AU2033699A - Hepatitis virus vaccines - Google Patents
Hepatitis virus vaccines Download PDFInfo
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- AU2033699A AU2033699A AU20336/99A AU2033699A AU2033699A AU 2033699 A AU2033699 A AU 2033699A AU 20336/99 A AU20336/99 A AU 20336/99A AU 2033699 A AU2033699 A AU 2033699A AU 2033699 A AU2033699 A AU 2033699A
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- Prior art keywords
- plasmid
- nucleic acid
- acid molecule
- phcv4
- individual
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Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
Regulationl 3.2(2)
AUSTRALIA
Patents Act 1990
ORIGINAL
COMPLETE
SPECIFICATION
STANDARD
PATENT
Application Number: Lodged: .4: Invention Title: HEPATITIS VIRUS VACCINES
I.'
The following statement is a full description of this invention, including the best method of performing it known to us
I-'
L A- P~Di.
1 m
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i 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 Science, 1992, 258, 135-140; and Alter, et al, New Eng. J. Med., 1992, 327, 1899-1905).
In addition, HCV infection is an independent risk factor for the development of hepatocellular carcinoma as shown by the prevalence of anti-HCV antibodies (Colombo, et Lancet, 1989, ii, 1006-1008; Saito, et al., Proc. Natl. Acad. Sci.
USA, 1990, 87, 6547-6549; Simonetti, et An. Int. Med., 1992, 116, 97-102; and Tsukuma, et al., New Eng. J. Med., 1993, 328,1797-1801).
20 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 ai., 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).
p i 1: .i 4- 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, etal., 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 Proc. Natal. Acad. Sci.
USA, 1991, 88, 1711-1715; and Bukh, et al., Proc. Natl. Acad. Sci. USA, 1992, I 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 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, etal., J. Viroi., 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, t 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. Sc. USA, 1992, 89, 3468-3472), the core protein is well I, 30 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 S: Houghton, etal., 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, etal., 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 i 15 infection and subsequent disease chronicity (Weiner, et al., Proc. Natl. Acad.
i Sci. USA, 1992, 89, 3468-3472; Kato, et al., Biochem. Biophys. Res. Comm., 1992, 189,119-127; and Alter, et 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 .llchronic 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, etal., J. Virol., 1993, 67, 7522-7532; and Koziel, et 25 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 Cemy, etal., Intl.
Symp. Viral Hepatitis Liver Dips. 1993, 83 (abstr).
Botarelli, et al. (Botarelli, et al., Gastroenterol., 1993, 104, 580-587) and Ferrari, et al. (Ferrari, et al., Hepatol., 1994, 19, 286-295) found HLA class II- Srestricted CD4+ T cell-mediated proliferative responses to several recombinant .f 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 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-a 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 20 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.
S- 30 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 I llrsrr~era~nrsnma--7u -;-cl-7-n 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 15 No. 62320/94 filed January 26, 1994, Australian Serial No. 62320/94 filed S.January 26, 1994, PCT Patent Application Serial Number PCT/US94/00899 filed January 26, 1994 and Australian Serial No. 22366/95 filed March 30, 1995 each contains descriptions of genetic immunisation protocols. Vaccines against HCV are disclosed in each.
The HCV core DNA-based vaccine expresses high levels of core antigen in vitro and induces a strong immune response in vivo.
There remains a need for vaccines useful to protect individuals against f hepatitis C virus infection.
SUMMARY OF THE INVENTION The present invention relates to nucleic acid molecules including an incomplete hepatitis C viral genome including SEQ ID NO:14.
The present invention relates to nucleic acid molecules including an incomplete hepatitis C viral genome including a nucleotide sequence that encodes the HCV core protein and at least the last 9 nucleotides of the untranslated region.
f u~arp lra~- r~-Clrr(ar.i 6 The present invention relates to pharmaceutical compositions that contain a nucleic acid molecule having an incomplete hepatitis C viral genome and at least the coding sequences of SEQ ID NO:14 operably linked to regulatory elements functional in human ceils.
The present invention relates to pharmaceutical compositions that include a nucleic acid molecule having an incomplete hepatitis C viral genome including a nucleotide sequence that encodes the HCV core protein and at least the last 9 nucleotides of the 5' untranslated region operably linked to regulatory elements functional in human cells.
The present invention relates to a method of immunizing an individual susceptible to or infected by hepatitis C virus including the step of administering to the individual, an amount of plasmid pHCV2-1 or plasmid pHCV4-2 effective to induce a protective or therapeutic immune response against hepatitis C virus i infection.
15 The present invention relates to the use of plasmid pHCV2-1 or plasmid pHCV4-2 for the manufacture of a pharmaceutical when used for the immunization of an individual susceptible to or infected by hepatitis C virus in an amount effective to induce a protective or therapeutic response against hepatitis C virus infection.
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.
Figure 2 depicts methods for constructing the HCV fusion constructs.
DETAILED DESCRIPTION OF THE INVENTION According to the present invention, compositions and methods are provided which prophylactically and/or therapeutically immunize an individual against HCV infection. For immunization against HCV infection, recombinant 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.
L'.
L f~ r 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 a ^either been native cell surface proteins or secreted proteins, such as, for example, influenza NP, HBsAg, and rabies virus glycoproteins. Although HCV core DNA-based vaccine expresses high levels of core antigen in vitro and I 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 sequence for the HCV polyprotein which is encoded by the HCV viral genome.
S "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 8 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.
S; The HCV core protein has been observed to exist as two distinct forms, the anchored core protein and the virion core protein. During HCV translation, a 15 precursor polypeptide is translated and subsequently processed by cellular Sil, proteases and viral proteases to yield the individual viral polypeptides. A cellular signal peptidase is believed to cleave at a site which separates the portion of the polyprotein that becomes the core protein from the portion that 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 recombinant nucleic acid molecule including a nucleotide coding sequence that i Wencodes 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.
i i; 9 As used herein, the term "genetic vaccine" refers to a pharmaceutial 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 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 S. which are useful to promote integration may also be included in the DNA 20 molecule. Alternatively, RNA may be administered to the cell. It is also Scontemplated to provide the gene construct as a linear minichromosome including a centromere, telomeres and an origin of replication.
S, According to other aspects of the invention, the gene construct includes Scoding sequences which encode the full-length HCV core protein. In some 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
L
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.
SIn 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.
S In some preferred embodiments, the vector used is selected from those S 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 i, 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 Sadministered.
i. 30 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 promo:er, 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 S 15 human metalothionein.
i' 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 include enhancers. The enhancer may be selected from the group including but 25 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.
o" s 12 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 .I 15 tissues associated with mucosal immunity. Thus, in some examples, the gene j 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 bombardment gene guns". Alternatively, the genetic vaccine may be introduced by various means into cells that are removed from the individual. Such means 1 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 nonimmunogenic cells that have gene constructs incorporated therein can be implanted into the individual even if the vaccinated cells were originally taken from another individual. i 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 jsimultaneously administered to an individual intradermally, subcutaneously and intramuscularly using a needleless injection device. Needleless injection i j c i device.- using injectio 1.r-.
I,
1, a 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 about 1000 micrograms of DNA. In some preferred embodiments, the vaccines 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 about 1 to about 350 micrograms of DNA. In some preferred embodiments, the 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 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.
ri"i i i i i r
I
ii~I" ~rra, 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 tiled 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: CaPO 4 DEAE dextran, amnionic lipids; extracellular matrix-active enzymes; saponins; lectins; estrogenic compounds and steroidal hormones; hydroxylated lower alkyls; dimethyl sulfoxide (DMSO); urea; and benzoic acid esters anilides, amidines, urethanes and the hydrochloride salts thereof such as those of the family of local anaesthetics. In addition, the gene constructs are encapsulated within/administered in conjunction with lipids/polycationic complexes.
EXAMPLES
1 20 EXAMPLE 1: Design and construction of full-length HCV expression plasmids Plasmids, pHCV2-1 and pHCV4-2, were both designed to express the Sanchored 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 I 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 NatI 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.
S 15 Both PCR products were digested with Natl and Sail (NED), gel purified and ligated into the NatI-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 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 S' 16 transfected. All analyses were conducted at 48 hours post-transfection. The transfection efficiency was determined by measuring the specific activity of Pgalactosidase 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 labelled anti-mouse IgG (Sigma) and FITC labelled anti human IgG (Sigma) were used respectively as the secondary antibodies.
Immunostaining was seen in cells that had been transfected with either pHCV2- 1 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 lysates 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 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 100 pg of pHCV4-2 plus 0.25% bupivacaine. All mice were shown to have seroconverted 21 days after DNA 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.
k r 17 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 I Norris STREET: One Liberty Place, 46th floor CITY: Philadelphia S(D) STATE:PA COUNTRY: USA S(F) 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: APPLICATION NUMBER: N/A FILING DATE: Herewith
CLASSIFICATION:
S(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/318,248 30 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:
N
I.
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: clDNA (ix) FEATURE: NAMEIKEY: CODS LOCATION: 342..914 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:-
GCCAGCCCCC
TCTTCACGCA
CCCCCCTCCC
GACGACCGGG
GCGAGACTGC
GTGCTTGCGA
GATTGGGGGC
GAAAGCGTCT
GGGAGAGCCA
TCCTTTCTTG
TAGCCGAGTA
GTGCCCCGGG
GACACTCCAC
AGCCATGGCG
TAGTGGTCTG
GATCAACCCG
GTGTTGGGTC
AGGTCTCGTA
CATAGATCAC
TTAGTATGAG
CGGP.ACCGGT
CTCAATGCCT
GCGAAAGGCC
GACCGTGCAC
TCCCCTGTGA
TGTCGTGCAG
GAGTACACCG
GGAGATTTGG
TTGTGGTACT
C ATG AGC Met Ser
GGAACTACTG
CCTCCAGGAC
GAATTGCCAG
GCGTGCCCCC
GCCTGATAGG
ACG AAT Thr Asn CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC PAC CGC CGC CCA CAG Pro Lys Pro Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gin 5 10 15 GAC CTC AAG TTC CCG GGC GGT GGT AspLeu Lys Phe Pro Gly Gly Gly CAG ATC GTT GGT Gin ie Val Gly GGA GTT TAC CTG Gly Val Tyr Leu ACT AGG AAG ACT Thr Arg Lys Thr so TTG CCG CGC Leu Pro Arg AGG GGC Arg Gly CCC AGG TTG GGT GTG CGC GCG Pro Arg Leu Gly Val Arg Ala 45 TCC GAG CGG Ser Giu Arg TCG CAA CCT Ser Gin Pro CGT GGA AGG Arg Gly Arg CGA CAA CCT ATC CCC AAG GAT Arg Gin Pro Ile Pro Lys Asp CGC CGG Arg Arg CCC GAG GGC AGG GCC TGG GCT CAA Pro Glu Gly Arg Ala Trp Ala Gin 75 CCT GGG Pro Gly TAC CCT TGG CCC Tyr Pro Trp Pro
SM
CTC TAT GGC AAC Leu Tyr Gly Asn CGT GGC TCC CG Arg Gly Ser Arg CGT AAT TTG GGT Arq Asn Leu Gly 120 CTC ATG GGG TAC Leu Met Gly Tyr 135 AGG GCC TTG GCG Arg Ala Leu Ala 150 GCA ACA GGG AAT Ala Thr Gly Asn 165 CTG CTG TCC TGT Leu Len Ser Cys GAG GGC Gin Gly 90 CCT AGT Pro Ser 105 AAA GTC Lys Val ATT CCG Ile Pro CAT GGC His Gly CTG CCC Leu Pro 170 TTG ACC Len Thr ATG 600 TGG Met Gly Trp TGG GGC CCC Trp Gly Pro ATC GAT ACC Ile Asp Thr 125 CTC GTC GGC Leu Val Gly 140 GTC CGG GTT Val Arg Val 155 GGT TGC TCT Gly Cys Ser ATC CCA GCT Ile Pro Ala GCA GGA TGG CTC CTG TCA CCC Ala Gly Trp Leu Len Ser Pro 95 100 AAT GAO CCC CGG CGT AGG, TCG Asn Asp Pro Arg Arg Arg Ser 110 115 CTT ACA TGC GGC TTC GCC GAC Len Thr Cys Gly Phe Ala Asp 130 GCT CCC ATG GGG GGC GCT GCC Ala Pro Met Gly Gly Ala Ala 145 CTG GAG GAC GGC GIG AAC TAT Len Gin Asp Gly Val Asn Tyr 160 TTC TCT ATC TTC CTC TTG GCT Phe Ser Ile Phe Leu Leu Ala 175 180 TCC GCT TAATAATAA Ser Ala 190 4A
J
185 INFORMATION FOR SEQ ID Met Ser 1 Arg Gly Val Thr Arg lie Pro Tyr Pro Wi SEQUENCE CHARACTERISTICS: LENGTH: 191 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE T YPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Asn Pro Ly s Pro Gin Arg Lys Thr Lys Arg Asn 5 Gin Asp Leu Lys Phe Pro Gly Gly Gly Gin Ile 25 Len Len Pro Arg Arg Gly Pro Arg Leu Giy Val 40 Thr Ser Gin Arg Ser Gin Pro Arg Gly Arg Arg 55 Asp Arg Argr Pro Gin Gly Arg Ala Trp Ala Gin 70 75 Pro Leu Tyr Gly Asn Gin Gly Met Gly Trp Ala 90 Thr Asn Val Gly Arg Ala Gin Pro Pro Gly Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp G1ly Pro Asn Asp Pro 100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 515120 125 Gly Phe Ala Asp Leu Mlet Gly Tyr Ile Pro Leu Val Gly Ala Pro Met 130 135 14J Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser ?he Ser Ile 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Lee Thr Ile Pro Ala Ser Ala IS0 185 190 t 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: AAAAAGGGAA. GGAAGGCGGC CGCCCGTGCA CCATGAGCAC GAATCCT 47 INFORMATICON 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 57 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cOMA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: AAAAAGGGAA GGAAGGCGGC CGCGCCAGCC CCCGATTGGG GGCGACA 47
Claims (22)
1. A nucleic acid molecule including an incomplete hepatitis C viral genome, said nucleic acid molecule encoding a protein having an amino acid sequence of SEQ ID
2. The nucleic acid molecule of claim 1 including SEQ ID NO:14.
3. The nucleic acid molecule of claim 1 further including a promoter and polyadenylation sequence, wherein coding sequence encoding a protein having amino acid sequence SEQ ID NO:15 is operably linked to said promoter and polyadenylation sequence.
4. The nucleic acid molecule of claim 1 wherein said nucleic acid molecule -Iis selected from the group consisting of plasmid pHCV2-1 and plasmid i" pHCV4-2. 31 5. The nucleic acid molecule of claim 1 wherein said nucleic acid molecule |i is plasmid pHCV2-1. S6. The nucleic acid molecule of claim 1 wherein said nucleic acid molecule is plasmid pHCV4-2.
7. A pharmaceutical composition containing a) a nucleic acid molecule including an incomplete hepatitis C viral genome, said nucleic acid molecule including a nucleotide sequence encoding a complete hepatitis C core protein having amino acid of SEQ ID operably linked to regulatory elements functional in human cells; and b) a pharmaceutically acceptable carrier or diluent.
8. The pharmaceutical composition of claim 7 wherein said nucleotide sequence encoding a complete hepatitis C core protein is coding sequence of SEQ ID NO:14.
9. The pharmaceutical composition of claim 7 wherein said nucleotide sequence encoding a complete hepatitis C core protein is coding sequence of SEQ ID NO:14 and further includes nucleotides 332-341 of SEQ ID NO:14. The pharmaceutical composition of claim 7 wherein said nucleic acid molecule comprises SEQ ID NO:14. li' -h~iaar~as'~ a S. i
11. The pharmaceutical composition of claim 7 wherein said nucleic acid molecule is selected from the group consisting of plasmid pHCV2-1 and pHCV4-2.
12. The pharmaceutical composition of claim 7 wherein said nucleic acid molecule is plasmid pHCV2-1.
13. The pharmaceutical composition of claim 7 wherein said nucleic acid molecule is pHCV4-2.
14. A pharmaceutical composition containing a) a nucleic acid molecule including an incomplete hepatitis C viral genome, said nucleic acid molecule including at least a fragment of the hepatitis C virus 5' untranslated region including the last 9 nucleotides and a nucleotide sequence encoding a complete hepatitis C core protein operably linked to regulatory elements functional in human cells; and b) a pharmaceutically acceptable carrier or diluent.
15. The pharmaceutical composition of claim 14 wherein said nucleotide molecule includes a complete hepatitis C virus 5' untranslated region.
16. A method of immunizing an individual susceptible to or infected by hepatitis C virus including the step of: administering an amount of plasmid pHCV2-1 or plasmid pHCV4-2 effective to induce a protective or therapeutic immune response against hepatitis C virus infection.
17. The method of claim 16 wherein plasmid pHCV2-1 is administered to said individual.
18. The method of claim 16 wherein plasmid pHCV4-2 is administered to said individual.
19. The method of claim 16 wherein bupivacaine is administered to said individual at the site of administration of plasmid pHCV 2-1 or plasmid pHCV4-2. The method of claim 16 wherein 100 pg of plasmid pHCV2-1 or plasmid pHCV4-2 is administered to said individual.
21. The method of claim 16 wherein said plasmid pHCV2-1 or said plasmid pHCV4-2 is administered to said individual with a needleless injection device intramuscularly, subcutaneously and intradermally. -cv- Fr i: i i 23
22. The use of plasmid pHCV2-1 or plasmid pHCV4-2 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.
23. The use according to claim 22 wherein plasmid pHCV2-1 is administered to said individual,
24. The use according to claim 22 wherein plasmid pHCV4-2 is administered to said individual. The use according to claim 22 wherein bupivacaine is administered to said individual at the site of administration of plasmid pHCV 2-1 or plasmid pHCV4-2.
26. The use according to claim 22 wherein 100 gg of plasmid pHCV2-1 or plasmid pHCV4-2 is administered to said individual.
27. The use according to claim 22 wherein said plasmid pHCV2-1 or said plasmid pHCV4-2 is administered to said individual with a needleless injection device intramuscularly, subcutaneously and intradermally. DATED this 21st day of December, 1998 APOLLON. INC. and THE MASSACHUSETTS GENERAL HOSPITAL WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA LCG/KMH/MEH VAX DOC 24 AU002928.WPC |I g .I 1
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU20336/99A AU741876B2 (en) | 1994-10-05 | 1999-03-11 | Hepatitis virus vaccines |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/318248 | 1994-10-05 | ||
US08/467859 | 1995-06-06 | ||
AU40061/95A AU701747B2 (en) | 1994-10-05 | 1995-10-05 | Hepatitis virus vaccines |
AU20336/99A AU741876B2 (en) | 1994-10-05 | 1999-03-11 | Hepatitis virus vaccines |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU40061/95A Division AU701747B2 (en) | 1994-10-05 | 1995-10-05 | Hepatitis virus vaccines |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU22986/02A Division AU2298602A (en) | 1994-10-05 | 2002-03-07 | Hepatitis virus vaccines |
Publications (2)
Publication Number | Publication Date |
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AU2033699A true AU2033699A (en) | 1999-05-20 |
AU741876B2 AU741876B2 (en) | 2001-12-13 |
Family
ID=3727171
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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AU20336/99A Ceased AU741876B2 (en) | 1994-10-05 | 1999-03-11 | Hepatitis virus vaccines |
Country Status (1)
Country | Link |
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AU (1) | AU741876B2 (en) |
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1999
- 1999-03-11 AU AU20336/99A patent/AU741876B2/en not_active Ceased
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