CN1289339A - Genetic Immunization Using Hepatitis C Virus Nonstructural Proteins - Google Patents

Abstract

The present invention discloses a nucleic acid molecule (including specifically disclosed DNA sequences) encoding hepatitis C nonstructural proteins. Also disclosed are pharmaceutical compositions comprising a nucleic acid molecule encoding a hepatitis C nonstructural protein of the nucleotide sequences NS3, NS4, and NS5, or a combination thereof, operably linked to regulatory elements functional in human cells. Also disclosed are methods of immunizing individuals susceptible to or infected with hepatitis C virus comprising administering these pharmaceutical compositions.

Description

Genetic Immunization Using Hepatitis C Virus Nonstructural Proteins

field of invention

The present invention relates to recombinant nucleic acid molecules and pharmaceutical compositions comprising, for example, the same useful vaccine components against hepatitis C virus in genetic immunization regimens; to methods of inducing an immune response against hepatitis C virus infection; and to the treatment of patients with hepatitis C Method for patients with hepatitis infection.

Background of the invention

Hepatitis C virus (HCV), the major causative agent of blood transfusion-acquired non-A, non-B hepatitis, causes approximately 150,000 new cases of acute viral hepatitis in the United States each year. (Choo, et al., Science, 1989, 244, 359-362.) In Western countries, there is a prevalence of 0.6% to 2.0%, and in some underdeveloped parts of the world it is as high as 15%. (Heintges, et al., Hepathology, 1997, 26, 521-526.) About half of these infections develop into chronic infections associated with cirrhosis and/or liver cancer. (Alter, et al., Science, 1992, 258, 135-140; and alter, et al., New England Journal of Medicine, 1992, 327, 1899-1905). Furthermore, the prevalence of anti-HCV antibodies showed that HCV infection is an independent risk factor for the development of hepatocellular carcinoma (Colombo, et al., Lancet, 1989, ii, 1006-1008; Saito, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 6547-6549; Simonetti , et al., An. Int. Med., 1992, 116, 97-102; and Tsukuma, et al., New England Journal of Medicine, 1993, 328, 1797-1801).

Hepatitis C virus is a capsid, positive-sense RNA virus approximately 9,500 nucleotides in length that was recently classified as a separate species in the genus Flavivirus. (Heinz, Arch. Virol. (Suppl.), 1992, 4, 163-171.) Different isolates show considerable nucleic acid sequence diversity, leading to a further division of the genome of HCV into eight genotypes. (Simmonds, et al., J.Gen.Virol., 1993,74,2391-2399.) In all genotypes, the viral genome contains a large open reading frame (ORF), encoding a sequence of 3010 to 3033 amino acids Consisting of a polyprotein precursor of approximately 330Kd. (Choo, etc., Proc.Natl.Acad.Sci.USA, 1991,88,2451-2455; Inchauspe, etc., Proc.Natl.Acad.Sci.USA, 1991,88,10292-10296; Kato, etc., Proc .Natl.Acad.Sci.USA, 1990, 87, 9524-9528; Okamoto, et al., Journal of Viral Genetics, 1991, 72, 2697-704; and Takamizawa, et al., J.Gen.Virol., 1991, 65, 1105-1113.)

Various HCV polypeptides are generated by proteolytic processing of this polyprotein precursor to generate core (C), capsid (E1, E2) and nonstructural (NS2-NS5) proteins. (Bartenschlager, et al., Journal of Viral Genetics, 1993, 67, 3835-3844; Grakoui, et al., Journal of Viral Genetics, 1993, 67, 2832-2843; and Selby, et al., Journal of Viral Genetics, 1993, 74, 1103-1113). This proteolysis is catalyzed by a combination of cellular and virally encoded proteases. The NS3 gene encodes a serine protease that cleaves the post-transcribed viral polyprotein precursor at several functional sites and also functions as a protein helicase. The NS5 region encodes the RNA-dependent RNA polymerase of this virus.

In addition to the transcribed region, the HCV genome contains a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR). The 5' untranslated region nucleic acid sequence from 324 to 341 represents the most conserved nucleic acid sequence among all HCV isolates reported so far. (Han, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 1711-1715; and Bukh, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 4942-4946). This 5' untranslated region is assumed to contain important regulatory elements for HCV RNA replication and/or translation. The 5' UTR also contains several small open reading frames (ORFs), but there is no evidence to date that these ORFs are actually translated.

The cellular immune mechanisms involved in liver injury and viral clearance during HCV infection are only partially described. In an attempt to examine the potential pathogenic role of liver-infiltrating lymphocytes in patients with chronic HCV infection, Koziel et al. examined cytotoxic T lymphocyte (CTL) responses to these cells and demonstrated that HLA class I-restricted CD8 ⁺ cytotoxic T lymphocytes Cellular responses are directed against structural and nonstructural regions of the HCV polypeptide. (Koziel, et al., J. Virol. 1993, 67, 7522-7532; and Koziel, et al., J. Immunol., 1992, 149, 3339-3344). Other investigators have also noted the presence of cytotoxic T lymphocytes in the peripheral blood mononuclear cell population that recognize epitopes on the viral core and other virus-associated proteins during chronic HCV infection. (Kita, et al., Heatol., 1993, 18, 1039-1044; and Cerny, et al., Intl. Symp. viral Hepatitis Liver Dis., 1993, 83 (abstr.).) Botarelli, et al., (Botarelli, et al., Gastroenterol ., 1993, 104, 580-587) and Ferrari, et al., (Ferrari, Hepatol., 1994, 19, 286-295) found HLA class II-restricted HLA class II-restricted proteins targeting different regions of HCV obtained from patients with chronic HCV infection CD4 ⁺ T cell-mediated proliferative response.

In effective HCV infection, humoral and cellular immune responses have been shown to be polyclonal, polyspecific. It is likely that the host immune response generated during persistent HCV infection partially induces hepatocellular injury. However, these immune responses are not broad or robust enough to promote viral clearance and protective immunity in patients with chronic HCV infection. (Chisari, J. Clin. Invest., 1997, 99, 1472-1477.) It was recently shown that those patients recovering from acute HCV infection can develop a strong proliferative response to nonstructural protein polypeptide CD4 ⁺ T cells. (Missale, et al., J.Clin.Invest., 1996, 98, 706-714; and Diepolder, et al., Lancet, 1995, 346, 1006-1007.) More importantly, the generation of HCV-specific CTL activity is associated with chronic hepatitis associated with the control of viral replication in an individual. (Rehermann, et al., J. Clin. Invest., 996, 98, 1432-1440; and Nelson, et al., J. Immunol., 1997, 158, 1473-1481.)

However, whether the nonstructural proteins NS3, NS4, and NS5 are sufficiently immunogenic to generate broad and robust CTL responses in vivo is unknown.

Currently there is no universal and highly effective treatment for chronic HCV infection. The development of HCV vaccine strategies is complicated not only by the significant heterogeneity of HCV isolates, but also by the mixing of heterologous genomes within a single viral isolate. (Martell, et al., J. Virol., 1992, 66, 3225.) Furthermore, viruses contain a highly variable capsid region. Effective treatment is limited to interferon. (Carithers, et al, Hepatology, 1997, 26, 83S-88S.) In fact, approximately 8-10% of patients treated with this formulation respond with HCV spread from the liver. However, recent studies have shown that patients recovering from acute HCV infection mount an adequate CD4 ⁺ T cell proliferative response to nonstructural proteins compared with those who acquire persistent HCV infection. (Missale, et al., J. Clin. Invest., 1996, 98, 706-714; and Diepolder, et al., Lancet, 1995, 346, 1006-1007)

Direct injection of DNA into animals is a promising approach for immunization to deliver specific immune antigens. (Barry, et al, Bio Techniques, 1994,16,616-619; Davis, et al, Hum. J.Virol.,1993,67,3338-3344; and Wolff, et al., Science, 1990,247,1465-1468.) This method has been successfully applied to produce influenza virus in mice and chickens, in mice and Protective immunity against bovine herpesvirus in calves and rabies virus in mice. (Cox, et al., J. Virol., 1993, 67, 5664-5667; Fynan, et al., DNA and Cell Biol., 1993, 12, 785-789; Ulmer et al., Science, 1993, 259, 1745-1749; and Xiang et al. , Virol., 1994, 199, 132-140.) In most cases, strong and highly diverse antibody and cytotoxic T cell responses are associated with control of infection. In fact, the possibility of generating long and persistent memory CTLs without the use of liver vectors, compared with those in which vaccines with killed viruses not only elicit protection against acute infection but also eradicate CTL responses to persistent viral infections , making this approach particularly attractive. (Wolff, et al., Science, 1990,247,1465-1468; Wolff, et al., Hum.Mol.Genet., 1992, I, 363-369; Manthorpe, et al., Human Gene Therapy, 1993,4,419-431; Ulmer et al. , Science, 1993, 259, 1745-1749; Yankauckas, etaal., DNA and Cell Biology, 1993, 12, 777-783; Montgomery, et al., DNA and Cell Biology, 1993, 12, 777-783; Fynan, et al., DNA and Cell Biology, 1993, 12, 777-783; Cell Biology, 1993, 12, 785-789; Wang, et al., Proc. et al., Virol., 1994, 199, 132-140; Davis, et al., Hum. Mol. Genet., 1993, II, 1847-1851; Donnelly, et al., Nat.Med., 1995, I, 583-587; Boyer, et al. , Nat. Med., 1997, 3, 526-532; Tascon, et al., Nat. Med., 1996, 2, 888-892; and Huygen, et al., Nat. Med. 1996, 2, 893-898.). Compared with fusion recombinant protein or polypeptide, the advantage of this method is that it can cause strong inflammatory CD4 ⁺ T cell response and cytotoxic T cell activity, presumably because viral protein is processed into polypeptide in the cell, and then loaded in the transfection On the MHC class I molecules of cells, it is caused by the interaction with antigen-presenting cells. In contrast, immunization with the fusion protein primarily results in a humoral immune response that is processed by the MHC class II pathway. Immunization with synthetic polypeptides has several drawbacks due to the fact that only a limited number of epitopes are capable of stimulating an immune response in the host. Conversely, for each protein encoded by the DNA construct of interest, naturally occurring B and T cell epitopes are likely to be reserved for recognition by T cell receptors and thus likely to generate a broad range of humoral and cellular immune responses. (McDonnell, et al., N. Engl. J. Med., 1996, 334, 42-45.)

Vaccination and immunization generally refer to the introduction of a non-toxic substance against which an individual's immune system stimulates an immune response, thereby protecting against attack by a pathogen. The immune system recognizes invading "foreign" components and substances primarily by recognizing proteins and other macromolecules that are not normally present in the individual. Foreign proteins are the targets of the immune response.

PCT patent application PCT/US90/01348 discloses sequence information of HCV genome clones, amino acid sequences of HCV viral proteins and methods of making and using components including anti-HCV vaccines containing HCV proteins and polypeptides produced therein.

US Patents 5,830,876, 5,593,972, 5,739,118 and PCT Patent Application Serial No. PCT/US94/00899, filed January 26, 1994, the entire contents of which are incorporated herein by reference. Each patent contains details of the genetic immunization protocol. Vaccines against HCV are also included in the various patents.

A need remains for a vaccine for protecting individuals against HCV infection, as does a need for methods of protecting individuals against HCV infection.

Summary of the invention

The present invention relates to a recombinant nucleic acid sequence molecule, which comprises a nucleotide sequence encoding an HCV non-structural region, such as NS3, NS4, or NS5, or a combination thereof.

The present invention relates to a pharmaceutical composition comprising recombinant nucleic acid molecule, which contains a nucleotide sequence encoding HCV non-structural protein. The nucleotide coding sequence encoding the HCV nonstructural protein is operably linked to functional regulatory elements in human cells. The pharmaceutical composition also comprises a pharmaceutically acceptable carrier or diluent, and optionally a facilitator, such as bupivacaine.

The invention relates to a method for immunizing HCV-susceptible individuals, comprising administering to the individuals, the pharmaceutical composition comprising recombinant nucleic acid molecules encoding HCV nonstructural protein nucleotide sequences. The nucleotide coding sequence encoding HCV nonstructural protein is operably linked to functional regulatory elements in human cells. The pharmaceutical composition also includes a pharmaceutically acceptable carrier or diluent. An amount of drug effective to induce a protective immune response against HCV infection is administered to the individual.

The present invention relates to a method for treating an individual suffering from HCV infection, comprising treating the individual with a pharmaceutical composition comprising a recombinant nucleic acid molecule encoding a nucleotide sequence of a nonstructural protein of HCV. The nucleotide coding sequence encoding the HCV non-structural protein is operably linked to the functional regulatory elements in human cells. The pharmaceutical composition also includes a pharmaceutically acceptable carrier or diluent. The individual is administered an amount of drug effective to induce a therapeutic immune response to HCV infection.

Brief description of the drawings

Figure 1A is a schematic diagram showing the single large open reading frame of HCV, which encodes a polyprotein precursor of approximately 3011-3030 amino acids. This protein precursor is cleaved by host signaling and viral proteases into different structural and nonstructural proteins, as indicated by the arrows. Figure IB shows exemplary autoradiograms of nonstructural protein expression after transient transfection of HuH-7 cells and stable transfection of SP2/0 cells. Lanes 1, 3, and 5 are mock DNA-transfected cells used as negative controls (Mock); lanes 2, 4, and 6 show specific bands, NS3 at about 70, NS4 at about 30, and NS5 at about 125KD. Lanes 7-10 show SP2/0 cells transfected with nucleic acid constructs containing NS3, NS4, and NS5 genes, respectively. Lanes 7 and 9 represent cell lysates obtained from cells stably expressing HCV-core protein as a negative control (SP2-10), while lanes 8 and 10 represent specific expression of NS3 and NS5.

Figure 2A is a histogram showing a representative humoral immune response to NS3, NS4 and NS5 elicited by DNA-based immunization; serum antibody levels were determined by an ELISA (per group: n=5). Figure 2B is a histogram showing a representative T cell proliferation measured in vitro after 3 days of stimulation with specific or non-specific recombinant proteins. Figures 2C, 2D, and 2E are bar graphs showing the secreted levels of representative cytokines IFN-γ, IL-2, and IL-4 in the supernatant, respectively, measured after stimulation in vitro for 48 hours.

Figures 3A and 3B are histograms showing representative cytotoxic T cells against NS3 and NS5 at different ratios of effector and target cells (100:1, 30:1, 10:1, 3:1). (CTL) response. Figure 3C is a histogram showing a representative chromium release experiment against a line of stably transfected target cells.

Figure 4A is a table showing representative results of tumor models evaluating CTL activity. Fig. 4B is a photograph showing from left to right that 1) mock DNA was inoculated and challenged with SP2/NS5-21 cells; 2) pApNS5 was challenged with SP2/NS5-21 cells; 3) pApNS5 was challenged with SP2-19 cells challenge; (stable expression of HCV core protein); and 4) animals inoculated three times intraperitoneally with recombinant NS5 protein and challenged with SP2/NS5-21 cells

Description of preferred examples of the invention

According to the present invention, there are provided methods of prophylactically and/or therapeutically immunizing, or treating an individual, against HCV infection. A recombinant nucleic acid molecule is inoculated into an individual, the recombinant nucleotide molecule comprises a nucleotide coding sequence encoding an HCV nonstructural protein, such as NS3, NS4, or NS5, or a combination thereof. The protein encoded by the recombinant nucleic acid gene construct is expressed by the cells of the individual, and acts as an immunogenic target to induce an anti-HCV immune response against it. The immune response elicited is extensive, with both pathways of cellular immunity activated in addition to the humoral immune response. The methods of the invention can be used to confer prophylactic and therapeutic immunity. In addition to being used for human beings, the method of the invention can also be used for biomedical research in mammals. Therefore, the method of the invention can be used not only for immunizing individuals against HCV challenge, but also for treating HCV-infected individuals.

The phrase "HCV nonstructural protein" as used herein refers to HCV nonstructural proteins NS3, NS4 and NS5, and their equivalents. Equivalent proteins include NS3, NS4 and NS5 peptide fragments that retain the biological activities described herein. Furthermore, the term HCV nonstructural protein means the corresponding HCV nonstructural protein from other HCV isolates, (possibly with variations in sequence). Those of ordinary skill in the art can readily identify nonstructural proteins of other HCV isolates. It is understood that substitutions of nucleotides in codons are acceptable when encoding the same amino acid. It should also be understood that when conservative amino acid substitutions result from nucleotide substitutions, nucleotide changes are also acceptable. It is to be understood that the phrase "HCV nonstructural protein" also includes fusion proteins comprising the nonstructural protein, as well as therapeutically and prophylactically active fragments thereof.

The phrase "genetic construct" as used herein refers to a recombinant nucleic acid molecule comprising a nucleotide coding sequence encoding a nonstructural protein of HCV, as well as start and stop signals, which include promoters, polyadenylation signals, and can direct Regulatory elements expressed in the cells of the seeded individual are operably linked. In some embodiments, the gene construct further comprises an enhancer, a Kozak sequence (GCCGCCATG; SEQ ID NO: 1), and at least one HCV 5'UTR fragment.

The phrase "genetic vaccine" as used herein refers to a pharmaceutical preparation that includes a genetic construct. Genetic vaccines comprise pharmaceutical agents for stimulating prophylactic and/or therapeutic immune responses to HCV.

The phrase "nucleic acid" as used herein refers to DNA, RNA, or chimeras formed therefrom.

According to the present invention, a genetic construct is introduced into and expressed in the cells of the individual, thereby producing at least one HCV nonstructural protein. More preferably, the regulatory elements of the genetic constructs of the invention are capable of directing expression in mammalian cells, especially human cells. The regulatory elements include promoters and polyadenylation signals. In addition, other elements, such as enhancers and Kozak sequences, may also be included in the gene construct.

After being taken up by the cells, the gene construct of the present invention can exist in the cells as extrachromosomal functional molecules or be integrated into the chromosomal DNA of the cells. Nucleic acids, such as DNA, can be introduced into cells where they exist as separate genetic material in the form of plasmids. In addition, linear nucleic acid molecules that can integrate into chromosomes can also be introduced into cells. When a nucleic acid is introduced into a cell, an agent that promotes integration of the nucleic acid into a chromosome may be added. DNA sequences that help facilitate integration can also be added to the DNA molecule. In addition, RNA can also be seeded into cells. It is also contemplated that the genetic constructs will be provided in the form of linear minichromosomes including centromeres, telomeres and origins of replication.

According to the present invention, the genetic construct comprises a recombinant nucleic acid molecule having a nucleotide coding sequence encoding a nonstructural protein of HCV. In some preferred embodiments, the recombinant nucleic acid molecule includes a nucleotide coding sequence encoding NS3. In other preferred embodiments, the recombinant nucleic acid molecule includes a nucleotide coding sequence encoding HCV nonstructural proteins including NS4. In other preferred embodiments, the recombinant nucleic acid molecule includes a nucleotide coding sequence encoding HCV nonstructural proteins including NS5. In other preferred embodiments, the recombinant nucleic acid molecule includes a nucleotide coding sequence encoding any combination of HCV non-structural proteins including NS3, NS4, and NS5.

In some preferred embodiments, the recombinant nucleic acid molecule includes a nucleotide coding sequence encoding HCV non-structural proteins including fragments of HCV NS3, NS4, and NS5 proteins or combinations thereof. Such fragments include, but are not limited to, fragments of 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acids of the corresponding nonstructural protein. In addition, these fragments may comprise a carboxy-terminal portion of the protein, an amino-terminal portion, or any portion in between. Researchers skilled in the art can readily prepare immunogenic fragments of HCV nonstructural proteins, or fusion proteins containing immunogenic fragments of nonstructural proteins in any combination. Thus, it is contemplated that recombinant nucleic acid molecules containing nucleotide coding sequences encoding HCV nonstructural proteins contain less than the entire HCV nonstructural gene product without substantially altering vaccine efficacy. It is also contemplated that at least one or more nucleotides may be substituted in the nucleotide coding sequence without affecting the amino acid sequence of the protein. In addition, it can also be considered that at least one or more conservative amino acid substitutions can be made in the entire protein without substantially reducing the immunogenicity of HCV nonstructural proteins.

In some preferred embodiments of the present invention, the recombinant nucleic acid molecule comprises the last 9 nucleotides of HCV5'UTR, the last 25 nucleotides of HCV5'UTR, the last 50 nucleotides of HCV5'UTR, and the last 75 nucleotides of HCV5'UTR Nucleotides, HCV5'UTR last 100 nucleotides, HCV5'UTR last 150 nucleotides, HCV5'UTR last 200 nucleotides, HCV5'UTR last 250 nucleotides, and HCV5'UTR last 300 nucleotides 5'UTR fragments of nucleotides. In some preferred embodiments, the genetic construct contains the entire HCV 5'UTR. In some preferred embodiments, the genetic construct contains the most 3' 9 nucleotides of the HCV 5' UTR.一个优选例中的整个HCV5’UTR是：GCCAGCCCCC GATTGGGGGC GACACTCCAC CATAGATCAC TCCCCTGTGA GGAACTACTGTCTTCACGCA GAAAGCGTCT AGCCATGGCG TTAGTATGAG TGTCGTGCAG CCTCCAGGACCCCCCCTCCC GGGAGAGCCA TAGTGGTCTG CGGAACCGGT GAGTACACCG GAATTGCCAGGACGACCGGG TCCTTTCTTG GATCAACCCG CTCAATGCCT GGAGATTTGG GCGTGCCCCCGCGAGACTGC TAGCCGAGTA GTGTTGGGTC GCGAAAGGCC TTGTGGTACT GCCTGATAGGGTGCTTGCGA GTGCCCCGGG AGGTCTCGTA GACCGTGCAC C  (SEQ ID NO:2)

Regulatory elements required for gene expression of DNA molecules include: promoters, initial codons, stop codons, and polyadenylic acid signals. In addition, enhancers are often required for gene expression. These elements need to be operably linked to sequences encoding HCV nonstructural proteins and need to be operable in the vaccinated individual. Start and stop codons are generally considered to be part of the nucleotide sequence encoding HCV nonstructural proteins.

The promoter and polyA signal used must be functional in the individual cell. To maximize protein production, regulatory elements well suited for gene expression in the cell to which the construct is administered can be selected. In addition, codons can be selected for the most efficient transcription in the cell. A researcher of ordinary skill in the field can construct DNA constructs that are functional in mammalian, especially human, cells.

Examples of promoters used to realize the present invention, especially for the production of genetic vaccines for humans, include but are not limited to: the promoter of Simian Virus 40 (SV40), the promoter of Murine Mammary Tumor Virus (MMTV) , human immunodeficiency virus (HIV), such as the promoter of the long last repeat (LTR) of HIV, leukemia (moloney) virus, ALV (avian leukemia (moloney) virus), cytomegalovirus (CMV), such as the immediate Early promoters, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and promoters from human genes such as human actin, human myosin, human hemoglobin, human creatinin, and human metallothionein .

Examples of polyA signals useful in the practice of the present invention, especially for the production of genetic vaccines for humans, include but are not limited to: the polyA signals of Simian Virus 40 (SV40) and LTR . The SV40 polyA signal specifically used was the SV40 polyA signal in the pCEP4 plasmid (Invitrogen, San Diego CA). In addition to the regulatory elements required for gene expression, other elements may also be contained in the gene construct. These other elements include enhancers. The enhancer can be selected from but not limited to the following group: human actin, human myosin, human hemoglobin, human creatinin and viral enhancers such as those of CMV, RSV and EBV.

A mammalian origin of replication can be provided to the genetic construct so that it remains extrachromosomal so that multiple copies of the construct are produced in the cell. Plasmids pCEP4, pREP4 (San Diego, CA) contain the replication origin of Epstein-Barr virus and the nucleic acid antigen EBNA-1 coding region that can produce high-copy episomal replication without integration.

Routes of administration include, but are not limited to: intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular and oral, as well as transdermally or by inhalation or suppositories. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. Delivery of genetic constructs encoding HCV nonstructural proteins confers mucosal immunity following immunization with substances present in tissues associated with mucosal immunity. Thus, in some embodiments, the genetic construct is delivered by oral vestibular inoculation in the oral cavity of the individual.

Genetic constructs can be administered by methods including, but not limited to: traditional injections, needle-free injection devices, or "microprojectile bombardment gene guns". In addition, genetic vaccines can also be introduced into cells removed from individuals by various methods. These methods include, for example, in vitro transfection, electroporation, microinjection, and microparticle bombardment. After the genetic construct has been taken up by the cells, the cells can be reimplanted into the individual. It is contemplated that otherwise non-immunogenic cells incorporating a genetic construct may be implanted into an individual, even if the inoculated cells originally came from another individual.

According to some preferred embodiments of the present invention, the gene construct is inoculated into the individual using a needle-free syringe. According to other preferred embodiments, the gene construct is inoculated into the individual simultaneously by intradermal, subcutaneous and intramuscular injection using a needle-free syringe. Needle-free injectors are well known and widely available. One of ordinary skill in the art would be able to use needle-free injectors to deliver genetic material to cells of an individual following the guidance herein. Needle-free injectors are ideal for delivering genetic material to all tissues. They are especially effective at transferring genetic material to skin and muscle cells. In some preferred embodiments, a needle-free injector can be used to push a liquid containing DNA molecules to the skin surface of an individual. The fluid is propelled with sufficient velocity that, due to the force of impact against the skin, the fluid penetrates the surface of the skin and penetrates into the underlying skin and muscle tissue. Thus, the genetic construct is simultaneously inoculated into the dermis, subcutaneously and muscle. In some preferred embodiments, for the introduction of nucleic acid molecules into the cells of an organ, the needle-free injector can be used to deliver genetic material to tissues of other organs.

According to the present invention, the genetic vaccine contains 1 ng to 1000 mg of nucleic acid, preferably DNA. In some preferred embodiments, the vaccine contains from 10 nanograms to about 800 milligrams of nucleic acid. In some preferred embodiments, the vaccine contains 0.1 to about 500 mg of nucleic acid. In some preferred embodiments, the vaccine contains 1 to about 350 mg of nucleic acid. In some preferred embodiments, the vaccine contains 25 to about 250 mg of nucleic acid. In some preferred embodiments, the vaccine contains about 100 mg of nucleic acid. Vaccines of nucleic acid in the required amount can be readily formulated by those skilled in the art.

The genetic vaccine of the present invention is configured according to the vaccination mode. Pharmaceutical compositions containing genetic constructs can be readily formulated by those of ordinary skill in the art. The pharmaceutical composition of the present invention comprises a single gene construct encoding NS3, NS4, or NS5, or a combination thereof. In addition, the pharmaceutical composition of the present invention comprises multiple gene constructs encoding NS3, NS4, or NS5, or combinations thereof. In addition, the pharmaceutical composition of the present invention comprises single or multiple genetic constructs encoding fragments of NS3, NS4, or NS5, or combinations thereof. In addition, the pharmaceutical compositions of the present invention comprise a single genetic construct encoding fusion proteins of all or any of the NS3, NS4, or NS5 protein fragments. In some instances, isotonic formulations are employed. Typically, isotonic additives may include sodium chloride, dextran, mannitol, sorbitol and lactose. In some instances, isotonic solutions such as phosphate buffered saline may be employed. Stabilizers include gelatin and albumin. In some preferred embodiments, a vasoconstrictor is added to the formulation. The pharmaceutical preparation of the present invention needs to be sterile and pyrogen-free.

The genetic constructs of the present invention may be formulated or co-vaccinated with agents that increase the uptake and/or expression of the genetic constructs, referred to herein as facilitators, which relate to the cells involved in the uptake and expression of the genetic constructs, and the cells that arose when the same genetic vaccine was vaccinated in the absence of these agents. These agents and protocols for co-vaccinating them with genetic constructs are described in US Pat. Examples of such agents include: CaPO4, DEAE, dextran, lipids, extracellular matrix active enzymes; saponins; lectins; estrogenic compounds and steroid hormones; carbonyl lower alkanes, dimethylsulfoxide (DMSO ); urea; benzoate anilides; amidines; urethanes and hydrochlorides of the local anesthetic family. In addition, the genetic construct can be encapsulated in or co-seeded with the lipid/polycation complex. A preferred facilitator is bupivacaine. The components may conveniently be presented in unit dosage and may be prepared by any of the methods well known in the art of pharmacy. For example, the method described in Remington's Phamaceutical Sciences (Mack Pub. Co., Easton, PA, 1980). The content disclosed in this article is incorporated by reference in its entirety.

In the Examples, the following DNA vaccination with plasmids encoding three different nonstructural proteins of HCV, shown to stimulate two components of the immune system capable of strong antigen-specific immune responses, is provided. After three immunizations, all animals developed a detectable antibody response. In this regard, these nonstructural proteins are much better antigens for stimulating humoral immune responses than previous studies using structural HCV core structural proteins. (Tokushige, et al., Hepatology, 1996, 24, 14-20; and Geissler, et al., J. Immunol., 1997, 158, 1231-1237.) In preferred embodiments, humoral immunity to nonstructural proteins can be achieved by adding energy Enhanced by compounds that activate antigen-presenting cells, for example, plasmids expressing cytokines such as IL-2 and GM-CSF. (Geissler, et al., J. Immunol., 1997, 158, 1231-1237; and Xiang, Immunity, 1995, 2, 129-135.) All three plasmids encoding NS3, NS4, NS5 were demonstrated to produce a dominant THI Phenotype of inflammatory CD4 ⁺ T cells. In addition, strong and specific CD8 ⁺ CTL responses were mainly directed against NS3 and NS5 produced by the lysed virus. It has previously been shown to induce protection against various pathogens in animal model systems. (Tascon, et al., Nat. Med., 1996, 2, 888-892; and Huygen, et al., Nat. Med., 1996, 2, 893-898.) Furthermore, it was to be determined whether the CTL responses generated by DNA vaccines could protect animals against the following Tumor formation in syngeneic SP2/0 tumor cells stably transfected with cDNA encoding NS5 protein. As a result, approximately 60% of the mice were protected from tumor formation, indicating that this immunization method produced high levels of CTL activity in vivo. In addition, compared with animals immunized with mock DNA or recombinant NS5 protein, tumor-bearing In those animals, tumor weight decreased significantly. This model also demonstrates the ability to assess high levels of cellular immune responses against flavivirus nonstructural proteins as a measure of tumor growth inhibition in animal models.

The results disclosed herein demonstrate that DNA-based immunization with genetic constructs encoding HCV nonstructural proteins, as described herein, can be used for the therapeutic treatment of individuals with HCV infection, as well as for prophylactic use against HCV. vaccine.

The present invention will be further described by the following examples in an attempt to clarify the invention. These examples are not meant, or construed, to limit the scope of the disclosure.

Example

Embodiment 1: design and construct HCV expression vector

Genes encoding individual nonstructural proteins were cloned into an expression plasmid with engineered start and stop codons, driven by the CMV promoter and RSV enhancer (pAp031). This expression vector (pcDNA3) including a selectable marker was also used to generate a stable SP2/0 transfected cell line. (See Figure 1A).

As the source of the virus gene, a plasmid named pBRTM/HCV1 covering the full-length HCV open reading frame is cloned into the expression vector of the present invention. (Grakoui, et al., J. Viol., 1993, 67, 1385-1395) The disclosure of which is incorporated herein by reference in its entirety. In addition, the nucleic acid sequence encoding HCV can be obtained from GenBank (GenBank) with accession numbers X61596 and D16435, the disclosures of which are fully incorporated herein. Constructs pAp031-NS3, pAp031-NS4, pAp031-NS5 were PCR clones after insertion of engineered start and stop codons and restriction sites using the following primers: For NS3: 5'-GGTCTAGATT GATGGCGCCC ATCACGGC-3'(XbaI) (SEQ ID NO:3), 5'-CACACGCGTT CACGTGACGA CCTCCAGGT-3'(MluI) (SEQ ID NO:4), for NS4: 5'GGTCTAGATG AGCACCTGGG TGCTC-3'(XbaI) (SEQ ID NO:5),5'-CCAGGATCCT CAGCATGGAG TGGTACA-3'(BamH1)(SEQ ID NO:6), and to NS5:5'-TCAGTCTAGA ATGTCCGGCT CCGGCTCCTG GCTAAGGGA-3'(XbaⅠ)(SEQ ID NO:7) , 5'-AGCTACGCGT TCACCGGTTG GGGAGGAGGT-3' (MluI) (SEQ ID NO: 8). After PCR amplification using a high-precision PCR system (Boehringer Mannheim, Indianapolis, IN), the cDNA fragment was inserted into a plasmid expression vector pAp031 containing an RSV enhancer element, driven by a CMV promoter (Apollon, Malvern, PA). The constructs were grown in DH5α cells and the plasmid DNA was purified by 2x cerium chloride centrifugation or Qiagen Giga kit using an endonuclease-free buffer system (Santa Clara, CA). Detection of insertions in nonstructural genes can be performed by sequence analysis using standard methods.

To establish stable expression cell lines of NS3, NS4, and NS5 as target cells for CTL assays, clone nonstructural protein-coding gene fragments into pcDNA3 and pcDNA3.I Zeo(-) expression vectors with neomycin selectable markers (Invitrogen, San Diego). First, the XbaI and MluI fragments of NS3 and NS5 were subcloned into the NheI/MluI sites of the Litmus-38 vector (New, England Biolabs, MA), cut with EcoRI and SalI, and religated into pcDNA3 and pcDNA3, respectively. EcoRI/XhoI multiple cloning site for Zeo(-). The XbaI and BamH1 fragments containing NS4 were religated into Litmus-20 (New, England Biolabs), cut again with KpnI and EcoRI, and then religated into the pcDNA3 vector. Plasmids were named: pcDNA3-NS3, pcDNA3-NS4 and pcDNA3.1/Zeo(1)-NS5.

Those skilled in the art can design primers to prepare any gene construct of the present invention if they have the DNA sequence encoding any HCV nonstructural protein. In addition, nucleotide base substitutions can be made without affecting primer binding. In addition, primers can be prepared with endonuclease restriction sites for cloning or ligation purposes, known to those skilled in the art. Therefore, those skilled in the art can prepare the gene construct of the present invention by designing appropriate primers and performing PCR amplification. The PCR product was ligated into an expression vector.

Plasmids Containing Nucleotide Coding Sequences of HCV Nonstructural Proteins Each contained nucleotide coding sequences for HCV nonstructural proteins placed under the transcriptional control of the CMV promoter and RSV enhancer elements, as described above.

Example 2: In vitro expression

The sequence of the plasmid construct spanning the gene insert was determined, and protein expression was confirmed in HuH-7 cells after transient transfection and in SP2/0 target cells after stable transfection, respectively. It was found that the protein bands of 70 for NS3, 30 for NS4 and 125kD for NS5 were expressed in the cells, but not secreted into the medium.

The HuH-7 human hepatoma cell line was transiently transfected with various constructs by the calcium phosphate method to assess the expression levels of HCV nonstructural proteins. Briefly, after 4 hours of metabolic labeling with 35-thio-methionine and cysteine, the cells were treated with modified RIPA buffer (0.15M NaCl, 1% NP-40, 50mMTris, 0.5% DOC and 1 %SDS) to prepare cell lysate. Cell lysates were pre-cleared with horse serum and then bound to Sepharose A by pre-incubation overnight with polyclonal antisera WU 110(NS3), W148/151(NS4) and WU 115(NS5). (Grakoui, et al., J. Virol., 1993, 67, 1385-1395, all incorporated herein by reference) After the proteins were separated by SDS-PAGE, the electrophoresis gel was left to dry and then autoradiographically developed. NS5 protein expression was also determined by Western blot and immunofluorescence analysis with a mouse monoclonal antibody (Biogenesis, Sandown, NH). To generate stably transfected cell lines expressing NS3, NS4 and NS5, the syngeneic BALB/c mouse myeloma cell line SP2/0 was electroporated with the pcDNA3 plasmid containing the viral gene insert of interest. Cells capable of growing under G418 selection were cloned by limiting dilution (0.3 cells/well) and screened as described above.

The pcD3 and pcD3.1 plasmids contain neomycin or zeomycin resistance genes, respectively, and are used to clone HCV nonstructural genes and generate stable homologous target cell lines. HuH-7 cells were transiently transfected with these constructs, and after controlling the transformation efficiency with β-galactosidase assay, the cells were starved for 30 min in medium without methionine and cystine, and treated with ^35- sulfur-methylthio Acine and cystine labeling for 4 hr. Cell lysates were immunoprecipitated with polyclonal rabbit serum specific for nonstructural proteins, captured on Sepharose A beads, electrophoresed by SDS-PAGE, and analyzed by autoradiography. Lanes 1, 3, and 5 are cells transfected with simulated DNA, serving as negative controls (mock). 2, 4 and 6 show specific bands around NS3 70, NS4 30, NS5 125kD. (lanes 7-10): SP2/0 cells were transfected with pcD3-based constructs containing NS3, NS4 and NS5, and after antibiotic selection, the cells were cloned and amplified by limiting dilution (0.3 cells/well), NS3 was analyzed by radiolabeling or immunoprecipitation, or NS5 by western blot as described above. Lanes 7 and 9 represent cell lysates obtained from cells stably expressing HCV-core protein as a negative control (SP2-19), and lanes 8 and 10 represent the specific expression of NS3 and NS5. These cells were used as in vitro stimulators and as target cells for CTL assays.

Example 3: Immunization Protocol

Female BALB/c(h-2d) mice were cultured in the animal facility of Massachusetts General Hospital under standard pathogen-free conditions. Mice were obtained from Charles River Laboratories (Wilmington, MA) and used for in vivo studies at 6 to 20 weeks of age. A total of 100 μg of plasmid DNA (prepared in 100 μl of 0.9% sodium chloride solution) was injected 2 and 3 times into the quadriceps muscle of mice at 5 different sites. Every 14 days, booster injections are given in the other leg. As a positive control for all immunological experiments, 5 μg of recombinant NS3, NS4, and NS5 nonstructural proteins (Mikrogen, Munich) in complete Freund's adjuvant (CFA) were injected intraperitoneally into mice on day 0, and treated with Equal amounts of protein formulated in 0.05% SDS were given as booster injections after 4 and 8 weeks. As negative controls for these experiments, blank plasmid vectors and recombinant hepatitis B virus surface antigen (HbsAg) (Energix, Smith Kline Beecham, Philadelphia) were used. All mice were sacrificed 10 days after the last immunization.

Example 4: Determination of Humoral Immune Response

Anti-NS3, NS4 and NS5 antibody levels in serum of each immunized animal were measured by established ELISA method. Briefly, a microtiter plate (Falcon, MicrotestIIIM Flexible Assay Plate) was coated with the above-mentioned recombinant protein at 4°C overnight. (0.5 μg/well). After blocking with fetal bovine serum at 20°C for 2 hours, add 1:50 mouse serum to the plate and incubate at 20°C for another hour. After washing 4 times with phosphate buffered saline (PBS) containing 0.05% Tween-20, a 1:2000 dilution of anti-mouse antibody (Amersham, Arlington Heights, IL) labeled with horseradish peroxidase was added. After incubating for 1 hour, wash the titer plate, add substrate to develop color, and read on an automatic reader.

After three immunizations, specific antibody responses against all three nonstructural proteins were found in all immunized animals by ELISA assay. No antigen-specific immune responses were detected in mice immunized with mock DNA (see Figure 2A). As a positive control, mice three times intraperitoneally inoculated with recombinant NS3, NS4 and NS5 nonstructural proteins conjugated to complete Freund's adjuvant (CFA) displayed strong humoral immune responses, as expected (data not shown).

Figure 2A shows humoral immune responses to NS3, NS4 and NS5 generated by DNA-based immunization. Serum antibody levels were determined by ELISA (each group: n=5). Controls included wells coated with BSA and serum from mock-immunized mice. Mice serving as positive controls were immunized intraperitoneally with recombinant protein (data not shown). Figure 2B shows T cell proliferation measured after 3 days of in vitro stimulation with specific or non-specific recombinant proteins. Cells were incubated with ^H3 -thymidine for 18 hours before harvesting. Δcpm is determined by subtracting the background activity (for example: culture without adding antigen). Cells incubated with 1 μg of recombinant NS3 protein were toxic, so no proliferation was observed. Mice immunized with the recombinant protein in combination with CFA had a 5- to 10-fold stronger response (data not shown).

Example 5: Analysis of Lymphocyte Proliferation and Cytokine Release

Mice were anesthetized with isoflurane (Aerrane, Anaquest, NJ), and splenocytes were collected. Red blood cells were removed by incubation with 0.83% NH ₄ Cl/0.17M Tris pH 7.4 at 25°C for 5 minutes. Splenocytes were washed twice, and 5×10 ⁵ cells per well were used in a 96-well round bottom titer plate, and 200 μl of complete DMEM (Minimum Essential Medium) (Mediatech, Washington, DC) containing 10% FBS and 2-mercaptoethanol (Mediatech, Washington, DC) was used as formula 3 part cultivation. Stimulation was performed with recombinant nonstructural proteins (NS3, NS4 and NS5) (Mikrogen, Munich) at different concentrations (0, 0.01, 0.1 and 1 μg/ml). As a negative control, effector cells were stimulated with recombinant HCV-core protein or HbsAg protein (Energix) at the same concentration. After 3 days of stimulation, 3H-thymidine (1 μgCi/well) was added. Cells were cultured for an additional 18 hours, harvested and assayed for 3H-thymidine incorporation into DNA. Incorporated radioactivity was corrected for background activity (Δcpm). To detect the release of cytokines, the effector cells were cultured as above, and the levels of IL-2, IL-4 and interferon-γ in the culture supernatant were measured with commercial kits according to the manufacturer's instructions (Endogen, Boston, MA).

To examine cell-mediated immune responses to nonstructural proteins, splenocytes were harvested and restimulated with recombinant antigens or with antigens expressed by stably transfected cell lines in vitro. All nonstructural proteins at different antigen concentrations induced substantial lymphocyte proliferation as determined ^by3H -thymidine incorporation (see Figure 2B). Peritoneal immunization with recombinant proteins, the method that produced the greatest stimulation, produced a 5- to 10-fold stronger lymphocyte proliferation rate for all three proteins (data not shown). The cytokine profile of the DNA-based post-immunization assay showed a T1 response with high levels of gamma-interferon (Fig. 2C) and IL-2 (Fig. 2D) secretion into the cell culture medium. In contrast, only little IL-4 production was detected after genetic immunization with genes encoding HCV nonstructural proteins (Fig. 2E).

Figures 2C, 2D and 2E show the cytokines secreted into the culture supernatant detected after 48 hours of in vitro stimulation. DNA constructs encoding NS3, NS4 and NS5 induced a T _H 1 -type cytokine profile. For comparison, the results of three times of peritoneal immunization of mice with recombinant protein (n=4) are shown. As a negative control, mice were immunized with recombinant HbsAg.

Example 6: Cytotoxic T-lymphocyte activity

Splenocytes from immunized mice were suspended in complete DMEM (5×10 ⁻⁵ M) containing 10% FCS and 2-mercaptoethanol and stimulated in vitro for 5 days to analyze cytotoxic activity. Recombinant mouse IL-2 was added once at a concentration of 5 U/ml, and the reaction cells (4×10 ⁷ ) were mixed with 2×10 ⁶ irradiated (10,000rad) homologous stable expression full-length NS3 or NS5 proteins (SP2/ NS3-3, SP2/NS5-21) SP2/0 cells were co-cultured. Five days later, the cytotoxic effector lymphocyte population was collected, and SP2/NS3-3 or SP2/NS5-21 labeled with ⁵¹ Cr was used to conduct a ⁵¹ Cr release test in a 96-well round-bottom titer plate for 4 hours. Parental SP2/0 or SP2-19 expressing HCV core protein were used as controls for antigen specificity and background activity of lysates. The CTL activity test was carried out with the ratios of effector lymphocytes to target cells (E:T) being 100:1, 30:1, 10:1 and 3:1, respectively. Use the anti-CD4 ⁺ or CD8 ⁺ monoclonal antibody containing the hybridoma supernatant GK1.5 (anti-CD4, rat); 3.155 (anti-CD8, rat) to incubate the effector cells for 30 minutes at 4°C, wash, and then Incubate with complement (1:5 dilution of low-toxicity rabbit complement (Cedarlane Laboratories, Ontario, Cananda)) at 37°C for T cell deficiency assay.

Since the CTL response is to eliminate virus from infected cells, the ⁵¹ Cr release assay was used to analyze the ability of splenocytes from immunized mice to lyse myeloma target cells from syngeneic SP2/0 mice stably transfected and expressing NS3 and NS5 proteins. After 5 days of in vitro stimulation, mice immunized with NS3 and NS5 displayed strong and specific cytotoxic T cell responses, whereas only SP2/0 or SP2-19 (stable expression of HCV core protein) as target cell-specific controls were observed to low activity (Table 3A and Table 3B). To illustrate the phenotype of specifically lysed cells, splenocytes were co-cultured with CD8 ⁺ or CD4 ⁺ reactive monoclonal antibodies (mAbs) in the presence of complement, CD8 ⁺ cell-mediated cytotoxic activity is shown in Table 3C. We were unable to establish an SP2/0 cell line stably expressing NS4 protein, so CTL activity was not measured for this HCV nonstructural protein.

Figure 3 shows the response of cytotoxic T cells (CTL) to NS3 (A) and NS5 (B) at different ratios of effector cells to target cells (100:1, 30:1, 10:1, 3:1). )Reaction. Splenocytes were co-cultured with irradiated stable NS3 and NS5 expressing mouse myeloma cells in vitro for 5 days (n=5). Then, a 4 hour ⁵¹ Cr release assay was performed to measure CTL activity against stably transfected target cell lines. Background activity to SP2/0 or SP2-19 (expressing HCV core protein) was subtracted to reveal specific lysis values. Figure 3C shows the T cell depletion experiment (n=3), cells were incubated with anti-CD8 ⁺ or CD4 ⁺ monoclonal antibody for 30 minutes on ice, and then incubated with complement for 30 minutes at 37°C. Control cells were incubated without complement and anti-CD8+ or anti-CD4+ monoclonal antibodies. Background activity was determined in SP2-19 cells stably transfected with an HCV core protein expression construct as an irrelevant negative control cell line.

Example 7: Evaluation of cytotoxic T lymphocyte activity in vivo

CTL assays are extremely difficult to perform because until now no one has been able to establish stable cell lines expressing nonstructural proteins for the measurement of CTL activity. Mice were immunized three times intramuscularly (im) with mock DNA or pApNS5 vector. Some animals were also immunized intraperitoneally with recombinant NS5 protein or a combination of both. Recombinant protein (5 μg intraperitoneal injection) as E. coli expressed protein (Mikogen, Munich, Germany) of NS5-4 (aa2622-2868) and NS5-12 (aa2007-2268), covering part of HCV-NS5a and HCV-NS5b (approximately 50% of the full length of NS5) is provided. One week after the last immunization with various plasmid constructs or recombinant proteins, 2 x ¹⁰⁶ syngeneic SP2/0-derived cells stably expressing NS5 were washed, resuspended in 200 μl of PBS, and inoculated subcutaneously on the right side. SP2/0 cells express HCV NS5 (SP2/NS5-21) or HCV core protein SP2/19. In this animal model, tumor formation was evaluated 15 days after subcutaneous inoculation, the number of animals with tumors and the weight of the tumors were determined.

Only 40% of mice immunized with mock DNA and challenged with an NS5-expressing mouse myeloma cell line (SP2/NS5-21) developed tumors after 15 days. In addition, the size of the tumor was determined by the determination of tumor weight, compared with mice immunized with mock DNA or recombinant NS5 protein, or with the same expression of different HCV structural proteins (HCV core protein), as a control homologous SP2/0 Compared to mice immunized with the cell line, the tumor size was smaller (Fig. 4A and 4B). In fact, 90-100% of mice immunized with mock DNA and challenged with SP2-19 cells showed tumor formation, thus showing the specificity of CTL activity in this small animal model. It is important to emphasize that immunization with recombinant NS5 protein in complete Freund's adjuvant did not protect animals from tumor formation. In order to evaluate the effect of combining DNA-based immunization and recombinant protein vaccine, a group of animals were immunized with both simultaneously, resulting in partial protection against tumor formation, but this method was not as good as immunizing animals three times with DNA encoding NS5 protein That works.

Figure 4A shows representative results obtained with one tumor model evaluating CTL activity. Mice were immunized three times with pApNS5 or mock DNA (100 μg) intramuscularly, or intraperitoneally with recombinant NS5 protein (5 μg). A final group of animals received combined DNA immunization and recombinant protein. Fifteen days after tumor challenge with SP2NS5-21 or SP2-10 cells, the number of mice with tumors and tumor weights were determined. Figure 4B, from left to right, shows animals immunized with mock DNA and challenged with SP2/NS5-21 cells; animals immunized with pApNS5 and challenged with SP2/NS5-21; animals immunized with pApNS5 and challenged with SP2-19 (stable expressing HCV core) challenged animals; and animals injected intraperitoneally three times with recombinant NS5 protein and challenged with SP2/NS5-21 cells. There was large tumor formation on the right flank of animals in groups 1, 3 and 4, but not in group 2, which was immunized with pApNS5 and challenged with a mouse myeloma cell line stably expressing NS5.

SEQUENCE LISTING <110> Wands, Jack R.

Encke, Jens <120> Genetic immunization using nonstructural proteins of hepatitis C virus <130> mgh-0002 <140> unassigned <141> 1999-01-28 <150> 60/073,156 <151> 1998-01- 9 <210>2<211>341<212>DNA<213>人工序列<220><220><223>人工序列的描述：合成序列<400>2gccagccccc gattgggggc gacactccac catagatcac tcccctgtga ggaactactg  60tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac 120cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag 180gacgaccggg tcctttcttg gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc 240gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac c                     341<210>3<211>28<212>DNA<213>人工序列<220><220><223>人工序列Description of the synthetic sequence <400>3ggtctagatt gatggcgccc atcacggc 28<210>4<211>29<212>DNA<213>Artificial sequence <220><220><223> Description of the artificial sequence: Synthetic sequence <400accgt>gaccgtga CCTCCAGT 29 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <220> <223> Artificial Sequence description: Synthetic sequence <400> 5GGTCTAGAGACCTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG <212>DNA<213>Artificial sequence<220><220><223>Description of artificial sequence: synthetic sequence<400>6ccaggatcct cagcatggag tggtaca 27<210>7<211>39<212>DNA<213>artificial sequence< 220><220><223>Description of artificial sequence: synthetic sequence<400>7tcagtctaga atgtccggct ccggctcctg gctaaggga 39<210>8<211>30<212>DNA<213>artificial sequence<220><220><223>artificial Description of the sequence: synthetic sequence <400>8agctacgcgt tcaccggttg gggaggaggt 30

Claims (33)

1. a recombinant nucleic acid molecules is characterized in that, it comprises the nucleotide sequence of coding hepatitis C Nonstructural Protein.

2. recombinant nucleic acid molecules as claimed in claim 1 is characterized in that, described Nonstructural Protein is selected from and comprises NS3, the group of NS4 and NS5.

3. recombinant nucleic acid molecules as claimed in claim 1 is characterized in that, described nucleotide sequence coded a kind of fusion rotein, this fusion rotein contain NS3, NS4 or NS5 or their combination.

4. recombinant nucleic acid molecules as claimed in claim 1 is characterized in that, described nucleotide sequence coded being selected from comprises NS3, at least 50 amino acid whose fragments of the Nonstructural Protein of the cohort of NS4 and NS5.

5. recombinant nucleic acid molecules as claimed in claim 2 is characterized in that, has functional controlling element operability to link to each other among described nucleotide sequence and the human cell.

6. recombinant nucleic acid molecules as claimed in claim 5 is characterized in that, described nucleotide sequence links to each other with promotor, enhanser, polyadenylic acid sequence and optional hepatitis C virus 5 ' UTR operability.

7. recombinant nucleic acid molecules as claimed in claim 6 is characterized in that described promotor is a cytomegalovirus promoter, and described enhanser is a Rous sarcoma virus enhanser.

8. recombinant host cell that comprises nucleic acid molecule described in the claim 1.

9. a pharmaceutical composition is characterized in that, comprises:

A) recombinant nucleic acid molecules as claimed in claim 1, wherein said nucleotide sequence links to each other with functional controlling element operability among the human cell; And

B) medicine is accepted carrier or thinner.

10. pharmaceutical composition as claimed in claim 9 is characterized in that, the described nucleotide sequence coded Nonstructural Protein that is selected from down group: NS3, NS4 and NS5.

11. pharmaceutical composition as claimed in claim 9 is characterized in that, described nucleotide sequence coded a kind of fusion rotein, this fusion rotein contain NS3, NS4 or NS5 or their combination.

12. pharmaceutical composition as claimed in claim 9 is characterized in that, the described nucleotide sequence coded NS3 that is selected from, at least 50 amino acid whose fragments of the Nonstructural Protein of NS4 and NS5.

13. pharmaceutical composition as claimed in claim 10 is characterized in that, described functional controlling element in the human cell comprises promotor, enhanser, polyadenylic acid sequence and optional hepatitis C virus 5 ' UTR.

14. pharmaceutical composition as claimed in claim 13 is characterized in that, described promotor is a cytomegalovirus promoter, and described enhanser is a Rous sarcoma virus enhanser.

15. pharmaceutical composition as claimed in claim 9, it also comprises promotor.

16. pharmaceutical composition as claimed in claim 15 is characterized in that, described promotor is bupivacaine.

18. in not infecting the human body of hepatitis C virus, induce method for one kind to the immunne response of hepatitis C virus, it is characterized in that, this method comprises uses a certain amount of at least a recombinant nucleic acid molecules as claimed in claim 1 to described people, and this consumption can effectively have been induced the immunne response to hepatitis C virus.

19. method as claimed in claim 18 is characterized in that, described Nonstructural Protein is selected from NS3, NS4 and NS5.

20. method as claimed in claim 18 is characterized in that, described nucleotide sequence coded a kind of fusion rotein, this fusion rotein contain NS3, NS4 or NS5 or their combination.

21. method as claimed in claim 18 is characterized in that, the described nucleotide sequence coded NS3 that is selected from, at least 50 amino acid whose fragments of the Nonstructural Protein of NS4 and NS5.

22. method as claimed in claim 19 is characterized in that, described nucleotide sequence links to each other with functional controlling element operability among the human cell.

23. method as claimed in claim 22 is characterized in that, described have functional controlling element to comprise promotor, enhanser, polyadenylic acid sequence and optional, hepatitis C virus 5 ' UTR in the human cell.

24. method as claimed in claim 23 is characterized in that, described promotor is a cytomegalovirus promoter, and wherein said enhanser is a Rous sarcoma virus enhanser.

25. method as claimed in claim 18 is characterized in that, described immunne response comprises cell response.

26. method as claimed in claim 18 is characterized in that, described immunne response comprises humoral response.

27. method as claimed in claim 18 is characterized in that, described recombinant nucleic acid molecules is present in and includes medicine and accept in the pharmaceutical composition of carrier or thinner.

28. method as claimed in claim 27 is characterized in that, described pharmaceutical composition also comprises promotor.

29. method as claimed in claim 28 is characterized in that, described promotor is bupivacaine.

30. the method to people's immunity of hepatitis C virus susceptible is characterized in that, this method comprises uses a certain amount of at least a pharmaceutical composition alleged as claim 9 to described people, and its consumption is induce immune response effectively.

31. method as claimed in claim 30 is characterized in that, gives bupivacaine at the position that gives described pharmaceutical composition.

32. the method to people's immunity of hepatitis C virus susceptible is characterized in that, this method comprises uses a certain amount of recombinant nucleic acid molecules as claimed in claim 1 to described people, and its consumption is induce immune response effectively.

33. method that the people who infects hepatitis C virus is treated, it is characterized in that, this method comprises and gives a certain amount of pharmaceutical composition as claimed in claim 9 to described people that its consumption can effectively be induced the therapeutic immunization of hepatitis C virus is replied.

34. as method as described in the claim 33, it is characterized in that, give described population's piperazine cacaine at the position that gives described pharmaceutical composition.

Images