Vaccines containing ribavirin and methods of use thereof6858590Abstract 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. Claims What is claimed is: Description FIELD OF THE INVENTION
TABLE 1
Amount Amount Antibody titre
ribavirin immunogen to rNS3 at indicated week
(mg/dose) (.mu.g/dose) Mouse ID 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 5:6 60 1500 1500
None 10 5:7 <60 7500 7500
None 10 5:8 300 37500 7500
Group mean titre (mean .+-. SD) 180 .+-. 7500 .+-. 3042 .+-.
139 12421 3076
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 mean titre (mean .+-. SD) 252 .+-. 28071 .+-. 36642 .+-.
107 16195 67565
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 7:5 300 37500 37500
3 10 7:6 300 37500 37500
3 10 7:7 60 7500 7500
3 10 7:8 60 37500 37500
Group mean titre (mean .+-. SD) 180 .+-. 30000 .+-. 22500 .+-.
128 13887 34637
10 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 mean titre (mean .+-. SD) 220 .+-. 18300 .+-. 18300 .+-.
139 18199 18199
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 .mu.g 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 25-30 g 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. EXAMPLE 2 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 20 .mu.g rNS3 alone or a mixture of 20 .mu.g rNS3 and 0.1 mg, 1 mg, or 10 mg 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 FIG. 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 (<1 mg) of ribavirin were found to enhance antibody levels at week one but not at week three, whereas, higher doses (1-10 mg) 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 .mu.l phosphate buffered saline and 20 .mu.g 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 25-30 g mouse.
TABLE 7
Amount (mg)
ribavirin
mixed with
the Mouse Endpoint titre of rNS3 IgG at indicated
week
Immunogen immunogen ID Week 1 Week 2 Week 3
20 .mu.g rNS3 None 1 60 360 360
20 .mu.g rNS3 None 2 360 360 2160
20 .mu.g rNS3 None 3 360 2160 2160
Mean 260 .+-. 173 960 .+-. 1039 1560 .+-. 1039
20 .mu.g rNS3 0.1 4 2160 12960 2160
20 .mu.g rNS3 0.1 5 60 60 60
20 .mu.g rNS3 0.1 6 <60 2160 2160
1110 .+-. 1484 5060 .+-. 6921 1460 .+-.
1212
20 .mu.g rNS3 1.0 7 <60 60 12960
20 .mu.g rNS3 1.0 8 <60 2160 2160
20 .mu.g rNS3 1.0 9 360 2160 2160
Mean 360 1460 .+-. 1212 5760 .+-. 6235
20 .mu.g rNS3 10.0 10 360 12960 77760
20 .mu.g rNS3 10.0 11 <60 2160 12960
20 .mu.g rNS3 10.0 12 360 2160 2160
Mean 360 5760 .+-. 6235 30960 .+-.
40888
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 .mu.g rNS3 with or without ribavirin and the IgG subclass responses to the antigen was monitored, as before. Accordingly, mice were immunized with 100 .mu.l phosphate buffered containing 10 .mu.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 Th1/Th2-balance. It appears that another mechanism may be responsible for the adjuvant effect of ribavirin.
TABLE 8
Amount (mg)
ribavirin
mixed with
the Endpoint titre of indicated NS3 IgG
subclass
Immunogen immunogen Mouse ID IgG1 IgG2a IgG2b IgG3
10 .mu.g rNS3 None 1 360 60 <60 60
10 .mu.g rNS3 None 2 360 <60 <60 60
10 .mu.g rNS3 None 3 2160 60 <60 360
Mean 960 .+-. 1039 60 -- 160 .+-.
173
10 .mu.g rNS3 0.1 4 360 <60 <60 60
10 .mu.g rNS3 0.1 5 60 <60 <60 <60
10 .mu.g rNS3 0.1 6 2160 60 60 360
860 .+-. 1136 60 60 210 .+-.
212
10 .mu.g rNS3 1.0 7 2160 <60 <60 60
10 .mu.g rNS3 1.0 8 360 <60 <60 <60
10 .mu.g rNS3 1.0 9 2160 <60 <60 60
Mean 1560 .+-. 1039 -- --
60
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. EXAMPLE 3 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.sup.+ T cell responses to a ribavirin-containing vaccine, groups of mice were immunized s.c. with either 100 .mu.g rNS3 in PBS or 100 .mu.g rNS3 and 1 mg ribavirin in PBS. The mice were sacrificed ten days post-immunization and their lymph nodes were harvested and drained. In vitro recall assays were then performed. (See e.g., Hultgren et al., J. Gen Virol. 79:2381-91 (1998) and Hultgren et al., Clin. Diagn. Lab. Immunol. 4:630-632 (1997), both of which are herein expressly incorporated by reference in their entireties). The amount of CD4.sup.+ T cell proliferation was determined at 96 h of culture by the incorporation of [.sup.3 H] thymidine. As shown in FIG. 3, mice that were immunized with 100.mu.g rNS3 mixed with 1 mg ribavirin had a much greater T cell proliferative response than mice that were immunized with 100 .mu.g 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. Nos. 5,589,466 and 6,235,888, both of which are herein expressly incorporated by reference in their entireties). 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 creatine 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, Calif.) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-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. EXAMPLE 4 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 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. One group of mice are injected with approximately 20 .mu.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 5 .mu.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 .mu.g of the expression vector having the gp-120 gene and the CMV promoter mixed with 1 mg of ribavirin and a fourth group of mice is injected with approximately 5 .mu.g of capped in vitro transcribed RNA mixed with 1 mg ribavirin. The vaccines are injected in 0.1 ml of solution (PBS) in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm 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 antigen, 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 of 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 200-226 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. NO.: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 preparation. EXAMPLE 5 The adjuvant effect of ribavirin was tested when mixed with two doses of a commercially available vaccine containing HBsAg and alum. (Engerix, SKB). Approximately 0.2 .mu.g or 2 .mu.g of Engerix vaccine was mixed with either PBS or 1 mg 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 2 .mu.g 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.
TABLE 9
Endpoint antibody titer to HBsAg in EIA
0.02 .mu.g Engerix 0.2 .mu.g Engerix
No ribavirin 1 mg ribavirin No ribavirin 1 mg ribavirin
Week #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 3 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, 400-800 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 NS5A/B (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 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, 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, 1658-1711, 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, 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 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 Ribavirin and 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 term 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 carrier 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 Remmington's Pharmaceutical Sciences, 15th Edition, Easton:Mack Publishing Company, pages 1405-1412 and 1461-1487(1975) and The National Formulary XIV, 14th Edition, Washington, American Pharmaceutical Association (1975), herein expressly incorporated by reference in their entireties. 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/US94/00899 filed Jan. 26, 1994, and U.S. Ser. No. 08/221,579 filed Apr. 1, 1994, which are each incorporated herein by reference in their entirety. Examples of such agents include: CaPO.sub.4, DEAE 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 and 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., ED.sub.50 (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 ED.sub.50 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 0.25-12.5 .mu.g/ml in the animal, preferably, about 2.5 .mu.g/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 0.1-6.0 mg/kg body weight. That is, some embodiments have an amount of ribavirin that corresponds to approximately 0.1-1.0 mg/kg, 1.1-2.0 mg/kg, 2.1-3.0 mg/kg, 3.1-4.0 mg/kg, 4.1-5.0 mg/kg, 5.1, and 6.0 mg/kg body weight of an animal. More conventionally, the vaccines contain approximately 0.25 mg-2000 mg of ribavirin. That is, some embodiments have approximately 250 .mu.g, 500 .mu.g, 1 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, and 2 g 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 0.25 mg-5 mg, 5-10 mg, 10-100 mg, 100-500 mg, and upwards of 2000 mg 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 ribavirin 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. EXAMPLE 6 The NS3/4A sequence was amplified from the serum of an HCV-infected patient (HCV genotype 1a) 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. Virol. 43:223-226 (1995), herein expressly incorporated by reference in its entirety). 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 codon and the primer NS4KR contained a XbaI 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 1a. 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 XbaI, and was inserted into a pcDNA3.1/His plasmid (Invitrogen) digested with the same enzymes. The NS3/4A-pcDNA3.1 plasmid was then digested with EcoRI and XbaI and the insert was purified using the QiaQuick kit (Qiagen, Hamburg, Germany) and was ligated to a EcoRI/Xba I digested pVAX vector (Invitrogen) so as to generate the NS3/4A-pVAX plasmid. The rNS3 truncated mutant was obtained by deleting NS4A sequence from the NS3/4A DNA. Accordingly, the NS3 gene sequence of NS3/4A-pVAX was PCR amplified using the primers NS3KF and 3'NotI (5'-CCA CGC GGC CGC GAC GAC CTA CAG-3' (SEQ. ID. NO.: 20)) containing EcoRI and Not I restriction sites, respectively. The NS3 fragment (1850 bp) was then ligated to a EcoRI 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 were 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 QUICKCHANGE.TM. 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 5'-ACCGAGCACCCAGGTAGGCGTGACGACCTCCAG-3' (SEQ. ID. NO.: 22) resulting in NS3/4A-TPT-pVAX. To generate the "RGT" mutation, the plasmid was amplified using the primers 5'-CTGGAGGTCGTCCGCGGTACCTGGGTGCTCGTT-3' (SEQ. ID. NO.: 23) and 5'-ACCGAGCACCCAGGTACCGCGGACGACCTCCAG-3' (SEQ. ID. NO.: 24) 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. 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.
TABLE 10
Plasmid Deduced amino acid sequence
*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)
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)
NS5-pVAX ASEDVVCCSMSYTWTG (SEQ. ID. NO.: 41)
NS5A/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 P1' positions.
Nucleic Acids Encoding HCV Peptides 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, 15, or 20 amino acids in length (e.g., SEQ. ID. 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 NaHPO.sub.4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 50.degree. C.) and washing in 0.2.times.SSC/0.2% SDS at 50.degree. 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 NaHPO.sub.4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 37.degree. C. and washing in 0.2.times.SSC/0.2% SDS at 37.degree. 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 15-30 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 0.5M NaHPO.sub.4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 42.degree. C. overnight and washing can be performed in 0.2.times.SSC/0.2% SDS at 42.degree. C. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., herein expressly incorporated by reference. HCV nucleic acids can also be isolated from patients infected with HCV using the nucleic acids described herein. (See also Example 6). 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), the disclosure of which is incorporated herein by reference in its entirety and the publication entitled "PCR Methods and Applications" (1991, Cold Spring Harbor Laboratory Press), the disclosure of which is incorporated herein by reference in its entirety. 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. Pat. No. 5,322,770, the disclosure of which is incorporated herein by reference in its entirety. Another technique involves the use of Reverse Transcriptase Asymmetric Gap Ligase Chain Reaction (RT-AGLCR), as described by Marshall R. L. et al. (PCR Methods and Applications 4:80-84, 1994), the disclosure of which is incorporated herein by reference in its entirety. 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 is 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 polymerase, such as Taq polymerase, Pfu polymerase, or Vent polymerase. 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 U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, the disclosures of which are incorporated herein by reference in their entirety. 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 are 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 bp to the entire length of the HCV genome. Amplification fragments from 25-1000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp 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 cDNA 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 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, herein expressly incorporated by reference in its entirety). 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, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification. (See Example 6). Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis. (Hutchinson et al., J. Biol. Chem., 253:6551 (1978), herein incorporated by reference in its entirety). Further, nucleic acids encoding other 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. EXAMPLE 7 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), herein expressly incorporated by reference). In brief, mice were injected intramuscularly with 50 .mu.l/TA of 0.01 mM cardiotoxin (Latoxan, Rosans, France) in 0.9% sterile NaCl. Five days later, each TA muscle was injected with 50 .mu.l 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 .mu.l/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 NS3 antibodies. These assays were performed essentially as described in (Chen et al., Hepatology 28(1): 219 (1998)). Briefly, rNS3 was passively adsorbed overnight at 4.degree. C. to 96-well microtiter plates (Nunc, Copenhagen, Denmark) at 1 .mu.g/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.degree. 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/5 ml of 1M Diethanol amine buffer with 0.5 mM MgCl.sub.2). 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 (FIG. 4). After six weeks, four out of five mice immunized with NS3/4A-pVAX had developed high levels (>10.sup.4) of NS3 antibodies (mean levels 10800.+-.4830) 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 1800.+-.805). 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. EXAMPLE 8 To test if the enhanced immunogenicity of NS3/4A 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.
TABLE 11
No. of antibody responders to the respective immunogen
Weeks from 1.sup.st after one 100 .mu.g i.m immunization
immunization NS3-pVAX NS3/4A-pVAX NS3/4A-TPT-pVAX
2 0/10 17/20 4/10
4 0/10 20/20 10/10
(<60) (2415 .+-. 3715) (390 .+-. 639)
55% > 10.sup.3 50% > 10.sup.2
10% > 10.sup.4 10% > 10.sup.3
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 (HCC). 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. EXAMPLE 9 To study whether the constructs described above were capable of eliciting a cell-mediated response against NS3, an in vivo tumor growth assay was perfomed. To this end, an SP2/0 tumor cell line stably transfected with the NS3/4A gene was made. The pcDNA3.1 plasmid containing the NS3/4A gene was linearized by BglII digestion. A total of 5 .mu.g linearized plasmid DNA was mixed with 60 .mu.g 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 800 .mu.g/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.times.10.sup.6 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.
TABLE 12
Mouse Tumor Maximum in vivo tumor size at indicated time
point
ID cell line 5 6 7 8 11 12 13
14 15
1 SP2/0 1.6 2.5 4.5 6.0 10.0 10.5 11.0
12.0 12.0
2 SP2/0 1.0 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 1.0 1.0 3.0 4.0 5.0 6.0 6.0
6.0 7.0
Group mean 1,92 3.3 5.0 6.2 9.3 10.1 10.7
11.6 12.1
6 NS3/4A- 1.0 2.0 3.0 3.5 4.0 5.5 6.0
7.0 8.0
SP2/0
7 NS3/4A- 2.0 2.5 3.0 5.0 7.0 9.0 9.5
9.5 11.0
SP2/0
8 NS3/4A- 1.0 2.0 3.5 3.5 9.5 11.0 12.0
14.0 14.0
SP2/0
9 NS3/4A- 1.0 1.0 2.0 6.0 11.5 13.0 14.5
16.0 18.0
SP2/0
10 NS3/4A- 3.5 6.0 7.0 10.5 15.0 15.0 15.0
15.5 20.0
SP2/0
Group mean 1,7 2.7 3.7 5.7 9.4 10.7 11.4
12.4 14.2
p-value of student's 0,7736 0.6918 0.4027 0.7903 0.9670
0.7986 0.7927 0.7508 0.4623
t-test comparison
between
group means
EXAMPLE 10 To examine whether a T-cell response is elicited by the NS3/4A immunization, the 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. Immunol. 161:4917 (1998), herein expressly incorporated by reference in its entirety). 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 one month intervals with either 100 .mu.g NS3-pVAX or 100 .mu.g NS3/4A-pVAX. Two weeks after the last immunization 2.times.10.sup.6 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 NS3/4A-SP2/0 tumor sizes in the NS3-pVAX immunized mice (See TABLE 13).
TABLE 13
Maximum tumor
Mouse ID Immunogen Dose (.mu.g) Tumor cell line Tumor growth size
(mm)
1 NS3-pVAX 100 SP2/0 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
5 NS3-pVAX 100 SP2/0 Yes 13
Group total 4/5 9.75 .+-. 4.992
6 NS3-pVAX 100 NS3/4A-SP2/0 Yes 9
7 NS3-pVAX 100 NS3/4A-SP2/0 Yes 8
8 NS3-pVAX 100 NS3/4A-SP2/0 Yes 7
9 NS3-pVAX 100 NS3/4A-SP2/0 No --
10 NS3-pVAX 100 NS3/4A-SP2/0 No --
3/5 8.00 .+-. 1.00
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 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 Std. Dev. Std. Err
NS3-sp2 4 9.750 24.917 4.992 2.496
NS3-spNS3 3 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 by NS3/4A-TVAX immunization was determined. (See TABLE 14). Thus, NS3/4A-pVAX immunization elicits CTLs that inhibit growth of cells expressing NS3/4A in 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.
TABLE 14
Maximum
tumor
Mouse ID Immunogen Dose (.mu.g) Tumor cell line Tumor 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 17.25 .+-.
8.421
16 NS3/4A-pVAX 100 NS3/4A-SP2/0 No --
17 NS3/4A-pVAX 100 NS3/4A-SP2/0 Yes 9
18 NS3/4A-pVAX 100 NS3/4A-SP2/0 Yes 7
19 NS3/4A-pVAX 100 NS3/4A-SP2/0 Yes 5
20 NS3/4A-pVAX 100 NS3/4A-SP2/0 Yes 4
4/5 6.25 .+-.
2.217
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 Diff. 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. Std. Err
NS3/4-sp2 4 17.250 70.917 8.421 4.211
NS3/4-spNS3 4 6.250 4.917 2.217 1.109
EXAMPLE 11 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 a 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.
TABLE 15
Dose Anti- Tumor Maximum
tumor
Mouse ID Immunogen (.mu.g) Tumor cell line NS3 growth size
(mm)
1 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 12.0
2 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 20.0
3 NS3-pVAX 10 NS3/4A-SP2/0 60 + 18.0
4 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 13.0
5 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 17.0
Group mean 60 5/5 16.0 .+-.
3.391
6 NS3-pVAX 100 NS3/4A-SP2/0 2160 + 10.0
7 NS3-pVAX 100 NS3/4A-SP2/0 <60 - --
8 NS3-pVAX 100 NS3/4A-SP2/0 <60 - --
9 NS3-pVAX 100 NS3/4A-SP2/0 360 - --
10 NS3-pVAX 100 NS3/4A-SP2/0 <60 + 12.5
Group mean 1260 2/5 11.25 .+-.
1.768
11 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 + 10.0
12 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 - --
13 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 - --
14 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 + 13.0
15 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 + 13.5
Group mean <60 3/5 12.167 .+-.
1.893
16 NS3/4A-pVAX 100 NS3/4A-SP2/0 60 + 10.0
17 NS3/4A-pVAX 100 NS3/4A-SP2/0 360 - --
18 NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 8.0
19 NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 12.0
20 NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 7.0
Group mean 1380 4/5 9.25 .+-.
2.217
36 p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 20.0
37 p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 7.0
38 p17-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 mean <60 5/5 14.20 .+-.
5.263
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-SP2/0 >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 mean 466560 4/5 17.333 .+-.
4.509
46 PBS -- NS3/4A-SP2/0 <60 + 10.0
47 PBS -- NS3/4A-SP2/0 <60 + 16.5
48 PBS -- NS3/4A-SP2/0 60 + 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 mean 60 5/6 15.50 .+-.
3.937
Note:
Statistical analysis (StatView): Student's t-test on maximum 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-sp3-4, NS3-100-sp3-4 2.950 5 .739 .4933
p17-sp3-4, NS3/4-10-sp3-4 2.033 6 .628 .5532
p17-sp3-4, NS3-10-sp3-4 -1.800 8 -.643 .5383
p17-sp3-4, NS3/4-100-sp3-4 4.950 7 1.742 .1250
p17-sp3-4, PBS-sp3-4 -1.300 8 -.442 .6700
p17-sp3-4, rNS3-sp3-4 -3.133 6 -.854 .4259
NS3-100-sp3-4, NS3/4-10-sp3-4 -.917 3 -.542 .6254
NS3-100-sp3-4, NS3-10-sp3-4 -4.750 5 -1.811 .1299
NS3-100-sp3-4, NS3/4-100-sp3-4 2.000 4 1.092 .3360
NS3-100-sp3-4, PBS-sp3-4 -4.250 5 -1.408 .2183
NS3-100-sp3-4, rNS3-sp3-4 -6.083 3 -1.744 .1795
NS3/4-10-sp3-4, NS3-10-sp3-4 -3.833 6 -1.763 .1283
NS3/4-10-sp3-4, NS3/4-100-sp3-4 2.917 5 1.824 .1277
NS3/4-10-sp3-4, PBS-sp3-4 -3.333 6 -1.344 .2274
NS3/4-10-sp3-4, rNS3-sp3-4 -5.167 4 -1.830 .1412
NS3-10-sp3-4, NS3/4-100-sp3-4 6.750 7 3.416 .0112
NS3-10-sp3-4, PBS-sp3-4 .500 8 .215 .8350
NS3-10-sp3-4, rNS3-sp3-4 -1.333 6 -.480 .6480
NS3/4-100-sp3-4, PBS-sp3-4 -6.250 7 -2.814 .0260
NS3/4-100-sp3-4, rNS3-sp3-4 -8.083 5 -3.179 .0246
PBS-sp3-4, rNS3-sp3-4 -1.833 6 -.607 .5662
EXAMPLE 12 To determine whether NS3-specific T-cells were elicited by the NS3/4A immunizations, an in vitro 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), herein expressly incorporated by reference in its entirety). Briefly, groups of five Balb/c mice were immunized three times with 100 .mu.g NS3/4A-pVAX i.m. Two weeks after the last injection the mice were sacrificed and splenocytes were harvested. Re-stimulation cultures with 3.times.10.sup.6 splenocytes and 3.times.10.sup.6 NS3/4A-SP2/0 cells were set. After five days, a standard Cr.sup.51 -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 FIGS. 5A 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. Am. Chem. Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA, 82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill. (1984), and Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y., herein expressly incorporated by reference. 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 transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in 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, which is incorporated by reference herein in its entirety. 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., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the HCV nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (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(1983), 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 Heeke & Schuster, J. Biol. Chem., 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, Autographa 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, herein expressly incorporated by reference in their entirety). 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.sup.-, hgprt.sup.- or aprt.sup.- 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. Sci. 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 (Colberre-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 Ni.sup.2+ 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. EXAMPLE 13 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, Wis.) according to the manufacturer's instructions. All in vitro translation reactions of the expression constructs were carried out at 30.degree. C. with .sup.35 S-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 61 kDa, 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 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 in 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 five 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 Chlamydomonas. Desirable insect systems having an HCV peptide include, but are not limited to, D. melanogaster and C. elegans. 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 de | ||||||