AU741876B2 - Hepatitis virus vaccines - Google Patents

Description

I/UU/U11 26/5/91 Regulation 3.2(2)

AUSTRALIA

f. 0I Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: HEPATITIS VIRUS VACCINES The following statement is a full description of this invention, including the best method of performing it known to us 4. ^1 *1 HEPATITIS VIRUS VACCINES FIELD OF THE INVENTION The present invention relates to gene constructs which are useful as antihepatitis C virus vaccine components in genetic immunization protocols, to methods of protecting individuals against hepatitis C virus infection, to use of gene constructs in protecting against hepatitis C virus.

BACKGROUND OF THE INVENTION Hepatitis C virus (HCV), the major etiologic agent of transfusion acquired non-A, non-B hepatitis, is responsible for approximately 150,000 new cases of acute viral hepatitis annually in the United States. Greenberger, 1983, "New Approaches for Hepatitis Contemporary Internal Medicine Feb:64.

Approximately half of these infections progress to a chronic infection that can be associated with cirrhosis and/or hepatocellular carcinoma (Alter, et al., Science, 1992, 258, 135-140; and Alter, etal., New Eng. J. Med., 1992, 327, 1899-1905).

15 In addition, HCV infection is an independent risk factor for the development of 0 hepatocellular carcinoma as shown by the prevalence of anti-HCV antibodies (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 Eng. J. Med., 1993, 328, 1797-1801).

HCV is an enveloped, positive stranded RNA virus, approximately 9,500 nucleotides in length, which has recently been classified as a separate genus within the Flavivirus family (Heinz, Arch. Virol. (Suppl.), 1992, 4, 163-171).

Different isolates show considerable nucleotide sequence diversity leading to the subdivision of HCV genomes into at least 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) that encodes a precursor polyprotein of 3010 to 3033 amino acids of approximately 330 Kd (Choo, et al., Proc. Natl. Acad. Sci.

USA, 1991, 88, 2451-2455; Inchauspe, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 10292-10296; Kato, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9524-9528; Okamoto, et al., J. Gen. Virol., 1991, 72, 2697-2704; and Takamizawa, et al., J.

Gen. Virol., 1991, 65, 1105-1113).

2 Individual HCV polypeptides are produced by proteolytic processing of the precursor polypeptide to produce core envelope (El, E2) and nonstructural (NS2-NS5) proteins (Bartenschlager, et al., J. Gen. Virol., 1993, 67, 3835-3844; Grakoui, et al., J. Gen. Virol., 1993, 67, 2832-2843; and Selby, et al., J. Gen. Virol., 1993, 74, 1103-1113). This proteolysis is catalyzed by a combination of both cellular and viral encoded proteases.

In addition to the translated region, the HCV genome also contains both a 5' untranslated region UTR) and a 3' untranslated region UTR) The UTR of 324 to 341 nucleotides represents the most highly conserved sequence among all HCV isolates reported to date (Han, et al., Proc. Natal. Acad. Sci.

USA, 1991, 88, 1711-1715; and Bukh, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 4942-4946). This 5' UTR has been postulated to contain important regulatory elements for replication and/or translation of HCV RNAs. The 5' UTR also contains several small open reading frames (ORF) but there is presently no 15 evidence to suggest that these ORF sequences are actually translated.

The HCV core gene may be an important target for nucleic acid-based antiviral approaches. The first 191 amino acids of the HCV polyprotein precursor are believed to represent the viral nucleocapsid protein. This protein is comprised of a basic, RNA-binding amino-terminal domain and a highly hydrophobic carboxy-terminal region (Bukh, et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 8239-8243; and Santolini, et al., J. Virol., 1994, 68, 3631-3641). The mature 21 kDa core protein is cleaved from the polyprotein precursor by cellular signal peptidase and there is evidence to suggest that the HCV nucleocapsid protein is stably associated with the cytoplasmic surface of the endoplasmic reticulum membrane (Hijikata, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 5547- 5551; and Santolini, et al., J. Virol., 1994, 68, 3631-3641). In contrast to the envelope glycoproteins which include a hypervariable region in the aminoterminal region of E2 (Weiner, et al., Virol., 1991, 180, 842-848; and Weiner, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3468-3472), the core protein is well conserved among the different HCV genotypes and generates a host immune response (Bukh, et al., Proc. Natal. Acad. Sci. USA, 1994, 91, 8239-8243; and Houghton, et al., Hepatology, 1991, 14, 381-388). Previous studies have shown 3 that the majority of HCV-infected individuals develop antibodies to the HCV core protein early in the course of infection (Chiba, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 4641-4645; Hosein, et al., Proc Natl. Acad. Sci. USA, 1991, 88, 3647-3651; Hsu, et al., Hepatology, 1993, 17, 763-771; Katayama, et al., Hepatology, 1992, 15, 391-394; Nasoff, etal., Proc. Natl. Acad. Sci. USA, 1991, 88, 5462-5466; and Okamoto, et al., Hepatology, 1992, 15, 180-186).

Furthermore, the nucleocapsid protein represents an important target for the cellular immune response against HCV (Botarelli, et al., Gastroenterol., 1993, 104, 580-587; Koziel, et al., J. Virol., 1993, 67, 7522-7532; and Shirai, et al., J.

Virol., 1993, 68, 3334-3342). Finally, there are recent observations to suggest that the HCV core protein may have certain gene regulatory functions as well (Shih, etal., J. Virol., 1993, 67, 5823-5832). The high mutational rate of the viral genome probably occurs during viral replication and through immune selection.

This phenomenon may be related to the establishment of persistent viral 15 infection and subsequent disease chronicity (Weiner, et al., Proc. Natl. Acad.

S. Sci. USA, 1992, 89, 3468-3472; Kato, et al., Biochem. Biophys. Res. Comm., 1992, 189, 119-127; and Alter, et al., New Eng. J. Med., 1992, 327, 1899-1905.

The cellular immune events involved in liver damage and viral clearance during HCV infection have only partially been defined. In an attempt to examine a potential pathogenic role of liver-infiltrating lymphocytes in patients with chronic HCV infection, Koziel, et al. examined the cytotoxic T lymphocyte (CTL) response of such cells and demonstrated an HLA class I-restricted CD8+ CTL response that was directed against both structural and non-structural regions of HCV polypeptides (Koziel, et al., J. Virol., 1993, 67, 7522-7532; and Koziel, et al., J. Immune., 1992, 149, 3339-3344). Other investigators have also noted the existence of CTLs in peripheral blood mononuclear cell populations that recognize epitopes on core and the other viral related proteins during chronic HCV infection (Kita, etal., Hepatol., 1993, 18, 1039-1044; and Cerny, et al., Intl.

Symp. Viral Hepatitis Liver Dips. 1993, 83 (abstr).

Botarelli, etal. (Botarelli, etal., Gastroenterol., 1993, 104, 580-587) and Ferrari, et al. (Ferrari, et al., Hepatol., 1994, 19, 286-295) found HLA class IIrestricted CD4+ T cell-mediated proliferative responses to several recombinant It 4 proteins derived from different regions of HCV in patients with chronic HCV infection. It is noteworthy that there was a correlation between T cell responses to HCV core protein, and a clinically benign course of the liver disease as well as subsequent eradication of the virus. However, a similar study showed the proliferative response to HCV core protein did not predict a benign clinical course with respect to the severity of the liver disease (Schupper, et al., Hepatol., 1993, 18, 1055-1060). Thus, it is important to clarify the association between active cellular immunity and the clinical course of the viral infection with respect to the type of liver injury and clinical response of HCV infection to IFN therapy. In this regard, studies involving peripheral blood mononuclear cell (PBMC) responses to a recombinant GST-HCV core fusion protein were conducted, and involved evaluating the ability of such cells to produce IFN-y correlations were made to different clinical outcomes of HCV infection. It was ft.

found that mononuclear cells from 24 of 46 patients with chronic liver disease responded to the core protein; asymptomatic HCV carriers demonstrated a lower response rate p<0.05). More important, individuals who had received IFN-c treatment and went into clinical and virologic emission had a higher response rate p<0.05) to HCV core protein compared to those with ongoing hepatitis who failed therapy Of 25 patients whose mononuclear cells responded to HCV core protein, 18 had a significant response to one or more peptides; 12 patients reacted to a peptide mixture containing hydrophilic sequences. The core peptide amino acid sequence 140- 160 was recognized by 9 patients. Interestingly, 7 of 8 patients bearing HLA DR4 and w53 haplotypes recognized the peptide sequence 141-160. Thus, the mononuclear cell response appeared to be HLA DR restricted and the responding cells were identified as CD4+ T cells. This study demonstrates the presence of immunodominant T cell epitopes within the HCV core protein in association with HLA DR phenotypes in patients with HCV associated liver disease.

Presently, there is no universal, highly effective therapy of chronic HCV infection. Development of a vaccine strategy for HCV is complicated not only by the significant heterogeneity among HCV isolates, but also by the mixture of heterogeneous genomes within an isolate (Martell, et al., J. Virol)., 1992, 66, 3225). In addition, the virus contains a highly variable envelope region.

Vaccination and immunization generally refer to the introduction of a non-virulent agent against which an individual's immune system can initiate an immune response which will then be available to defend against challenge by a pathogen. The immune system identifies invading "foreign" compositions and agents primarily by identifying proteins and other large molecules which are not normally present in the individual. The foreign protein represents a target against which the immune response is made.

PCT Patent Application PCT/US90/01348 discloses sequence information of clones of the HCV genome, amino acid sequences of HCV viral proteins and methods of making and using such compositions including anti- HCV vaccines comprising HCV proteins and peptides derived therefrom.

Australian Serial No. 62320/94 filed January 26, 1994, Australian Serial No. 62320/94 filed January 26, 1994, Australian Serial No. 62320/94 filed January 26, 1994, PCT Patent Application Serial Number PCT/US94/00899 filed January 26, 1994 and Australian Serial No. 22366/95 filed March 1995 each contains descriptions of genetic immunization protocols. Vaccines against HCV are disclosed in each.

20 The HCV core DNA-based vaccine expresses high levels of core Oeo.: antigen in vitro and induces a strong immune response in vivo.

There remains a need for vaccines useful to protect individuals against hepatitis C virus infection.

SUMMARY OF INVENTION 25 The present invention relates to an isolated and purified nucleic acid molecule having a nucleotide sequence comprising a coding sequence encoding an incomplete hepatitis C viral genome, wherein nucleotide sequence is free of the 5' untranslated region of hepatitis C virus except the S* last 9 nucleotides of the 5' untranslated region. Particularly where the coding sequence encodes a hepatitis C core protein, such as that disclosed in SEQ ID NO: 15 or SEQ ID NO: 14. The nucleic acid molecule will be usefully employed where it has a promoter and polyadenylation sequence and wherein said coding sequence is operably linked to said promoter and polyadenylation f 6 sequence. A usual example is where the nucleic acid molecule is the plasmid called pHCV2-1.

The present invention further relates to an isolated and purified nucleic acid molecule having a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 15 and a portion of the untranslated region of hepatitis C virus.

In another aspect the present invention relates to pharmaceutical compositions comprising the nucleic acid molecule mentioned above and a pharmaceutically acceptable carrier or diluent.

The present invention also relates to a method of inducing a protective immune response against hepatitis C virus in a individual uninfected by hepatitis C virus comprising administering to an individual an amount of the nucleic acid molecule having an nucleotide sequence comprising a coding sequence encoding an incomplete hepatitis C viral genome, wherein said nucleotide sequence is free of the 5' untranslated region of hepatitis C virus except the last 9 nucleotides of the said 5' untranslated region, in an infective amount to induce an immune response against hepatitis C virus.

Immunization is effectively carried out where the nucleic acid molecule is in a pharmaceutically acceptable carrier or diluent and where the administration is 20 carried out conjunctively with bupivacaine. Particularly the administration of •ooo 100p g of nucleic acid molecule, and where administration is carried out with a needless injection device intramuscularly, subcutaneously or intradermally.

The present invention also relates to a pharmaceutical composition comprising any of the nucleic acids mentioned above with a pharmaceutically 25 effective carrier or diluent, and the administration thereof.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a diagram of an expression vector into which HCV coding sequence is inserted to produce plasmids pHCV2-1 and pHCV4-2.

Oi o Figure 2 depicts method for constructing the HCV fusion constructs.

DETAILS DESCRIPTION OF THE INVENTION According to the present invention, compositions and methods are provided which prophylactically and/or therapeutically immunize an individual Sagainst HCV infection. For immunization against HCV infection, recombinant 6a nucleic acid molecules including a nucleotide coding sequence that encodes a fusion protein that comprises HBV S gene product linked to amino acids 1-69 of HCV core protein are administered to the individual. The fusion protein encoded by the gene construct is expressed by the individual's cells and serves as an immunogenic target against which an anti-HCV immune responses are elicited.

e ee eo For immunization against HCV infection, genetic material constituting an incomplete hepatitis C viral genome but which encodes HCV core protein is administered to the individual. The protein encoded by the gene construct is expressed by the individual's cells and serves as an immunogenic target against which an anti-HCV immune response is elicited. The resulting immune responses are broad based; in addition to a humoral immune response, both arms of the cellular immune response are elicited. The methods of the present invention are useful for conferring prophylactic and therapeutic immunity. Thus, a method of immunizing includes both methods of protecting an individual from HCV challenge as well as methods of treating an individual suffering from HCV infection.

It has been shown that many proteins previously known to induce a humoral and cellular immune responses following DNA immunization have ~either been native cell surface proteins or secreted proteins, such as, for 15 example, influenza NP, HBsAg, and rabies virus glycoproteins. Although HCV core DNA-based vaccine expresses high levels of core antigen in vitro and induces an immune response in vivo, it is likely that the HCV core protein remains anchored in the cytoplasm and is not expressed on the surface of the cell or secreted from the cell. Therefore, the immune response may be increased by providing for cell surface expression and/or secretion of the protein.

As used herein, the term "incomplete hepatitis C viral genome" is meant to refer to a nucleic acid molecule that does not contain the complete coding oe*lO sequence for the HCV polyprotein which is encoded by the HCV viral genome.

.oo.oi 25 Incorporation of an incomplete HCV genome into a cell does not constitute introduction of sufficient genetic information for the production of infectious virus.

As used herein the term "HCV core protein" is meant to refer to truncated HCV core proteins, such as those having the full-length HCV core protein having the amino acid sequence disclosed in SEQ ID NO:14 and SEQ ID NO:15 which is the amino acid sequence of the HCV core protein of a specific HCV isolate. The full-length HCV core protein normally has 191 amino acids, but for the purposes of the present invention relating to the truncated HCV core protein, the C-terminal 37 amino acids (amino acids 155-191) have been deleted to enhance the ability of the fusion protein to be secreted. The Cterminal twenty amino acids of the full-length HCV core protein are postulated to contain the binding sites to the endoplasmic reticulum, resulting in the retention of the core protein inside the cell cytoplasm. In addition, the term HCV core protein is meant to refer to corresponding HCV core proteins, full-length or truncated, from additional HCV isolates which may vary. Those having ordinary skill in the art can readily identify the HCV core protein from additional HCV isolates. It is understood that nucleotide substitutions in the codon may be acceptable when the same amino acid in encoded. In addition, it is also to be used that nucleotide changes may be acceptable wherein a conservative amino acid substitution results from the nucleotide substitution.

The HCV core protein has been observed to exist as two distinct forms, anchored core protein and the virion core protein. During HCV translation, a 15 precursor polypeptide is translated and subsequently processed by cellular proteases and viral proteases to yield the individual viral polypeptides. A cellular signal peptidase is believed to cleave at a site which separates the 0 portion of the polyprotein that becomes the core protein from the portion that 0.:0 becomes the envelope protein, thereby releasing anchored core protein from the envelope protein. Anchored core protein, which is associated with the endoplasmic reticulum, is further processed to mature virion core. Anchored core protein differs from virion core in that it contains an extra 18 amino acids at the C terminus. Virtually no anchored core is found on viral particles.

As used herein, the term "gene construct" is meant to refer to a el•.il 25 recombinant nucleic acid molecule including a nucleotide coding sequence that encodes a fusion protein that includes the full-length HCV core protein, as well as initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the vaccinated individual. In some at least a fragment of the HCV 5' UTR.

As used herein, the term "genetic vaccine" refers to a pharmaceutical preparation that contains a gene construct. Genetic vaccines include pharmaceutical preparations useful to invoke a prophylactic and/or therapeutic immune response to HCV.

According to the present invention, gene constructs are introduced into the cells of an individual where it is expressed, thus producing a HCV core or a full-length HCV core protein. The regulatory elements of the gene constructs of the invention are capable of directing expression in human cells. The regulatory elements include a promoter and a polyadenylation signal. In addition, other elements, such as an enhancer and a Kozak sequence, may also be included in the gene construct.

When taken up by a cell, the gene constructs of the invention may remain *.":present in the cell as a functioning extrachromosomal molecule or it may integrate into the cells chromosomal DNA. DNA may be introduced into cells 15 where it remains as separate genetic material in the form of a plasmid.

Alternatively, linear DNA which can integrate into the chromosome may be introduced into the cell. When introducing DNA into the cell, reagents which promote DNA integration into chromosomes may be added. DNA sequences which are useful to promote integration may also be included in the DNA molecule. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the gene construct as a linear minichromosome including a centromere, telomeres and an origin of replication.

According to other aspects of the invention, the gene construct includes ooo coding sequences which encode the full-length HCV core protein. In some 25 preferred embodiments, the gene construct encodes an anchored form of the full-length HCV core protein. Anchored core protein is preferred in some embodiments for the following reasons. First, it is believed to be desirable to correctly compartmentalize the core protein to the endoplasmic reticulum where the core protein is first found in naturally infected cells. If a protein is incorrectly compartmentalized, there is a risk of lowering total expression of that protein due to degradation or poisoning of the cell. Further, since a cellular protease is thought to cleave anchored core to virion core, both species may be present in cells expressing the anchored form. If there are differences in the antigenicity between the two species, a broader immune response against core is potentially generated by expressing the anchored form of full-length core protein. Moreover, the extra 18 amino acids in the anchored core may contain either antibody epitopes or cytotoxic T cell epitopes or both. Expression of the anchored form provides the presence of these regions which may broaden the immune response to core protein.

In some preferred embodiments, the gene construct encodes a fulllength HCV core protein which consists of the amino acid sequence of SEQ ID NO:14 and SEQ ID NO:15. In some preferred embodiments, the gene construct comprises the coding sequence in SEQ ID NO:14.

In some preferred embodiments, the gene construct comprises SEQ ID NO:14.

In some preferred embodiments, the vector used is selected from those 15 described in Figure 1. In some embodiments, nucleotides 342 to 923 of SEQ ID NO:14 is inserted into backbone A to form plasmid pHCV2-1. In some embodiments, SEQ ID NO:14 is inserted into backbone A to form plasmid pHCV4-2.

In plasmids pHCV2-1 and pHCV4-2, coding sequence encoding the fulllength HCV core protein is under the regulatory control of the CMV immediate early promoter and the SV40 minor polyadenylation signal. Constructs may optionally contain the SV40 origin of replication.

The regulatory elements necessary for gene expression of a DNA molecule include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression. It is necessary that these elements be operably linked to the sequence that encodes the fusion protein or the full-length HCV core protein and that the regulatory elements are operable in the individual to whom they are administered.

Initiation codons and stop codon are generally considered to be part of a nucleotide sequence that encodes the fusion protein or the full-length HCV core protein.

11 Promoters and polyadenylation signals used must be functional within the cells of the individual. In order to maximize protein production, regulatory sequences may be selected which are well suited for gene expression in the cells the construct is administered into. Moreover, codons may be selected which are most efficiently transcribed in the cell. One having ordinary skill in the art can produce DNA constructs which are functional in the cells.

Examples of promoters useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from 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 15 human metalothionein.

S. Examples of polyadenylation signals useful to practice the present invention, 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 CA), 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 'i 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, CA) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region which produces high copy episomal replication without integration.

In some preferred embodiments, the gene construct used is selected from the vectors described in Figure 1. In some embodiments, nucleotide coding sequence encoding the fusion protein is inserted into backbone A.

In expression vectors of the invention, nucleotide coding sequence encoding the fusion protein is under the regulatory control of the CMV immediate early promoter and the SV40 minor polyadenylation signal.

Constructs may optionally contain the SV40 origin of replication.

Routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterially, intraoccularly and oral as well as transdermally or by inhalation or suppository.

Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. Delivery of gene constructs which encode full-length HCV core protein can confer mucosal immunity in individuals .immunized by a mode of administration in which the material is presented in 15 tissues associated with mucosal immunity. Thus, in some examples, the gene construct is delivered by administration in the buccal cavity within the mouth of *an individual.

Gene constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, or "microprojectile .9 bombardment gene guns". Alternatively, the genetic vaccine may be introduced by various means into cells that are removed from the individual. Such means include, for example, ex vivo transfection, electroporation, microinjection and microprojectile bombardment. After the gene construct is taken up by the cells, they are reimplanted into the individual. It is contemplated that otherwise non- 25 immunogenic cells that have gene constructs incorporated therein can be implanted into the individual even if the vaccinated cells were originally taken from another individual.

According to some embodiments of the present invention, the gene construct is administered to an individual using a needleless injection device.

According to some embodiments of the present invention, the gene construct is simultaneously administered to an individual intradermally, subcutaneously and intramuscularly using a needleless injection device. Needleless injection 13 devices are well known and widely available. One having ordinary skill in the art can, following the teachings herein, use needleless injection devices to deliver genetic material to cells of an individual. Needleless injection devices are well suited to deliver genetic material to all tissue. They are particularly useful to deliver genetic material to skin and muscle cells. In some embodiments, a needleless injection device may be used to propel a liquid that contains DNA molecules toward the surface of the individual's skin. The liquid is propelled at a sufficient velocity such that upon impact with the skin the liquid penetrates the surface of the skin, permeates the skin and muscle tissue therebeneath. Thus, the genetic material is simultaneously administered intradermally, subcutaneously and intramuscularly. In some embodiments, a needleless injection device may be used to deliver genetic material to tissue of other organs in order to introduce a nucleic acid molecule to cells of that organ. The genetic vaccines according to the present invention comprise about 1 nanogram to I 0 15 about 1000 micrograms of DNA. In some preferred embodiments, the vaccines o* contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccines contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the vaccines contain of l about 1 to about 350 micrograms of DNA. In some preferred embodiments, the 0 20 vaccines contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the vaccines contain about 100 micrograms DNA.

The genetic vaccines according to the present invention are formulated according to the mode of administration to be used. One having ordinary skill in the art can readily formulate a pharmaceutical composition that contains a gene 000 ~25 construct. In some cases, an isotonic formulation is used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. The pharmaceutical preparations according to the present invention are provided sterile and pyrogen free.

14 The gene constructs of the invention 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 Australian Application No. 62320/94 filed January 26, 1994, Australian Application No. 62320/94 filed January 1994, Australian Application No.

62320/94 filed January 26 1994, PCT Patent Application Serial Number PCT/US94/00899 filed January 26 1994 and Australian Application No.

22366/95 filed March 30 1995, which are each incorporated herein by reference. Examples of such agents include: CaP0 4 DEAE dextran, amnionic lipids; extracellular matrix-active enzymes; saponins; lectins; estrogenic compounds and steroidal hormones; hydroxylated lower alkyls; dimethyl 15 sulfoxide (DMSO); urea; and benzoic acid esters anilides, amidines, urethanes and the hydrochloride salts thereof such as those of the family of local anaesthetics. In addition, the gene constructs are encapsulated within/administered in conjunction with lipids/polycationic complexes.

EXAMPLES

20 EXAMPLE 1: Design and construction of full-length HCV expression plasmids *Plasmids, pHCV2-1 and pHCV4-2, were both designed to express the anchored core protein of HCV. Anchored core, the cellular precursor to virion core, remains membrane associated by means of a hydrophobic carboxy terminus.

Each plasmid construct contains the full-length HCV core coding region placed under the transcriptional control of the CMV promoter and the RSV enhancer element. Plasmid pHCV2-1 contains a 9 nucleotide fragment of the UTR of HCV which includes the 9 most 3' nucleotides of the 5' UTR of HCV. Plasmid pHCV4-2 differs from plasmid pHCV2-1 in that it also contains the entire 5' UTR of HCV.

HCV specific sequences were amplified by Polymerase Chain Reaction (PCR) using pUC-T7-HCVTH (HCV Type 2) as a template and oligonucleotides comprises of the following sequences as PCR primers SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

The 5' PCR primers, SEQ ID NO:16 and SEQ ID NO:18 have Natl sites and the 3' PCR primer SEQ ID NO:17 has a Sail site. Three stop codons engineered into SEQ ID NO:17. SEQ ID NO:18 and SEQ ID NO:17 were used as primers to generate a PCR product which maps from position 1 to position 810 of the HCV genome and which contains the entire 5' HCV untranslated region and core coding region.

The use of SEQ ID NO:16 and SEQ ID NO:17 as primers resulted in a PCR product mapping from position 333 to position 810 of the HCV genome.

This product contains the last 9 nucleotides of the HCV 5' untranslated region and the entire core coding region.

15 Both PCR products were digested with Natl and Sail (NED), gel purified and ligated into the Natl-Sall sites of plasmid Backbone A shown in Figure 1 to generate pHCV4-2 and pHCV2-1.

Plasmid backbone A is 3969 base pairs in length. It contains a PBR origin of replication for replicating in E. coli. It also contains a kanamycin 20 resistance gene so that the plasmid can be selected in E. coli. Inserts such as the full-length HCV core gene, are cloned into a polylinker region which places the insert between and operably linked to the promoter and polyadenylation signal. Transcription of the cloned inserts is under the control of the CMV promoter and the RSV enhancer elements. A polyadenylation signal is provided by the presence of an SV40 poly A signal situated just 3' of the cloning site.

Example 2: In vitro expression analysis Expression of full-length HCV core from pHCV2-1 and pHCV4-2 was assayed in vitro in a rhabdomyosarcoma (RD) cell line. Cells were cotransfected with pHCV2-1 and a P-galactosidase reporter plasmid, pCMVB (Clonetech), pHCV4-2 and pCMVB, vector plasmid and pCMVB, or mock transfected. All analyses were conducted at 48 hours post-transfection. The transfection efficiency was determined by measuring the specific activity of 13galactosidase in cell lysates.

Transfected cells were analyzed by indirect immunofluorescence using either an anti-core monoclonal antibody or pooled HCV patient sera as the primary antibodies. FITC labeled anti-mouse IgG (Sigma) and FITC labeled anti human IgG (Sigma) were used respectively as the secondary antibodies.

Immunostaining was seen in cells that had been transfected with either pHCV2-1 or pHCV4-2 but not in cells transfected with the vector plasmid or mock transfected. Immunofluorescence in fixed cells appeared as punctate staining localized to the cytoplasm when either the monoclonal antibody or the pooled patient's sera was used as the source of primary antibody. There appeared to be no difference in the level or type of staining between pHCV2-1 and pHCV4-2 transfected cells. No staining was seen in unfixed cells indicating that the expressed core protein is probably not associated with the cell's surface membrane.

Transfected cell lystates were also analyzed by Western blot and immunoprecipitation. The presence or absence of the HCV 5' UTR seemed to have little effect upon the expression of core since pHCV2-1 and pHCV4-2 20 expressed equivalent amounts of core in the cell lines tested.

Example 3: DNA Inoculation Humoral immunity in a group of six mice was assessed following a single intramuscular inoculation of 100pg of pHCV4-2 plus 0.25% bupivacaine. All mice were shown to have seroconverted 21 days after DNA 25 inoculation. The humoral response persisted for several months. Studies are currently underway to evaluate the effect of multiple dosing.

This HCV core DNA-based vaccine expresses high levels of core antigen in vitro and induces a strong immune response in vivo.

"Comprises/comprising" when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Wands, Jack Tokushige, Katsutoshi Wakita, Takaji Pachuk, Catherine J.

Zurawski, Jr., Vincent R.

Coney, Leslie R.

(ii) TITLE OF INVENTION: HEPATITIS VIRUS VACCINES (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Woodcock, Washburn, Kurtz, Mackiewicz Norris STREET: One Liberty Place, 46th floor CITY: Philadelphia S: STATE: PA COUNTRY: USA ZIP: 19103 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA: 25 APPLICATION NUMBER: N/A FILING DATE: Herewith

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/318,248 FILING DATE: 05-OCT-1994

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/467,859 FILING DATE: 06-JUN-1995

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: DeLuca, Mark REGISTRATION NUMBER: 33,229 REFERENCE/DOCKET NUMBER: APOL-0238 18 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (215) 568-3100 TELEFAX: (215) 568-3439 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 923 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: ODS LOCATION: 342..914 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 4:

GOOAGCCCCC

TCTTCACGCA

CCCCOTCCO

GACGACCGGG

GCGAGACTGC

20 GTGCTTGCGA

GATTGGGGGC

GAAAGCGTCT

GGGAGAGCCA

TOOTTTCTTG

TAGCCGAGTA

GACACTCCAC

AGOOATGGCG

TAGTGGTCTG

GATCAACCCG

GTGTTGGGTC

CATAGATOAC

TTAGTATGAG

CGGAACCGGT

CTCAATGCCT

GCGAAAGGCC

TCCCCTGTGA

TGTCGTGCAG

GAGTACAOOG

GGAGATTTGG

TTGTGGTACT

GGAACTACTG

CTCAGGAC

GAATTGCCAG

GCGTGCOCC

GCCTGATAGG

GTGCCCCGGG AGGTCTCGTA GACCGTGCAC C ATG AGO ACG AAT Met Ser Thr Asn COT AAA OCT CAA AGA AAA ACC AAA CGT AAC ACC AAC CGC CGC CCA Pro Lys Pro Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro 5 10 15

CAG

Gin GAC CTC AAG TTC Asp Leu Lys Phe

COG

Pro GGO GGT GGT CAG Gly Giy Gly Gin ATO GTT Ile Val 30 GGT GGA GTT Gly Gly Val TAO CTG Tyr Leu TTG COG OGO Leu Pro Arg TOO GAG CGG Ser Giu Arg 3 5 OGO OGG CCC Arg Arg Pro GGO COO AGG Giy Pro Arg TTG GGT Leu Giy GGA AGG Giy Arg GTG OGO GOG ACT Vai Arg Ala Thr AGG AAG ACT Arg Lys Thr CCC AAG GAT Pro Lys Asp TOG CAA CCT CGT Ser Gin Pro Arg OGA CAA COT Arg Gin Pro GAG GGC AGG Giu Giy Arg TGG GOT CAA COT Trp Ala Gin Pro TAO COT TGG COO Tyr Pro Trp Pro 19 CTC TAT GGC AAC GAG GGC ATG GGG TGG GCA GGA TGG CTC CTG TCA CCC Leu

CGT

Arg

CGT

Arg

CTC

Leu

AGG

Arg 15

GCA

Ala 165 20 CTG Leu (2) Tyr Gly Asn GGC TCC CGG Gly Ser Arg AAT TTG GGT Asn Leu Gly 120 ATG GGG TAC Met Gly Tyr 135 GCC TTG GCG Ala Leu Ala 150 ACA GGG AAT Thr Gly Asn CTG TCC TGT Leu Ser Cys INFORMAT ION (i) Giu

CCT

Pro 105

AAA

Lys

ATT

Ile

CAT

His

CTG

Leu

TTG

Leu 185 Gly 90

AGT

Ser

GTC

Val1

CCG

Pro

GGC

Gly

CCC

Pro 170

ACC

Thr Met

TGG

Trp

ATC

Ile

CTC

Leu

GTC

Val1 155

GGT

Gly

ATC

Ile Gly

GGC

Gly

GAT

Asp

GTC

Val1 140

CGG

Arg

TGC

Cys

CCA

Pro Trp Ala CCC AAT Pro Asn 110 ACC CTT Thr Leu 125 GGC GCT Gly Ala GTT CTG Val Leu TCT TTC Ser Phe GCT TCC Ala Ser 190 Gly 95

GAG

Asp

ACA

Thr

CCC

Pro

GAG

Glu

TCT

Se r 175

GCT

Ala Trp Leu Leu CCC CGG CGT Pro Arg Arg TGC GGC TTC Cys Gly Phe 130 ATG GGG GGC Met Gly Gly 145 GAG GGC GTG Asp Gly Vai 160 ATC TTC CTC Ile Phe Leu

TAATAATAA

Se r

AGG

Arg 115

GCC

Ala

GCT

Ala

AAC

Asn

TTG

Leu Pro 100

TCG

Ser

GAC

Asp

GCC

Ala

TAT

Tyr

GCT

Ala 180 Met 1 Arg Gly Th r Ile Tyr Thr Pro Tyr 35 Lys Lys T rp (ii) (xi) Asn Gin Leu Thr Asp Pro FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 191 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID Pro Lys Pro Gin Arg Lys Thr Lys Arg Asn 5 10 Asp Leu Lys Phe Pro Gly Gly Giy Gin Ile 25 Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 40 Ser Giu Arg Ser Gin Pro Arg Gly Arg Arg Arg Arg Pro Giu Gly Arg Ala Trp Ala Gin 70 Leu Tyr Gly Asn. Giu Gly Met Gly Trp Ala 90 Th r Val1 Arg Gin Pro Gly 9 Leu Leu Ser Arg Arg Arg 115 Pro 100 Arg Gly Ser Arg Pro 105 Ser Trp Gly Pro Asn Asp Pro 110 Leu Thr Cys Ser Arg Asn Leu Gly 120 Lys Val Ile Asp Gly Phe 130 Ala Asp Leu Met Gly 135 Tyr Ile Pro Leu Val 140 Gly Ala Pro Met Gly 145 Gly Ala Ala Arg Ala 150 Leu Ala His Gly Val 155 Arg Val Leu Glu Asp 160 Ile 175 Gly Val Asn Tyr Thr Gly Asn Leu Pro 170 Gly Cys Ser Phe Ser Phe Leu Leu Ala 180 Leu Leu Ser Cys Leu 185 Thr Ile Pro Ala Ser Ala 190 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GGAAGGCGGC CGCCCGTGCA CCATGAGCAC GAATCCT AAAAAGGGAA INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AAAAAGGGAA GGAAGGTCGA CTTATTATTA TTAAGCGGAA GCTGGGATGG TCAAACA INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: AAAAAGGGAA GGAAGGCGGC CGCGCCAGCC CCCGATTGGG GGCGACA

Claims (18)

1. An isolated and purified nucleic acid molecule having an incomplete hepatitis C viral genome, said nucleic acid molecule comprising a coding sequence, wherein said nucleic acid molecule is free of the 5' untranslated region of hepatitis C virus except the last 9 nucleotides of said 5' untranslated region.

2. The nucleic acid molecule of claim 1 wherein said coding sequence encodes a hepatitis C virus core protein.

3. The nucleic acid molecule of claim 2 wherein said coding sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:

4. The nucleic acid molecule of claim 2 wherein said coding sequence comprises SEQ ID NO: 14. S

5. The nucleic acid molecule of claim 2 comprising a promoter and polyadenylation sequence, wherein said coding sequence is operably linked to said promoter and polyadenylation sequence.

6. The nucleic acid molecule of claim 1 wherein said nucleic acid molecule is plasmid pHCV2-1. S..

7. A pharmaceutical composition comprising the nucleic acid molecule of S: claim 1 and a pharmaceutically acceptable carrier, diluent or excipient. S S.

8. A method of inducing a protective immune response against hepatitis C virus in an individual uninfected by hepatitis C virus comprising administering to said individual an amount of the nucleic acid molecule of claim 5 effective to induce an immune response against hepatitis C virus.

9. The method of claim 8 wherein said nucleic acid molecule is plasmid pHCV2-1. The method of claim 8 wherein bupivacaine is administered to said individual at the site of administration of said nucleic acid molecule.

11. The method of claim 10 wherein 100, g of said nucleic acid molecule is administered to said individual.

12. The method of claim 11 wherein said nucleic acid molecule is administered to said individual with a needleless injection device intramuscularly, subcutaneously or intradermally.

13. A method of immunizing an individual susceptible to or infected by hepatitis C virus comprising administering an amount the nucleic acid molecule of claim 1 effective to induce a protective or therapeutic immune :response against hepatitis C virus infection.

14. The method of claim 13 wherein said nucleic acid molecule is plasmid pHCV2-1. The method of claim 13 wherein bupivacaine is administered to said individual at the site of administration of said nucleic acid molecule.

16. The method of claim 15 wherein 100tg of said nucleic acid molecule is administered to said individual.

17. The method of claim 16 wherein said nucleic acid molecule is administered to said individual with a needleless injection device intramuscularly, subcutaneously or intradermally. 23

18. The use of plasmid pHCV2-1 for the manufacture of a pharmaceutical when used for the immunization of an individual susceptible to or infected by hepatitis C virus by administering to said individual an amount effective to induce a protective or therapeutic response against hepatitis C virus infection.

19. The use according to claim 18 wherein bupivacaine is administered to said individual at the site of administration of plasmid pHCV2-1. The use according to claim 18 wherein 100tg of plasmid pHCV2-1 is administered to said individual. 0*

21. The use according to claim 18 wherein said plasmid pHCV2-1 is 0 administered to said individual with a needleless injection device intramuscularly, subcutaneously and intradermally. DATED 22nd day of October 2001 S" APOLLON, INC, AND THE GENERAL HOSPITAL CORPORATION doing business as THE MASSACHUSETTS GENERAL HOSPITAL WATERMARK PATENT AND TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA CJH:ALJ:PXT P6774AU00