proteins are obtained from biological samples and are then labeled by conventional approaches (e.g., radioactivity, colorimetrically, or fluorescently). The labeled samples are then applied to the array under conditions that permit binding. If a protein in the sample binds to an antibody probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the antibody-protein complex.
Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence, concentration, and/or expression level can be rapidly determined. That is, by employing labeled standards of a known concentration of HCV
peptide, an investigator can accurately determine the protein concentration of the particular peptide in a tested sample and can also assess the expression level of the HCV
peptide. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of the HCV peptide. These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic analysis.
In another embodiment, an opposite approach to that presented above can be employed.
Proteins present in biological samples can be disposed on a support so as to create an addressable array. Preferably, the protein samples are disposed on the support at known positions that do not overlap. The presence of an HCV peptide in each sample is then determined by applying labeled antibody probes that recognize epitopes specific for the HCV peptide. Because the identity of the biological sample and its position on the array is known, an identification of the presence, concentration, and/or expression level of an HCV peptide can be rapidly determined.
That is, by employing labeled standards of a known concentration of HCV
peptide, an investigator can accurately determine the concentration of peptide in a sample and from this information can assess the expression level of the peptide. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of the HGV
peptide. These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis. As detailed above, any addressable array technology known in the art can be employed. The section below describes some of the compositions that can have one or mode of the embodied HCV nucleic acids or HCV peptides.
Cornpositio~s comprisi~ag the enabodied HCV nucleic acids or peptides Sorne embodiments contain at least one of the HCV nucleic acids or peptides joined to a support. Preferably, these supports are manufactured so as to create a multimeric agent. These multimeric agents provide the HCV peptide or nucleic acid in such a form or in such a way that a sufficient affinity to the molecule is achieved. A multimeric agent having an HCV nucleic acid or peptide can be obtained by joining the desired molecule to a macromolecular support. A "support"
can be a termed a carrier, a protein, a resin, a cell membrane, or any macromolecular structure used to join or immobilize such molecules. Solid supports include, but are not limited to, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, animal cells, Duracyte~, artificial cells, and others. An HCV nucleic acid or peptide can also be joined to inorganic carriers, such as silicon oxide material (e.g., silica gel, zeolite, diatomaceous earth or aminated glass) by, for example, a covalent linkage through a hydroxy, carboxy or amino group and a reactive group on the carrier.
In several multimeric agents, the macromolecular support has a hydrophobic surface that interacts with a portion of the HCV nucleic acid or peptide by a hydrophobic non-covalent interaction. In some cases, the hydrophobic surface of the support is a polymer such as plastic or any other polymer in which hydpophobic groups have been linked such as polystyrene, polyethylene or polyvinyl. Additionally, HCV nucleic acid or peptide can be covalently bound to carriers including proteins and oligo/polysaccarides (e.g. cellulose, starch, glycogen, chitosane or aminated sepharose). In these later multimeric agents, a reactive group on the molecule, such as a hydroxy or an amino group, is used to join to a reactive group on the carrier so as to create the covalent bond. Additional multimeric agents comprise a support that has other reactive groups that are chemically activated so as to attach the HCV nucleic acid or peptide. For example, cyanogen bromide activated matrices, epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxy succinimide chlorformate linkages, or oxirane acrylic supports are used. (Sigma).
Carriers for use in the body, (i.e. for prophylactic or therapeutic applications) are desirably physiological, non-toxic and preferably, non-immunoresponsive. Suitable carriers for use in the body include poly-L-lysine, poly-D, L-alanine, liposomes, and Chromosorb~
(Johns-Manville Products, Denver Co.). Ligand conjugated Chromosorb~ (Synsorb-Pk) has been tested in humans for the prevention of hemolytic-uremic syndrome and was reported as not presenting adverse reactions. (Armstrong et al. J. Infectious Diseases 171:1042-1045 (1995)). For some embodiments, a "naked" carrier (i.e., lacking an attached HCV nucleic acid or peptide) that has the capacity to attach an HCV nucleic acid or peptide in the body of a organism is administered. By this approach, a "prodrug-type" therapy is envisioned in which the naked carrier is administered separately from the HCV nucleic acid or peptide and, once both are in the body of the organism, the carrier and the HCV nucleic acid or peptide are assembled into a multimeric complex.
The insertion of linkers, such as linkers (e.g., "~, linkers" engineered to resemble the flexible regions of ~, phage) of an appropriate length between the HCV nucleic acid or peptide and the support are also contemplated so as to encourage greater flexibility of the HCV peptide, hybrid, or binding partner and thereby overcome any steric hindrance that can be presented by the support.
The determination of an appropriate length of linker that allows for an optimal cellular response or lack thereof, can be determined by screening the HCV nucleic acid or peptide with varying linkers in the assays detailed in the present dASClosure.
A composite support comprising more than one type of HCV nucleic acid or peptide is also envisioned. A "composite support" can be a carrier, a resin, or any macromolecular structure used to attach or immobilize two or more different HCV nucleic acids or peptides.
As above, the insertion of linkers, such as ~, linkers, of an appropriate length between the HCV nucleic acid or peptide and the support is also contemplated so as to encourage greater flexibility in the molecule and thereby overcome any steric hindrance that can occur. The determination of an appropriate length of linker that allows for an optimal cellular response or lack thereof, can be determined by screening the HCV nucleic acid or peptide with varying linkers in the assays detailed in the present disclosure.
In other embodiments, the multimeric and composite supports discussed above can have attached multimerized HCV nucleic acids or peptides so as to create a "multimerized-multimeric support" and a "multimerized-composite support", respectively. A multimerized ligand can, for example, be obtained by coupling two or more HCV nucleic acids or peptides in tandem using conventional techniques in molecular biology. The multimerized form of the HCV
nucleic acid or peptide can be advantageous for many applications because of the ability to obtain an agent with a higher affinity, for example. The incorporation of linkers or spacers, such as flexible ~, linkers, between the individual domains that make-up the multimerized agent can also be advantageous for some embodiments. The insertion of ~, linkers of an appropriate length between protein binding domains, for example, can encourage greater flexibility in the molecule and can overcome steric hindrance. Similarly, the insertion of linkers between the multimerized HCV
nucleic acid or peptide and the support can encourage greater flexibility and limit steric hindrance presented by the support. The determination of an appropriate length of linker can be determined by screening the HCV nucleic acids or peptides in the assays detailed in this disclosure.
Embodiments of the invention also include genetic vaccines, as described above.
Preferably these compositions contain ribavirin and a nucleic acid encoding NS3/4A (SEQ. ID.
NO.: 17), NS3 (SEQ. ID. NO.: 29), or a mutant (e.g., SEQ. ID. NOs.: 30 - 32 and 43-49) or a fragment thereof (e.g., SEQ. ID. NOs.: 25-27, and 33-42). The following example describes the preparation of a genetic vaccine suitable for use in humans.

An HCV expression plasrnid is designed to express the NS3/4A peptide. The coding sequence of NS3/4A-pVAX is removed by digestion with EcoRl and Xbal, and the isolated i fragment is inserted into plasmid A so that it is under the transcriptional control of the CMV
promoter and the RSV enhancer element. (See U.S. Pat. No. 6,235,888 to Pachuk, et al.). Plasmid backbone A is 3969 base pairs in length; it contains a PBR origin of replication for replicating in E.
coli and a kanamycin resistance gene. Inserts such as the NS3/4A, are cloned into a polylinker region, which places the insert between and operably linked to the promoter and polyadenylation signal. Transcription of the cloned inserts is under the control of the CMV
promoter and the RSV

enhancer elements. A polyadenylation signal is provided by the presence of an SV40 poly A signal situated just 3' of the cloning site. An NS3/4A containing vaccine composition is then made by mixing SOO~,g of the rNS3/4A construct with lmg of ribavirin.
Said vaccine composition can be used to raise antibodies in a mammal (e.g., mice or rabbits) or can be injected intramuscularly into a human so as to to raise antibodies, preferably a human that is chronically infected with the HCV virus. The recipient preferably receives three immunization boosts of the mixture at 4-week intervals, as well. By the third boost, the titer of antibody specific for HCV will be significantly increased. Additionally, at this time, said subject will experience an enhanced antibody and T-cell mediated immune response against NS3, as evidenced by an increased fraction of NS3 specific antibodies as detected by EIA, and a reduction in viral load as detected by RT-PCR.
Embodiments also include NS3/4A fusion proteins or nucleic acids encoding these r molecules. For instance, production and purification of recombinant protein may be facilitated by the addition of auxiliary amino acids to form a "tag". Such tags include, but are not limited to, His-6, Flag, Myc and GST. The tags may be added to the C-terminus, N-terminus, or within the NS3/4A amino acid sequence. Further embodiments include NS3/4A fusion proteins with amino or carboxy terminal truncations, or internal deletions, or with additional polypeptide sequences added to the amino or carboxy terminal ends, or added internally. Other embodiments include NS3/4A fusion proteins, or truncated or mutated versions thereof, where the residues of the NS3/4A profeolytic cleavage site have been substituted. Such substitutions include, but are not limited to, sequences where the Pl' site is a Ser, Gly, or Pro, or the Pl position is an Arg, or where the P8 to P4' sequence is Ser-Ala-Asp-Leu-Glu-Val-Val-Thr-Ser-Thr-Trp-Val (SEQ. ID.
NO.: 28).
Other embodiments concern an immunogen comprising the NS3/4A fusion protein, or a truncated or modified version thereof, capable of eliciting an enhanced immune response against NS3. The immunogen can be prov,~ded in a substantially purified form, which means that the immunogen has been rendered substantially free of other proteins, lipids, carbohydrates or other compounds with which it naturally associates. Embodiments also include vaccine compositions comprising the NS3/4A fusion protein (SEQ. ID. NO.: 17), or a truncated or mutated version thereof (e.g., SEQ. ID. NOS.: 29- 32 and 43-49) or a fragment thereof (e.g., SEQ. ID. NOs.: 25-27, and 33-42), and an adjuvant, such as ribavirin. The following example describes one approach to prepare a vaccine composition comprising the NS3/4A fusion protein and an adjuvant.

Example 15 To generate a tagged NS3/4A construct, the NS3/4A coding sequence of NS3/4A-pVAX is removed by digestion with EcoRl and Xbal, and the isolated fragment is inserted into an Xpress vector (Invitrogen). The Xpress vector allows for the production of a recombinant fusion protein having a short N-terminal leader peptide that has a high affinity for divalent cations. Using a nickel-chelating resin (Invitrogen), the recombinant protein can be purified in one step and the leader can be subsequently removed by cleavage with enterokinase. A preferred vector is the pBlueBacHis2 Xpress. The pBIueBacHis2 Xpress vector is a Baculovirus expression vector containing a multiple cloning site, an ampicillin resistance gene, and a lac z gene. Accordingly, the digested amplification fragment is cloned into the pBIueBacHis2 Xpress vector and SF9 cells are infected. The expression protein is then isolated or purified according to the maufacturer's instructions. An NS3/4A
containing vaccine composition is then made by mixing 100p,g of the rNS3/4A
with lmg of ribavirin.
Said vaccine composition can be used to raise antibodies in a mammal (e.g., mice or rabbits) or can be injected intramuscularly into a human so as to to raise antibodies, preferably a human that is chronically infected with the HCV virus. The recipient preferably receives three immunization boosts of the mixture at 4-weak intervals. By the third boost, the titer of antibody specific for HCV will be significantly increased. Additionally, at this time, said subject will experience an enhanced antibody and T-cell mediated immune response against NS3, as evidenced by an increased fraction of NS3 specific antibodies as detected by EIA, and a reduction in viral load as detected by RT-PCR. The section below provides more explanation concerning the methods of using the compositions described herein.
Methods of using cot~apositio as cornp~isihg f~ibavirih and ah antigen Routes of administration of the vaccines described herein include, but are not limited to, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar.
Transdermal administration can be accomplished by application of a cream, rinse, gel, or other compounds capable of allowing ribavirin and antigen to penetrate the skin. Parenteral routes of administration include, but are not limited to, electrical or direct injection such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.
Gastrointestinal routes of administration include, but are not limited to, ingestion and rectal.
Transbronchial and transalveolar routes of administration include, but are not limited to, inhalation, either via the mouth or intranasally.
Compositions having ribavirin and an antigen that are suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams, and ointments applied directly to the skin or incorporated into a protective carrier such as a transdermal device ("transdermal patch"). Examples of suitable creams, ointments, etc. can be found, for instance, in the Physician's Desk Reference. Examples of suitable transdermal devices are described, for instance, in U.S. Patent No. 4,818,540 issued April 4, 1989 to Chinen, et al.
Compositions having ribavirin and an antigen that are suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, bnt are not limited to, saline, phosphate buffered saline and oil preparations for injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.
Compositions having ribavirin and an antigen that are suitable for transbronchial and transalveolar administration include, but not limited to, various types of aerosols for inhalation.
Devices suitable for transbronchial and transalveolar administration of these are also embodiments.
Such devices include, but are not limited to, atomizers and vaporizers. Many forms of currently available atomizers and vaporizers can be readily adapted to deliver vaccines having ribavirin and an antigen.
Compositions having ribavirin and an antigen that are suitable for gastrointestinal administration include, but not limited to, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration.
The gene constructs described herein, in particular, may be administered by means including, but not limited to, traditional syringes, needleless injection devices, or "microprojectile bombardment gene guns". Alternatively, the genetic vaccine may be introduced by various means into cells that are removed from the individual. Such means include, for example, ex vivo transfection, electroporation, microinjection and microprojectile bombardment.
After the gene construct is taken up by the cells, they are reimplanted into the individual.
It is contemplated that otherwise non-immunogenic cells that have gene constructs incorporated therein can be implanted into the individual even if the vaccinated cells were originally taken from another individual.
According to some embodiments, the gene construct is administered to an individual using a needleless injection device. According to some embodiments, the gene construct is simultaneously administered to an individual intradermally, subcutaneously and intramuscularly using a needleless injection device. Needleless injection devices are well known and widely available. One having ordinary skill in the art can, following the teachings herein, use needleless injection devices to deliver genetic material to cells of an individual.
Needleless injection devices are well suited to deliver genetic material to all tissue. They are particularly useful to deliver genetic material to skin and muscle crells. In some embodiments, a needleless injection device may be used to propel a liquid that contains DNA molecules toward the surface of the individual's skin.
The liquid is propelled at a sufficient velocity such that upon impact with the skin the liquid penetrates the surface of the skin, permeates the skin and muscle tissue therebeneath. Thus, the genetic material is simultaneously administered intradermally, subcutaneously.
and intramuscularly. In some embodiments, a needleless injection device may be used to deliver genetic material to tissue of other organs in order to introduce a nucleic acid molecule to cells of that organ.
The vaccines containing ribavirin and an antigen can be used to treat and prevent a vast spectrum of diseases and can enhance the immune response of an animal to an antigen. As one of skill in the art will appreciate, conventional vaccines have been administered to subjects in need of treatment or prevention of bacterial diseases, viral diseases, fungal diseases, and cancer. Because the vaccines described herein include conventional vaccines, which have been modified by the addition of ribavirin, the methods described herein include the treatment and prevention of a disease using a vaccine that comprises an antigen and ribavirin.
Preferred embodiments concern methods of treating or preventing hepatitis infection. In these embodiments, an animal in need is provided a hepatitis antigen (e.g., a peptide antigen or nucleic acid-based antigen) and an amount of ribavirin sufficient to exhibit an adjuvant activity in said animal. Accordingly, an animal"can be identified as one in need by using currently available diagnostic testing or clinical evaluation. The range of hepatitis viral antigens that can be used with these embodiments is diverse. Preferred hepatitis viral antigens include an HBV antigen, an HAV
antigen, an HCV antigen, nucleic acids encoding these antigens, or any combination thereof.
Highly preferred embodiments include an HBV antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), hepatitis core antigen (HBcAg), and hepatitis E antigen (HBeAg), in particular, the peptide and nucleic acid-based antigens described supra. The ribavirin and antigen can be provided separately or in combination, and other adjuvants (e.g., oil, alum, or other agents that enhance an immune response) can also be provided to the animal in need. Thus, preferred embodiments include methods of treating or preventing hepatitis in an animal (e.g., HBV) by identifying an infected animal or an animal at risk of infection and providing said animal a hepatitis antigen (e.g., HBsAg, HBcAg, and HBeAg) and an amount of ribavirin sufficient to exhibit adjuvant activity.
Other embodiments include methods of enhancing an immune response to an antigen by f providing an animal in need with an amount of ribavirin that is effective to enhance said immune response. In these embodiments, an animal in need of an enhanced immune response to an antigen is identified by using currently available diagnostic testing or clinical evaluation. Oftentimes these individuals will be suffering from a disease (e.g., bacterial, fungal, mold, viral, or cancer) or are at risk from contracting the disease. However, an animal in need of an enhanced immune response can be an animal that has been poisoned (e.g., bit by a poisonous insect or animal) or that has been exposed to a toxin or other toxic compound. Once identified, these animals are provided an appropriate antigen and an amount of-ribavirin effective to enhance an immune response in the animal.
As above, the hepatitis viral antigens that can be used with these embodiments include, but are not limited to, an HBV antigen, an HAV antigen, an HCV antigen, a nucleic acid encoding these molecules, or any combination thereof. Highly preferred embodiments include an HBV
antigen selected from the group consisting of hepatitis B surface antigen (HBsAg), hepatitis core antigen (HBcAg), and hepatitis E antigen (HBeAg), in particular, the peptide and nucleic acid based antigens described supra. The ribavirin and antigen can be provided separately or in combination, and other adjuvants (e.g., oil, alum, or other agents that enhance an immune response) can also be provided to the animal in need. Thus, preferred embodiments include methods of enhancing an immune response to a hepatitis antigen (e.g., HBV) by identifying an animal in need and providing the arfimal a hepatitis antigen (e.g., HBsAg, HBcAg, and HBeAg) and an amount of ribavirin that is effective to enhance an immune response in the animal.
By one approach, for example, an uninfected individual is provided with the above mentioned vaccine compositions in an amount sufficient to elicit a cellular and humoral immune response to NS3 so as to protect said individual from becoming infected with HCV. In another embodiment, an HCV-infected individual is identified and provided with a vaccine composition comprising ribavirin and NS3 in an amount sufficient to enhance the cellular and humoral immune response against NS3 so as to reduce or eliminate the HCV infection. Such individual may be in the chronic or acute phase of the infection. In yet another embodiment, an HCV-infected individual suffering from HCC is provided with a composition comprising ribavirin and the NS3/4A fusion gene in an amount sufficient to elicit a cellular and humoral immune response against NS3-expressing tumor cells.
Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

SEQUENCE LISTING
<110> TRIPEP AB
Matti SALLBERG
Catharina HULTGREN
<120> VACCINES CONTAINING RIBAVIRIN AND
METHODS OF USE THEREOF
<130> TRIPEP,023VPC
<150> US 09/705,547 <151> 2000-11-03 <150> US 60/229,175 <151> 2000-08-29 <150> US 60/225,767 <151> 2000-OS-17 <160> 49 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 3011 <212> PRT
<213> Artificial Sequence <220>
<Z23> Hepatitis C virus sequence <400> 1 Met Ser Thr Asn Pro Lys Pro G1n Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro I1e Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Sex Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe A1a Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr 180 1.85 190 Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser Val Val Tyr Glu Ala Ala Asp Ala Ile Leu His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala Val Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Thr Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser val Phe Leu Val Gly Gln Leu Phe Thr Phe Ser Pro Arg His His Trp Thr Thr Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asn Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ala Gln Leu Leu Arg Ile Pro Gln Ala Ile Met Asp Met Ile Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile Lys Tyr Phe Ser Met Val Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr His Val Thr Gly Gly Asn Ala Gly Arg Thr Thr Ala Gly Leu Val Gly Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn.Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe Ala Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly Leu Asp Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Va1 Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly.Thr Thr Asps Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe 545 550 ~ 555 560 Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly Asn Asn Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys Tyr Pro Glu Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Arg Ile Thr Pro Arg Cys Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp°Ala Ile Lys Trp Glu Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu Trp Met Met Leu Leu Ile Ser Gln Ala G~.u Ala Ala Leu Glu Asn Leu Val Ile Leu Asn Ala Ala.Ser Leu A1a Gly Thr His Gly Leu Va1 Ser Phe Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Arg Trp Val Pro Gly Ala Val Tyr Ala Leu Tyr Gly Met Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Val Ala Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu Ser Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Met Trp Trp Leu Gln Tyr Phe Leu Thr Arg Val Glu Ala Gln Leu His Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu Leu Thr Cys Val Val His Pro Ala Leu Val Phe Asp Ile Thr Lys Leu Leu Leu Ala Ile Phe 885 ~ 890 895 Gly Pro Leu Trp Ile Leu Gln Ala Ser Leu Leu Lys Val Pro Tyr Phe Val Arg Val Gln Gly Leu Leu Arg Ile Cys Ala Leu Ala Arg Lys Ile Ala Gly Gly His Tyr Val Gln Met Ala I1e Ile Lys Leu Gly Ala Leu Thr Gly Thr Cys Val Tyr Asn His Leu Ala Pro Leu Arg Asp Trp Ala His Asn Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Arg Met Glu Thr Lys Leu Ile Thr Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Asn Gly Leu Pro Val Ser Ala Axg Arg Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Val Ser Lys Gly Trp Arg Leu Leu A1a Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Thr Gln Thr Phe Leu Ala Thr 1060 ~ 1065 1070 Cys Ile Asn Gly Val Cys Trp Thr~Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Thr Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Sex Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Thr Gly His Ala Val Gly Leu Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Va1 Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro A1a Ala Tyr Ala Ala Lys Gly Tyr Lys Val Leu Val Leu Asn Pro Sex Val Ala Ala Thr Leu Gly Phe 1250 1255 r 1260 Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro Asn I1e Arg Thr G1y Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Ala Gly Cps Ser Gly Gly A1a Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Ser Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly A1a Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Ser His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu I1e Pro Phe Tyr Gly Lys Ala Tle Pro Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ser Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe~ Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile 1460 1465. 1470 Glu Thr Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser,Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys 1505 1510 ~ 1515 1520 Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Gly Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Arg Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln 1620 1625 1630 , Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly 1650 1655 ~ 1660 Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu G1n Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg His Ala Glu Val Ile Thr Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Val Phe Trp Ala Lys His Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Gly Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Leu Asp Ser Val Gly Leu Gly Lys Val Leu~ Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Ala Val Gly Val Val Phe Ala Ser Ile Leu Arg Arg Arg Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg Gly Tyr Arg Gly Val Trp Arg Gly Asp Gly Ile Met His Thr Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Lys Asn Met Trp Ser Gly Thr Phe Phe Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn Tyr Lys Phe Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Val Glu Ile Arg Arg Val Gly Asp Phe His Tyr val Ser Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Ile Pro Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Met Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Lei Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Glu Val Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Pro Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Leu Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Pro Arg Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Pro Thr Ala Leu Ala Glu Leu Ala Thr Lys Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro Asp Ser Asp Val Glu Sex Tyr Ser Ser Met Pro.Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Gly Ala Asp Thr Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg Lys Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu 2465 2470 . 2475 2480 Asp Ser His Tyr Gln Asp val Leu Lys Glu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser Leu Ala Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Ala His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Ser Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Ser Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu 2610 2615 ~ 2620 Val Gln Ala Trp Lys Ser Lys Lys Thr Pro Met Gly Leu Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Va1 Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Arg Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Arg Tyr Ile Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Val Gln Glu Asp Ala A1a Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Arg _ 7 _ Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile I1e Met Phe Ala Pro Thr Leu Trod Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Val Leu Ile Ala Arg Asp Gln Leu Glu Gln Ala Leu Asn Cys Glu Ile Tyr Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Trp Ser Val Arg Ala Arg Leu Leu Ala Arg Gly Gly Lys Ala Ala Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Thr Ala Ala Gly Arg Leu Asp Leu Ser Gly Trp Phe Thr Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val Ser His Ala Arg Pro Arg Trp Phe Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg <210> 2 <211> 182 < 212 > PI2T
<213> Artificial Sequence <220>
<223> Hepatitis C virus core protein sequence <400> 2 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg A1a Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp A1a Gln Pro Gly 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 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 _ 125 _ g _ Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 l40 Gly Gly Ala Ala Arg'Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu <210> 3 <211> 197 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus E1 protein sequence <400> 3 Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser Val Val Tyr 20 ~ 25 30 Glu Ala Ala Asp Ala Ile Leu His Thr Pro Gly Cys VaI Pro Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala Val Thr Pro Thr Val 50 55 _ 60 Ala Thr Arg Asp Gly Lys Leu Pro Thr Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Gly Gln Leu Phe Thr Phe Ser Pro Arg His His Trp Thr Thr Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asn Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val Ala Gln Leu Leu Arg Ile Pro Gln 145 150 l55 160 Ala Ile Met Asp Met Ile Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile Lys Tyr Phe Ser Met Val Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala <210> 4 <211> 350 <212> PRT
<213> Artificial Sequence <22O>
<223> Hepatitis C virus E2 protein sequence <400> 4 Gly Val Asp Ala Glu Thr His Val Thr Gly Gly Asn Ala Gly Arg Thr 1 5 l0 15 Thr Ala Gly Leu Val Gly Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr Asp Phe Ala Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly Leu Asp Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly I1e Val Pro Al~y Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu 145 150 ~ 155 160 Asn Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly G1y Val Gly Asn Asn Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys Tyr Pro Glu Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Arg Ile Thr Pro Arg Cys Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr G1n Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala <210> 5 <211> 315 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS2 protein sequence <400> 5 Arg Val Cys Ser Cys Leu Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr His Gly Leu Val Ser Phe Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Lys Gly Arg Trp Val Pro Gly Ala Val Tyr Ala Leu Tyr Gly Met Trp Pro Leu Leu Leu Leu Leu Lei, Ala Leu Pro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Val Ala Ala Ser Cys Gly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu Ser Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Met Trp Trp Leu Gln Tyr Phe Leu Thr Arg Val Glu Ala Gln Leu 115 ~ 120 125 His Val Trp Val Pro Pro Leu Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu Leu Thr Cys Val Val His Pro Ala Leu Val Phe Asp Ile Thr Lys Leu Leu Leu Ala Ile Phe Gly Pro Leu Trp Ile Leu Gln Ala Ser Leu Leu Lys Val Pro Tyr Phe Val Arg Val Gln Gly Leu Leu Arg Ile Cys Ala Leu Ala Arg Lys Ile Ala Gly Gly His Tyr Val Gln Met Ala Ile Ile Lys Leu Gly Ala Leu Thr Gly Thr Cys Val Tyr Asn His Leu Ala Pro Leu Arg Asp Trp Ala His Asn Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Val Val Phe Ser Arg Met Glu Thr Lys Leu Ile Thr 245 ~ 250 255 Trp G1y Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Asn Gly Leu Pro Val Ser Ala Arg Arg Gly Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Val Ser Lys Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile <210> 6 <211> 613 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS3 protein sequence <400> 6 Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Thr Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Thr Tyr Thr Asn Val Asp Gln Asp Leu 50 55 ~ 60 Val Gly Trp Pro Ala Pro Gln Gly Ser Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Thr Gly His Ala Val Gly Leu Phe Arg Ala Ala Val Cys Thr.Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val G1u Asn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Lys Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Val Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Th~ Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Ala Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp G1u Cys His Ser Thr Asp Ala Thr Ser Ile Ser Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Ser His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ser Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pr~ Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Sex Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg 485 _ 490 495 Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Gly Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Arg Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly A1a Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr <210> 7 <211> 54 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS4A protein sequence <400> 7 Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu Glu Cys <210> 8 <211> 260 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS4B protein sequence <400> 8 Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg His Ala Glu Val Ile Thr Pro Ala Val Gln Thr Asn Trp Gln Lys Leu Glu Va1 Phe Trp Ala Lys His.Met Trp Asn Phe I1e Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Gly Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala Gly Ala Ala Leu Asp Ser Val Gly Leu Gly Lys Val Leu Va1 Asp I1e Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu 165 '~ 170 175 Ser Pro Gly Ala Leu Ala Val Gly Val Val Phe Ala Ser Ile Leu Arg Arg Arg Val Gly Pro Gly G1u Gly Ala Val Gln Trp Met Asn Arg Leu 195 200 _ 205 Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro <210> 9 <211> 1040 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NSSA/B protein sequence <400> 9 Cys Ser Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cys Gln Arg Gly Tyr Arg Gly Val Trp Arg Gly Asp Gly Ile Met His Thr Arg Cys His Cys Gly Ala Glu Tle Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Lys Asn Met Trp Ser Gly Thr Phe Phe Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn Tyr Lys Phe Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Val Glu Ile Arg Arg Val Gly Asp Phe His Tyr Val Ser Gly Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Ile Pro Ser Pro Glu Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val A1a Val Lei Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Met Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu 225 230 ~ 235 240 Lys Ala Thr Cys Thr Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val G1u Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Glu Val Ser Val Pro Ala Glu Ile Leu Arg Lys Sex Arg Arg Phe Ala Pro Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Leu Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Pro Arg Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg Thr Val Val Leu Thr Glu Ser Thr Leu Pro Thr Ala Leu Ala Glu Leu Ala Thr Lys Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro Asp Ser Asp Val Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Gly Ala Asp Thr Glu Asp Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Ile Asn A1a Leu Ser Asn Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg Lys Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Ser His Tyr Gln Asp Val Leu Lys Glu Val Lys Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala Cys Ser Leu Ala Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val Ala His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Ser Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Sex Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys Thr Pro Met Gly Leu Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Arg Val Leu Thr Thr Sex Cys Gly Asn Thr Leu Thr Arg Tyr Ile Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys Glu Ser Alai Gly Val Gln Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly 785 7g0 795 800 Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Val Leu Ile Ala Arg Asp Gln Leu Glu G1n Ala Leu Asn Cys Glu Ile Tyr Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile I1e Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Trp Ser Val Arg Ala Arg Leu 945 950 ' 955 960 Leu Ala Arg Gly Gly Lys Ala Ala Tle Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Thr Ala Ala Gly Arg Leu Asp Leu Ser Gly Trp Phe Thr Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val Ser His Ala Arg Pro Arg Trp Phe Trp Phe Cys 1010 1015 ' 1020 Leu Leu Leu Leu Ala Ala Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg <210> 10 <211> 226 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis B virus S antigen (HBsAg) sequence <400> 10 Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asr~ Phe Leu Gly Gly Thr Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro G1y Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 ~ 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val g5 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Tle <210> 11 <211> 212 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis B virus C antigen and a antigen (HBcAg/HBeAg) sequence <400> 11 Met Gln Leu Phe His'Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Val Asr), Leu Glu Asp Pro Ala Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 ' 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln Cys <210> 12 <211> 2227 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis A virus sequence <400> 12 Met Asn Met Ser Lys Gln Gly Ile Phe Gln Thr Val Gly Ser Gly Leu Asp His Ile Leu Ser Leu Ala Asp Ile Glu Glu Glu Gln Met Ile Gln Ser Val Asp Arg Thr Ala Val Thr Gly Ala Ser Tyr Phe Thr Ser Val Asp Gln Ser Ser Val His Thr Ala Glu Val Gly Ser His Gln Ile Glu Pro Leu Lys Thr Ser Val Asp Lys Pro Gly Ser Lys Lys Thr Gln Gly 65 70 75 g0 Glu Lys Phe Phe Leu Ile His Ser Ala Asp Trp Leu Thr Thr His Ala Leu Phe His Glu Val Ala Lys Leu Asp Val Val Lys Leu Leu Tyr Asn Glu Gln Phe Ala Val Gln Gly Leu Leu Arg Tyr His Thr Tyr Ala Arg 115 l20 125 Phe Gly Ile Glu Ile Gln Val Gln Ile Asn Pro Thr Pro Phe Gln Gln Gly Gly Leu Ile Cys Ala Met Val Pro Gly Asp Gln Ser Tyr Gly Ser Ile Ala Ser Leu Thr Val Tyr Pro His Gly Leu Leu Asn Cys Asn Ile Asn Asn Val Val Arg Ile Lys Val'~Pro Phe Ile Tyr Thr Arg Gly Ala Tyr His Phe Lys Asp Pro Gln Tyr Pro Val Trp Glu Leu Thr Ile Arg Val Trp Ser Glu Leu Asn Ile Gly '~hr Gly Thr Ser Ala Tyr Thr Ser Leu Asn Val Leu Ala Arg Phe Thr Asp Leu Glu Leu His Gly Leu Thr Pro Leu Ser Thr Gln Met Met Arg Asn Glu Phe Arg Val Ser Thr Thr Glu Asn Val Val Asn Leu Ser Asn Tyr Glu Asp Ala Arg A1a Lys Met Ser Phe Ala Leu Asp Gln Glu Asp Trp Lys Ser Asp Pro Ser Gln Gly Gly Gly Ile Lys Ile Thr His Phe Thr Thr Trp Thr Ser Ile Pro Thr Leu Ala Ala Gln Phe Pro Phe Asn Ala Ser Asp Ser Val Gly Gln Gln Ile Lys Val I1e Pro Val Asp Pro Tyr Phe Phe Gln Met Thr Asn Thr Asn Pro Asp Gln Lys Cys Ile Thr A1a Leu Ala Ser Ile Cys Gln Met Phe Cys Phe Trp Arg Gly Asp Le~ Val Phe Asp Phe Gln Val Phe Pro Thr Lys Tyr His Ser Gly Arg Leu Leu Phe Cys Phe Val Pro Gly Asn Glu Leu Ile Asp Val Thr Gly Ile Thr Leu Lys Gln Ala Thr Thr Ala 385 390 ' 395 400 Pro Cys Ala Val Met Asp Ile Thr Gly Val Gln Ser Thr Leu Arg Phe Arg Val Pro Trp Ile Ser Asp Thr Pro Tyr Arg Val Asn Arg Tyr Thr Lys Ser Ala His Gln Lys Gly Glu Tyr Thr Ala Ile Gly Lys Leu Ile Val Tyr Cys Tyr Asn Arg Leu Thr Ser Pro Ser Asn Val Ala Ser His Val Arg Val Asn Val Tyr Leu Ser Ala Ile Asn Leu Glu Cys Phe Ala Pro Leu Tyr His Ala Met Asp Val Thr Thr Gln Val Gly Asp Asp Ser Gly Gly Phe Ser Thr Thr Val Ser Thr Glu Gln Asn Val Pro Asp Pro Gln Val Gly Ile Thr Thr Met Arg Asp Leu Lys Gly Lys Ala Asn Arg 515 . 520 525 Gly Lys Met Asp Val Ser Gly Val Gln Ala Pro Arg Gly Ser Tyr Gln Gln Gln Leu Asn Asp Pro Val Leu Ala Lys Lys Val Pro Glu Thr Phe Pro Glu Leu Lys Pro Gly Glu Ser Arg His Thr Ser Asp His Met Ser Ile Tyr Lys Phe Met Gly Arg Ser His Phe Leu Cys Thr Phe Thr Phe Asn Ser Asn Asn Lys Glu Tyr Thr Phe Pro Ile Thr Leu Ser Ser Thr Ser Asn Pro Pro His G1y Leu Pro 8er Thr Leu Arg Trp Phe Phe Asn Leu Phe Gln Leu Tyr Arg Gly Pro Leu Asp Leu Thr Ile Ile Ile Thr Gly Ala Thr Asp Val Asp Gly Met Ala Trp Phe Thr Pro Val Gly Leu Ala Val Asp Pro Trp Val Glu Lys Glu Ser Ala Leu Ser Ile Asp Tyr Lys Thr Ala Leu Gly Ala Val Arg Phe Asn Thr Arg Arg Thr Gly Asn Tle Gln Ile Arg Leu Pro Trp Tyr Ser Tyr Leu Tyr Ala Val Ser Gly Ala Leu Asp Gly Leu Gly Asp Lys Thr Asp Ser Thr Phe Gly Leu Phe Leu Phe Glu Ile Ala Asn Tyr Asn His Ser Asp Glu Tyr Leu Ser Phe Ser Cys Tyr Leu Ser Val Thr Glu Gln Ser Glu Phe Tyr Phe Pro Arg Ala Pro Leu Asn Ser Asn Ala Me~Z Leu Ser Thr Glu Ser Met Met Ser Arg Ile Ala Ala Gly Asp Leu Glu Ser Ser Val Asp Asp Pro Arg Ser Glu Glu Asp Arg Arg Phe Glu Ser His Ile Glu Cys Arg Lys Pro Tyr 785 790 ~ 795 800 Lys Glu Leu Arg Leu Glu Val Gly Lys Gln Arg Leu Lys Tyr Ala Gln Glu Glu Leu Ser Asn Glu Val Leu Pro Pro Pro Arg Lys Met Lys Gly Leu Phe Ser Gln Ala Lys Ile Ser Leu Phe Tyr Thr Glu Glu His Glu Ile Met Lys Phe Ser Trp Arg Gly Val Thr Ala Asp Thr Arg Ala Leu g50 855 860 Arg Arg Phe Gly Phe Ser Leu Ala Ala Gly Arg Ser Val Trp Thr Leu Glu Met Asp Ala Gly Val Leu Thr Gly Arg Leu Ile Arg Leu Asn Asp Glu Lys Trp Thr Glu Met Lys Asp Asp Lys Ile Val Ser Leu I1e Glu Lys Phe Thr Ser Asn Lys Tyr Trp Ser Lys Val Asn Phe Pro His Gly Met Leu Asp Leu Glu Glu Ile Ala Ala Asn Ser Lys Asp Phe Pro Asn 930 935 f 940 Met Ser Glu Thr Asp Leu Cys Phe Leu Leu His Trp Leu Asn Pro Lys Lys Ile Asn Leu Ala Asp Arg Met Leu Gly Leu Ser Gly Val Gln Glu 965 _ 970 975 Ile Lys Glu Gln Gly Val Gly Leu Ile Ala Glu Cys Arg Thr Phe Leu Asp Ser Ile Ala Gly Thr Leu Lys Ser Met Met Phe Gly Phe His His Ser Val Thr Val Glu Ile Ile Asn 'Phr Val Leu Cys Phe Val Lys Ser Gly Ile Leu Leu Tyr Val Ile Gln Gln Leu Asn Gln Asp Glu His Ser His Ile Ile Gly Leu Leu Arg Val Met Asn Tyr Ala Asp Ile Gly Cys Ser Val Ile Ser Cys Gly Lys Val Phe Ser Lys Met Leu Glu Thr Val Phe Asn Trp Gln Met Asp Ser Arg Met Met Glu Leu Arg Thr Gln Ser Phe Ser Asn Trp Leu Arg Asp Ile Cys Ser Gly Ile Thr Ile Phe Lys Ser Phe Lys Asp Ala Ile Tyr Trp Leu Tyr Thr Lys Leu Lys Asp Phe Tyr Glu Val Asn Tyr Gly Lys Lys Lys Asp Ile Leu Asn Ile Leu Lys Asp Asn Gln Gln Lys Ile Glu Lys Ala Ile Glu Glu Ala Asp Asn Phe Cys Ile Leu Gln Ile Gln Asp Val~ Glu Lys Phe Asp Gln Tyr Gln Lys G1y Val Asp Leu Ile Gln Lys Leu Arg Thr Val His Ser Met Ala Gln 1170 7.175 1180 Val Asp Pro Asn Leu Gly Val His Leu Ser Pro Leu Arg Asp Cys Ile 1185 1190 ' 1195 1200 Ala Arg Val His Gln Lys Leu Lys Asn Leu Gly Ser Ile Asn Gln Ala Met Val Thr Arg Cys Glu Pro Val Val Cys Tyr Leu Tyr Gly Lys Arg Gly Gly Gly Lys Ser Leu Thr Ser Ile Ala Leu Ala Thr Lys Ile Cys Lys His Tyr Gly Val Glu Pro Glu Lys Asn Ile Tyr Thr Lys Pro Val Ala Ser Asp Tyr Trp Asp Gly Tyr Ser Gly Gln Leu Val Cys Ile Ile Asp Asp Ile Gly Gln Asn Thr Thr Asp Glu Asp Trp Ser Asp Phe Cys Gln Leu Val Ser Gly Cys Pro Met Arg Leu Asn Met Ala Ser Leu Glu Glu Lys Gly Arg His Phe Ser Ser Pro Phe Ile Ile Ala Thr Ser Asn Trp Ser Asn Pro Ser Pro Lys Thr Val Tyr Val Lys Glu Ala Ile Asp Arg Arg Leu His Phe Lys Val Glu Val Lys Pro Ala 5er Phe Phe Lys Asn Pro His Asn Asp Met Leu Asn Val Asn Leu Ala Lys Thr Asn Asp Ala Ile Lys Asp Met Ser Cys Val Asp Leu Ile Met Asp Gly His Asn Ile Ser Leu Met Asp Leu Leu Ser Ser Leu Val Met Thr Val Glu Ile Arg Lys Gln Asn Met Ser Glu Phe Met Glu Leu Trp Ser Gln Gly Ile Ser Asp Asp Asp Asn Asp Ser Ala Val Ala Glu Phe Phe Gln Ser Phe Pro Ser Gly Glu Pro Ser Asn Trp Lys Leu Ser Ser Phe Phe Gln Ser Val Thr Asn His Lys Trp Val Ala Val Gly Ala Ala Val Gly Ile Leu Gly Val Leu Val Gly Gly Trp Phe Val Tyr Lys His Phe Ser Arg Lys Glu Glu Glu Pro Ile Pro Ala Glu Gly Val Tyr His Gly Val Thr Lys Pro Lys Gln Val Ile Lys Leu Asp Ala Asp Pro Val Glu Ser Gln Ser Thr Leu Glu Tle Ala Gly Leu Val Arg Lys Asn Leu Val Gln Phe Gly Val Gly Glu Lys Asn Gly Cys Val Arg Trp Val Met Asn Ala Leu Gly Val Lys Asp Asp Trp Leu Leu Valf Pro Ser His Ala Tyr Lys Phe Glu Lys Asp Tyr Glu Met Met Glu Phe Tyr Phe Asn Arg Gly Gly Thr Tyr Tyr Ser Ile Ser Ala Gly Asn Val Val Ile Gln Ser Leu Asp Val Gly 1585 1590 ' 1595 1600 Phe Gln Asp Val Val Leu Met Lys Val Pro Thr Ile Pro Lys Phe Arg Asp Ile Thr Gln His~Phe I1e Lys Lys Gly Asp Val Pro Arg Ala Leu Asn Arg Leu Ala Thr Leu Val Thr Thr Val Asn Gly Thr Pro Met Leu Ile Ser Glu Gly Pro Leu Lys Met Glu Glu Lys Ala Thr Tyr Val His Lys Lys Asn Asp Gly Thr Thr Val Asp Leu Thr Val Asp Gln Ala Trp Arg Gly Lys Gly Glu Gly Leu Pro Gly Met Cys Gly Gly Ala Leu Val Ser Ser Asn Gln Ser Ile Gln Asn Ala Ile Leu Gly Ile His Val Ala Gly Gly Asn Ser Ile Leu Val Ala Lys Leu Val Thr Gln Glu Met Phe Gln Asn Ile Asp Lys Lys Ile Glu Ser Gln Arg Ile Met Lys Val Glu 1735 ~ 1740 1730 ' Phe Thr Gln Cys Ser Met Asn Val Val Ser Lys Thr Leu Phe Arg Lys Ser Pro Ile His His His Ile Asp Lys Thr Met Ile Asn Phe Pro Ala Ala Met Pro Phe Ser Lys Ala Glu~Ile Asp Pro Met Ala Met Met Leu Ser Lys Tyr Ser Leu Pro Ile Val Glu Glu Pro Glu Asp Tyr Lys Glu Ala Ser Val Phe Tyr Gln Asn Lys Ile Val Gly Lys Thr Gln Leu Val Asp Asp Phe Leu Asp Leu Asp Met Ala Ile Thr Gly Ala Pro Gly Ile Asp Ala Ile Asn Met Asp Ser Ser Pro Gly Phe Pro Tyr Val Gln Glu Lys Leu Thr Lys Arg Asp Leu Ile Trp Leu Asp Glu Asn Gly Leu Leu _ 22 _ ls6o 18s5 1870 Leu Gly Val His Pro Arg Leu Ala Gln Arg Ile Leu Phe Asn Thr Val Met Met Glu Asn Cys Ser Asp Leu Asp Val Val Phe Thr Thr Cys Pro Lys Asp Glu Leu Arg Pro Leu Glu Lys Val Leu Glu Ser Lys Thr Arg Ala Ile Asp Ala Cys Pro Leu Asp Tyr Thr Ile Leu Cys Arg Met Tyr Trp Gly Pro Ala Ile Ser Tyr Phe His Leu Asn Pro Gly Phe His Thr Gly Val Ala Ile Gly Ile Asp Pro~Asp Arg Gln Trp Asp Glu Leu Phe Lys Thr Met Ile Arg Phe Gly Asp Val Gly Leu Asp Leu Asp Phe Ser Ala Phe Asp Ala Ser Leu Ser Pro Pie Met Ile Arg Glu Ala Gly Arg Ile Met Ser Glu Leu Ser Gly Thx Pro Ser His Phe Gly Thr Ala Leu Ile Asn Thr Ile Ile Tyr Ser Lys His Leu Leu Tyr Asn Cys Cys Tyr His Val Cys Gly Ser Met Pro Ser Gly Ser Pro Cys Thr Ala Leu Leu Asn Ser Ile Ile Asn Asn Ile Asn Leu Tyr Tyr Val Phe Ser Lys Ile Phe Gly Lys Ser Pro Val Phe Phe Cys Gln Ala Leu Arg Ile Leu Cys Tyr Gly Asp Asp Val Leu Ile Val Phe Ser Arg Asp Val Gln Ile Asp Asn Leu Asp Leu I1e Gly Gln Lys Ile Val Asp Glu Phe Lys Lys Leu Gly Met Thr Ala Thr Ser Ala Asp Lys Asn Val Pro Gln Leu Lys Pro Val Ser Glu Leu Thr.Phe Leu Lys Arg Ser Phe Asn Leu Val Glu Asp 2130 2135 '~ 2140 Arg Ile Arg Pro Ala Ile Ser Glu Lys Thr Ile Trp Ser Leu Met Ala Trp Gln Arg Ser Asn Ala Glu Phe Glu Gln Asn Leu Glu Asn Ala Gln Trp Phe Ala Phe Met His Gly Tyr Glu Phe Tyr Gln Lys Phe Tyr Tyr Phe Val Gln Ser Cys Leu Glu Lys Glu Met Ile Glu Tyr Arg Leu Lys 21g5 2200 2205 Ser Tyr Asp Trp Trp Arg Met Arg Phe Tyr Asp Gln Cys Phe Ile Cys Asp Leu Ser <2l0> 13 <211> 9416 <212> DNA
<213> Artificial Sequence <220>

<223> Hepatitis C virus sequence <400> 13 gCCagCCCCC tgatgggggc gacactccac catgaatcac tcccctgtga ggaactactg 60 tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac 120 CCCCCCtCCC gggagagcca tagtggtct~g cggaaccggt gagtacaccg gaattgccag 180 gacgaccggg tcctttcttg gataaacccg ctcaatgcct ggagatttgg gcgtgccccc 240 gcaagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300 gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac 360 ctcaaagaaa aaccaaacgt aacaccaacc gtcgcccaca ggacgtcaag ttcccgggtg 420 gcggtcagat cgttggtgga gtttacttgt-tgccgcgcag gggccctaga ttgggtgtgc'480 gcgcgacgag gaagacttcc gagcggtcgc aacctcgagg tagacgtcag cctatcccca 540 aggcacgtcg gcccgagggc aggacctggg ctcagcccgg gtacccttgg cccctctatg 600 gcaatgaggg ttgcgggtgg gcgggatggc tCCtgtCtCC CCgtggCtCt cggcctagct 660 ggggccccac agacccccgg cgtaggtcgc gcaatttggg taaggtcatc gataccctta 720 cgtgcggctt cgccgacctc atggggtaca taccgctcgt cggcgcccct cttggaggcg 780 ctgccagggc cctggcgcat ggcgtccggg ttctggaaga cggcgtgaac tatgcaacag 840 ggaaccttcc tggttgctct ttCtCtatCt tCCttCtggC CCtgCtCtCt tgCCtgaCtg 900 tgcccgcttc agcctaccaa gtgcgcaatt cctcggggct ttaccatgtc accaatgatt 960 gccctaactc gagtgttgtg tacgaggcgg ccgatgccat cctgcacact ccggggtgtg 1020 tccettgcgt tcgcgagggt aacgcctcga ggtgttgggt ggcggtgacc cccacggtgg 1080 ccaccaggga cggcaaactc cccacaacgc agcttcgacg tcatatcgat ctgcttgtcg 1140 ggagcgccac cctctgctcg gccctctacg tgggggacct gtgcgggtct gtctttcttg 1200 ttggtcaact gtttaccttc tctcccaggc accactggac gacgcaagac tgcaattgtt 1260 ctatctatcc cggccatata acgggtcatc gcatggcatg gaatatgatg atgaactggt 1320 cccctacggc agcgttggtg gtagctcagc tgctccgaat cccacaagcc atcatggaca 1380 tgatcgctgg cgcccactgg ggagtcctgg cgggcataaa gtatttctcc atggtgggga 1440 actgggcgaa ggtcctggta gtgctgctgc tatttgccgg cgtcgacgcg gaaacccacg 1500 tcaccggggg aaatgccggc cgcaccac~g ctgggcttgt tggtctcctt acaccaggcg 1560 ccaagcagaa catccaactg atcaacacca acggcagttg gcacatcaat agcacggcct 1620 tgaactgcaa tgaaagcctt aacaccggct ggttagcagg gctcttctat cagcacaaat 1680 tcaactcttc aggctgtcct gagaggttgg ccagctgccg acgccttacc gattttgccc 1740 agggctgggg tcctatcagt tatgccaacg gaagcggcct cgacgaacgc ccctactgct 1800 ggcactaccc tccaagacct tgtggcattg'tgcccgcaaa gagcgtgtgt ggcccggtat 1860 attgcttcac\tCCCagCCCC gtggtggtgg gaacgaccga caggtcgggc gcgcctacct 1920 acagctgggg tgcaaatgat acggatgtct tcgtccttaa caacaccagg ccaccgctgg 1980 gcaattggtt cggttgtacc tggatgaact caactggatt caccaaagtg tgcggagcgc 2040 ccccttgtgt catcggaggg gtgggcaaca acaccttgct ctgccccact gattgcttcc 2100 gcaaatatcc ggaagccaca tactctcggt gcggctccgg tcccaggatt acacccaggt 2160 gcatggtcga ctacccgtat aggctttggc actatccttg taccatcaat tacaccatat 2220 tcaaagtcag gatgtacgtg ggaggggtcg agcacaggct ggaagcggcc tgcaactgga 2280 cgcggggcga acgctgtgat ctggaagaca gggacaggtc cgagctcagc ccgttgctgc 2340 tgtccaccac acagtggcag gtccttccgt gttctttcac gaccctgcca gccttgtcca 2400 ccggcctcat ccacctccac cagaacattg tggacgtgca gtacttgtac ggggtagggt 2460 caagcatcgc gtcctgggcc attaagtggg agtacgtcgt tctcctgttc cttctgcttg 2520 cagacgcgcg cgtctgttcc tgcttgtgga tgatgttact catatcccaa gcggaggcgg 2580 ctttggagaa cctcgtaata ctcaatgcag catccctggc cgggacgcat ggtcttgtgt 2640 CCttCCtCgt gttCttCtgC tttgcgtggt atctgaaggg taggtgggtg cccggagcgg 2700 tctacgccct ctacgggatg tggcctctcc tcctgctcct gctggcgttg cctcagcggg 2760 catacgcact ggacacggag gtggccgcgt cgtgtggcgg cgttgttctt gtcgggttaa 2820 tggcgctgac tctgtcgcca tattacaagc gctatatcag ctggtgcatg tggtggcttc 2880 agtattttct gaccagagta gaagcgca~ac tgcaCgtgtg ggttCCCCCC CtCaaCgtCC 2940 ggggggggcg cgatgccgtc atcttactca cgtgtgtagt acacccggcc ctggtatttg 3000 aCatCaCCaa aCtaCtCCtg gccatcttcg gaCCCCtttg gattcttcaa gccagtttgc 3060 ttaaagtccc ctacttcgtg cgcgttcaag gccttctccg gatctgcgcg ctagcgcgga 3120 agatagccgg aggtcattac gtgcaaatgg ccatcatcaa gttaggggcg cttactggca 3180 cctgtgtgta taaccatctc gctcctcttc gagactgggc gcacaacggc ctgcgagatc 3240 tggccgtggc tgtggaacca gtcgtcttct cccgaatgga gaccaagctc atcacgtggg 3300 gggcagatac cgccgcgtgc ggtgacatca tcaacggctt gcccgtctct gcccgtaggg 3360 gccaggagat actgcttggg ccagccgacg-gaatggtctc caaggggtgg aggttgctgg 3420 cgcccatcac ggcgtacgcc cagcagacga gaggcctcct agggtgtata atcaccagcc 3480 tgactggccg ggacaaaaac caagtggagg gtgaggtcca gatcgtgtca actgctaccc 3540 agaccttcct ggcaacgtgc atcaatgggg tatgctggac tgtctaccac ggggccggaa 3600 cgaggaccat cgcatcaccc aagggtcctg tcatccagac gtataccaat gtggatcaag 3660 acctcgtggg CtggCCCgCt CCtCaaggtt cccgctcatt gacaccctgc acctgcggct 3720 cctcggacct ttacctggtc acgaggcacg ccgatgtcat tcccgtgcgc cggcgaggtg 3780 atagcagggg tagcctgctt tcgccccggc ccatttccta cttgaaaggc tcctcggggg 3840 gtccgctgtt gtgccccacg ggacacgccg tgggcctatt cagggccgcg gtgtgcaccc 3900 gtggagtggc taaggcggtg gactttatcc ctgtggagaa cctagagaca accatgagat 3960 ccccggtgtt cacggacaac tCCtCtccac cagcagtgcc ccagagcttc caggtggccc 4020 acctgcatgc tcccaccggc agcggtaaga gcaccaaggt cccggctgcg tacgcagcca 4080 agggctacaa ggtgttggtg ctcaacccct ctgttgctgc aacactgggc tttggtgctt 4140 acatgtccaa ggcccatggg gttgatccta atatcaggac cggggtgaga acaattacca 4200 ctggcagccc catcacgtac tccacctacg gcaagttcct tgccgacgcc gggtgctcag 4260 gaggtgctta tgacataata atttgtgacg agtgccactc cacggatgcc acatccatct 4320 cgggcatcgg cactgtcctt gaccaagcag agactgcggg ggcgagactg gttgtgctcg 4380 CCaCtgCtaC CCCtCCgggC tccgtcactg tgtcccatcc taacatcgag gaggttgctc 4440 tgtccaccac cggagagatc cccttttar~g gcaaggctat ccccctcgag gtgatcaagg 4500 ggggaagaca tctcatcttc tgccactcaa agaagaagtg cgacgagctc gcegcgaagc 4560 tggtcgcatt gggcatcaat gccgtggcct actaccgcgg tcttgacgtg tctgtcatcc 4620 cgaccagcgg cgatgttgtc gtcgtgtcga ccgatgctct catgactggc tttaccggcg 4680 acttcgactc tgtgatagac tgcaacacgt gtgtcactca gacagtcgat tttagccttg 4740 accctacctt taccattgag acaaccacgc'tcccccagga tgctgtctcc aggactcaac 4800 gccggggcag gactggcagg gggaagccag gcatctatag atttgtggca ccgggggagc 4860 gcccctccgg catgttcgac tcgtccgtcc tctgtgagtg ctatgacgcg ggctgtgctt 4920 ggtatgagct cacgcccgcc gagactacag ttaggctacg agcgtacatg aacaccccgg 4980 ggcttcccgt gtgccaggac catcttggat tttgggaggg cgtctttacg ggcctcactc 5040 atatagatgc ccactttcta tcccagacaa agcagagtgg ggagaacttt ccttacctgg 5100 tagcgtacca agccaccgtg tgcgctaggg ctcaagcccc tcccccatcg tgggaccaga 5160 tgcggaagtg tttgatccgc cttaaaccca ccctccatgg gccaacaccc ctgctataca 5220 gactgggcgc tgttcagaat gaagtcaccc tgacgcaccc aatcaccaaa tacatcatga 5280 catgcatgtc ggccgacctg gaggtcgtca cgagcacctg ggtgctcgtt ggcggcgtcc 5340 tggctgctct ggccgcgtat tgcctgtcaa caggctgcgt ggtcatagtg ggcaggatcg 5400 tcttgtccgg gaagccggca attatacctg acagggaggt tctctaccag gagttcgatg 5460 agatggaaga gtgctctcag cacttaccgt acatcgagca agggatgatg ctcgctgagc 5520 agttcaagca gaaggccctc ggCCtCCtgC agaCCgCgtC CCgCCatgCa gaggttatca 5580 cccctgctgt ccagaccaac tggcagaaac tcgaggtctt ttgggcgaag cacatgtgga 5640 atttcatcag tgggatacaa tacttggcgg gcctgtcaac gctgcctggt aaccccgcca 5700 ttgcttcatt gatggctttt acagctgccg tcaccagccc actaaccact ggccaaaccc 5760 tcctcttcaa catattgggg gggtgggtgg CtgCCCagCt CgCCgCCCCC ggtgccgcta 5820 ccgcctttgt gggcgctggc ttagctgg~g ccgcactcga cagcgttgga ctggggaagg 5880 tcctcgtgga cattcttgca ggctatggcg cgggcgtggc gggagctctt gtggcattca 5940 agatcatgag cggtgaggtc ccctccacgg aggacctggt caatctgctg cccgccatcc 6000 tctcacctgg agcccttgca gtcggtgtgg tctttgcatc aatactgcgc cggcgtgttg 6060 gcccgggcga gggggcagtg caatggatga accggctaat agccttcgcc tcccggggga 6120 aCCatgtttC CCCCdCaCaC tacgtgccgg'agagcgatgc agccgcccgc gtcactgcca 6180 tactcagcag cctcactgta acccagctcc tgaggcgact gcatcagtgg ataagctcgg 6240 agtgtaccac tccatgctcc ggttcctggc taagggacat ctgggactgg atatgcgagg 6300 tgctgagcga ctttaagacc tggctgaaag ccaagctcat gccacaactg cctgggattc 6360 cctttgtgtc ctgccagcgc gggtataggg gggtctggcg aggagacggc attatgcaca 6420 ctcgctgcca ctgtggagct gagatcactg ~gacatgtcaa aaacgggacg atgaggatcg 6480 tcggtcctag gacctgcaag aacatgtgga gtgggacgtt cttcattaat gcctacacca 6540 cgggcccctg tactcccctt cctgcgccga actataagtt cgcgctgtgg agggtgtctg 6600 cagaggaata cgtggagata aggcgggtgg gggacttcca ctacgtatcg ggcatgacta 6660 ctgacaatct caaatgcccg tgccagatcc catcgcccga atttttcaca gaattggacg 6720 gggtgcgcct acataggttt gcgccccctt gcaagccctt gctgcgggag gaggtatcat 6780 tcagagtagg actccacgag tacccggtgg ggtcgcaatt accttgcgag cccgaaccgg 6840 acgtagccgt gttgacgtcc atgctcactg atcCCtcCCa tataacagca gaggcggccg 6900 ggagaaggtt ggcgagaggg tcaccccctt ctatggccag ctcctcggct agccagctgt 6960 CCgCtCCatC tctcaaggca acttgcaccg CCaaCCatga CtCCCCtgaC gccgagctca 7020 tagaggctaa cctcctgtgg aggcaggaga tgggcggcaa catcaccagg gttgagtcag 7080 agaacaaagt ggtgattctg gactccttcg atccgcttgt ggcagaggag gatgagcggg 7140 aggtctccgt acccgcagaa attctgcgga agtctcggag attcgcccca gccctgcccg 7200 tctgggcgcg gccggactac aaccccctgc tagtagagac gtggaaaaag cctgactacg 7260 aaccacctgt ggtccatggc tgCCCgCtaC CdCCtCCaCg gtCCCCtCCt gtgcctccgc 7320 ctcggaaaaa gcgtacggtg gtcctcaccg aatcaaccct acctactgcc ttggccgagc 7380 ttgccaccaa aagttttggc agctcctcaa cttccggcat tacgggcgac aatacgacaa 7440 catcctctga gCCCgCCCCt tCtggCtgCC CCCCCgaCtC cgacgttgag tCCtattCtt 7500 ccatgccccc cctggagggg gagcctggg~,g atccggatct cagcgacggg tcatggtcga 7560 cggtcagtag tggggccgac acggaagatg tcgtgtgctg ctcaatgtct tattcctgga 7620 caggcgcact cgtcaccccg tgcgctgcgg aggaacaaaa actgcccatc aacgcactga 7680 gcaactcgtt gctacgccat cacaatctgg tgtattccac cacttcacgc agtgcttgcc 7740 aaaggaagaa gaaagtcaca tttgacagac tgcaagttct ggacagccat taccaggacg 7800 tgctcaagga ggtcaaagca gcggcgtcaa~aagtgaaggc taacttgcta tccgtagagg 7860 aagcttgcag CCtggCg'CCC CCaCattCag ccaaatccaa gtttggctat ggggcaaaag 7920 acgtccgttg ccatgccaga aaggccgtag cccacatcaa ctccgtgtgg aaagaccttc 7980 tggaagacag tgtaacacca atagacacta ccatcatggc caagaacgag gttttctgcg 8040 ttcagcctga gaaggggggt cgtaagccag ctcgtctcat cgtgttcccc gacctgggcg 8100 tgcgcgtgtg cgagaagatg gccctgtacg acgtggttag caagctcccc ttggccgtga 8160 tgggaagctc ctacggattc caatactcac caggacagcg ggttgaattc ctcgtgcaag 8220 cgtggaagtc caagaagacc ccgatggggc tctcgtatga tacccgctgt tttgactcca 8280 cagtcactga gagcgacatc cgtacggagg aggcaattta ccaatgttgt gacctggacc 8340 CCCaagCCCg cgtggccatc aagtccctca ctgagaggct ttatgttggg ggccctctta 8400 ctaattcaag gggggaaaac tgcggctacc gcaggtgccg cgcgagcaga gtactgacaa 8460 ctagctgtgg taacaccctc actcgctaca tcaaggcccg ggcagcctgt cgagccgcag 8520 ggctccagga ctgcaccatg ctcgtgtgtg gcgacgactt agtcgttatc tgtgaaagtg 8580 cgggggtcca ggaggacgcg gcgagcctga gagccttcac ggaggctatg accaggtact 8640 CCgCCCCCCC CggggaCCCC CCaCaaCCag aataCgaCtt ggagcttata acatcatgct 8700 cctccaacgt gtcagtcgcc cacgacggcg ctggaaagag ggtctactac cttacccgtg 8760 accctacaac ccccctcgcg agagccgcgt gggagacagc aagacacact ccagtcaatt 8820 cctggctagg caacataatc atgtttgccc ccacactgtg ggcgaggatg atactgatga 8880 cccacttctt tagcgtcctc atagccaggg atcagcttga acaggctctc aactgcgaga 8940 tctacggagc ctgctactcc atagaaccac tggatctacc tccaatcatt caaagactcc 9000 atggcctcag cgcattttca ctccacagtt actctccagg tgaaattaat agggtggccg 9060 catgcctcag aaaacttggg gtcccgccct tgcgagcttg gagacaccgg gcctggagcg 9120 tccgcgctag gcttctggcc agaggaggca aggctgccat atgtggcaag tacctcttca 9180 actgggcagt aagaacaaag ctcaaactca'ctccgataac ggccgctggc cggctggact 9240 tgtccggctg gttcacggct ggctacagcg ggggagacat ttatcacagc gtgtctcatg 9300 cccggccccg ctggttctgg ttttgcctac tcctgcttgc tgcaggggta ggcatctacc 9360 tcctccccaa ccgatgaaga ttgggctaac cactccaggc caataggcca ttccct 9416 <210> 14 <211> 3182 <212> DNA
<213> Artificial Sequence <220>
<223> Hepatitis B virus sequence <400> 14 aattccacaa ccttccacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60 gctggtggct ccagttcagg aacagtaaac cctgttctga ctactgcctc tcccttatcg 120 tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180 ctaggacccc ttctcgtgtt acaggcggg~g tttttcttgt tgacaagaat cctcacaata 240 ccgcagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac taccgtgtgt 300 cttggccaaa attcgcagtc CCCaaCCtcc aatcactcac caacctcttg tCCtccaact 360 tgtcctggtt ategctggat gtgtctgcgg cgttttatca tcttcctctt catcctgctg 420 ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480 ctaattccag gatcctcaac aaccagcacg ggaccatgcc ggacctgcat gactactgct 540 caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac cttcggacgg aaattgcacc 600 tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660 cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780 ttgagtCCCt ttttaCCgCt gttaCCaatt ttCttttgtC tttgggtata catttaaacc 840 ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 900 atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 960 ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 1020 ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 1080 ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 1140 aCCtttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 1200 ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 1260 tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 1320 acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 1380 tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 1440 cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 1500 gtctgccgtt CCgaCCgaCC aCggggCg~a CCtCtCttta CgCggaCtCC CCgtCtgtgC 1560 CttCtCatct gccggaccgt gtgcacttcg CttCaCCtCt gCdCgtCgCa tggagaccac 1620 cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 1680 aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 1740 gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 1800 CtgCgCaCCa gCaCCatgCa aCtttttCaC~CtCtgCCtaa tcatctcttg ttcatgtcct 1860 actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat cgacccttat 1920 aaagaatttg gagctactgt ggagttactc tcgtttttgc cttctgactt ctttccttca 1980 gtacgagatc ttctagatac cgcctcagct ctgtatcggg aagccttaga gtctcctgag 2040 cattgttcac ctcaccatac tgcactcagg caagcaattc tttgctgggg ggaactaatg 2100 actctagcta Cctgggtggg tgttaatttg gaagatccag cgtctagaga cctagtagtc 2160 agttatgtca acactaatat gggcctaaag ttcaggcaac tcttgtggtt tcacatttct 2220 tgtctcactt ttggaagaga aacagttata gagtatttgg tgtctttcgg agtgtggatt 2280 cgcactcctc cagcttatag accaccaaat gcccctatcc tatcaacact tccggagact 2340 actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400 aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc 2460 ttggactcat aaggtgggga actttactgg gctttattct tctactgtac ctgtctttaa 2520 tcctcattgg aaaacaccat cttttcctaa tatacattta caccaagaca ttatcaaaaa 2580 atgtgaacag tttgtaggcc cactcacagt taatgagaaa agaagattgc aattgattat 2640 gcctgccagg ttttatccaa aggttaccaa atatttacca ttggataagg gtattaaacc 2700-ttattatcca gaacatctag ttaatcatta cttccaaact agacactatt tacacactct 2760 atggaaggcg ggtatattat ataagagaga aacaacacat agcgcctcat tttgtgggtc 2820 accatattct tgggaacaag atctacagca tggggcagaa tctttccacc agcaatcctc 2880 tgggattctt tcccgaccac cagttggatc cagccttcag agcaaacacc gcaaatccag 2940 attgggactt caatcccaac aaggacacct ggccagacgc caacaaggta ggagctggag 3000 cattcgggct gggtttcacc ccaccgcacg gaggcctttt ggggtggagc cctcaggctc 3060 agggcatact acaaactttg ccagcaaatc cgcctcctgc ctccaccaat cgccagtcag 3120 gaaggcagcc taccccgctg tctccacc~t tgagaaacac tcatcctcag gccatgcagt 3180 gg <210> 15 <211> 7478 <212> DNA
<213> Artificial Sequence <220>
<223> Hepatitis A virus sequence <400> l5 ttcaagaggg gtctccggag gtttccggag cccctcttgg aagtccatgg tgaggggact 60 tgatacctca ccgccgtttg ectaggctat aggctaaatt tCCCtttCCC tgtCCCtCCC 120 ttatttccct ttgttttgct tgtaaatatt aattcctgca ggttcagggt tctttaatct 180 gtttctctat aagaacactc aattttcacg CtttCtgtCt tCtttCttCC agggCtCtCC 240 ccttgcccta ggctctggcc gttgcgcccg gcggggtcaa ctccatgatt agcatggagc 300 tgtaggagtc taaattgggg acgcagatgt ttgggacgtc accttgcagt gttaacttgg 360 ctctcatgaa cctctttgat cttccacaag gggtaggcta cgggtgaaac ctcttaggct 420 aatacttcta tgaagagatg ctttggatag ggtaacagcg gcggatattg gtgagttgtt 480 aagacaaaaa ccattcaacg ccggaggact ggctctcatc cagtggatgc attgagtgga 540 ttgattgtca gggctgtctc taggtttaat ctcagacctc tctgtgctta gggcaaacac 600 catttggcct taaatgggat cctgtgagag ggggtccctc cattgacagc tggactgttc 660 tttggggcct tatgtggtgt ttgactct~qa ggtactcagg ggcatttagg tttttcctca 720 ttcttaaaca ataatgaata tgtccaaaca aggaattttc cagactgttg ggagtggcct 780 tgaccacatc ctgtctttgg cagatattga ggaagagcaa atgattcagt ccgttgatag 840 gactgcagtg actggagctt cttacttcac ttctgtggac caatcttcag ttcatactgc 900 tgaggttggc tcacatcaaa ttgaaccttt gaaaacctct gttgataaac ctggttctaa 960 gaaaactcag ggggaaaagt ttttcctgat-tcattctgct gattggctca ctacacatgc 1020 tctctttcat gaagttgcaa aattggatgt ggtgaaacta ctgtataatg agcagtttgc 1080 cgtccaaggt ttgttgagat accatacata tgcaagattt ggcattgaga ttcaagttca 1140 gataaatccc acaccctttc agcaaggagg actaatttgt gccatggttc ctggtgacca 1200 aagttatggt tcaatagcat ccttgactgt ttatcctcat ggtctgttaa attgcaatat 1260 caacaatgta gttagaataa aggttccatt tatttatact agaggtgctt atcattttaa 1320 agatccacag tacccagttt gggaattgac aatcagagtt tggtcagagt tgaatattgg 1380 aacaggaact tcagcttaca cttcactcaa tgttttagct aggtttacag atttggagtt 1440 gcatggatta actcctcttt ctacacagat gatgagaaat gaatttaggg tcagtactac 1500 tgaaaatgtt gtaaatttgt caaattatga agatgcaagg gcaaaaatgt cttttgcttt 1560 ggatcaggaa gattggaagt ctgatccttc ccaaggtggt ggaattaaaa ttactcattt 1620 tactacctgg acatccattc caaccttagc tgctcagttt ccatttaatg cttcagattc 1680 agttggacaa caaattaaag ttattccagt ggacccatac tttttccaaa tgacaaacac 1740 taatcctgat caaaaatgta taactgcctt ggcctctatt tgtcagatgt tctgcttttg 1800 gaggggagat cttgtttttg attttcaggt ttttccaacc aaatatcatt caggtagact 1860 gttgttttgt tttgttcctg ggaatgagtt aatagatgtt actggaatta cattaaaaca 1920 ggcaactact gctccttgtg cagtgatgga cattacagga gtgcagtcaa ccttgagatt 1980 tcgtgttcct tggatttctg atacacctta tcgagtgaat aggtacacga agtcagcaca 2040 tcaaaaaggt gagtacactg ccattggg~a gcttattgtg tattgttata acagactgac 2100 ttctccttct aatgttgcct ctcatgttag agttaatgtt tatctttcag caattaattt 2160 ggaatgtttt gctcctcttt accatgctat ggatgttact acacaggttg gagatgattc 2220 aggaggtttc tcaacaacag tttctacaga gcagaatgtt cctgatcccc aagttgggat 2280 aacaaccatg agggatttaa aaggaaaagc caatagggga aagatggatg tttcaggagt 2340 _ 28 _ gcaagcacct cgtgggagct atcagcaac~a attgaacgat ccagttttag caaagaaagt 2400 acctgagaca tttcctgaat tgaagcctgg agagtccaga catacatcag atcacatgtc 2460 tatttataaa ttcatgggaa ggtctcattt tttgtgcact tttactttca attcaaataa 2520 taaagagtac acatttccaa taaccctgtc ttcgacttct aatcctcctc atggtttacc 2580 atcaacatta aggtggttct tcaatttgtt_tcagttgtat agaggaccat tggatttaac 2640 aattataatc acaggagcca ctgatgtgga tggtatggcc tggtttactc cagtgggcct 2700 tgctgtcgac ccttgggtgg aaaaggagtc agctttgtct attgattata aaactgccct 2760 tggagctgtt agatttaata caagaagaac aggaaacatt caaattagat tgccgtggta 2820 ttcttatttg tatgccgtgt ctggagcact ggatggcttg ggggataaga cagattctac 2880 atttggattg tttctattcg agattgcaaa ttacaatcat tctgatgaat atttgtcctt 2940 cagttgttat ttgtctgtca cagagcaatc agagttctat tttcctagag ctccattaaa 3000 ttcaaatgct atgttgtcca ctgaatccat gatgagtaga attgcagctg gagacttgga 3060 gtcatcagtg gatgatccca gatcagagga ggatagaaga tttgagagtc atatagaatg 3120 taggaaacca tacaaagaat tgagactgga ggttgggaaa caaagactca aatatgctca 3180 ggaagagtta tcaaatgaag tgcttccacc tcctaggaaa atgaaggggt tattttcaca 3240 agctaaaatt tctctttttt atactgagga gcatgaaata atgaagtttt cttggagagg 3300 agtgactgct gatactaggg ctttgagaag atttggattc tctctggctg ctggtagaag 3360 tgtgtggact cttgaaatgg atgctggagt tcttactgga agattgatca gattgaatga 3420 tgagaaatgg acagaaatga aggatgataa gattgtttca ttaattgaaa agttcacaag 3480 caataaatat tggtctaaag tgaattttcc acatggaatg ttggatcttg aagaaattgc 3540 tgccaattct aaggattttc caaatatgtc tgagacagat ttgtgtttcc tgttacattg 3600 gctaaatcca aagaaaatca atttagcaga tagaatgctt ggattgtctg gagtgcagga 3660 aattaaggaa cagggtgttg gactgata~c agagtgtaga actttcttgg attctattgc 3720 tgggactttg aaatctatga tgtttggg t tcatcattct gtgactgttg aaattataaa 3780 tactgtgctt tgttttgtta agagtggaat cctgctttat gtcatacaac aattgaacca 3840 agatgaacac tctcacataa ttggtttgtt gagagttatg aattatgcag atattggctg 3900 ttcagttatt tcatgtggta aagttttttc caaaatgtta gaaacagttt ttaattggca 3960 aatggattct agaatgatgg agctgaggac~tcagagcttc tctaattggt taagagatat 4020 ttgttcagga attactattt ttaaaagttt taaggatgcc atatattggt tatatacaaa 4080 attgaaggat ttttatgaag taaattatgg caagaaaaag gatattctta atattctcaa 4140 agataatcag caaaaaatag aaaaagccat tgaagaagca gacaattttt gcattttgca 4200 aattcaagat gtagagaaat ttgatcagta tcagaaaggg gttgatttaa tacaaaagct 4260 gagaactgtc cattcaatgg cgcaagttga ccccaatttg ggggttcatt tgtcacctct 4320 cagagattgc atagcaagag tccaccaaaa gctcaagaat cttggatcta taaatcaggc 4380 catggtaaca agatgtgagc cagttgtttg ctatttgtat ggcaaaagag ggggagggaa 4440 aagcttgact tcaattgcat tggcaaccaa aatttgtaaa cactatggtg ttgaacctga 4500 gaaaaatatt tacaccaaac ctgtggcctc agattattgg gatggatata gtggacaatt 4560 agtttgcatt attgatgata ttggccaaaa cacaacagat gaagattggt cagatttttg 4620 tcaattagtg tcaggatgcc caatgagatt gaatatggct tctctagagg agaagggcag 4680 acatttttcc tctcctttta taatagcaac ttcaaattgg tcaaatccaa gtccaaaaac 4740 agtttatgtt aaggaagcaa ttgatcgtag gcttcatttt aaggttgaag ttaaacctgc 4800 ttcatttttt aaaaatcetc acaatgatat gttgaatgtt aatttggcca aaacaaatga 4860 tgcaattaag gacatgtctt gtgttgattt aataatggat ggacacaata tttcattgat 4920 ggatttactt agttccttag tgatgacagt tgaaattagg aaacagaata tgagtgaatt 4980 catggagttg tggtctcagg gaatttcaga tgatgacaat gatagtgcag tggctgagtt 5040 tttccagtct tttccatctg gtgaaccaAtc aaattggaag ttatctagtt ttttccaatc 5100 tgtcactaat cacaagtggg ttgctgtggg agctgcagtt ggcattcttg gagtgcttgt 5160 gggaggatgg tttgtgtata agcatttttc ccgcaaagag gaagaaccaa ttccagctga 5220 aggggtttat catggcgtga ctaagcccaa acaagtgatt aaattggatg cagatccagt 5280 agagtcccag tcaactctag aaatagcagg attagttagg aaaaatctgg ttcagtttgg 5340 agttggtgag aaaaatggat gtgtgagatg ggtcatgaat gccttaggag tgaaggatga 5400 ttggttgtta gtaccttctc atgcttataa atttgaaaag gattatgaaa tgatggagtt 5460 ttacttcaat agaggtggaa cttactattc aatttcagct ggtaatgttg ttattcaatc 5520 tttagatgtg ggatttcaag atgttgtttt aatgaaggtt cctacaattc ccaagtttag 5580 agatattact caacacttta ttaagaaagg agatgtgcct agagccttaa atcgcttggc 5640 aacattagtg acaaccgtta atggaactcc tatgttaatt tctgagggac cattaaagat 5700 ggaagaaaaa gccacttatg ttcataagaa gaatgatggt actacagttg atttgactgt 5760 agatcaggca tggagaggaa aaggtgaagg tcttcctgga atgtgtggtg gggccctagt 5820 gtcatcaaat cagtccatac agaatgcaat tttgggtatt catgttgctg gaggaaattc 5880 aattcttgtg gcaaagctgg ttactcaaga aatgtttcaa aacattgata agaaaattga 5940 aagtcagaga ataatgaaag tggaatttac tcaatgttca atgaatgtag tctccaaaac 6000 gctttttaga aagagtccca ttcatcacca cattgataaa accatgatta attttcctgc 6060 agctatgcct ttctctaaag ctgaaattga tccaatggct atgatgttgt ccaaatattc 6120 attacctatt gtggaggaac cagaggatta caaggaagct tcagtttttt atcaaaacaa 6180 aatagtaggc aagactcagc tagttgatga ctttttagat cttgatatgg ctattacagg 6240 ggctccaggc attgatgcta tcaatatgga ttcatctcct gggtttcctt atgttcaaga 6300 aaaattgacc aaaagagatt taatttggtt ggatgaaaat ggtttgctgt taggagttca 6360 cccaagattg gcccagagaa ttttatttaa tactgtcatg atggaaaatt gttctgactt 6420 agatgttgtt tttacaactt gtccaaaaga tgaattgaga ccattagaga aagttttgga 6480 atcaaaaaca agagccattg atgcttgtcc tttggattat acaattctat gtcgaatgta 6540 ttggggtcca gctatcagtt atttccattt gaatccaggg tttcacacag gtgttgctat 6600 tggcatagat cctgatagac agtgggatga attatttaaa acaatgataa gatttggaga 6660 tgttggtctt gatttagatt tctctgcttt tgatgccagt cttagtccat ttatgattag 6720 ggaagcaggt agaatcatga gtgaatta~c tggaacacca tctcattttg gaacagctct 6780 tatcaatact atcatttatt ctaaacatct gctgtacaac tgttgttatc atgtttgtgg 6840 ttcaatgcct tctgggtctc cttgcacagc tttgttgaat tcaattatta ataatattaa 6900 tctgtattat gtgttttcta aaatatttgg aaagtctcca gttttctttt gtcaagcttt 6960 gaggatcctt tgttacggag atgatgtttt.gatagttttt tccagagatg ttcaaattga 7020 caatcttgac ttgattggac agaaaattgt agatgagttc aaaaaacttg gcatgacagc 7080 cacctcagct gataaaaatg tgcctcaact gaagccagtt tcagaattga cttttctcaa 7140 aagatctttc aatttggtgg aggatagaat tagacctgca atttcagaaa agacaatttg 7200 gtctttgatg gcttggcaga gaagtaacgc tgagtttgag cagaatttag aaaatgctca 7260 gtggtttgct tttatgcatg gctatgagtt ctatcagaaa ttttattatt ttgttcagtc 7320 ctgtttggag aaagagatga tagaatatag acttaaatct tatgattggt ggagaatgag 7380 attttatgac cagtgtttca tttgtgacct ttcatgattt gtttaaacaa attttcttac 7440 tctttctgag gtttgtttat ttcttttgtc cgctaact 7478 <210> 16 <211> 2061 <212> DNA
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS3/4A coding region <400> 16 atggcgccta tcacggccta tgcccagcag acaaggggcc ttttgggatg cataatcacc 60 agcttgaccg gccgggacaa aaaccaggtg gagggtgagg ttcagatcgt gtcaactgct 120 gcccagactt tcttggcaac ctgcattaaG ggggtgtgtt ggactgtcta ccatggagcc 180 ggaacaagga ccattgcgtc acctaagggt cctgttatcc agatgtacac caatgtggac 240 CaagaCCtCg taggCtggCC cgctccccaa ggtgcccgct Cattaacacc atgcacttgc 300 ggctcctcgg acctttacct ggtcacgagg cacgccgatg tcattcctgt gcgccgacgg 360 ggtgatggca ggggcagcct gctttcgccc cggcctatct cttacttgaa aggctcctcg 420 ggaggccctc tgctgtgccc cgcaggacat gccgtaggca tattcagagc cgcggtatgc 480 acccgtggag tggctaaggc ggtggacttc atccccgtag agagcttaga gacaaccatg 540 aggtccccgg tgttCtCaga CaaCtCCtCC CCaCCagCag tgCCCCagag ctaccaagtg 600 gcccacctgc atgctcccac cggcagcggt aagagcacca aggtcccggc cgcatacgca 660 gctcagggct acaaggtgct ggtgctcaac ccctccgttg ctgcaacaat gggctttggt 720 gcttacatgt ccaaggccca tgggattgat cctaacatca ggactggggt gaggacaatt 780 actactggca gcccgatcac gtattccacc tacggcaagt tccttgccga cggcgggtgt 840 tcagggggtg cttatgacat aataatttgt gacgagtgcc actccacgga tgcaacatcc 900 atcttgggca ttggcactgt ccttgaccaa gcagagaccg cgggggcgag actgactgtg 960 CtCgCCaCCg CtaCCCCtCC gggctccgtc actgtgcccc atCCtaacat cgaggaggtt 1020 gctctgtcca ctaccggaga gatccccttt tatggcaagg ctattcccct tgaagcaatt 1080 aaggggggga gacatctcat cttctgccac tcaaagaaga agtgcgacga gctcgccgca 1140 aaactggtcg cgttgggcgt caatgccgtg gcttactacc geggccttga tgtgtccgtc 1200 atcccgacca gtggtgacgt tgtcgtcgtg gcaactgacg ccctcatgac cggctttacc 1260 ggcgacttcg attcggtgat agactgcaac acgtgtgtca cccagacagt cgacttcagc 1320 cttgacccta ccttcaccat tgagacaatc acgcttcccc aggatgctgt ctcccgtact 1380 caacgtcggg gtaggactgg cagagggaag ccaggcatct acagatttgt ggcaccgggg 1440 gagcgtcctt ctggcatgtt tgactcgtot gtcctctgcg agtgctatga cgcgggttgt 1500 gcttggtatg agcttacgcc cgccgagacc acagttaggc tacgagcata catgaacacc 1560 ccgggacttc ccgtgtgcca agaccatct~'t gaattttggg agggcgtctt tacgggtctc 1620 acccacatag acgcccactt cctatcccag acaaagcaga gtggggaaaa ccttccctat 1680 ctggtagcgt accaagccac cgtgtgcgct agagctcaag CCCCtCCCCC gtcgtgggac 1740 cagatgtgga agtgcttgat ccgtctcaag CCCdCCCtCC atgggCCaaC aCCtCtgCta 1800 tatagactgg gcgctgtcca gaatgaagtc,accctgacgc acccagtcac caagtatatc 1860 atgacatgta tgtcggctga cctggaggtc gtcacgagta cctgggtgct cgttggcggc 1920 gttctggctg ctttggccgc gtattgccta tccacaggct gcgtggtcat agtaggtagg 1980 attgtcttgt ccggaaagcc ggcaatcata cccgacaggg aagtcctcta ccgggagttc 2040 gatgaaatgg aagagtgctg a 2061 <210> 17 <2l1> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS3/4A peptide <400> 17 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1 5 10 l5 Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr g5 90 95 Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val A1a His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr 210 215 ~ 220 Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Fro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Va1 Leu Asp Gln A1a Glu Thr Ala Gly A1a Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Va~r Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Va1 Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 435 440 ~ 445 Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro .

Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr 580 f 585 590 Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met 610 6l5 I 620 Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala 21e Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> Z8 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<223> cloning oligonucleotide <400> 18 ccgtctagat cagcactctt ccatttcatc 30 <210> 19 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<223> cloning oligonucleotide <400> 19 cctgaattca tggcgcctat cacggcctat 30 <210> 20 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> cloning oligonucleotide <400> 20 ccacgcggcc gcgacgacct acag 24 <210> 21 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> cloning oligonucleotide <400> 21 ctggaggtcg tcacgcctac ctgggtgctc gtt 33 <210> 22 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> cloning oligonucleotide <400> 22 accgagcacc caggtaggcg tgacgacctc cag 33 <210> 23 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> cloning oligonucleotide <400> 23 ctggaggtcg tccgcggtac ctgggtgctc gtt 33 <210> 24 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> cloning oligonucleotide <400> 24 accgagcacc caggtaccgc ggacgacctc cag 33 <210> 25 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS3/4A peptide <400> 25 Thr Lys Tyr Met Thr Cys Met Ser A1a Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu 20 ~5 <210> 26 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 26 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr Gly Thr Trp Val Leu Val Gly Gly Val Leu <210> 27 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus~N53/4A peptide <400> 27 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Arg Gly Thr Trp Val Leu Val Gly Gly Val Leu <210> 28 <211> 12 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS3/4A peptide <400> 28 Ser Ala Asp Leu Glu Val Val Thr Ser Thr Trp Va1 <210> 29 <211> 632 <212> PRT
<213> Hepatitis C virus NS3 peptideArtificial Sequence <220>
<223> Hepatitis C virus N53 peptide <400> 29 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys G1y Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Va7.~Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly 195 200 ~ 205 Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Va'1 Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Tle Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Va1 Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys.Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val A1a Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr.Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr 545 550 ~, 555 560 Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr <210> 30 <211> 54 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS4A peptide <400> 30 Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser G1y Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> 31 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A
<400> 31 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys 35 40f 45 Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly A1a Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly,Trp Pro Ala' Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyx Lys Va1 Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly 260 ~ 265 270 Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg G1y Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Va1 Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Al~a Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp her Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Va1 Val Thr Gly Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp 660 ' 665 670 Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> 32 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A
<400> 32 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp 65 70 ,~ 75 80 Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr G1y Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly I1e Asp Pro Asn Ile Arg Thr Gly 245 ' 250 255 Val Arg Thr I1e Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile 290 295 f 300 Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Ash Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly G1u Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln A1a Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Arg Gly Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> 33 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 33 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Va1 Val Thr Pro Thr Trp Val Leu Val Gly Gly Val Leu <210> 34 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 34 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Arg Pro Thr Trp Val Leu Val Gly Gly Val Leu <210> 35 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 35 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Arg Pro Ala Trp Val Leu Val Gly Gly Val Leu <210> 36 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus~,NS3/4A peptide <400> 36 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Cys Ser Thr Trp Val Leu Val Gly Gly Val Leu <210> 37 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 37 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Val Cys Cys Ser Thr Trp Val Leu Val Gly Gly Val Leu <210> 38 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 38 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Leu Glu Val Ser Ser Ser Thr Trp Val Leu Val Gly Gly Val Leu <210> 39 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 39 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Ser Ser Ser Ser Cys Ser Thr Trp Val Leu Val Gly Gly Val Leu <210> 40 <211> 25 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A peptide <400> 40 Thr Lys Tyr Met Thr Cys Met Ser Ala Asp Val Val Val Val Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu <210> 41 <211> 16 <212> PRT
<213> Artificial Sequence <220>
<223> Hepatitis C virus NS5 pe~5tide <400> 41 Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly <210> 42 <211> 18 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NSSA/B peptide <400> 42 Ser Ser Glu Asp Val Val Cys Cys Ser Met Trp Val Leu Val Gly Gly Val Leu <210> 43 <211> 686 <212> PRT
<213> Artificial Sequence <220> -<223> Mutant Hepatitis C virus NS3/4A

<400> 43 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1 5 , 10 15 Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys 35 40 45 , Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp G1y Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Vaf Asp Phe Ile Pro Val Glu Sex Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Tle Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val Leu A1a Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Th~r Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala A1a Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Tle Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro 565 '~ 570 575 Pro Ser Trp Asp Gln Met Trp Lys Cys Leu I1e Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn 5g5 600 _ 605 Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Thr Pro Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> 44 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus~NS3/4A
<400> 44 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1 5 _ 10 15 Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly A1a Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp G1n Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser G1y Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Va1 Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro G1n Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly lle Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr G1y Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe 355 . 360 365 Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser G1y Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys G1n Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln.Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Tle Met Thr Cys Met 610 615 p 620 Ser Ala Asp Leu Glu Val Val Arg Pro Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val 645 , 650 655 Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> 45 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A
<400> 45 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1 5 10 15~
Cys Ile Ile Thr Ser Leu Thr G1y Arg Asp Lys Asn Gln Val Glu Gly 20 ~ 25 30 Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu A1a Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr 50 55 . 60 Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile 290 .295 300 Gly Thr Val Leu Asp Gln Ala Glu T~hr Ala Gly Ala Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly ~Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Al~, Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr 485 ' 490 495 Asp Ala Gly Cys A1a Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Arg Pro Ala Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Sex Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys 675 680f 685 <210> 46 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A
<400> 46 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly A1a Arg Ser Leu Thr 85 p 90 95 Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu ~ys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cy~ Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val 305 310 ~ 315 320 Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Z;ys Gly Gly Arg His heu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala 370 . 375 380 Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr G1y Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr G1~ Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp 530 535 ~ 540 Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val Cys Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys 675 68~b 685 <210> 47 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A
<400> 47 Met Ala Pro Ile Thr Ala Tyr Ala G1n Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Sex Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys 145 150 f 155 160 Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln G1y Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Tle Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln A1a Glu Thr Ala Gly Ala Arg Leu Thr VaI

Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn 325 r 330 335 Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Plie Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp ~Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys 420 x.25 430 val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val 'Fhr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Cys Cys Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Va1 Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> 48 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virusfNS3/4A
<400> 48 Met Ala Pro Tle Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly ~.lrg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu A1a Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp 65 70 ' 75 80 Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr g5 90 95 Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala l00 105 110 Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr 210 215 p 220 Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile 2gp 295 300 Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly A1a Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Vaa Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu 435 440 ' 445 Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His I1e Asp Ala His Phe Leu Ser Gln Thr Lys Gln Sex Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Ser Ser Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val 645 650 ~ 655 Ile Val Gly Arg Ile Val Leu Seer Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys <210> 49 <211> 686 <212> PRT
<213> Artificial Sequence <220>
<223> Mutant Hepatitis C virus NS3/4A
<400> 49 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys Gly Pro Va~l. Ile Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Gly Arg Gly Ser Leu Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Leu Glu Thr Thr Met Arg Ser Pro Val Phe Ser Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Tyr Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Met Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile 290 295 f 300 Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr G1y Lys Ala Ile Pro Leu Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val A1a Leu Gly Val Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile G1u Thr Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg G1y Arg Thr Gly Arg G1y Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly 465 470 ~ 475 480 Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr fro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Ser Ser Ser Ser Cys Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu Cys

Claims (20)

WHAT IS CLAIMED IS:

1. A composition comprising ribavirin and the nucleic acid of SEQ. ID. NO.:
16.

2. A composition comprising Ribavrin and the peptide of SEQ. ID. NO.: 17.

3. A composition comprising Ribavrin and the nucleic acid of SEQ. ID. NO.: 13 or a fragment thereof at least 18 consecutive nucleotides in length.

4. A composition comprising ribavirin and the peptide of SEQ. ID. NO.: 1 or a fragment thereof at least 6 consecutive amino acids in length.

5. A composition comprising ribavirin and an antigen.

6. The composition of Claim 5, wherein said antigen is a nucleic acid.

7. The composition of Claim 5, wherein said antigen is a peptide.

8. The composition of Claim 6, wherein said nucleic acid is derived from a virus selected from the group consisting of hepatitis A virus (HAV), hepatitis B
virus (HBV), and hepatitis C virus (HCV).

9. The composition of Claim 7, wherein said peptide is derived from a virus selected from the group consisting of hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C
virus (HCV).

10. The composition of Claim 5, wherein said antigen is a nucleic acid or a peptide corresponding to an antigen selected from the group consisting of hepatitis B
surface antigen (HBsAg), hepatitis core antigen (HBcAg), and hepatitis E antigen (HBeAg).

11. The composition of Claim 7, wherein said peptide comprises at least three consecutive amino acids of a sequence selected from the group consisting of SEQ. ID. NOs.: 1-12.

12. The composition of Claim 6, wherein said nucleic acid comprises at least 9 consecutive nucleotides of a sequence selected from the group consisting of SEQ. ID. NOs.: 13-15.

13. A method of enhancing an immune response to a hepatitis C antigen comprising:
identifying an animal in need of an enhanced immune response to a hepatitis C
antigen; and providing to said animal a composition comprising ribavirin and the nucleic acid of SEQ. ID. NO.: 16.

14. A method of enhancing an immune response to a hepatitis C antigen comprising:
identifying an animal in need of an enhanced immune response to a hepatitis C
antigen; and providing to said animal a composition comprising ribavirin and the peptide of SEQ. ID. NO.: 17.

15. A method of enhancing an immune response to a hepatitis C antigen comprising:
identifying an animal in need of an enhanced immune response to a hepatitis C
antigen; and providing to said animal a composition comprising ribavirin and the nucleic acid of SEQ. ID. NO.: 13 or a fragment thereof at least 18 consecutive nucleotides in length.

16. A method of enhancing an immune response to a hepatitis C antigen comprising:
identifying an animal in need of an enhanced immune response to a hepatitis C
antigen; and providing to said animal a composition comprising ribavirin and the peptide of SEQ. ID. NO.: 1 or a fragment thereof at least 6 consecutive amino acids in length.

17. A method of making a vaccine comprising:
providing ribavirin;
providing the nucleic acid of SEQ. ID. NO.: 16; and mixing said ribavirin and said nucleic acid so as to formulate said vaccine.

18. A method of making a vaccine comprising:
providing ribavirin;
providing the peptide of SEQ. ID. NO.: 17; and mixing said ribavirin and said peptide so as to formulate said vaccine.

19. A method of making vaccine comprising:
providing ribavirin;
providing the nucleic acid of SEQ. ID. NO.: 13 or a fragment thereof at least at least 18 consecutive nucleotides in length; and mixing said ribavirin and said nucleic acid so as to formulate said vaccine.

20. A method of making a vaccine comprising:
providing ribavirin;
providing the nucleic acid of SEQ. ID. NO.: 1 or a fragment thereof at least 6 consecutive amino acids in length; and mixing said ribavirin and said nucleic acid so as to formulate said vaccine.