WO2014093602A1 - Compositions and methods for treating and preventing hepatitis c virus infection - Google Patents

Abstract

A recombinant vesicular stomatitis virus (rVSV) immunogenic composition useful in the treatment or prophylaxis of Hepatitis C virus infection involves a selected, sequential, modified order of VSV genes, as well as a specific selection of HCV nonstructural proteins. Methods of treatment or prevention of a mammalian subject for HCV using the recombinant VSV include administration of only the rVSV immunogenic composition. Alternatively, a method for treating or preventing HCV infection can include administering to the mammalian subject an effective amount of a priming composition including a plasmid comprising a single open reading frame encoding selected HCV nonstructural proteins under the control of regulatory sequences directing expression thereof by the plasmid and a pharmaceutically acceptable diluent. The priming composition is administered before and/or after administration of the above-described immunogenic rVSV composition.

Description

COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING

HEPATITIS C VIRUS INFECTION

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. 1R43CA165293- 01, awarded by the National Institutes of Health. The government has certain rights in this invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

The Sequence Listing material filed in electronic form herewith is hereby incorporated by reference. This file is labeled "PBI1001PCT_ST25.txt", was created on December 9, 2013, and is 35KB (35,000 bytes).

BACKGROUND OF THE INVENTION

The World Health Organization estimates the global prevalence of Hepatitis C virus (HCV) infections to be 3%, making it the most common chronic blood-borne infection of humans. HCV infection is spread predominantly through the parenteral route; but iatrogenic, nosocomial, occupational, sexual, and perinatal transmission accounts for approximately 40% of new cases in the US¹'².

Chronic hepatitis caused by HCV is a serious public health problem in many parts of the world. Surveillance studies conducted by the Centers for Disease Control and Prevention (CDCP) and the NIH show that HCV accounts for 40% to 60% of chronic liver disease in the US, which is currently the tenth leading cause of death among adults. HCV is also the most frequent indication for liver transplantation in US; the number of patients on transplant waiting lists has doubled in the past 5 years, and about 50 percent of these patients die while awaiting an organ³'⁴. Estimates of morbidity and mortality resulting from HCV disease project that by 2023 the rates of hepatic decomposition, hepatocellular carcinoma (HCC), and liver related death will increase by 81 - 180%> ⁵'⁶. Without improved therapies, 11 ,000 to 13,000 annual cases of HCV-related HCC will occur in the US through 2040. Globally, HCV disease is projected to cause an unmanageable burden on health care over the next 10 to 20 years.

Twenty per cent (20%) of genotype 1 HCV (HCV1, the serotype that predominates in the US and Europe) infected subjects spontaneously cure due to a robust and sustained cell- mediated immune (CMI) response that targets a broad number of epitopes in various non- structural (NS) viral proteins and results in the intra-hepatic production of interferons (IFNs).

Both CD4⁺ T helper cells and CD8⁺ cytotoxic T lymphocytes (CTL) are required for resolution of the acute infection and for protection against re-infection. In contrast, the 80% of individuals that progress to chronic infection initially mount an early CMI response, but lose the CD4⁺ component of this response which results in the return of virus to the blood circulation and the onset of chronic disease.^7"23

The current standard of care for HCV1 infection with evidence of advancing cirrhosis is a 48 week course of treatment with pegylated interferon-alpha (PEG-IFN) and the nucleoside analog Ribavirin. Both patients and physicians are hesitant to undertake this treatment option until absolutely necessary because it is expensive, the success rate with HCV1 infection is only

42-46%, and the 48 week course of treatment involves debilitating side effects that result in a

10-14%o drop out rate in well controlled studies^24"26.

Thus, there is substantial need for compositions and methods that slow disease progression, and increase the efficacy rate of current treatment without adding to the negative side effect profile.

SUMMARY OF THE INVENTION

Described herein are therapeutic compositions and methods that meet the need in the art by inducing a robust CMI response, permitting administration at an immune inductive site distant from the liver, and overcoming the immunologic anergy that characterizes chronic HCV infection.

In one aspect, a recombinant vesicular stomatitis virus (rVSV) comprises the nucleic acid sequence construct of 3'-Pi-M2-(G-CT)₃-N4-X₅-L₆ -5' or 3'-Pi-M2-(G-CT)₃-N4-L₅-X₆-5'. According to these formulae, X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A-like peptide positioned therebetween. The subscript numbers indicate the genomic position of each VSV gene, P (encoding the phosphoprotein), M (encoding the matrix protein), G (encoding the attachment protein with a cytoplasmic tail truncation (G-CT)), N (encoding the nucleocapsid protein) and L (encoding the polymerase protein). In some aspects, NS5b lacks its polymerase and retinoblastoma protein (Rb) binding activity.

In another aspect, an rVSV comprises the nucleic acid sequence 3'-Pi-M₂-(G-CT)₃-N₄- (X/L)₅-5\ According to this aspect, X/L is a single open reading frame, and X is a nucleic acid sequence comprising (i) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b ; or (ii) sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A-like peptide positioned therebetween. The subscript numbers indicate the genomic position of each VSV gene, P, M, G (with a cytoplasmic tail truncation (G-CT)), N and L. In some aspects, NS5b lacks its polymerase and Rb binding activity.

In another aspect, the 2A-like peptide is the T2A sequence from Thosea asigna virus.

In still other aspects, an rVSV described herein further comprises a sequence encoding a 2A-like peptide positioned between the HCV NS5b nucleic acid sequence and the VSV L gene.

In still other aspects, an rVSV described herein further comprises as part of X a nucleic acid sequence encoding an AAY peptide motif inserted between NS3 and the 2A-like sequence.

In still a further aspect, an rVSV described herein is one of rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)-T2A-NS5b(pol-)]5 or rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)-T2A-

NS5b(pol-)-T2A-VSV_in LJ5 or rVSV_in -N4(G CT1)3- [HCV la NS3-NS4a-NS5b(pol-)-T2A-VSV_in LJ5.

In another aspect, an immunogenic composition comprises an rVSV as described herein and a pharmaceutically acceptable diluent.

In yet another aspect, a method of treating or preventing Hepatitis C Virus infection includes administering to a mammalian subject in need thereof an immunogenic composition comprising an rVSV as described herein and above.

In a further aspect, a method of treating or preventing Hepatitis C Virus infection includes administering to a mammalian subject in need thereof an immunogenic composition comprising an rVSV as described herein and above and administering to the subject an effective amount of a priming composition. The priming composition comprises a plasmid comprising a single open reading frame encoding HCV nonstructural proteins NS3, NS4a and NS5b; or HCV NS3 lacking the NS3/4a protease function and HCV NS5b with a 2A-like peptide positioned therebetween under the control of regulatory sequences directing expression thereof by the plasmid and a pharmaceutically acceptable diluent. The HCV NS5b in the priming composition or the rVSV can be HCV NS5b lacking its polymerase and Rb binding activity. The method includes administering the priming composition at least once prior to administering the immunogenic rVSV composition. In another aspect, the method includes repeating the priming composition administration following the immunogenic rVSV composition.

Similarly, use of an rVSV in the preparation of a medicament used for the treatment or prevention of Hepatitis C Virus infection is provided.

Still another aspect involves a method of generating an rVSV as described above by introducing into a host cell a viral cDNA expression vector comprising a nucleic acid sequence 3'-Pi-M₂-(G-CT)₃-N₄-X5-L₆ -5' or 3 '-Pi-M₂-(G-CT)₃-N4-L₅-X6-5 ', or 3 '-Pi-M₂-(G-CT)₃-N₄- (X/L)₅-5', as defined above and herein, flanked by a T7 promoter sequence upstream and a hepatitis delta virus ribozyme site (HDV Rz) and T7 terminator sequence downstream. The T7 promoter directs the synthesis of viral RNA anti-genome transcripts from the cDNA expression vector, in the presence of T7 RNA polymerase. The essential VSV proteins N, P and L, and optionally M and G, are co-expressed in the host cell from plasmid DNAs (pDNAs) under the control of the hCMV promoter. Assembled, infectious rVSV RNA virus is then recovered from the host cells.

These and other embodiments and advantages of the invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of plasmid pPBS-HCV-081, which contains the HCV NS3, NS4a and NS5b genes encoding the HCV polyprotein. In this construct, the nucleic acid sequence encoding the NS5b protein has been mutated to ablate its polymerase activity and

retinoblastoma protein binding activity (pol-). Other conventional components of the plasmid, e.g., the hCMV promoter (hCMV pro.), the bovine growth hormone polyadenylation site (BGH poly A), the kanamycin resistance gene (Kan), as well as numerous enzyme restriction sites and the direction of translation (arrows) are identified.

FIG. 2A is a diagram depicting the proteolytic processing of the HCV polyprotein

NS3-4a-5b(pol-) into the individual NS3, NS4a and NS5b(pol-) proteins.

FIG. 2B is a diagram depicting the processing of NS3 (prot-)-T2A-5b(pol-) which results in the expression of two separate proteins, the NS3(prot-) protein and the NS5b(pol-) protein, with the T2A peptide sequence attached to the C-terminus of the NS5b(pol-) protein.

FIG. 3 is a schematic of the gene order of the plasmid pPBS-VSV-HCV-003, which contains between the T7 promoter (T7 pro.) and the HDV Rz/T7 termination (T7 term.) gene sequences in the order shown in Table 1 below. NS5b(pol-) represents a mutation in the NS5b encoding sequence that ablates the polymerase and Rb binding activity of HCV NS5b.

FIG. 4 is a schematic of the gene order of the plasmid pPBS-VSV-HCV-008, which contains between T7 pro. and T7 term, genes in the order shown in Table 1 below. ETU means empty transcription unit.

FIG. 5 is a schematic of the gene order of the plasmid pPBS-VSV-HCV-032, which contains between T7 pro. and T7 term, genes in the order shown in Table 1 below.

FIG. 6 is a schematic of the gene order common to the plasmids pPBS-VSV-HCV-048 and pPBS-VSV-HCV-034, which each contain between T7 pro. and T7 term, genes in the order shown in Table 1 below. The difference between pPBS-VSV-HCV-048 and pPBS-VSV-HCV- 034 are several non-coding changes in the HCV antigen, as indicated in Table 9 herein.

FIG. 7 is a schematic of the gene order of the plasmid pPBS-VSV-HCV-043 which contains between the T7 pro. and T7 term, genes in the order shown in Table 1 below. NS3 (prot-) is an NS3 with an inactivated protease catalytic site.

FIG. 8 is a schematic of the gene order of the plasmid pPBS-VSV-HCV-044 which contains between the T7 pro. and T7 term, genes in the order shown in Table 1 below.

FIG. 9A is a Western blot showing HCV NS3 protein immunodetection in lysates from

Vero cells infected with rVSV-HCV-008 (lane 1), rVSV-HCV-032 (lane 2), rVSV-HCV-034 (lane 3), rVSV-HCV-043 (lane 4), rVSV-HCV-044 (lane 5).

FIG. 9B is a Western blot showing HCV NS5b protein immunodetection in lysates from Vero cells infected with rVSV-HCV-008 (lane 1), rVSV-HCV-032 (lane 2), rVSV-HCV- 034 (lane 3), rVSV-HCV-043 (lane 4), rVSV-HCV-044 (lane 5).

FIG. 1 OA is a Western blot showing HCV NS3 protein immunodetection in lysates from uninfected Vero cells (lane 1), from Vero cells infected with rVSV_!n -Gag 1, a negative control virus (lane 2), from Vero cells infected with rVSV-HCV-032, in working stock (lane 3), from Vero cells infected with rVSV-HCV-034, working stock (lane 4), from Vero cells infected with rVSV-HCV-034 passage 1 (lane 5), from Vero cells infected with rVSV-HCV-034 passage 10 (lane 6), from Vero cells infected with rVSV-HCV-043 passage 10 (lane 7), from Vero cells infected with rVSV-HCV-043 working stock (lane 8). Expected molecular sizes for the processed proteins: for NS3, 67kDa and for NS5b, 65kDa. FIG. 1 OB is a Western blot showing HCV NS5b protein immunodetection in lysates from uninfected Vero cells (lane 1), from Vero cells infected with rVSV_in -Gag 1, a negative control virus (lane 2), from Vero cells infected with rVSV-HCV-032 working stock (lane 3), from Vero cells infected with rVSV-HCV-034, working stock (lane 4), from Vero cells infected with rVSV-HCV-034 passage 1 (lane 5), from Vero cells infected with rVSV-HCV-034 passage 10 (lane 6), from Vero cells infected with rVSV-HCV-043 passage 10 (lane 7), from Vero cells infected with rVSV-HCV-043 working stock (lane 8).

FIG. 11 is a bar graph depicting the mean HCV-specific interferon-γ ELISpot responses in spot-forming cells (SFC) /million splenocytes of groups of mice one week after immunization with rVSV-HCV-032 (bar 1), rVSV-HCV-034 (bar 2), rVSV-HCV-043 (bar 3) and rVSV-HCV-044 (bar 4). The results are provided in Table 2 below.

FIG. 12 is a bar graph depicting the mean HCV-specific interferon-γ ELISpot responses in SFC/ million splenocytes of groups of mice two weeks after priming immunization with pPBS-HCV-081 (bar 1), pPBS-HCV-080 (bar 2) and pPBS-HCV-016 (bar 3), as described in Table 3 below.

FIG. 13 depicts the mean HCV-specific interferon-γ ELISpot responses in groups of mice treated with a pDNA prime followed by an rVSV boost administration three weeks later. These results were observed two weeks following the rVSV boost (five weeks after pDNA priming) and reported as SFC/million splenocytes. The prime/boost protocols are pPBS-HCV- 081 primdrVSV-HCV-043 boost (bar 1), pPBS-HCV-081 pnme/rVSV-HCV-048 boost (bar 2) and pPBS-HCV-080 pnmdrVSV-HCV-043 boost (bar 3).

FIG. 14 is the rVSV-HCV-043 nucleotide sequence SEQ ID NO: 1 of plasmid pPBS- VSV-HCV-043. SEQ ID NO: 1 shows the VSV leader (nucleotides 1-50 ), VSV P sequence (nucleotides 61-860), VSV M sequence (nucleotides 915-1606), truncated VSV_in G-CT1 sequence (nucleotides 1743-3194), VSV N sequence (nucleotides 3227-4495), HCV NS3(prot-) sequence (nucleotides 4583-6477), T2A sequence (nucleotides 6488-6541), HCV NS5b(pol-) sequence (nucleotides 6542-8317), VSV L sequence (nucleotides 8458 - 14787) and VSV trailer (nucleotides 14830-14886). The VSV leader and trailer sequences are depicted in bold. The HCV polygene, i.e., HCVla NS3(prot-)-T2A-NS5b(pol-) (nucleotides 4583-8317 of SEQ ID NO: 1) are marked in bold/italic. In addition, gene start and stop signals in the VSV intergenic regions are marked in'Ttalic" and "Italic/Underlined" respectively. The VSV intergenic regions are unmarked sequences between the identified genes. The complementary nucleotide sequence is shown underneath the sequence encoding the plasmid.

FIG. 15 is the HCV NS4a nucleotide sequence SEQ ID NO:42 (with its amino acid sequence SEQ ID NO:43) in, for example, pPBS-HCV-081.

FIG. 16 is a bar graph depicting the total HCV-antigen specific interferon-γ ELISpot responses in groups of rhesus macaques treated with three doses of a pDNA prime at weeks 0, 4 and 8, followed by an rVSV boost administration eight weeks later. Doses are indicated by arrows. These results were observed at weeks 0, 2, 6, 8, 10, 16 and 17, and reported as SFC/million peripheral blood mononuclear cells (PBMCs). The prime/boost protocols for the three groups are:

i. pPBS-HCV-081 prime plus plasmid rhesus IL-12 (pILl2)/rVSV-HCV- 043 boost (indicated as pHCV + pIL12/rVSV),

ii. pPBS-HCV-081 prime/ rVSV-HCV-043 boost (indicated as pHCV/rVSV), and

iii. pPBS-HCV-081 prime plus≠Ll2/rVSV-HCV-043 boost, where at study week 14 the macaques were placed on a five week course of PEGASYS® pegylated alpha-interferon 2 (PEG-interferon alfa-2a, Hoffmann-LaRoche, Inc.; indicated as pHCV + pIL12/rVSV + PEGASYS®). FIG. 17 depicts the total HCV-antigen specific interferon-γ ELISpot responses over time of the three groups of rhesus macaques treated as described in the legend for FIG. 16.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, therapeutic and immunogenic compositions and methods are provided to overcome previously unresolved complications in the design of HCV vaccines and therapies. The compositions and methods provide a prime/boost therapy for the prevention or treatment of HCV based upon use of an rVSV containing a specific selection and assembly of HCV genes encoding the selected antigens and VSV genes to provide both anti-HCV immunogenic efficacy and pharmacological stability, and upon the use of a plasmid expressing HCV antigens essential to the induction of a useful immune response. Further, the methods of treatment or prevention of HCV infection and the immunogenic compositions useful therein should lack serious side effects characteristic of known treatments. I. Definitions

All scientific and technical terms used herein have their known and normal meaning to a person of skill in the fields of biology, biotechnology and molecular biology and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. However, for clarity, the following terms are defined as follows:

The terms "2A", "2A peptide or "2A-like peptide" refer to peptides that have been used successfully to generate multiple proteins from a single open reading frame. These peptides are small (18-22 amino acids) and have divergent amino -terminal sequences, but all contain a PGP motif at the C-terminus. Through a ribosomal skip mechanism, the 2A peptide prevents normal peptide bond formation between a glycine and a proline residue at the C-terminus of the peptide. These 2A and 2A-like sequences are known in the art and may be readily selected for such use. See, e.g., Szymczak- Workman et al, in Cold Spring Harbor Protocols 2012, doi 10.1101/pdb.ip067876; and Friedmann and Rossi (eds), Gene Transfer: Delivery and

Expression of DNA and RNA., CSHL Press, Cold Spring Harbor, NY USA, 2007, among others. One such 2A peptide is the peptide T2A, which has the sequence

EGRGSLLTCGDVEENPGP SEQ ID NO: 8 (nucleic acids 6488-6541 of SEQ ID NO: 1) as shown in FIG. 14. T2A is isolated from Thosea asigna virus.

By "rVSV" is meant recombinant vesicular stomatitis virus, a member of the taxonomic Order Mononegavirales, which comprises an approximately 11 kb non-segmented, negative-strand RNA genome that encodes five major viral proteins abbreviated N, P, M, G and L. In 3' to 5' genomic order in the wild-type VSV, the genes encode proteins designated the nucleocapsid (N), phosphoprotein (P), matrix protein (M), transmembrane glycoprotein (G) and polymerase (L), i.e., 3'- N-P-M-G-L-5'. The nucleotide sequences encoding VSV G, M, N, P and L proteins are known in the art ²⁹'³⁰. A number of VSV serotypes are known and have been sequenced. The genomic sequence of VSV (Indiana) is set out under Accession No. NC001560 in the NCBI database. Other sequences for VSV, including VSV (Chandipura) sequences, are available in that database; for example, see Accession Nos. Ay382603, Afl28868, V01208, V01207, V01206, M16608, M14715, M14720 and J04350, among others. VSV serotypes, such as New Jersey, among others are also available from depositories such as the American Type Culture Collection, Rockville, Maryland (see, e.g., Accession Nos. VR-1238 and VR-

1239). Other known VSV sequences and serotypes are described in the art or referenced in the documents cited throughout this specification, see, e.g., International Patent Application No. WO2004/093906 and US Patent No. 8,287,878. Variants of these viruses having the entire complement of genes in a rearranged format have not been observed in nature.

By "rVSV" is also meant a replication-competent, attenuated, recombinant VSV that comprises a nucleic acid sequence, which may further encode one or more selected heterologous antigens under the control of viral regulatory sequences directing expression thereof in a mammalian host cell. Such an rVSV may be designed to reduce the N mRNA protein synthesis in cells infected with virus by shuffling the N (nucleocapsid protein) gene to a position in the genome that is further away (distal) from the native 3' transcription promoter.

Because VSV is not considered a human pathogen, and pre-existing immunity to VSV is rare in the human population, the development of VSV-derived vectors has been a focus in areas such as immunogenic compositions and gene therapy. For example, studies have established that VSV can serve as an effective vector for immunogenic compositions, expressing influenza virus haemagglutinin³³, measles virus H protein³⁴ and HIV-1 env and gag proteins³⁵.

As used herein, "G-CT" refers to a mutated VSV G gene wherein the encoded G protein is truncated or deleted of some of the amino acids in its cytoplasmic domain (carboxy- terminus), also referred to as the "cytoplasmic tail region" of the G protein. G-CT1 SEQ ID NO: 4 is truncated of its last carboxy terminal 28 amino acids, resulting in a protein product that retains only one amino acid from the twenty -nine amino acid wild-type cytoplasmic domain. Other G gene truncations are identified in US Patent No. 8,287,878, e.g., G-CT9, having the last twenty carboxy-terminal amino acid residues of the cytoplasmic domain deleted, relative to the wild-type. Among known methods for altering the G protein of rVSV are the technologies described in International Publication No. W099/32648 and Rose, N. F. et al. 2000 J. Virol. , 74:10903-10. As used herein, HCV can be any HCV serotype, including HCV serotype la, isolate H77. The H77 polyprotein sequence is publically available under GENBANK Accession No. AAB67037. That isolate's nucleic acid sequence is provided at GENBANK Accession No. JF343780.2.

As used herein, the term "HCV NS3 lacking the NS3/4a protease function" or "NS3

(prot-)" can be used interchangeably to refer to the HCV NS3 in the absence of the NS4a protein, or a wild-type NS3 having at least one mutation that eliminates its protease catalytic site; or NS3 which is both in the absence of the NS4a protein and has at least one mutation that destroys the protease catalytic site. In one embodiment used herein, NS3 (prot-) (e.g., SEQ ID NO: 6) is encoded by the native HCV la isolate H77 sequence (see NCBI reference sequence NP_803144.1), in which the amino acid (aa) at position 57 is changed. In certain embodiments, aa57 is changed or mutated, for example, from histidine to threonine.

As used herein, "NS5b(pol-)" (e.g., SEQ ID NO: 9) refers to the HCV NS5b gene in which the wild-type nucleic acid sequence is mutated to eliminate, inactivate or ablate the RNA polymerase function and retinoblastoma protein binding (Rb) function. In one embodiment, the HCV NS5b nucleic acid sequence has mutations which change the aspartic acid residues at positions 318 and 319 in the RNA polymerase catalytic domain of the encoded protein. In another embodiment, the HCV NS5b nucleic acid sequence has mutations which change the aspartic acid residues at positions 318 and 319 in the RNA polymerase catalytic domain of the encoded protein to alanine residues. In one embodiment, NS5b(pol-) is generated by inserting two coding changes in the HCV la, isolate H77 NS5b sequence (NCBI ref: 2XI3_B) which changes the encoded aspartic acid residues at amino acid positions 318-319 in the RNA polymerase catalytic domain to alanine residues.

As defined herein, the terms "gene shuffling", "shuffled gene", "shuffled", "shuffling", "gene rearrangement" and "gene translocation" are used interchangeably, and refer to a change (mutation) in the order of the VSV genes in the viral genome.

By the term "polygene" is meant the assembly of two or more genes in a single open reading frame.

By "mammalian subject" is meant primarily a human, but also domestic animals, e.g., dogs, cats, horses, livestock, such as cattle, pigs, etc.; and common laboratory mammals, such as primates, rabbits, and rodents, and including the mice and Rhesus macaques which were the subjects of the examples below.

The terms "a" or "an" refers to one or more, for example, "an immunogenic composition" is understood to represent one or more such compositions. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. As used herein, the term "about" means a variability of 10 % from the reference given, unless otherwise specified.

While various embodiments in the specification are presented using "comprising" language, under other circumstances, a related embodiment is also intended to be interpreted and described using "consisting of or "consisting essentially of language. The words "comprise", "comprises", and "comprising" are to be interpreted inclusively, rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. II. Immunogenic compositions

In one aspect, the compositions of this invention are an rVSV capable of expressing certain HCV antigens. In another aspect, the compositions of this invention include at least two immunogenic compositions: a DNA plasmid capable of expressing certain selected HCV antigens, and an rVSV capable of expressing certain HCV antigens. Together, these two compositions can be utilized in a prime/boost immunogenic regimen.

A. Selected Hepatitis C Virus Antigens

In one embodiment of the invention, the immunogenic compositions express a subset of HCV non-structural proteins, namely NS3, NS5b, and optionally NS4a. The HCV NS3 gene is a wild-type sequence which encodes a protease. The HCV NS4a is a necessary co-factor to permit the HCV NS3 to function as a protease. The HCV NS5b gene is a wild-type sequence which encodes an RNA-dependent RNA polymerase.

In some embodiments the NS5b gene is mutated to NS5b(pol-) for use in the DNA plasmid and/or rVSV vector. NS5b(pol-) (e.g., SEQ ID NO: 9) lacks this antigen's polymerase and retinoblastoma protein (Rb) binding activity. Throughout this specification, where reference is made to NS5b, it should be understood to be interchangeable with NS5b(pol-), unless stated otherwise.

In some embodiments, the NS3 gene is mutated to NS3(prot-) (e.g., SEQ ID NO: 6) to destroy its protease catalytic site. Throughout this specification, where reference is made to NS3, it should be understood to be interchangeable with NS3(prot-), unless stated otherwise.

Thus, in one embodiment, when the wild-type NS3 gene is present in the immunogenic compositions, e.g., plasmid DNA or rVSV, the single HCV polygene is NS3-NS4a-NS5b or NS3-NS4a-NS5b(pol-) to permit the resulting NS3/4a protease to express NS3, NS4a and NS5b as separate proteins. In another embodiment, in which the NS3 gene is mutated to NS3(prot-) to destroy its protease catalytic site, the single HCV polygene is NS3(prot-)-NS4a-NS5b or NS3(prot-)-NS4a-NS5b(pol-). In yet a further embodiment, the NS4a sequence can be eliminated from the polygene and a sequence encoding a 2A-like peptide such as T2A is inserted between the NS3(prot-) and NS5b sequences. Thus, in one embodiment, the single HCV polygene is NS3-T2A-NS5b, NS3(prot-)-T2A-NS5b, NS3-T2A-NS5b(pol-), or NS3(prot-)-T2A-NS5b(pol-) (e.g., SEQ ID NO: 7).

The inventors selected only these NS3-T2A-NS5b or NS3-NS4a-NS5b HCV antigens, because in one embodiment, the decreased size of the HCV NS3/NS4a/NS5b polygene insert permitted by this selection of HCV genes allows for the use of a single DNA plasmid. In another embodiment, this polygene insert and/or the even further decreased size of the HCV insert containing only HCV genes NS3 and NS5b allows for the use of a single rVSV construct, thereby providing a practical formulation. The selection of the subset of antigens (NS3, NS4a and NS5b, or NS3 and NS5b) in the methods and compositions of this invention direct the entire HCV-specific immune response to viral structures that are more conserved across HCV genotypes and thus more efficacious in the resulting immunogenic compositions.

These embodiments containing only selected HCV genes permit the immunogenic composition and consequent method to be based on either a single rVSV immunogenic composition, or the combination of a single plasmid DNA (pDNA) prime component and a single rVSV vector as a boost component. Thus one embodiment of an immunogenic composition of this invention is an rVSV construct expressing only NS3 or NS3(prot-), NS4a, and NS5b or NS5b(pol-). Another embodiment of an immunogenic composition of this invention is an rVSV construct expressing only NS3 or NS3(prot-) and NS5b or NS5b(pol-), with a 2A-like peptide interposed between the NS3 and NS5b protein encoding sequences. In still another embodiment, an rVSV described herein further comprises a sequence encoding a 2A-like peptide positioned between the HCV NS5b nucleic acid sequence and the VSV L gene.

In another embodiment, wherein the immunogenic composition comprises a pDNA prime composition and rVSV boost immunogenic composition, the pDNA constructs express NS3 or NS3(prot-), NS4a, and NS5b or NS5b (pol-). In another embodiment, wherein the immunogenic composition comprises a pDNA prime composition and rVSV boost immunogenic composition, the pDNA constructs express NS3 or NS3(prot-), T2A, and NS5b or NS5b(pol-). See, for example, pPBS-HCV-080 described herein.

Nucleotide and protein sequences for the above-listed, known HCV antigens are readily publicly available through databases such as NCBI, or may be available from other sources such as the American Type Culture Collection and universities.

B. rVSV Immunogenic Composition

An immunogenic composition useful in this invention is a replication-competent, attenuated, recombinant vesicular stomatitis virus (rVSV). In certain embodiments, the immunogenic composition contains any of the rVSV described below in a pharmaceutically acceptable carrier.

The rVSV comprises a nucleic acid sequence encoding the selected HCV antigens discussed above under the control of regulatory sequences directing expression thereof in the cells of the immunized mammalian subject.

VSV genomes have been shown to accommodate more than one foreign gene, with expansion to at least three kilobases. The rVSV backbone is stable in its attenuation, and the virus does not undergo detectable recombination. In addition, since viral replication is cytoplasmic and viral genomes are comprised of RNA, virus is incapable of integrating into the genomes of infected host cells. Also, these negative-strand RNA viruses possess relatively simple, well-characterized transcriptional control sequences, which allow for efficient foreign gene expression. Finally, the level of foreign gene expression can be modulated by changing the position of the foreign gene relative to the viral transcription promoter (see, e.g., US Patent No. 6,136,585 and 8,287,878, among others). The 3' to 5' gradient of gene expression reflects the decreasing likelihood that the transcribing viral RNA-dependent RNA polymerase will traverse successfully each intergenic gene stop/gene start signal encountered as it progresses along the genome template. Thus, foreign genes placed in proximity to the 3 ' terminal transcription initiation promoter are expressed abundantly, while those inserted in more distal genomic positions are less so.

VSV replicates to high titers in a large array of different cell types, and viral proteins are expressed in great abundance. This not only means that VSV will act as a potent functional foreign gene delivery vehicle, but also, that relevant rVSV vectors can be scaled to manufacturing levels in cell lines approved for the production of human biologicals. This replication-competent virus gene delivery vehicle is safe, since wild-type VSV produces little to no disease symptoms or pathology in healthy humans, even in the face of substantial virus replication³⁹. Additionally human infection with, and thus pre-existing immunity to, VSV is rare. Therefore, rVSV is useful as a vector.

While a variety of rVSVs have been disclosed in the art with their genes "shuffled" to genome positions different from those of wild-type VSV (see US Patent No. 8,287,878; US Patent No. 6,596,529, and references cited therein), it may be useful for the N gene to be in the fourth position (N4) in the VSV gene order as part of a combination of mutations, so that the virus is sufficiently attenuated. In order to further attenuate rVSV, the cytoplasmic tail of the G protein is truncated (G-CT).

However, as described in detail below and in the examples, the inventors discovered and overcame unexpected difficulties in obtaining a sufficiently stable and effective rVSV for use as an immunogenic composition. The various embodiments of this invention overcame such difficulties through selective design of both the rVSV genome and the design of the selected HCV polygene insert.

In one aspect, an rVSV as described herein comprises the nucleic acid sequence 3 '-Pi-M₂-(G-CT)₃-N₄-X5-L₆ -5 ' or 3 '-Pi-M₂-(G-CT)₃-N4-L₅-X6-5 '.

In these embodiments, the subscript numbers indicate the genomic position of each VSV gene, and the genes are described as follows: P gene encodes the VSV phosphoprotein (e.g., SEQ ID NO: 2); M gene encodes the VSV matrix protein (e.g., SEQ ID NO: 3); G-CT gene encodes a VSV attachment glycoprotein with a truncated cytoplasmic tail (e.g., SEQ ID NO: 4); N gene encodes the VSV nucleocapsid protein (e.g., SEQ ID NO: 5); and L gene encodes VSV RNA- dependent RNA polymerase protein (e.g., SEQ ID NO: 10). In these embodiments of the rVSV, X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 with an inactivated protease catalytic site, HCV NS5b , and a 2A-like peptide positioned therebetween. Thus in one embodiment, X is the HCV polygene comprising NS3(prot-)-2A-NS5b(pol-) (e.g., SEQ ID NO: 7). The inclusion of the T2A or 2A-like peptide encoding sequences in X enables the expression of NS3 or NS3(prot-) and NS5b (pol-) as separate proteins from one open reading frame. In some aspects, NS5b lacks its polymerase and Rb binding activity.

In one aspect, a recombinant vesicular stomatitis virus (VSV) as described herein comprises the nucleic acid sequence 3 '-Pi-M2-(G-CT)₃-N₄-(X/L)₅-5 '. In these embodiments, the subscript numbers indicate the genomic position of each VSV gene, and the genes are described as follows: P gene encodes the VSV phosphoprotein; M gene encodes the VSV matrix protein; G-CT gene encodes a VSV attachment glycoprotein with a truncated cytoplasmic tail; N gene encodes the VSV nucleocapsid protein; and L gene encodes the VSV RNA-dependent RNA polymerase protein. However, in these embodiments, X and the VSV L protein encoding sequences form a single open reading frame. In one such embodiment X is a nucleic acid sequence encoding the HCV nonstructural proteins NS3, NS4a, and NS5b . In another such embodiment, X is a sequence encoding HCV NS3 with an inactivated protease catalytic site, HCV NS5b, and a 2A-like peptide positioned therebetween. Thus in one embodiment, X/L is a polygene comprising NS3- NS4a- NS5b(pol-)-2A-L. In another embodiment, X/L is the polygene comprising NS3(prot-) - 2A - NS5b(pol-)-2A-L. The inclusion of the T2A or 2A-like peptide encoding sequences in X or X/L enables the expression of NS3(prot-) and NS5b (pol-) and VSV L as separate proteins from one open reading frame.

In one rVSV embodiment, G-CT is G-CT 1 , which represents a sequence that encodes a truncated VSV attachment glycoprotein having a deletion of its last 28 carboxy-terminal amino acids. The use of G-CT1, in combination with shuffling of the N gene, ensures that the virus is sufficiently attenuated. In still further embodiments of these rVSV vectors, the carboxy- terminal coding sequence for the 29 amino acid cytoplasmic domain of the VSV G may be alternatively truncated by deleting 20 amino acids from the C-terminus of the VSV G, resulting in G-CT9. It is further contemplated that other truncated G constructs, such as those described in Schnell et ah, 1998⁴⁰ and US Patent No. 8,287,878, are useful in the contracts of this invention. Such other constructs may be readily selected by one of skill in the art.

Still another modification of any of the rVSV embodiments described above includes a nucleic acid sequence encoding an AAY peptide motif inserted between the HCV NS3 gene or NS3(prot-) gene and the T2A sequence. The insertion of the AAY motif provides a proteasomal cleavage site⁴¹, therefore limiting the generation of potentially harmful antigenic peptides containing amino acids from NS3 or NS3(prot-) and T2A.

Various embodiments of the rVSV described above employ VSV sequences derived from VSV serotype Indiana. Various embodiments of the rVSV described above also employ HCV sequences derived from HCV serotype la. However, it is anticipated that other known

VSV serotypes and HCV serotypes may be readily substituted for the exemplified sequences of the described embodiments by one of skill in the art, given the teachings of this specification.

Thus, one specific embodiment of an rVSV as described herein is rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)-T2A-NS5b(pol-)J 5. Another specific embodiment of an rVSV as described herein is rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)-T2A-NS5b(pol-)-T2A-VSV_in L]5. Still another exemplary embodiment of an rVSV as described herein is rVSV_in -N4(G CT1)3- [HCV la NS3-NS4a-NS5b(pol-)-T2A-VSV_!n L]5. These embodiments are described specifically in the examples which follow. In some embodiments, an immunogenic rVSV composition includes one or more of rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)-T2A-NS5b(pol-)] 5, rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)-T2A-NS5b(pol-)-T2A-VSV_in L], and rVSV_in -N4(G CT1)3 -[HCV la NS3-NS4a-NS5b(pol-)-T2A-VSV_in L ]5.

Techniques used in designing the rVSV with the selected HCV gene inserts and the appropriate regulatory sequences inserted into the above-indicated positions of the rVSV genome under the control of the viral transcription promoter described herein include those previously described⁴². Cloning to produce the corresponding shuffled recombinant cDNA sequences involves modification of a pDNA containing the original rVSV genome. The cloning strategy used to create these plasmids employs a previously described method⁴³. This technique takes advantage of the fact that the gene-end/gene-start signals found between each coding sequence are conserved, and allows gene rearrangements to be constructed without introducing any nucleotide substitutions. Alternatively, a few strategic point mutations may be introduced into noncoding sequences to create convenient restriction sites that facilitate genome rearrangements. The rVSV design is described in detail in the examples below.

To overcome any potential problem of diminished vector replication efficiencies with sequential administration due to neutralization by serotype-specific antibody (although sequential administration of rVSV Indiana may not be subject to such diminution), a vector set of similar design, each carrying a G gene from a different VSV serotype, permits successful booster immunizations. The primary amino acid sequences of the G proteins from VSV Indiana, New Jersey, and Chandipura, are sufficiently divergent such that preexisting immunity to one does not preclude infection and replication of the others. Thus, the neutralizing antibody response generated by rVSV (Indiana) should not interfere with replication of either rVSV (New Jersey) or rVSV (Chandipura). A vector set that can permit successful sequential immunizations can be prepared by replacing the G gene from VSV Indiana with either the divergent homolog from VSV Chandipura or from VSV New Jersey, forming three immunologically distinct vectors. Other suitable VSV serotypes include, without limitation, VSV San Juan and VSV Glasgow.

It is also desirable in selection and use of the antigenic sequences for design of the DNA plasmids and rVSV constructs of this invention to alter codon usage of the selected antigen-encoding gene sequence, and/or to remove inhibitory sequences therein. The removal of inhibitory sequences can be accomplished by using the technology discussed in detail in US Patent Nos. 5,972,596; 6,174,666; 6,291,664; 6,414,132; and 5,786,464; and in International Patent Publication No. WOO 1/46408, incorporated by reference herein. Briefly described, this technology involves mutating identified inhibitor/instability sequences in the selected gene, in some instances with multiple point mutations.

Suitable promoters for use in any of the components of this invention may be readily selected from among constitutive promoters, inducible promoters, tissue-specific promoters and others. Examples of constitutive promoters that are non-specific in activity and employed in the expression of nucleic acid molecules of this invention include, without limitation, those promoters identified in International Patent Application No. WO2004/093906 and US Patent No. 8,287,878. For use in the various plasmids to either express HCV antigens in pDNA compositions or to express VSV proteins for rVSV rescue purposes, the hCMV promoter is exemplified. Other pol II promoters that may be used include, inter alia, the ubiquitin C (UbiC) promoter, the phosphoglycerate kinase (PGK) promoter, the bovine cytomegalovirus (bCMV) promoter, a beta-actin promoter with an upstream CMV IV enhancer (CAGGS), and the elongation factor 1 alpha promoter (EF1A). In certain other embodiments, the T7 RNA polymerase promoter is used as noted in the examples and figures herein. C. Production and Rescue of the rVSV

A method of generating an attenuated rVSV useful for an HCV vaccine or HCV immunogenic composition, comprises the steps of rearranging the virus' gene order as described above by moving the N gene away from its wild- type 3' promoter-proximal position to position 4 in the genome, and placing the heterologous HCV antigen gene or polygene coding for the selected HCV immune response-inducing antigens in position 5 or higher of the gene order.

Briefly described is a method of generating an rVSV, which comprises introducing into a host cell a viral cDNA expression vector comprising a nucleic acid sequence specifically as described herein. In one embodiment the vector comprises the nucleic acid sequence 3'-Pi-M₂- (G-CT)₃-N₄-X₅-L₆ -5 ' or 3'-Pi-M2-(G-CT)₃-N4-L₅-X₆-5' as described above, wherein X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking a functional protease catalytic site, HCV NS5b lacking its polymerase and Rb binding activity, and a 2A-like peptide positioned therebetween. In another embodiment, the vector comprises the nucleic acid sequence 3'-Pi-M2-(G-CT)3-N₄-(X/L)₅-5' as described above, wherein X/L is a single open reading frame, and X is a nucleic acid sequence comprising (a) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b lacking its polymerase and Rb binding activity; or (b) sequences encoding HCV NS3 lacking a functional protease catalytic site, HCV NS5b lacking its polymerase and Rb binding activity, and a 2A- like peptide positioned therebetween. The selected nucleic acid sequence is flanked by a T7 promoter upstream of Pi and a hepatitis delta virus ribozyme site (HDV Rz) and T7 terminator sequence downstream of the last genomic position of the nucleic acid sequence. The T7 promoter directs synthesis of viral RNA anti-genome transcripts from the cDNA expression vector, in the presence of the T7 RNA polymerase.

In some embodiments, this method further comprises transiently-transfecting the host cells with a plasmid expressing the T7 RNA polymerase. In other embodiments, the method further involves co -trans fecting the host cell with one or more plasmids expressing at least the viral proteins N, P and L of VSV (and optionally M and G). In some embodiments, these VSV proteins are expressed in the host cell using an RNA polll-dependent expression system. Other embodiments include steps such as heat-shocking the host cells containing the cDNA vector, T7 polymerase and viral proteins of rVSV after pDNA transfection. The transfected host cells or supernatant obtained from the transfected host cells may be transferred into a culture of fresh expansion cells, and assembled, infectious rVSV is recovered from the culture.

In more detail, a replication-competent rVSV may be isolated and "rescued" using techniques known in the art⁴³'⁵¹'⁵². See, also, e.g., US Patent Nos. 8,287,878; 6,168,943; and 6,033,886; International Patent Publication No. WO99/02657. Methods of producing recombinant RNA virus are referred to in the art as "rescue" or "reverse genetics" methods. Exemplary rescue methods for VSV are described in U.S. Pat. Nos. 6,033,886, 6,596,529 and WO 2004/1 13517, each incorporated herein by reference. The transcription and replication of negative-sense, single stranded, non-segmented, RNA viral genomes are achieved through the enzymatic activity of a multimeric protein complex acting on the ribonucleoprotein core (nucleocapsid). The viral sequences are recognized only when they are entirely encapsidated by the N protein into the nucleocapsid structure, in which context the genomic and antigenomic terminal promoter sequences are recognized to initiate the transcriptional or replication pathways.

A cloned DNA equivalent of the VSV genome is placed between a suitable DNA- dependent RNA polymerase promoter (e.g., the T7 RNA polymerase promoter) and a self- cleaving ribozyme sequence (e.g., the hepatitis delta ribozyme), which is inserted into a suitable transcription vector (e.g., a propagatable bacterial plasmid). This transcription vector provides the readily manipulable DNA template from which the RNA polymerase (e.g., T7 RNA polymerase) can faithfully transcribe a single-stranded RNA copy of the VSV anti-genome (or genome) with the precise, or nearly precise, 5' and 3' termini. The orientation of the VSV genomic DNA copy and the flanking promoter and ribozyme sequences determine whether anti-genome or genome RNA equivalents are transcribed. Also required for rescue of new VSV progeny are the VSV-specific trans-acting support proteins needed to encapsidate the naked, single-stranded VSV anti-genome or genome RNA transcripts into functional nucleocapsid templates: the viral nucleocapsid (N) protein, the polymerase-associated phosphoprotein (P) and the polymerase (L) protein. These proteins comprise the active viral RNA-dependent RNA polymerase which must engage this nucleocapsid template to achieve transcription and replication.

Thus, a genetically modified and attenuated rVSV as described above is produced according to rescue methods known in the art and more specifically as described in the examples below. Any suitable VSV strain or serotype may be used, including, but not limited to, VSV Indiana, VSV New Jersey, VSV Chandipura, VSV San Juan, VSV Glasgow, and the like. As described above, in addition to polynucleotide sequences encoding attenuated forms of VSV, the polynucleotide sequence also encodes the heterologous HCV polynucleotide sequences or open reading frames (ORFs) encoding the selected HCV antigens.

The typical (although not necessarily exclusive) circumstances for rescue include an appropriate mammalian cell milieu in which T7 polymerase is present to drive transcription of the antigenomic (or genomic) single-stranded RNA from the viral genomic cDN A- containing transcription vector. Either co-transcriptionally or shortly thereafter, this viral anti-genome (or genome) RNA transcript is encapsidated into functional templates by the nucleocapsid protein and engaged by the required polymerase components produced concurrently from co- transfected expression plasmids encoding the required virus-specific trans-acting proteins. These events and processes lead to the prerequisite transcription of viral mRNAs, the replication and amplification of new genomes and, thereby, the production of novel VSV progeny, i.e., rescue.

The transcription vector and expression vector are typically plasmid vectors designed for expression in the host cell. The expression vector which comprises at least one isolated nucleic acid molecule encoding the trans-acting proteins necessary for encapsidation, transcription and replication expresses these proteins from the same expression vector or at least two different vectors.

Additional techniques for conducting rescue of viruses such as VSV are described in U.S. Pat. No. 6,673,572 and U.S. published patent application Number US2006/0153870, which are hereby incorporated by reference.

The host cells used in the rescue of VSV are those which permit the expression from the vectors of the requisite constituents necessary for the production of recombinant VSV. Such host cells can be selected from a eukaryotic cell, such as a vertebrate cell. In general, host cells are derived from a human cell, such as a human embryonic kidney cell (e.g., 293). Vero cells, as well as many other types of cells are also used as host cells as described in the above noted US patents and published application cited above. In certain embodiments, a transfection facilitating reagent is added to increase DNA uptake by cells. Many of these reagents are known in the art (e.g., calcium phosphate), LIPOFECTACE cationic lipid (Life Technologies, Gaithersburg, Md.) and EFFECTENE cationic lipid (Qiagen, Valencia, Calif).

The rescued rVSV is then tested for its desired phenotype (plaque morphology and transcription and replication attenuation), first by in vitro means. The rVSV is also tested in vivo in an animal neurovirulence model. For example, mouse and/or ferret models are established for detecting neurovirulence. Briefly, groups of ten mice are injected intra-cranially (IC) with each of a range of virus concentrations that span the anticipated LD ₅₀ dose (a dose that is lethal for 50% of animals). For example, IC inoculations with virus at 10 ², 10 , 10 ⁴ and 10 ⁵ pfu are used where the anticipated LD ₅₀ for the virus is in the range 10 -10 ⁴pfu. Virus formulations are prepared by serial dilution of purified virus stocks in PBS. Mice are then injected through the top of the cranium with the requisite dose, in 50-100 μΐ of PBS. Animals are monitored daily for weight loss, morbidity and death. The LD ₅₀ for a virus vector is then calculated from the cumulative death of mice over the range of concentrations tested. To determine immunogenicity or antigenicity by detecting binding to antibody, various immunoassays known in the art are used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, immunoprecipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and

Immunoelectrophoresis assays, neutralization assays, etc. In one embodiment, antibody binding is measured by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by measuring binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay. In one embodiment for detecting immunogenicity, T cell-mediated responses are assayed by standard methods, e.g., in vitro or in vivo cytotoxicity assays, tetramer assays, ELISpot assays or in vivo delayed-type

hypersensitivity assays.

D. DNA Plasmid Immunogenic Composition

In another embodiment an immunogenic composition includes a DNA plasmid comprising a DNA sequence encoding the selected HCV antigens to which an immune response is desired. In the pDNA, the HCV selected antigens are under the control of regulatory sequences directing expression thereof in a vertebrate or mammalian cell.

Non-viral, plasmid vectors useful in this invention contain isolated and purified DNA sequences comprising DNA sequences that encode the selected immunogenic antigens. In one embodiment, the pDNA comprises a single open reading frame encoding HCV nonstructural proteins NS3, NS4a and NS5b, wherein the HCV NS5b can be HCV NS5b lacking its polymerase and Rb binding activity. In another embodiment, the plasmid comprises a single open reading frame encoding HCV NS3 lacking the protease function and HCV NS5b with a 2A-like peptide positioned therebetween. The open reading frame is under the control of regulatory sequences directing expression of the HCV genes by the plasmid. As described in detail above, the HCV NS5b in the plasmid can be HCV NS5b lacking its polymerase and Rb binding activity. Similarly the HCV NS3 lacking the NS3/4a protease function can be selected from NS3 in the absence of the NS4a protein; NS3 having at least one mutation that destroys the protease catalytic site; or NS3 in the absence of the NS4a protein, and having at least one mutation that destroys the protease catalytic site. The components of the pDNA backbone itself are conventional. As described in the examples below, to generate the plasmids described herein, modifications were made to the pT7Blue bacterial cloning vector (Novagen). A variety of non-viral vectors are known in the art and may include, without limitation, plasmids, bacterial vectors, bacteriophage vectors, "naked" DNA and DNA condensed with cationic lipids or polymers.

The promoter and other regulatory sequences that drive expression of the selected HCV antigens in the desired mammalian or vertebrate host may similarly be selected from a wide list of known promoters known to be useful for that purpose. Examples of suitable DNA plasmid constructs for use in immunogenic compositions are described in detail in the following patent publications, International Patent Publication Nos. W098/17799 and W099/43839; and United States Patent Nos. 5,593,972; 5,817,637; 5,830,876; and 5,891,505, which are incorporated by reference herein for such disclosures, among others. A variety of such promoters are described in the documents incorporated by reference herein, as noted above and herein. In an embodiment of the immunogenic DNA plasmid composition described below, useful promoters are the human cytomegalovirus (HCMV) promoter/enhancer (described in, e.g., US Patent Nos. 5,168,062 and 5,385,839, incorporated herein by reference).

Additional regulatory sequences for inclusion in a nucleic acid sequence, molecule or vector of this invention include, without limitation, an enhancer sequence, a polyadenylation sequence, a splice donor sequence and a splice acceptor sequence, a site for transcription initiation and termination positioned at the beginning and end, respectively, of the polypeptide to be translated, a ribosome binding site for translation in the transcribed region, an epitope tag, a nuclear localization sequence, an IRES element, a Goldberg-Hogness "TATA" element, a restriction enzyme cleavage site, a selectable marker and the like. Enhancer sequences include, e.g., the 72 bp tandem repeat of SV40 DNA or the retroviral long terminal repeats or LTRs, etc. and are employed to increase transcriptional efficiency. These other components useful in DNA plasmids, including, e.g., origins of replication, polyadenylation sequences (e.g., BGH poly A, SV40 poly A), drug resistance markers (e.g., kanamycin resistance), and the like may also be selected from among widely known sequences, including those described in the examples, in FIG. 1 , and mentioned specifically herein.

Selection of promoters and other common vector elements are conventional and many such sequences are available with which to design the plasmids useful in this invention. All components of the plasmids useful in this invention may be readily selected by one of skill in the art from among known materials in the art and available from the pharmaceutical industry. Selection of plasmid components and regulatory sequences are not considered a limitation on this invention. The antigen sequence and other components of the DNA plasmid may be optimized, such as by codon selection appropriate to the intended host and by removal of any inhibitory sequences, also discussed below with regard to antigen preparation.

This immunogenic composition may include therefore one plasmid encoding the selected HCV antigens for expression in the host. To reduce the size of the DNA insert, each HCV antigen is under the control of the same regulatory elements. In still another embodiment, the DNA plasmid composition may contain multiple plasmids, wherein each DNA plasmid encodes the same or a different antigen. In still a further embodiment, the DNA plasmid immunogenic composition may further contain, as an individual DNA plasmid component or as part of the HCV antigen-containing DNA plasmid, a nucleotide sequence that encodes a desirable cytokine, lymphokine or other genetic adjuvant.

In another embodiment, the DNA priming composition further consists of a second plasmid encoding a selected cytokine. For example, as one embodiment, a DNA priming composition contains a plasmid encoding a codon-optimized HCV polyprotein NS3-NS4a- NS5b(pol-) gene (see pPBS-HCV-081 of FIG. 1); and a second plasmid encoding two human IL-12 subunits p35 and p40, under individual control of two promoters.

E. Other Components for use in the Immunogenic Compositions

The immunogenic compositions useful in this invention, whether the DNA plasmid or rVSV compositions, further comprise an immunologically or pharmaceutically acceptable diluent, excipient or carrier, such as sterile water or sterile isotonic saline. The antigenic compositions may also be mixed with such diluents or carriers in a conventional manner. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other vertebrate hosts. The appropriate carrier is evident to those skilled in the art and will depend in large part upon the route of administration.

Still additional components that may be present in the immunogenic compositions of this invention are adjuvants, preservatives, surface active agents, and chemical stabilizers, suspending or dispersing agents. Typically, stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target human or animal.

In still a further embodiment, the immunogenic compositions may further contain or be administered with, a cytokine, lymphokine or genetic adjuvant. A host of such suitable adjuvants for which nucleic acid sequences are available are identified below.

/. Adjuvants An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen. A number of cytokines or lymphokines have been shown to have immune modulating activity, and thus may be used as adjuvants, including, but not limited to, the interleukins 1-a, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Patent No.

5,723,127), 13, 14, 15, 16, 17, 18 (and its mutant forms) and 33, the interferons-a, and y, granulocyte-macrophage colony stimulating factor (GM-CSF, see, e.g., U.S. Patent No.

5,078,996 and ATCC Accession Number 39900), macrophage colony stimulating factor, granulocyte colony stimulating factor (G-CSF), and the tumor necrosis factors a and β. Still other adjuvants useful in this invention include a chemokine, including without limitation, MCP-1, ΜΙΡ-Ια, ΜΙΡ-Ιβ, and RANTES. Adhesion molecules, such as a selectin, e.g., L- selectin, P-selectin and E-selectin may also be useful as adjuvants. Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM-1, a member of the integrin family such as LFA-1, VLA-1, Mac-1 and pl50.95, a member of the immunoglobulin superfamily such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3, co-stimulatory molecules such as CD40 and CD40L, growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1, and vascular endothelial growth factor, receptor molecules including Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still another adjuvant molecule includes Caspase (ICE). See, also International Patent Publication Nos. W098/17799 and

W099/43839, incorporated herein by reference.

Suitable adjuvants used to enhance an immune response include, without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094, which is hereby incorporated by reference. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from GlaxoSmithKline

(Hamilton, MT), and which are described in United States Patent No. 6,113,918, which is hereby incorporated by reference. One such AGP is 2-[(R)-3- Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-0-phosphono-3-0-[(R)-3- tetradecanoyoxytetradecanoy 1] -2 - [(R)-3 -tetradecanoyloxytetradecanoyl-amino] -β-D- glucopyranoside, which is also known as 529 (formerly known as RC529). This 529 adjuvant is formulated as an aqueous form or as a stable emulsion.

Still other adjuvants include mineral oil and water emulsions, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic polyols, muramyl dipeptide, killed Bordetella, saponins, such as Stimulon™ QS-21 (Agenus, Framingham, MA.), described in U.S. Patent No. 5,057,540, which is hereby incorporated by reference, and particles generated therefrom such as ISCOMS (immunostimulating complexes), Mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Patent No. 6,207,646, which is hereby incorporated by reference), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302 and WO 92/19265, incorporated herein by reference.

Also useful as adjuvants are cholera toxins and mutants thereof, including those described in published International Patent Application number WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid), preferably a histidine). Similar CT toxins or mutants are described in International Patent Publication No. WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid). Other CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36).

A cytokine may be administered as a protein or in a plasmid in which a nucleic acid sequence encoding the cytokine is under the control of a regulatory sequence directing expression thereof in mammalian cells. In the embodiments exemplified in this invention, a desirable cytokine for administration with the DNA plasmid composition of this invention is Interleukin-12 or one or both of its subunits. In one embodiment exemplified below, the desired adjuvant is IL-12, which is expressed from a plasmid. See, e.g., US Patent Nos.

5,457,038; 5,648,467; 5,723,127 and 6,168,923, incorporated by reference herein. In one embodiment, the cytokine nucleic acid composition comprises a nucleic acid sequence that encodes the IL-12 p35 subunit operably linked to a first regulatory sequence directing expression thereof in mammalian cells; and a second nucleic acid sequence that encodes the IL- 12 p40 subunit operably linked to a second regulatory sequence directing expression thereof in mammalian cells. In another embodiment, these nucleotide sequences encoding each IL-12 subunit are present on the same plasmid. In another embodiment, the nucleotide sequence encoding an IL-12 subunit is present on a plasmid different from the plasmid encoding the HCV proteins. In one embodiment, the IL-12 expressing plasmid(s) is incorporated into the immunogenic priming composition of the examples. However, it should be noted that this plasmid could be administered to the mammalian host with the rVSV composition or alone, between the priming and boosting compositions. In still another useful embodiment, the cytokine-expressing plasmid is administered with the DNA composition. In yet another step, the cytokine is administered with the boosting step. In still another embodiment, the cytokine is administered with both priming and boosting compositions.

2. Facilitating Agents or Co-Agents

In addition to a carrier as described above, pDNA immunogenic compositions desirably contain optional polynucleotide facilitating agents or "co-agents", such as a local anesthetic, a peptide, a lipid including cationic lipids, a liposome or lipidic particle, a polycation such as polylysine, a branched, three-dimensional polycation such as a dendrimer, a carbohydrate, a cationic amphiphile, a detergent, a benzylammonium surfactant, or another compound that facilitates polynucleotide transfer to cells. Such a facilitating agent includes the local anesthetic bupivacaine or tetracaine (see U.S. Patent Nos. 5,593,972; 5,817,637;

5,380,876 and 5,981,505 and International Patent Publication No. W098/17799, which are hereby incorporated by reference). Other non-exclusive examples of such facilitating agents or co-agents useful in this invention are described in U. S. Patent Nos. 5,703,055; 5,739,118; 5,837,533; International Patent Publication No. WO96/10038, published April 4, 1996; and International Patent Publication No W094/16737, published August 8, 1994, which are each incorporated herein by reference.

In still other embodiments, additional additives included in the immunogenic compositions are preservatives, stabilizing ingredients, surface active agents, and the like. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.

Suitable stabilizing ingredients that may be used include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk. Suitable surface active substances include, without limitation, Freund's incomplete adjuvant, quinone analogs, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide), methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl peptide and dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum phosphate, etc. and immune stimulating complexes (ISCOMS). The plasmids and rVSVs may also be incorporated into liposomes for use as an immunogenic composition. The immunogenic compositions may also contain other additives suitable for the selected mode of administration of the composition. The compositions of the invention may also involve lyophilized polynucleotides, which can be used with other pharmaceutically acceptable excipients for developing powder, liquid or suspension dosage forms. See, e.g., Remington: The Science and Practice of Pharmacy, Vol. 2, 19^th edition (1995), e.g., Chapter 95 Aerosols; and International Patent Publication No. W099/45966, the teachings of which are hereby incorporated by reference.

These immunogenic compositions can contain additives suitable for administration via any conventional route of administration. In some embodiments, the immunogenic composition of the invention is prepared for administration to human subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Thus, the immunogenic compositions may also include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Other useful parenterally- administrable formulations include those which comprise the active ingredient in

microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

The immunogenic compositions described herein are not limited by the selection of the conventional, physiologically acceptable carriers, adjuvants, or other ingredients useful in pharmaceutical preparations of the types described above. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art.

F. Treating Subjects for HCV Infection

A method for treating or preventing Hepatitis C Virus infection comprises administering to a mammalian subject in need thereof an immunogenic composition as described herein. In one embodiment, the administration is of an rVSV alone. In another embodiment, the plasmid DNA and rVSV immunogenic compositions are combined in a prime/boost regimen.

/. rVSV Administration In one embodiment of this invention, a method for treating or preventing Hepatitis C virus infection comprises administering to a mammalian subject in need thereof an immunogenic composition comprising an rVSV comprising the nucleic acid sequence 3'-Pi- M₂-(G-CT)₃-N₄-X5-L₆ -5' or 3 '-Pi-M₂-(G-CT)₃-N4-L₅-X6-5' . In these rVSVs, the subscript numbers indicate the genomic position of each VSV gene, and the VSV genes P, M, G-CT, N and L are as described above. X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b (which may lack its polymerase and Rb binding activity), and a 2A-like peptide positioned therebetween.

In another embodiment of this invention, a method for treating or preventing Hepatitis

C virus infection comprises administering to a mammalian subject in need thereof an immunogenic composition comprising an rVSV comprising the nucleic acid sequence 3'-Pi- M₂-(G-CT)3-N₄-(X/L)5-5' . In these rVSV vectors, the subscript numbers indicate the genomic position of each VSV gene, and the VSV genes P, M, G-CT, N and L are as described above. X/L is a single open reading frame, and X is a nucleic acid sequence comprising (i) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b (which may lack its polymerase and Rb binding activity); or (ii) sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b (which may lack its polymerase and Rb binding activity), and a 2A-like peptide positioned therebetween.

As described above, any of the embodiments of the rVSVs may be used in these methods of treatment. Desirably, this composition in admixed with a pharmaceutically acceptable diluent or other components as described above. In one embodiment, the treatment or prevention of HCV involves administration of one or more effective amounts of one or a combination of the rVSVs described herein.

2. Prime/Boost Administration

In the second embodiment of the methods described herein, the administration of the rVSV as described is preceded by administering to said mammalian subject an effective amount of a priming composition comprising a plasmid (pDNA) comprising a single open reading frame encoding the HCV nonstructural proteins NS3, NS4a and NS5b, or HCV NS3 lacking the NS3/4a protease function and HCV NS5b with a 2A-like peptide positioned therebetween. The HCV NS5b can be HCV NS5b lacking its polymerase and Rb binding activity and the NS3 can lack a functional protease catalytic site. The pDNA further contains the open reading frame under the control of regulatory sequences directing expression thereof by the pDNA. Desirably, this pDNA composition in admixed with a pharmaceutically acceptable diluent or other components as described above. In one embodiment, the pDNA composition includes an additional plasmid encoding a selected cytokine, such as IL-12.

According to the present invention, the rVSV immunogenic composition may be administered as a boosting composition subsequent to the administration of the priming pDNA immunogenic composition that presents the selected HCV antigen subset to the host. The mammalian subject is administered an effective amount of a priming composition comprising a plasmid comprising a single open reading frame encoding HCV nonstructural proteins NS3,

NS4a and NS5b, or NS3(prot-) and NS5b (pol-) under the control of regulatory sequences directing expression thereof by the plasmid and a pharmaceutically acceptable diluent prior to the immunogenic rVSV composition. When used as a priming composition, this DNA plasmid composition is administered once or more than once prior to the boosting rVSV composition.

In another embodiment of the prime/boost method, the priming composition is administered at least once following the immunogenic rVSV composition, or administered both prior to and after the rVSV immunogenic composition.

As discussed above, the rVSV composition can express the same antigens, NS3, NS4a and NS5b as expressed by the priming pDNA composition. In another embodiment, the rVSV is any one of the above described rVSV, such as that expressing only NS3(prot-) and

NS5b(pol-).

In still further embodiments of the prime/boost regimen, multiple rVSV compositions are administered as later boosters. In one embodiment at least two rVSV compositions are administered following the priming compositions.

Each subsequent rVSV composition may have a different serotype selected from among known naturally occurring serotypes and from among any synthetic serotypes provided by manipulation of the VSV G protein. For example one rVSV may be the Indiana serotype and the other may be the Chandipura serotype or the New Jersey serotype. In another embodiment, additional rVSV boosters are of the same serotype. When used as a boosting composition, the rVSV compositions are administered serially, after the priming pDNA immunogenic compositions. rVSVs displaying a desired balance of attenuation and immunogenicity are useful in this invention.

In one embodiment, a method and immunogenic composition of this invention employs one of these rVSV constructs: rVSV_in-N4(G CTl)3-[HCVla NS3(prot-)-T2A-NS5b(pol-)]5, rVSV_in-N4(G CTl)3-[HCVla NS3(prot-)-T2A-NS5b(pol-)-T2A-VSV_in LJ5, or rVSV_in-N4(G CT1)3-[HCV la NS3-NS4a-NS5b(pol-)-T2A-VSV_in LJ5. Alternatively, the HCV genes may be inserted downstream of L in the sixth position of the VSV genome. In still another embodiment, administration of one or more of the plasmid DNA immunogenic compositions is followed by one or more administrations of the rVSV immunogenic compositions, and then followed by one or more additional administrations of the plasmid DNA immunogenic compositions.

3. Administration

The antigenic or immunogenic compositions of this invention are administered to a human or to a non-human vertebrate by a variety of routes including, but not limited to, intramuscular, intraperitoneal, subcutaneous, intravenous and intraarterial, intranasal, oral, vaginal, rectal, parenteral, intradermal, transdermal (see, e.g., International patent publication No. WO 98/20734, which is hereby incorporated by reference). The appropriate route is selected depending on the nature of the immunogenic composition used, and an evaluation of the age, weight, sex and general health of the patient and the antigens present in the immunogenic composition, and similar factors by an attending physician. Although the composition may be administered by any selected route of administration, in one embodiment a desirable method of administration is coadministration intramuscularly of a composition comprising the plasmids with bupivacaine as the facilitating agent.

In the examples provided below, both the immunogenic pDNA compositions and rVSV compositions are administered intramuscularly (i.m.) for either rVSV alone or prime/ boost regimens. In other embodiments, it is desirable to administer the pDNA compositions and rVSV compositions by different routes. However, the selection of dosages and routes of administration are not limitations upon this invention.

For example, the rVSV composition may be administered by conventional means, including intramuscular and intranasal administration.

The pDNA composition may be administered by conventional means, including intramuscular, subcutaneous and intradermal administration. The pDNA composition may be delivered by in vivo electroporation. Electroporation (EP) is a technique for intracellular delivery based on the propagation of electrical fields within a target region of tissue. The application of electrical signals of sufficient magnitude and duration induces a transient increase in membrane permeability in cells exposed to threshold level electrical fields, allowing enhanced intracellular uptake of agents distributed within the interstitium of the local tissue.

Numerous pre-clinical studies have demonstrated that the application of EP can enhance the delivery of DNA vectors, resulting in increased gene expression and downstream biological response.

One such EP administration system is the TRIGRID™ Delivery System, developed by Ichor Medical Systems, Inc., San Diego, CA. Examples of EP devices are described in U.S. Patent Nos. 5,873,849, 6,041,252, 6,278,895, 6,319,901, 6,912,417 and 8,187,249, which are hereby incorporated by reference.

Similarly, the order of immunogenic composition administration and the time periods between individual administrations may be selected by the attending physician or one of skill in the art based upon the physical characteristics and precise responses of the host to the application of the method. Such optimization is expected to be well within the skill of the art.

In general, selection of the appropriate "effective amount" or dosage for the components of the immunogenic composition(s) of the present invention will also be based upon whether the administration is rVSV only or prime/boost with a DNA composition, as well as the physical condition of the subject, most especially including the general health, age and weight of the immunized subject. The method and routes of administration and the presence of additional components in the immunogenic compositions may also affect the dosages and amounts of the plasmid and rVSV compositions. Such selection and upward or downward adjustment of the effective dose is within the skill of the art. The amount of plasmid and rVSV required to induce an immune response, such as a protective response, or produce a therapeutic effect in the patient without significant adverse side effects varies depending upon these factors.

A suitable dose is formulated in a pharmaceutical composition, as described above (e.g., dissolved in about 0.1 ml to about 2 ml of a physiologically compatible carrier) and delivered by any suitable means. Dosages are typically expressed in a "unit dosage", which is defined as dose per subject, e.g., a unit dosage of 1 mg immunogen. Alternatively dosages can be expressed as amount per body weight of the subject or patient, using the norm for therapeutic conversions as 80 kg body weight. For example, a 1 mg unit dose per subject is equivalent to about 12.5 μg/kg body weight.

In one embodiment, the dosage for an initial therapeutic administration or for a first priming therapeutic or prophylactic immunogenic composition is a "unit dosage" of less than about 0.01 mg to 100 mg of DNA plasmid immunogenic composition. In one embodiment, the single or boosting dosages for rVSV are the same. Such dosages are generally between lxlO⁷ pfu (or measured as viral particles) and lxl0⁹pfu/viral particles/ml. However, any suitable dose is readily determined by persons skilled in the art.

G. Kit Components

In still another embodiment, the present invention provides a pharmaceutical kit for ready administration of an immunogenic, prophylactic, or therapeutic regimen for treatment of HCV infection. This kit is designed for use in a method of inducing a high level of antigen- specific immune response in a mammalian or vertebrate subject. The kit contains at least one immunogenic composition comprising a DNA plasmid composition as described herein. For example, multiple prepackaged dosages of the DNA immunogenic composition are provided in the kit for multiple administrations. The kit also contains at least one immunogenic composition comprising a replication-competent, attenuated rVSV immunogenic composition as described herein. In one embodiment, multiple prepackaged dosages of the rVSV immunogenic composition are provided in the kit for multiple administrations.

Where the above-described immunogenic compositions do not also contain DNA plasmids that express a cytokine, such as IL-12, the kit also optionally contains a separate cytokine composition or multiple prepackaged dosages of the cytokine composition for multiple administrations. These cytokine compositions are generally nucleic acid compositions comprising a nucleic acid sequence encoding the selected cytokine under the control of regulatory sequences directing expression thereof in a mammalian or vertebrate cell.

The kit also contains instructions for using the immunogenic compositions in a prime/boost method as described herein. The kits may also include instructions for performing certain assays, various carriers, excipients, diluents, adjuvants and the like above-described, as well as apparatus for administration of the compositions, such as syringes, electroporation devices, spray devices, etc. Other components may include disposable gloves, decontamination instructions, applicator sticks or containers, among other compositions.

H. Illustrative Embodiments

In order that this invention may be better understood, the following examples are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention. Negative results, as well as the identified vector components necessary for a useful rVSV vector for successful HCV prevention or treatment, are discussed in detail in the examples below.

The examples discussed below further support the unexpected and unpredictable nature of the rVSV and plasmids forming the immunogenic compositions and methods of this invention. Despite all that is currently known of the use of rVSV as a vaccine or immunogenic vector and all that is known of HCV, the selection of the correct components and their orientation and presentation in the virus vector and plasmid were not predictable. The examples below show, among other things, that certain rVSV constructs did not work as anticipated. For example, because it was desirable that the N gene be in position 4 for attenuation purposes, an rVSV N4CTl incorporating the HCV polygene NS3-4a-5b(pol-) upstream of the N-gene was constructed. However, attempts to rescue this virus were unsuccessful (see Example 2). To overcome this problem, while maintaining an attenuated rVSV N4CT1 vector backbone, two additional approaches were pursued, but also proved unsuccessful:

In the first alternative design, the N gene was moved upstream by one position, to nominal position 3. In turn, just upstream of the translocated N gene, an empty transcriptional unit (ETU) was inserted. The ETU is in accordance with the teaching of Whelan et al., 2000 J. Virology, Vol. 74, pages 8268-8276, in that the ETU in position 3 of rVSV has 75 nucleotides, which exceeds the minimum of 70 nucleotides taught as necessary for termination of a preceding gene in order to express the downstream gene (see Whelan at al. at page 8274). The insertion of the ETU had the effect of returning the N gene to position 4. The HCV NS3-NS4a- NS5b(pol-) polygene was inserted upstream of the L gene in position 6 of the rVSV gene order. The gene order was thus as follows: 3'-P-M-ETU-N-G-HCV polygene-L-5'.

The resulting construct, designated rVSV_in-N4CTl-ETU3-[HCV la NS3-NS4a- NS5b(pol-)]6, was able to be rescued and amplified (see Example 4). However, this rVSV showed genetic instabilities in the rVSV vector backbone during extensive passage in cell culture.

In the second alternative design, the N gene and G CT1 gene exchanged positions within the attenuated rVSV N3CT1, moving N to nominal position 4 and G CT1 to nominal position 3. The HCV NS3-NS4a-NS5b(pol-) polygene was inserted into an engineered ETU upstream of the L gene in position 5 of the rVSV gene order. The gene order was thus as follows: 3 ' -P-M-G-N-HCV polygene-L-5 ' . The resulting construct, designated rVSVin-N4(G CT1)3-[HCV la NS3-NS4a-NS5b(pol-)J5, was able to be rescued and amplified (see Example 4). However, this rVSV showed genetic instabilities for HCV antigen expression during extensive passage in cell culture.

Further alternative designs which proved successful retained the (G CT1)3-N4 gene order, but required the use of still other elements.

Also as shown in the examples below, prime/boost immunization regimens were evaluated in both mice and non-human primates using a heterologous pDNA prime/rVSV boost and compositions described herein. In the primate example (Example 6), three groups of macaques administered a DNA prime/rVSV boost protocol exhibited a clear IL-12 pDNA adjuvant effect and increased IFN γ ELISpot responses after an rVSV boost.

EXAMPLE 1 : METHODS AND MATERIALS

A. rVSV Rescue Protocol

The following protocol is exemplary of that used in each attempt to rescue recombinant VSV in Examples 2 and 4 below. For each transfection reaction (confluent monolayer of Vero cells in a T150 cell culture flask), a total volume of 250μ1 aqueous solution (distilled water for irrigation) was mixed with the amounts of pDNA as listed in Table 4.

* pCI-T7-Neo is pCI-Neo (Promega, Madison, WI), modified to express T7 RNA polymerase.

** All viral proteins were expressed from optimized nucleotide sequences and transcription was under control of a CMV promoter.

*** Amounts given in microgram, using abbreviation μg.

Thereafter, 50μ1 3M sodium acetate and 750ul 100% EtOH were added and the mixture was stored overnight at -20°C. The mixture was then centrifuged at 14000 rpm, 4°C, 30 minutes and the supernatant removed. A quick spin was performed to collect remaining supernatant and remove it. Pellets of pDNA were air-dried in a cell culture hood and resuspended in 50μ1 H₂0.

The following three media were prepared containing the components listed in Table 5 below:

The T150 flask with Vero cells was washed with PBS^{" "} (lacking calcium and magnesium) and trypsinized with 5ml of trypsin-EDTA solution, and then incubated at 37°C for up to 5 minutes. After knocking the sides of the flask to dislodge cells, the cells were transferred to a single 50 ml conical tube and the volume brought up to 25ml with Medium 1 The cells were collected by centrifugation at 1000 rpm for 5 minutes and the supernatant discarded. Cells were then washed with 10 ml of Medium 2, followed by centrifuging at 1000 rpm/5 minutes with the supernatant discarded. Cell pellets were resuspended in 0.7 ml of Medium 2 and the cell suspension transferred to a tube containing 50 μΐ DNA solution.

Incubation occurred for 6 minutes at room temperature and the cell/DNA suspension was transferred into an electro cuvette. The cuvette was placed in a BTX820 Electroporator and electroporated with 4 pulses at 140V each, followed by incubation at room temperature for 6 minutes.

Thereafter, 1 ml Medium 3 was added and the electroporated cells were transferred using a sterile pipet to a 15ml conical tube containing 10 ml of Medium 3. Cells were pelleted for 5 minutes at 1000 rpm and the supernatant was discarded. Cells were resuspended in 5 ml of Medium 3 and transferred to a T-150 cell culture flask containing 25 ml of Medium 3. The cells were incubated at 37°C and 5% CO₂ for 3 hours, followed by heat shocking at 43°C and 5% CO₂ for 3 hours. Long-term incubation occurred at 32°C and 5% CO₂. After overnight incubation supernatant was replaced with 25 ml of fresh Medium 3.

Positive rVSV rescues showed a cytopathic effect (CPE) after 5-10 days, when virus- containing supernatant was collected and flash-frozen at -78 °C. A single virus clone was isolated by plaque picking, and amplified on Vero monolayers to generate a virus working stock.

B. Protein Expression Experiments (Western Blotting)

Monolayers of Vero cells in a well of a 6-well plate, which had just reached confluence, were infected with the virus stock with a high multiplicity of infection (MOI). After 16 hours, all cells were collected and pelleted. Cell lysis was performed by resuspension in 200 μΐ lysis buffer (0.05M Tris/HCl pH 8.0, 0.05M NaCl, 1% Triton X100) and 10 minutes incubation on ice. The lysate was cleared by centrifugation (20000xg, 5 minutes) and mixed with 200 μΐ 2x Laemmli buffer. Proteins in 10 μΐ lysate were electrophoretically separated by SDS-PAGE and transferred to a nitrocellulose membrane using the iBlot® Dry Blotting System from Invitrogen. Immunodetection was performed using anti-HCV NS5b (Virostat, #1825) and anti-HCV NS3 (Virostat, #1859) monoclonal antibodies. The signal was developed using an anti-mouse-AP conjugate (Promega, #S372B) and Western Blue Substrate (Promega, #S3841).

C. IFN-YELISPOT Assay

The enzyme-linked immunospot assay (ELISpot) takes advantage of the relatively high concentration of a given protein (such as a cytokine) in the environment immediately surrounding the protein-secreting cell. These cell products are captured and detected using high-affinity antibodies. The ELISpot assay utilizes two high-affinity cytokine-specific antibodies directed against different epitopes on the same cytokine molecule: either two monoclonal antibodies or a combination of one monoclonal antibody and one polyvalent antiserum. ELISpot generates spots based on a colorimetric reaction that detects the cytokine secreted by a single cell. The spot represents a "footprint" of the original cytokine -producing cell. Spots (i.e., spot forming cells or SFC) are permanent and can be quantitated visually, microscopically or electronically.

The ELISpot assay was performed as follows: At various times after immunization, mice were sacrificed and spleen cells were harvested. For the determination of immunization- elicited IFN-γ ELISpot responses in individual mice, a mouse IFN-γ ELISpot kit (Catalog No. 551881, BD Biosciences, San Diego CA) was used. Ninety-six-well flat-bottom ELISpot plates (ImmunoSpot, Cellular Technology Limited, Cleveland OH) were coated overnight with a purified anti-mouse γ-interferon (mIFN-γ) monoclonal antibody (Catalog No. 51-2525KC, BD-Biosciences, San Diego CA) at a concentration of 5 μί*/πι1, after which the plates were washed three times with sterile lx phosphate buffered saline (lx PBS) and then blocked for two hours with RIO complete culture medium (RPMI-1640 containing 10% FCS and 2 mM L- glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin sulfate, 1 mM sodium pyruvate, 1 mM HEPES, 0.1 mM non-essential amino acids). Mouse spleens were homogenized by grinding the spleens between the frosted ends of two sterile microscope slides.

The resulting homogenate was lysed with 1 ml ACK lysing buffer (Invitrogen, Chicago, IL) on ice for one minute, then stopped with 10 ml cold R10 culture medium. The cells were then suspended in 5 ml of complete R05 culture medium (RPMI 1640 medium supplemented with 5%> FCS, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin sulfate, 1 mM sodium pyruvate, 1 mM HEPES, 0.1 mM non-essential amino acids) and splenocytes were subsequently isolated by Ficoll-Hypaque density gradient centrifugation (1500 rpm for 30 minutes) and resuspended in complete R10 culture medium containing either 1 μg/ml Con-A (Sigma), peptide pools (15mers overlapping by 11 amino acids; 1 μΜ each final peptide concentration) spanning HCV NS3, NS4a, NS5b, or medium alone. Input cell numbers were 4 x 10⁵ splenocytes per well (4 x 10⁶ splenocytes/ml) and cells were assayed in duplicate wells.

Splenocytes were incubated for 18-20 hours at 37°C and 5%> C0₂, and then removed from the ELISpot plate by first washing three times with deionized water followed by incubation on ice for 10 minutes. Then plates were washed six times with lx PBS containing 0.1% Tween-20. Thereafter, plates were treated with an anti -mouse IFN-γ biotinylated detection antibody (2.0 μg/ml, Material No. 51-1818KZ, BD-Biosciences, San Diego CA) diluted with PBS/10%> FCS and incubated for 18 hours at 4°C. ELISpot plates were then washed six times with lx PBS containing 0.1% Tween-20 and treated with 100 μΐ per well of streptavidin-horseradish peroxidase conjugate (Catalog No. 557630, BD-Biosciences, San Diego CA) diluted 1 : 100 with PBS/10% FCS and incubated for an additional one hour at room temperature. Unbound conjugate was removed by rinsing the plate six times with lx PBS containing 0.1% Tween-20 and 3 times with lx PBS. AEC Chromogen was diluted to 20 μΐ/ml in AEC substrate solution (Catalog No. 551951, BD-Biosciences, San Diego CA) and was then added (100 μΐ/well) for 3-5 minutes before being rinsed away with water, after which the plates were air-dried, and the resulting spots counted using an Immunospot Reader (CTL Inc., Cleveland, OH).

Peptide-specific IFN-γ ELISpot responses were considered positive if the response (minus media background) was > 3 fold above the media response and > 50 SFC/10⁶ splenocytes.

EXAMPLE 2 - ATTEMPTS TO RESCUE rVSV EXPRESSING HCV ANTIGENS

Using the protocol set forth in Example 1, the following rVSVs in Table 6 were attempted to be rescued:

* For some rescue experiments, small foci of CPE were observed, however, virus could not be amplified for further analysis.

** In contrast to rVSV-HCV-001, the HCV antigen is encoded by RNA-optimized

sequence.

With the exception of pPBS-VSV-HCV-012, all HCV antigens were encoded by a codon optimized nucleotide sequence. None of these constructs included an ETU in position 3. Therefore, no rVSVs could be rescued which expressed a large HCV polyprotein containing both NS3 and NS5b(pol-) from the third transcriptional unit in a live attenuated N4CT1 rVSV vector. Rescue was achieved in the fifth position of an N3CT1 or N3CT9 rVSV, but as discussed above, an N3 construct is not recommended.

EXAMPLE 3 - GENERATION OF HCV pDNA CANDIDATES

This example describes illustrative plasmids useful in one embodiment of this invention. These plasmids are not a limitation on the present invention, but have been optimized for use in the subsequent experiments. The following DNA immunogenic compositions were designed utilizing standard recombinant DNA techniques. The DNA backbone vector expressing HCV genes utilizes the human cytomegalovirus promoter, the bovine growth hormone (BGH) polyadenylation termination sequence, and a kanamycin resistance gene for selection.

A. Primers

The primers used in this example are identified in Table 7 below and referenced in the description by the Primer ID Ref.

M 5 ' -GCGCGCGTCGACGCCGCC ACC ATGGCCCCC ATC ACCG-3 ' 23

N 5 ' -GTGACACAGGTGTTGCAGTC-3 ' 24

O 5 ' -GCGAGATCCCCTTCTACG-3 ' 25

P 5 ' -AGGACGCACTAGTCGTCTCCGTAGGCGGCGGTG ACG AC 26

CTCCAGGTCGG-3 '

Q 5 ' -GCGAGATCCCCTTCTACG-3 ' 27

R 5 ' -AGGACGCACTAGTCGTCTCCGT AGGCGGCGGTG ACG AC 28

CTCCAGGTCGG-3'

S 5 ' -CT ACGAGGGC AGAGGAAGTCTGCTAACATGCGGTGAC 29

GTCGAGGAGAATCCTGGCCCA-3 '

T 5 ' -TGCTTGGGCC AGGATTCTCCTCGACGTCACCGC ATGTTAG 30

CAGACTTCCTCTGCCCTC-3 '

u 5 ' -TGAAAAGGCGCGCCCGTCTCCGAAGTCC ACGATTTTG-3 ' 31

V 5'- GGAACCGCGGCCGCGCATATGTTCGTTCCATGAG -3' 32 w 5 ' -GTGTGCACGCGTCTCAGCGGTTGGGGAGGAGG-3 ' 33

X 5'- GCGAGATCCCCTTCTACG -3' 34

Y 5 ' -CCGCGCCGCCT ACG AGGGC AGAGGAAGTCTGCTAAC 35

ATGCGGTGACGTC GAGGAGAATCCTGGCCCA -3'

z 5 ' -CTTCTGGGCC AGG ATTCTCCTCGACGTCACCGC ATGT 36

TAGCAGACTTCCT CTGCCCTCGTAGGCGGC-3 '

AA 5 ' -GACCCGCTACTCCGCCCCTCCCGGAG ACCCTCCCC AGCCC 37

GAATACGACC-3 '

AB 5 ' -CGT ATTCGGGCTGGGGAGGGTCTCCGGGAGGGGCGG AGT 38

AGCGGGTCATG-3 '

AC 5 ' -CCCGCGCCC AGGCCCCTCCTCCC AGCTGGGACC AGATG 39

TGG-3'

AD 5 ' -CATCTGGTCCC AGCTGGGAGG AGGGGCCTGGGCGCG 40

GGCGC-3'

B. HCV plasmid DNAs

DNA plasmid wHCV21 is a single promoter pDNA expressing HCV subtype la NS3- 4a-4b-5a-5b(pol-). The HCV polyprotein was derived from the HCV la isolate H77 (Hong et al. 1999 Virology 256, 36-44) and the corresponding nucleotide sequence was codon-optimized and de novo assembled. For an additional margin of safety, the native polymerase and Rb binding activity in NS5b was inactivated by two coding changes (the GDD motif in the RNA polymerase catalytic domain was changed to GAA). DNA plasmid wHCV12 is an almost identical single promoter pDNA expressing HCV subtype la NS3(prot-)-4a-4b-5a-5b(pol-), in which the native NS3/4a protease function is ablated due to a H57T amino acid change in NS3. Starting from wHCV21, a PCR fragment containing NS3-4a was generated using primers A and B. This fragment was inserted into a modified pT7Blue cloning vector (Novagen) via Sall/Spel-restriction sites (to generate pPBS-HCV-013).

A second PCR fragment containing NS5b was generated using primers C and D. This fragment was inserted into pPBS-HCV-013 immediately downstream of NS3-4a via Kpnl/Spel- restriction sites (to generate pPBS-HCV-014). Restriction digest of pPBS-HCV-0 '14 with BsmBI and ligation of the two larger fragments resulted in pPBS-HCV-015, containing a complete HCVla NS3-4a-5b(pol-) ORF.

The HCVla NS3-4a-5b(pol-) ORF in pPBS-HCV-015 was then isolated using Sall/MluI-restriction sites and inserted into the protein expression cassette of wHCV21. The resulting pDNA pPBS-HCV-016 is a single promoter pDNA expressing HCVla NS3-4a- 5b(pol-). In addition to the codon-optimized nucleotide sequence, high levels of HCV polyprotein expression were obtained by optimizing translation initiation (by using a Kozak signal) and termination efficiency (by the use of a TGA as the stop codon, followed by an additional A nucleotide).

In addition to pPBS-HCV-016,pPBS-HCV-081 (FIG. 1) was generated, which carries silent point mutations in the HCV NS3-NS4a-NS5b(pol-) ORF compared to pPBS-HCV-016. These non-coding (silent) mutations in pDNA constructs pPBS-HCV-016 and pPBS-HCV-081 are listed in Table 8 below.

TABLE 8 - Non-coding (silent) mutations in pDNA constructs pPBS-HCV-016

and pPBS-HCV-081

pDNA construct amino acid position in codon/nucleotide position in pDNA

NS3-4a-5b(pol-) protein construct

pPBS-HCV-016 GCC (nt 1701 -1703)

Ala at position 284

pPBS-HCV-081 GCT (nt 1701-1703)

pPBS-HCV-016 CCC (nt 2574-2576)

Pro at position 575

pPBS-HCV-081 CCT (nt 2574-2576)

pPBS-HCV-016 CCC (nt 2577-2579)

Pro at position 576

pPBS-HCV-081 CCT (nt 2577-2579)

pPBS-HCV-016 CCC (nt 3954-3956)

Pro at position 1055

pPBS-HCV-081 CCT (nt 3954-3956)

pPBS-HCV-016 GGG (nt 3960-3962)

Gly at position 1057

pPBS-HCV-081 GGA (nt 3960-3962)

pPBS-HCV-016 CCC (nt 3966-3968)

Pro at position 1059

pPBS-HCV-081 CCT (nt 3966-3968)

pPBS-HCV-016 CGC (nt 3966-3968)

Arg at position 1203

pPBS-HCV-081 CGG (nt 3966-3968)

pPBS-HCV-016 TAATGA (nt 4683-4688)

Stop codon(s)

pPBS-HCV-081 TGAAGA (nt 4683-4688)

pPBS-HCV-016 ACGCGT (nt 4695-4700)

pPB S-HCV-081 n/a Deletion of Mlul restriction site (nt

4695-4700)

Construction of pPBS-HCV-081 started from pPBS-HCV-015. First, the internal Notl site at the 3 '-end of HCV NS5b was removed for cloning purposes: A PCR fragment was generated using primers E and F and pPBS-HCV-015 as a template and was inserted into pPBS- HCV-015 via KpnI/BsmBI and Kpnl/Notl-restriction sites, respectively, to generate pPBS- HCV-017.

A mutagenesis reaction was performed using pPBS-HCV-017 as template and the primers G and H. The resulting construct pPBS-HCV-054 has different codons encoding amino acids 349, 351 and 353 of NS5b(pol-) (Table 8). In a second mutagenesis step, pPBS-HCV-066 was generated using pPBS-HCV-054 as template and the following primers I and J resulting in different codon usage for amino acid positions 574 and 575 in NS3 (Table 8). In a final cloning step, the HCVla NS3-4a-5b(pol-) ORF in pPBS-HCV-066 was isolated by Sall/Ascl- restrictions and inserted into the Sall/AscI-vector fragment of pPBS-HCV-016 to generate pPBS-HCV-081. The cloning procedure added another silent nucleotide change in NS3 (at amino acid position 283), which was accepted.

Protein expression of the HCV polyprotein as well as the expected posttranslational proteolytic processing was confirmed by Western Blot analysis on lysates of 293 cells transiently transfected with pPBS-HCV-016 and pPBS-HCV-081. In the natural HCV polyprotein, the NS3/4a protease is responsible for the protein cleavage in trans at the following junctions: NS3/4a, NS4a/4b, NS4b/5a and NS5a/b. The consensus sequence for all trans cleavage sites is (D/E)XXXXC(A/S) SEQ ID NO: 41 , with the scissile bond being located between Cys and Ala or Ser. Fusing the C-terminus of NS4a to the N-terminus of NS5b maintains the consensus sequence. Therefore, the NS3-4a-5b polyprotein is proteolytically processed into the individual protein components NS3, NS4a and NS5b, as confirmed in protein expression studies (FIG. 2A). The NS3, NS4a and NS5b proteins had the expected sizes of 631, 54 and 591 amino acids, respectively.

As an alternative to the NS3-4a-5b(pol-) antigen design used in construct pPBS-HCV-

081, an HCV polyprotein NS3(prot-)-T2A-NS5b(pol-) was generated, in which the amino acid sequence of the T2A peptide sequence from Thosea asigna virus, i.e.,

EGRGSLLTCGDVEENPGP SEQ ID NO: 8 (nucleic acids 6488-6541 of SEQ ID NO: 1) replaces NS4a SEQ ID NO: 43 between NS3(prot-) and NS5(pol-). Although this antigen is expressed from one open reading frame, the T2A peptide sequence results in the expression of NS3(prot-) and NS5b(pol-) as two separate protein entities (See FIG. 2B). Insertion of an AAY motif between NS3(prot-) and the T2A peptide sequence serves the purpose of a proteasomal cleavage site and therefore limits the generation of potentially harmful antigenic peptides containing amino acids from both NS3(prot-) and T2A. In addition, the introduced change at the catalytic site of NS3 (H57T) directly ablates the protease function of this serine protease NS3(prot-).

Starting from wHCV21, a PCR fragment containing NS5b(pol-) was generated using primers K and L, and inserted into pPBS-HCV-013 via Kpnl/Spel-restriction sites (to generate pPBS-HCV-014). In addition, starting from wHCV12, a PCR fragment containing NS3(prot-)- 4a was generated using primers M and N, and inserted into pPBS-HCV-017 via Sall/BamHI- restriction sites (to generate pPBS-HCV-019).

pPBS-HCV-019 and pPBS-HCV-014 were combined by swapping the Sphl/BamHI- insert from pPBS-HCV-019 into the corresponding vector fragment of pPBS-HCV-014, so as to generate pPBS-HCV-020. The NS4a protein was removed by inserting a PCR fragment, which was generated by using pPBS-HCV-020 as template and the primers O and P into the

XhoI/Spel-vector fragment of pPBS-HCV-020. The resulting pDNA pPBS-HCV-021 and pPBS-HCV-066 were combined by swapping the Aatll/Kpnl -insert from pPBS-HCV-021 into the corresponding vector fragment of pPBS-HCV-066, so as to generate pPBS-HCV-067. Starting from pPBS-HCV-066, a PCR fragment containing NS5b(pol-) was generated using primers Q and R, and inserted into pPBS-HCV-067 via Xhol/Spel -restriction sites, so as to generate pPBS-HCV-068.

The NS3(prot-)-T2A-NS5b(pol-) antigen was then assembled by inserting an oligonucleotide linker generated from primers S and T into the BsmBI-vector fragment of pPBS-HCV-068, so as to generate pPBS-HCV-022. The corresponding protein expression pDNA pPBS-HCV-080 was generated by inserting the NS3(prot-)-T2A-NS5b(pol-) antigen from pPBS-HCV-022 into pPBS-HCV-016 via Sall/Ascl restriction sites.

Additional plasmids expressing the two interleukin 12 subunits p35 and p40 under the control of separate regulatory elements are generated as described in International Patent Application No. WO2004/093906.

EXAMPLE 4: GENERATION OF RECOMBINANT VESICULAR STOMATITIS VIRUS (rVSV)

Starting from pPBS-HCV-017 of Example 3, the HCVla NS3-4a-5b(pol-) ORF was isolated by Sall/Notl -restrictions and inserted into the XhoI/Notl-vector fragment of pPBS- VSV-HIV-006. Plasmid pPBS-VSV-HIV-006 contains the anti-genome of a live -attenuated (N3CT1) recombinant VSV expressing HIV-1 full length gpl60 from the fifth transcriptional unit (rVSVin-N3CTl -[HIV-1 6101 gpl60]5).

As with typical rVSV rescue plasmids, the rVSV anti-genome in pPBS- VSV-HIV-006 was flanked by aT7-promoter upstream and a hepatitis delta virus (HDV) ribozyme site plus a T7 terminator downstream, which allowed the expression of the rVSV anti-genome in the presence of T7 polymerase during virus rescue. The HIV-1 gpl60 gene in pPBS-VSV-HIV-006 was originally inserted into an additional transcriptional unit at the fifth position via XhoI/NotI restriction sites. Replacing this HIV gene with the HCVla NS3-4a-5b(pol-) ORF as described above created plasmid pPBS-VSV-HCV-003 (depicted in FIG. 3). The plasmid pPBS-VSV- HCV-003 was used in a rescue procedure to generate rVSV-HCV-003, also called rVSVin- N3CTl-[HCVla NS3-4a-5b(pol-)]5.

In order to further attenuate an HCVla NS3-4a-5b(pol-) expressing rVSV, the position of all genes in the rVSV-HCV-003 genome downstream of M was shifted one position down in the anti-genome by combining pPBS-VSV-HCV-003 and pVSVin-N4CTl-MCS3 without Nhe. The latter pDNA contains an anti-genome of a live-attenuated (N4CT1) recombinant VSV (rVSV) with an empty transcriptional unit (ETU) in the rVSV anti-genome at position 3 immediately upstream of VSV M. The ETU is also referred to as a multiple cloning site (MCS), and consists of 75 nucleotides. The combination cloning step was achieved by using Xbal and M restriction sites and resulted in pPBS-VSV-HCV-008 (see FIG. 4). The plasmid pPBS-VSV-HCV-008 was used in a rescue procedure to generate the corresponding recombinant virus rVSV-HCV-008, also called rVSVin-N4CTl-ETU3-[HCVla NS3-4a- 5b(pol-)]6. However, rVSV-HCV-008 showed genetic instabilities in the rVSV vector backbone at the ETU- containing transcriptional cassette during extensive passaging in Vero cell culture.

Therefore, a further modified viral vector rVSVin-N4(G CT1J3-MCS5 was generated with the gene order as follows: 3'-P-M-N-G-ETU-L-5'. Then the HCVla NS3-4a-5b(pol-) ORF was isolated from pPBS-HCV-054 by Sall/Notl -restrictions and inserted into the XhoI/Notl-vector fragment of pVSVin-N4(G CT1J3-MCS5, so as to generate pPBS-VSV-HCV- 032. The plasmid pPBS-VSV-HCV-032 (see FIG. 5) was used in a rescue procedure to successfully generate the corresponding recombinant virus rVSV-HCV-032, also called rVSVin- N4(G CTl)3-[HCVla NS3-4a-5b(pol-)] 5. However, rVSV-HCV-032 showed genetic instabilities in HCV antigen during extensive passaging in Vero cell culture.

Therefore the open reading frame containing the HCV NS3-4a-5b(pol-) antigen was linked to the ORF of rVSV L using the T2 A signal sequence. Starting from pPBS- VSV-HCV- 032, a PCR fragment containing the very 5 '-part of rVSVin L was generated using primers U and V, and inserted into pPBS-HCV-017 via Notl/Ascl -restriction sites, so as to generate pPBS- HCV-034. A PCR fragment was generated by using pPBS-HCV-034 as template and the primers W and X, cut with Mlul/Xhol restriction enzymes, and inserted into the Ascl/Xhol vector fragment of pPBS-HCV-034, so as to generate pPBS-HCV-035.

An oligonucleotide linker containing the T2A signal sequence was generated from the primers Y and Z, and inserted into the BsmBI-vector fragment of pPBS-HCV-035, so as to generate pPBS-HCV-049.

A mutagenesis reaction was performed using pPBS-HCV-049 as template and the primers AA and AB. The resulting plasmid construct, designated, pPBS-HCV-056, has different codons encoding amino acids 349, 351 and 353 of NS5b(pol-).

In addition, a mutagenesis reaction was performed using pPBS-HCV-054 as template and the primers AC and AD. The resulting construct, designated pPBS-HCV-066, has different codons encoding amino acids 349, 574 and 575 of NS3. Then the 3'-portion of HCVla NS3- 4a-5b(pol-) ORF was isolated from pPBS-HCV-066 by XhoI/Notl-restrictions and inserted into the XhoI/Notl-vector fragment of pPBS-VSV-HCV-032, so as to generate pPBS-VSV-HCV-041.

In a final cloning step, pPBS-HCV-056 and pPBS-VSV-HCV-041 were combined by swapping the SanDI/Hpal-insert from pPBS-HCV-056 into the corresponding vector fragment of pPBS-VSV-HCV-041, so as to generate pPBS-VSV-HCV-048. The plasmid pPBS-VSV-HCV-048 (FIG. 6) is related to another plasmid pPBS-VSV- HCV-034 (pDNA construction not described herein). These two plasmids differ in that they contain several non-coding changes in the HCV antigen, as indicated in Table 9 below.

The plasmids pPBS-VSV-HCV-048 and pPBS-VSV-HCV-034 were used in rescue procedures to successfully generate the corresponding recombinant viruses rVSV-HCV-048 and rVSV-HCV-034, both also called rVSVin-N4(G CTl)3-[HCVla NS3-4a-5b(pol-)-T2A-VSVin L]5. The vector rVSV-HCV-048 was extensively tested for genetic stability by passaging in cell culture and showed no signs of genetic instabilities regarding vector attenuation or antigen expression. The vector rVSV-HCV-034 appeared to be genetically stable; however, it could not be sufficiently characterized because of problems with the RT-PCR/Consensus Nucleotide Sequence Analysis at a polyC nucleotide sequence stretch in the HCV antigen. The vector rVSV-HCV-034, which is considered to be equivalent with rVSV-HCV-048, was selected for inclusion in the rVSV-only mouse immunization study (FIG. 11), while rVSV-HCV-048 was selected as the boost component in the pDNA prime/rVSV boost study (FIG. 13).

The genetic instabilities of rVSV-HCV-032 {rVSVin-N4(G CTl)3-[HCVla NS3-4a- 5b(pol-)]5), resulting from HCV antigen expression during extensive passaging in cell culture, were also overcome by the different HCV antigen design described for the construction of pDNA pPBS-HCV-080. Starting from pPBS-HCV-022, the HCV1 a NS3(prot-)-T2A-5b(pol-) ORF was isolated by Sall/Notl-restrictions and inserted into the XhoI/Notl-vector fragment of pVSVin-N4(G CT1J3-MCS5, so as to generate the plasmid pPBS-VSV-HCV-043 (depicted in FIG. 7). The corresponding virus, rVSV-HCV-043, also called rVSVin-N4(G CTl)3-[HCVla NS3(prot-)-T2A-5b(pol-)] 5, was extensively tested for genetic stability by passaging in cell culture and showed no signs of genetic instabilities regarding the vector attenuation or antigen expression.

The HCVla NS3(prot-)-T2A-5b(pol-) antigen design (as in pPBS-VSV-HCV-043) was also combined with the technique of linking the HCV antigen and VSV L expression via the T2A peptide sequence (as in pPBS-VSV-HCV-048). Starting from the plasmid pPBS-VSV-HCV- 048, the SanDI/Hpal-fragment was inserted into pPBS-VSV-HCV-043, so as to generate pPBS- VSV-HCV-044 (FIG. 8). The corresponding virus, rVSV-HCV-044, also called rVSVin-N4(G CTl)3-[HCVla NS3(prot-)-T2A-5b(pol-)-T2A-VSVin L]5, was extensively tested for genetic stability by passaging in cell culture and showed no signs of genetic instabilities regarding the vector attenuation or antigen expression.

For virus rescue of each of rVSV-HCV-032, -034, -043, -044 and -048, following the protocol of Example 1, the corresponding plasmids pVSV-HCV-032, -034, -043, -044 and -048 were transfected into Vero cells by electroporation in conjunction with six support plasmids expressing T7 polymerase and all five viral proteins of VSV. After the electroporation, transfected cells were heat-shock treated at 43°C before the final long-term incubation at 32°C. Positive rVSV rescues showed a cytopathic effect (CPE) after 5-10 days, when virus-containing supernatant was collected and flash-frozen at -78°C. A single virus clone was isolated by plaque picking, and amplified on Vero monolayers to generate a virus working stock. The rVSV-HCV-032, -034, 043, -044 and -048 working stocks were titered on Vero cell monolayers.

Protein expression from the [HCVla NS3-4a-5b(pol-)] or [HCVla NS3(prot-)-T2A- 5b(pol-)] ORFs was confirmed by Western blot analysis as described in Example IB (FIGS. 9 and 10).

The expected molecular sizes for the proteolytically processed proteins were as follows: NS3 - 67kDa, NS3-T2A - 69KDa, NS5b - 65kDa, NS4a - not tested because it is small. The presence of NS5b was an inference that NS4a was present, because NS4a expression is necessary for the processing of the polyprotein to produce NS3 and NS5b.

Virus used in immunogenicity experiments was further amplified from the working stock on BHK cell monolayers, purified by centrifugation through a 10% (wt/vol) sucrose cushion and resuspended in phosphate-buffered saline (PBS^{~ ~}).

The stability of antigen expression from rVSV-HCV-043, rVSV-HCV-044, and rVSV- HCV-048 was investigated by passaging the virus 10 times at low MOI on Vero cells. Stable antigen expression during passaging was demonstrated by Western blot analysis (FIGS. 9 and 10).

Thus, three recombinant VSV vectors - rVSV-HCV-043, rVSV-HCV-044, and rVSV-

HCV-048 - were shown to be genetically stable and expressed HCV NS3(native or prot-) and NS5b(pol-) proteins.

EXAMPLE 5 -MOUSE IMMUNIZATION STUDIES To assess to what extent various immunization regimens, that is, rVSV alone or a heterologous pDNA prime/rVSV boost, affect the magnitude and the quality of the resulting cell-mediated immune response in mice, the following protocol was used.

A. rVSV Immunization Study

Six to eight (6-8) week old female C57BL/6 mice were purchased from Charles River

Laboratories (Wilmington, MA) and maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academic Press,

Washington, DC, 1996). In addition, procedures for the use and care of the mice were approved by New York Medical College's Institutional Animal Care and Use Committee.

Groups of eight mice each were immunized once by intramuscular injection in the calf muscles (0.05 ml total injection volume, one site) using a 30-gauge needle and a 0.3 ml Insulin syringe (Becton-Dickinson, Franklin Lakes, NJ) with a dose of 10⁷ pfu as shown in Table 10.

An IFN-γ ELISpot assay was performed as described in Example 1C. As shown in FIG. 11, mice immunized once with an rVSV which expressed varying subsets of HCV nonstructural proteins elicited varying levels of HCV NS3, 4a and 5b-specific interferon-γ ELISpot responses one week post-immunization, as measured in the number of spot-forming cells (SFC) per million splenocytes:

rVSV-HCV-032 (expressing NS3-4a-5b(pol-), but genetically unstable during extensive cell culture passaging), demonstrated a mean total HCV-specific interferon-γ ELISpot response of 245 SFC/10⁶ splenocytes, with values of 28 against NS3, 2 against NS4a and 215 against NS5b.

rVSV-HCV-034 (expressing NS3-4a-5b(pol-) is linked to the amino -terminus of VSV L via T2A, but cannot be sufficiently characterized because of problems with the RT- PCR/Consensus Nucleotide Sequence Analysis at a polyC nucleotide sequence stretch in the HCV antigen). rVSV-HCV-034 demonstrated a mean total HCV-specific interferon-γ ELISpot response of 606 SFC/10⁶ splenocytes, with values of 107 against NS3, 34 against NS4a and 465 against NS5b.

Genetically stable rVSV-HCV-043 (expressing NS3(prot-)-T2A-NS5b(pol-)), demonstrated a mean total HCV-specific interferon-γ ELISpot response of 1233 SFC/10⁶ splenocytes, with values of 130 against NS3, 6 against NS4a and 1097 against NS5b.

Genetically stable rVSV-HCV-044 (expressing NS3(prot-)-T2A-NS5b(pol-), which is linked via a second T2A to the amino-terminus of VSV L, demonstrated a mean total HCV- specific interferon-γ ELISpot response of 300 SFC/10⁶ splenocytes, with values of 61 against NS3, 34 against NS4a and 205 against NS5b.

Based on these results, rVSV-HCV-043, together with the subsequently-generated rVSV-HCV-048, was chosen for use as the boost component in a pDNA prime/rVSV boost experiment in mice, although rVSV-HCV-044 is also suitable for such use.

B. pDNA Prime/rVSV Boost Immunization Study

Immediately prior to immunization, the indicated pDNA expression vectors were mixed and administered by intramuscular (IM) injection into the Tibialis anterior muscle (0.02 ml total injection volume, 0.02 ml per site) using a 30 gauge needle and 0.3 ml syringe, or IM injection with in vivo electroporation (IM/EP). Electroporation was performed under anesthesia using the TRIGRID™ Delivery System (TDS-IM) device (Ichor Medical Systems, Inc., San Diego, CA) in accordance to the supplier's instruction manual and written instructions. Electrical stimulation was delivered via a 2.5 mm electrode array at amplitude of 250 volts/centimeter of electrode spacing. The total duration of electrical stimulation was 40 mS, applied over a 400 mS interval (a 10% duty cycle).

Groups of mice as indicated in Table 11 were immunized once intramuscularly with electroporation at week 0 with a plasmid encoding the indicated HCV antigens and a plasmid encoding murine IL-12, then boosted at week 3 with an intramuscular immunization of rVSV encoding the indicated HCV antigens.

TABLE 11

GROUP # IMMUNOGENS DOSE ROUTE

# MICE

1 5 Prime: pPBS-HCV-081 + pmIL-12 1.0 μ_§ IM/EP

Boost: rVSV-HCV-043 10⁷ pfu IM

2 10 Prime: pPBS-HCV-081 + pmIL-12 1.0 μ_§ IM/EP

Boost: rVSV-HCV-048 = rVSVin-N4(G CT1)3- 10⁷ pfu IM

[HCVla NS3-4a-5b(pol-)-T2A-VSVin LJ5

3 10 Prime: pPBS-HCV-080 + pmIL-12 1.0 μ_§ IM/EP

Boost: rVSV-HCV-043 10⁷ pfu IM

4 5 Prime: pPBS-HCV-016 + pmIL-12 1.0 μ_§ IM/EP

(No Boost)

An IFN-γ ELISpot assay was performed as described in Example 1C. As shown in FIG. 12, mice immunized once with a pDNA, which expressed varying subsets of HCV nonstructural proteins, elicited varying levels of HCV NS3, 4a and 5b specific interferon-γ ELISpot responses two weeks post-prime immunization, as measured in the number of spot-forming cells (SFC) per million splenocytes:

pPBS-HCV-081 (expressing NS3-NS4a-NS5b(pol-)), demonstrated a mean total HCV- specific interferon-γ ELISpot response of 1862 SFC/10⁶ splenocytes, with values of 1463 against NS3 and 399 against NS5b (no significant responses against NS4a were measured). pPBS-HCV-080 (expressing NS3(prot-)-T2A-NS5b(pol-)), demonstrated a mean total

HCV-specific interferon-γ ELISpot response of 668 SFC/10⁶ splenocytes, with values of 108 against NS3 and 560 against NS5b (no significant responses against NS4a were measured). pPBS-HCV-016 (expressing NS3-NS4a-NS5b(pol-), the parent plasmid for pPBS- HCV-081), demonstrated a mean total HCV-specific interferon-γ ELISpot response of 2759 SFC/10⁶ splenocytes, with values of 2284 against NS3 and 475 against NS5b (no significant responses against NS4a were measured).

The plasmids pPBS-HCV-081 and pPBS-HCV-080 were chosen for use as the prime component in a pDNA prime/rVSV boost experiment in mice, in groups 1-3 shown in Table 11. The results of the prime/boost study, as measured by HCV NS3, NS4a and NS5b-specific interferon-γ ELISpot responses two weeks post-boost immunization, are shown in FIG. 13.

Group 1 - pPBS-HCV-081 (expressing NS3-4a-5b(pol-)) primdrVSV-HCV-043 (expressing NS3(prot-)-T2A-NS5b(pol-)) boost, demonstrated a mean total HCV-specific interferon-γ ELISpot response of 7918 SFC/10⁶ splenocytes, with values of 5748 against NS3 and 2170 against NS5b (no significant responses against NS4a were measured). This represented a four- fold increase in the total response versus the pDNA prime alone, with a four- fold increase in the NS3 response and a five-fold increase in the NS5b response versus the pDNA prime alone.

Group 2 - pPBS-HCV-081 (expressing NS3-4a-5b(pol-)) primdrVSV-HCV-048 (expressing NS3-NS4a-NS5b(pol-)) boost, demonstrated a mean total HCV-specific interferon- γ ELISpot response of 8608 SFC/10⁶ splenocytes, with values of 6549 against NS3 and 2059 against NS5b (no significant responses against NS4a were measured). This represented a more than four-fold increase in the total response versus the pDNA prime alone, with a more than four-fold increase in the NS3 response and a five-fold increase in the NS5b response versus the pDNA prime alone.

Group 3 - pPBS-HCV-080 (expressing NS3(prot-)-T2A-NS5b(pol-)) pnmdrVSV-

HCV-043 (expressing NS3(prot-)-T2A-NS5b(pol-)) boost, demonstrated a mean total HCV- specific interferon-γ ELISpot response of 4702 SFC/10⁶ splenocytes, with values of 2185 against NS3 and 2517 against NS5b (no significant responses against NS4a were measured). This represented a seven-fold increase in the total response versus the pDNA prime alone, with a 20-fold increase in the NS3 response and a more than four- fold increase in the NS5b response versus the pDNA prime alone.

EXAMPLE 6 - NON-HUMAN PRIMATE IMMUNIZATION STUDY

To assess an immunization regimen in non-human primates using a heterologous pDNA prime/rVSV boost, the following protocol was used.

Groups of rhesus macaques as indicated in Table 12 were primed by immunizing three times intramuscularly with electroporation at weeks 0, 4 and 8 with a plasmid encoding the indicated HCV antigens and, for groups 1 and 3, with a plasmid encoding rhesus IL-12. The groups were then boosted at week 16 with an intramuscular immunization of rVSV encoding the indicated HCV antigens.

Group 3 macaques were boosted with the indicated rVSV while receiving a short course of PEGASYS® (1.5 μg/kg, s.c, once a week). At study weeks 0, 4 and 8, the appropriate pDNA expression vector(s): 1000 μg pPBS-HCV-081 (expressing NS3-4a-5b(pol-)) alone (group 2), or 1000 μg pPBS-HCV-081 plus 300 μg plasmid rhesus IL-12 (groups 1 and 3), were mixed immediately prior to immunization and administered by intramuscular injection into the left quadriceps muscle (0.6cc/ injection) using a lcc syringe with a 27G x 1/2 inch needle and immediately followed by in vivo electroporation using the TRIGRID™ Delivery System device (Ichor Medical Systems, Inc., San Diego, CA) in accordance with the supplier's instruction manual and written instructions.

At study week 14, group 3 macaques were placed on a five week course of

PEGASYS® (PEG-interferon alfa-2a, Hoffmann LaRoche, Inc.), which was administered once a week at 1.5 μg/kg by subcutaneous injection.

At study week 16, the rVSV-HCV-043 (expressing NS3(prot-)-T2A-5b(pol-)) immunogenic composition (1 X 10⁷ pfu) was thawed and immediately administered by standard intramuscular injection in both left and right quadriceps (dose volume 0.5mL/site).

An IFN-γ ELISpot assay was performed as described in Example 1C. As shown in

FIGS. 16 and 17, macaques immunized three times with a pDNA, which expressed varying subsets of HCV non-structural proteins, elicited varying levels of HCV NS3, 4a and 5b specific interferon-γ ELISpot responses, as measured in the number of spot-forming cells (SFC) per million PBMCs; however, the levels increased after each of the three priming pDNA immunizations:

Group 1 - pPBS-HCV-081 (expressing NS3-4a-5b(pol-)) plus pIL- 12, demonstrated a mean total HCV-specific interferon-γ ELISpot response two weeks after the third

immunization of 6,833 SFC/10⁶ PBMCs, with values of 5,445 against NS3, 111 against NS4a and 1 ,277 against NS5b.

Group 2 - pPBS-HCV-081 (expressing NS3-4a-5b(pol-)) without pIL-12, demonstrated a mean total HCV-specific interferon-γ ELISpot response of 3,014 SFC/10⁶ PBMCs, with values of 2,217 against NS3, 62 against NS4a and 734 against NS5b.

Group 3 - pPBS-HCV-081 (expressing NS3-4a-5b(pol-)) plus pIL-12, demonstrated a mean total HCV-specific interferon-γ ELISpot response of 6,261 SFC/10⁶ PBMCs, with values of 5,300 against NS3, 197 against NS4a and 765 against NS5b..

The macaques were then boosted with a single dose of rVSV-HCV-043, which elicited varying levels of HCV NS3 and 5b specific interferon-γ ELISpot responses, as measured in the number of spot- forming cells (SFC) per million PBMCs (FIGs. 16 and 17): Group 1 - demonstrated a mean total HCV-specific interferon-γ ELISpot response one week after the rVSV boost immunization of 5,392 SFC/10⁶ PBMCs, with values of 3,975 against NS3, 26 against NS4a and 1,391 against NS5b.

Group 2 - demonstrated a mean total HCV-specific interferon-γ ELISpot response of 3,014 SFC/10⁶ PBMCs, with values of 2,217 against NS3, 62 against NS4a and 734 against NS5b.

Group 3 - demonstrated a mean total HCV-specific interferon-γ ELISpot response of 2,683 SFC/10⁶ PBMCs, with values of 2,422 against NS3, 23 against NS4a and 238 against NS5b.

The current standard of care for HCV1 infection includes pegylated interferon-alpha.

Interferon-alpha is thought to shut down viral replication. At week 17, one week post-rVSV boost administration, a blunting of the interferon-gamma ELISpot response was seen: Group 1 (no PEGASYS® treatment) 5,392 SFC/10⁶ PBMCs versus Group 3 (PEGASYS® treated) 2,683 SFC/10⁶ PBMCs. Future HCV treatment modalities may not include pegylated interferon-alpha, so this blunting effect may not be relevant.

The study also evaluated the effect of the inclusion of IL-12 in the pDNA priming formulation. A clear adjuvant effect was seen with the inclusion of IL-12. At week 10, two weeks post-third pDNA priming, a two-fold increase in the interferon-gamma ELISpot response was seen in the macaques receiving IL-12: Group 1 (received pIL-12) 6,833 SFC/10⁶ PBMCs versus Group 2 (no pIL-12) 3,014 SFC/10⁶ PBMCs. This adjuvant effect persisted after administration of the rVSV boost: At week 17, one week post-rVSV boost, an almost three-fold increase in the interferon-gamma ELISpot response was seen in the macaques receiving IL-12: Group 1 (received pIL- 12) 5,392 SFC/10⁶ PBMCs versus Group 2 (no pIL- 12) 1,855 SFC/10⁶ PBMCs.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compositions of the present invention and practice the claimed methods. While the invention has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. All patents, patent applications, and other publications cited throughout this application including US provisional patent applications No. 61/737,467 and No. 61/778,604, and publically available sequences cited throughout the disclosure and the sequence listing, are herein incorporated by reference in their entirety, is expressly incorporated herein by reference in its entirety. (Sequence Listing Free Text)

The following information is provided for sequences containing free text under numeric identifier <223>.

SEQ ID NO: Free text under <223>

(containing free text)

1 <213> Artificial Sequence

<223> plasmid sequence containing vesicular stomatitis virus and hepatitis C virus sequences

11 <213> Artificial Sequence

<223> primer

12 <213> Artificial Sequence

<223> primer

13 <213> Artificial Sequence

<223> primer

14 <213> Artificial Sequence

<223> primer

15 <213> Artificial Sequence

<223> primer

16 <213> Artificial Sequence

<223> primer

17 <213> Artificial Sequence

<223> primer

18 <213> Artificial Sequence

<223> primer

19 <213> Artificial Sequence

<223> primer

20 <213> Artificial Sequence

<223> primer

21 <213> Artificial Sequence

<223> primer

22 <213> Artificial Sequence

<223> primer

23 <213> Artificial Sequence

<223> primer

24 <213> Artificial Sequence <223> primer

25 <213> Artificial Sequence

<223> primer

26 <213> Artificial Sequence

<223> primer

27 <213> Artificial Sequence

<223> primer

28 <213> Artificial Sequence

<223> primer

29 <213> Artificial Sequence

<223> primer

30 <213> Artificial Sequence

<223> primer

31 <213> Artificial Sequence

<223> primer

32 <213> Artificial Sequence

<223> primer

33 <213> Artificial Sequence

<223> primer

34 <213> Artificial Sequence

<223> primer

35 <213> Artificial Sequence

<223> primer

36 <213> Artificial Sequence

<223> primer

37 <213> Artificial Sequence

<223> primer

38 <213> Artificial Sequence

<223> primer

39 <213> Artificial Sequence

<223> primer

40 <213> Artificial Sequence

<223> primer

41 <211> 7 <212> PRT

<213> Hepatitis C virus

<220>

<221> MISC FEATURE

<222> (1)..(1)

<223> can be Asp or Glu

<220>

<221> MISC FEATURE <222> (2)..(2)

<223> can be any amino acid

<220>

<221> MISC FEATURE <222> (3)..(3)

<223> can be any amino acid

<220>

<221> MISC FEATURE <222> (4)..(4)

<223> can be any amino acid

<220>

<221> MISC FEATURE <222> (5)..(5)

<223> can be any amino acid

<220>

<221> MISC FEATURE

<222> (7)..(7)

<223> can be Ala or Ser References Cited:

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10.1 101/pdb.ip067876.

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0. Gallione et al., 1981 J. Virol., 39:529-535

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Claims

CLAIMS:

1. A recombinant vesicular stomatitis virus (rVSV) comprising the nucleic acid sequence

(a) 3'-P₁-M₂-(G-CT)₃-N₄-X₅-L₆ -5 ',

or

3 ' __Pl -M₂-(G-CT)₃-N₄-L₅-X6-5 ' ,

wherein

X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A- like peptide positioned therebetween;

or

(b) 3 ' -Pi -M₂-(G-CT)3-N₄-(X/L)₅-5 ' ,

wherein

X/L is a single open reading frame, and X is a nucleic acid sequence comprising (i) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b; or (ii) sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A-like peptide positioned therebetween;

wherein the subscript numbers indicate the genomic position of each VSV gene, and

P gene encodes the VSV phosphoprotein;

M gene encodes the VSV matrix protein;

G-CT gene encodes a VSV attachment glycoprotein with a truncated cytoplasmic tail;

N gene encodes the VSV nucleocapsid protein; and

L gene encodes the VSV RNA-dependent RNA polymerase protein.

2. The rVSV according to claim 1 , wherein HCV NS3 lacking the NS3/4a protease function is:

NS3 in the absence of the NS4a protein;

NS3 having at least one mutation that eliminates its protease catalytic site; or NS3, in the absence of the NS4a protein, and having at least one mutation that eliminates its protease catalytic site.

3. The rVSV according to claim 1, wherein HCV NS5b lacks its polymerase and retinoblastoma protein (Rb) binding activity.

4. The rVSV according to claim 1, wherein G-CT is G-CT1, wherein the VSV attachment glycoprotein encoded by the truncated G gene has a deletion of its last 28 carboxy-terminal amino acids, or G-CT9, wherein the VSV attachment glycoprotein encoded by the truncated G gene has a deletion of its last 20 carboxy-terminal amino acids

5. The rVSV according to claim 1, which further comprises a sequence encoding a 2A- like peptide positioned between the HCV NS5b nucleic acid sequence and the VSV L gene.

6. The rVSV according to claim 1, wherein the VSV sequences are derived from VSV serotype Indiana.

7. The rVSV according to claim 1 , wherein the HCV sequences are derived from HCV serotype la.

8. The rVSV according to claim 2, wherein the HCV NS3 nucleic acid sequence has a mutation which changes the amino acid histidine at position 57.

9. The rVSV according to claim 8, wherein the HCV NS3 nucleic acid sequence has a mutation which changes the amino acid histidine at position 57 to a threonine.

10. The rVSV according to claim 1, wherein the HCV NS5b nucleic acid sequence has mutations which change the aspartic acid residues at positions 318 and 319 in the RNA polymerase catalytic domain of the encoded protein.

11. The rVSV according to claim 10, wherein the HCV NS5b nucleic acid sequence has mutations which change the aspartic acid residues at positions 318 and 319 in the RNA polymerase catalytic domain of the encoded protein to alanine residues.

12. The rVSV according to claim 1 , wherein X further comprises a nucleic acid sequence encoding an AAY peptide motif inserted between NS3 and the 2A-like sequence.

13. The rVSV according to claim 1, wherein the 2A-like sequence is T2A from Thosea asigna virus.

14. The rVSV according to claim 1, which is rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)- T2A-NS5b(pol-)]5.

15. The rVSV according to claim 1, which is rVSV_in -N4(G CT1)3- [HCVla NS3(prot-)- T2A-NS5b(pol-)-T2A-VSV_in LJ5.

16. The rVSV according to claim 1, which is rVSV_in -N4(G CT1)3- [HCV la NS3-NS4a- NS5b(pol-)-T2A-VSV_in LJ5.

17. An immunogenic composition comprising a recombinant vesicular stomatitis virus of any of claims 1-16 and a pharmaceutically acceptable diluent.

18. A method of treating or preventing Hepatitis C Virus infection comprising

administering to a mammalian subject in need thereof an immunogenic composition comprising a recombinant vesicular stomatitis virus (VSV) comprising the nucleic acid sequence

(a) 3'-P₁-M₂-(G-CT)₃-N₄-X₅-L₆ -5',

or

3 ' __Pl -M₂-(G-CT)₃-N₄-L₅-X6-5 ' ,

wherein

X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A- like peptide positioned therebetween;

or

(b) 3'-P₁-M₂-(G-CT)₃-N₄-(X/L)₅-5',

wherein

X/L is a single open reading frame, and X is a nucleic acid sequence comprising (i) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b; or (ii) sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b and a 2A- like peptide positioned therebetween;

wherein the subscript numbers indicate the genomic position of each VSV gene, and

P gene encodes the VSV phosphoprotein;

M gene encodes the VSV matrix protein;

G-CT gene encodes a VSV attachment glycoprotein with a truncated cytoplasmic tail; N gene encodes the VSV nucleocapsid protein; and

L gene encodes the VSV RNA-dependent RNA polymerase protein.

19. The method according to claim 18, wherein HCV NS5b lacks its polymerase and Rb binding activity.

20. The method according to claim 18, further comprising administering to said mammalian subject an effective amount of a priming composition comprising

(a) plasmid comprising a single open reading frame encoding

(i) HCV nonstructural proteins NS3, NS4a and NS5b; or

(ii) HCV NS3 lacking the NS3/4a protease function and HCV NS5b with a 2A-like peptide positioned therebetween;

said open reading frame under the control of regulatory sequences directing expression thereof by the plasmid; and

(b) a pharmaceutically acceptable diluent.

21. The method according to claim 20, wherein HCV NS5b lacks its polymerase and Rb binding activity.

22. The method according to claim 20, wherein HCV NS3 lacking the NS3/4a protease function is:

NS3 in the absence of the NS4a protein;

NS3 having at least one mutation that eliminates its protease catalytic site; or NS3, in the absence of the NS4a protein, and having at least one mutation that eliminates its protease catalytic site.

23. The method according to claim 20, wherein the priming composition is administered at least once prior to the immunogenic rVSV composition.

24. The method according to claim 20, wherein the priming composition is administered at least once following the immunogenic rVSV composition.

25. The method according to claim 20, wherein said priming composition further comprises a transfection- facilitating agent.

26. The method according to claim 25, wherein the transfection facilitating agent is a local anesthetic.

27. The method according to claim 26, wherein the local anesthetic is bupivacaine.

28. The method according to claim 18, wherein the immunogenic rVSV composition comprises a single rVSV selected from the group consisting of:

rVSVi_n -N4(G CT1)3- [HCV NS3(prot-)-T2A-NS5b(pol-)] 5,

rVSV_in -N4(G CT1)3- [HCV NS3(prot-)-T2A-NS5b(pol-)-T2A-VSV LJ5, and rVSVi_n -N4(G CT1)3- [HCV NS3-NS4a-NS5b(pol-)-T2A-VSV L] 5.

29. The method according to claim 20, wherein the priming composition further comprises an effective amount of a cytokine.

30. The method according to claim 29, wherein said cytokine is administered as a nucleic acid composition comprising a nucleic acid sequence encoding said cytokine under the control of a regulatory sequence directing expression thereof in mammalian cells.

31. The method according to claim 29, wherein said cytokine is interleukin-12 (IL-12).

32. The method according to claim 31 , wherein the cytokine nucleic acid composition comprises

(a) a nucleic acid sequence that encodes an IL-12 p35 subunit operably linked to a first regulatory sequence directing expression thereof in mammalian cells; and

(b) a second nucleic acid sequence that encodes an IL-12 p40 subunit operably linked to a second regulatory sequence directing expression thereof in mammalian cells.

33. The method according to claim 32, wherein the nucleotide sequences (a) and (b) encoding an IL-12 subunit are present on the same plasmid.

34. The method according to claim 32, wherein the nucleotide sequence encoding an IL-12 subunit is present on a plasmid different from the plasmid encoding the HCV proteins.

35. The method according to claim 18, wherein said mammalian subject is a human.

36. A method of generating an rVSV comprising:

(A) introducing into a host cell a viral cDNA expression vector comprising a nucleic acid sequence

(i) 3'-P₁-M₂-(G-CT)₃-N₄-X₅-L₆ -5'

or

3 ' __Pl -M₂-(G-CT)₃-N₄-L₅-X6-5 ' ,

wherein

X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A-like peptide positioned therebetween;

or

(ii) 3'-P₁-M₂-(G-CT)₃-N₄-(X L)₅-5 ',

wherein

X/L is a single open reading frame, and X is a nucleic acid sequence comprising (a) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b; or (b) sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A-like peptide positioned therebetween;

wherein the subscript numbers of (i) and (ii) indicate the genomic position of each VSV gene, and

P gene encodes the VSV phosphoprotein;

M gene encodes the VSV matrix protein;

G-CT gene encodes a VSV attachment glycoprotein with a truncated cytoplasmic tail;

N gene encodes the VSV nucleocapsid protein; and

L gene encodes the VSV RNA-dependent RNA polymerase protein, said nucleic acid sequence (i) or (ii) flanked by a T7 promoter upstream of Pi and a hepatitis delta virus ribozyme site and T7 terminator sequence downstream of the last genomic position of the nucleic acid sequence,

wherein the T7 promoter directs synthesis of viral RNA anti-genome transcripts from the cDNA expression vector, in the presence of the T7 RNA polymerase and VSV proteins P, M, G, N and L in the host cell; and

(B) recovering assembled infectious rVSV from said host cells.

37. The method according to claim 36, wherein HCV NS5b lacks its polymerase and Rb binding activity.

38. The method according to claim 36, further comprising transiently-transfecting the host cells with a plasmid expressing the T7 RNA polymerase.

39. The method according to claim 36, further comprising co -trans fecting the host cell with a plasmid expressing at least one viral protein of VSV.

40. The method according to claim 36, further comprising heat-shocking the host cells containing the cDNA vector, T7 polymerase and viral proteins of VSV prior to incubation.

41. The method according to claim 36, further comprising transferring said host cells or supernatant obtained from the host cells into a culture of plaque expansion cells; and recovering assembled infectious, non-segmented, negative-stranded rVSV from said culture.

42. Use of a recombinant vesicular stomatitis virus (VSV) comprising the nucleic acid sequence

(a) 3'-P₁-M₂-(G-CT)₃-N₄-X₅-L₆ -5',

or

3 ' __Pl -M₂-(G-CT)₃-N₄-L₅-X6-5 ' ,

wherein

X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A- like peptide positioned therebetween;

or

(b) 3'-P₁-M₂-(G-CT)₃-N₄-(X/L)₅-5',

wherein

X/L is a single open reading frame, and X is a nucleic acid sequence comprising (i) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b; or (ii) sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b and a 2A- like peptide positioned therebetween;

wherein the subscript numbers indicate the genomic position of each VSV gene, and

P gene encodes the VSV phosphoprotein;

M gene encodes the VSV matrix protein; G-CT gene encodes a VSV attachment glycoprotein with a truncated cytoplasmic tail;

N gene encodes the VSV nucleocapsid protein; and

L gene encodes the VSV RNA-dependent RNA polymerase protein, for treating or preventing Hepatitis C Virus infection.

43. Use of a recombinant vesicular stomatitis virus (VSV) comprising the nucleic acid sequence

(a) 3 ' -Pj -M₂-(G-CT)₃-N4-X₅-L₆ -5 ' ,

or

3 ' __Pl -M₂-(G-CT)₃-N₄-L₅-X6-5 ' ,

wherein

X is a nucleic acid sequence comprising, in a single open reading frame, sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b, and a 2A- like peptide positioned therebetween;

or

(b) 3 ' -Pj -M₂-(G-CT)3-N₄-(X/L)₅-5 ' ,

wherein

X/L is a single open reading frame, and X is a nucleic acid sequence comprising (i) sequences encoding the HCV nonstructural proteins NS3, NS4a, and NS5b; or (ii) sequences encoding HCV NS3 lacking the NS3/4a protease function, HCV NS5b and a 2A- like peptide positioned therebetween;

wherein the subscript numbers indicate the genomic position of each VSV gene, and

P gene encodes the VSV phosphoprotein;

M gene encodes the VSV matrix protein;

G-CT gene encodes a VSV attachment glycoprotein with a truncated cytoplasmic tail;

N gene encodes the VSV nucleocapsid protein; and

L gene encodes the VSV RNA-dependent RNA polymerase protein, in the preparation of a medicament for treating or preventing Hepatitis C Virus infection.