HK1198941B - Improved hcv vaccines and methods for using the same - Google Patents

Description

Improved HCV vaccines and methods of use thereof

Technical Field

The present invention relates to improved HCV antigens and vaccines made therefrom, as well as improved methods for inducing an immune response against HCV and prophylactically and/or therapeutically immunizing individuals.

Background

Applicants disclose co-pending U.S. patent application No. 13/127,008, filed on 29/4/2011, the disclosures of which are each incorporated herein by reference.

Hepatitis C (HCV) is a small enveloped positive-stranded RNA virus that represents a major health problem worldwide, with more than 1.7 million individuals currently infected with the virus [ Thomson, B.J. and R.G.Finch, hepatitis C Virus infection. Clin Microbiol Infect,2005.11(2): pages 86-94 ]. One of the most successful of all human viruses, HCV, potentially infects hepatocytes and can persist in up to 70% of the liver of all infected individuals [ Bowen, d.g., and c.m.walker, adaptivemunepressessinsaceuticandchronichepetisic infection, nature,2005.436(7053): pages 946-52 ]. It is estimated that up to 30% of chronically infected individuals will develop progressive liver disease during their life span, including cirrhosis and hepatocellular carcinoma (HCC), making HCV infection the leading cause of liver transplantation worldwide. In addition, HCV and HBV infection, which is the third leading cause of cancer death worldwide, has been implicated in 70% of all HCC cases [ Levrero, M., Viralhepatitatswandlever: the caseofhepatits C. oncogene,2006.25(27): pages 3834-47 ].

HCV infection can be very difficult and expensive to treat due to the persistent nature of the virus. Most infected individuals do not receive treatment. However, individuals receiving treatment pay an average of $ 17,700 to $ 22,000 for standard treatment regimens [ Salomon, J.A. et al, Cost-effective news recovery for chronic hepatitis Cinfectiona clinical laboratory. Jama,2003.290(2): pages 228-37 ]. The most prevalent genotype 1 infections in europe and north america have the worst prognosis, with only 42% of individuals responding to standard therapy [ Manns, m.p., et al, pegiontrona fa-2bplus vironin compounded with interferon alfa-2bplus vironin for initiating the diagnosis of chronic hepatitis c: arandomizactual lancet,2001.358(9286): pages 958-65 ].

Thus, the high prevalence of infection, the lack of effective treatment, and the economic burden of chronic HCV suggest that there is an urgent need to develop new immunotherapeutic strategies to combat this disease. There is currently no prophylactic or therapeutic vaccine against HCV.

Understanding adaptive immunity to this virus is crucial for designing strategies such as DNA vaccines to combat viral infections. Although virus-specific antibodies were detected 7-8 weeks after HCV infection [ Pawlotsky, J.M., diagnostics for hepatitis C.JHepatol,1999.31Suppl1: pages 71-9 ], they did not protect against reinfection [ Farci, P. et al, Lackfurt protective immunity against infection with hepatitis Cvirus. science,1992.258(5079): pages 135-40; lai, m.e., et al, hepatiss cvirrunmultiple multiplepresodes of acetylhepatisis not and 1994.343(8894): pages 388-90 ], and they are completely absent after the infection has subsided [ Cooper, s. et al, analytes of fungal fermentation infection of the tissues cvirrus, immunity,1999.10(4): pages 439-49; post, J.J., et al, Clearanceof hepatitis Cviremia assicated with a cellular immunity in a sorbent isolation in a Jinfect, 2004.189(10): pages 1846-55 ].

Thus, one of the major challenges in developing vaccines against HCV is that protection against HCV infection does not appear to be antibody mediated, unlike other hepatitis viruses such as hepatitis a and hepatitis b in which successful antibody-based vaccines have been produced. Although the exact relevance of immune protection still needs to be elucidated, extensive studies on acutely infected patients and chimpanzees have provided strong evidence that targeting a strong helper T1(Th1) response against the more genetically conserved non-structural regions of the virus is associated with clearance of HCV infection. See, Missale, G., et al, Differenclinica vacuorfacia hepaticati Cvirucinfectinfunction-viralcell-meditated immunmunepressure, JClininvest,1996.98(3): pages 706-14; and Diepolder, h.m. et al, podsiemans minor volatile t-lymphocyeresponson-structular protein3 invirallelearanananceacceacephates cvirrunction, lancet,1995.346(8981): pages 1006-7. Furthermore, it is important that HCV-specific T cells have been shown to localize in the liver rather than in the peripheral blood, which is critical for both reduction of viral load and clearance of acute infections. See Thimme, R. et al, Determinantsof Viralclearanane and DPersistention Virus filtration section JEXPmed,2001.194(10): page 1395-; and Shoukry, N.H., et al, memory CD8+ Tcellsarerequire for detecting from a structural section Cvirucinfection. JEXPmed,2003.197(12), pages 1645-55

Furthermore, it appears that infected individuals who produce early multispecific intrahepatic CD4+ helper and CD8+ cytotoxic T cell responses tend to show elimination of HCV infection [ Lechner, F. et al, analysis of the liver cancer cell proliferation infection cultured with hepatitis Cvirus. JEXPmed,2000.191(9): page 1499-512; gerlach, J.T., et al, Currence of hepatitis Cvirusafterloo of virus-specific CD4(+) T-cell responseinascitetheratitis C.gastroenterology,1999.117(4) pages 933-41; thimme, R. et al, Determinantsof Virallelearananance and dpstiststrendustin Virus filtration section, JEXPmed,2001.194(10), page 1395-; grakoui, A. et al, HCVpersistenceindumevasion intheabescen of memoryttcellhell. science,2003.302(5645): pages 659-62 ].

DNA vaccines have many conceptual advantages over more traditional vaccination methods, such as live attenuated viruses and recombinant protein-based vaccines. DNA vaccines are safe, stable, easy to produce and well tolerated in humans in clinical trials, which indicates little evidence of plasmid integration [ Martin, T. et al, plasmid DNA molecules: the genetic engineering of genetic integration into human genetic theragraphroditiser, 1999.10(5): pages 759-68; nichols, W.W., et al, PotentialDNAvaccetineinterationinghostcellgene. AnnNYACAdSci,1995.772: pages 30-9 ]. In addition, DNA vaccines are well suited for repeated administration, since the effect of the vaccine is not actually affected by the pre-existing antibody titer to the vector [ Chattergoon, M., J.Boyer and D.B.Weiner, geneticinmunization: anewerainvascinedimmenetherapeutics. FASEBJ,1997.11(10): pages 753-63 ]. However, one major obstacle to clinical adoption of DNA vaccines when moving to larger animals is the reduction of platform immunogenicity [ Liu, M.A. and J.B.Ulmer, Humanicalistrialsopropamidgenes. AdvGenet,2005.55: pages 25-40 ]. Recent technological advances in the engineering of DNA vaccine immunogens such as codon optimization, RNA optimization and addition of immunoglobulin leader sequences have improved the expression and immunogenicity of DNA vaccines [ Andre, S. et al, Increase immummunoresponse permissibly bDNAsvactationationwith the expression of synthetic p120sequence with optimized timing codon usage. JVirol,1998.72(2): page 1497-503; dell, L, et al, Multipleefffect of coding using optionalizing expression and dimmunogenetics of DNACandendidated vacutaine coding of the humanim nondeficien virotype1 Gagprotein. JVirol,2001.75(22) page 10991 and 1001; add, D.J., et al, immunogenictylofvnovlconsensus-basedDNAvaccinesainainanflunza. vaccine,2007.25(16): page 2984-9; frelin, L.et al, Codonotimization and RNAamplification efficiency of electroporation and their use in therapy, recently developed techniques in plasmid delivery systems such as electroporation [ Hirao, L.A. et al, Intradmail/subcategorization and electroporation and their use in therapy, vaccine,2008.26(3): pages 440-8; in Luckay, A. et al, Effect of plasmid DNA vaccine of protein and of vitamin electric and of protein production of vaccine-specific immune response of bacteria in the family of the culture of JV, 2007.81(10) pages 5257-69; ahlen, G., et al, Invivoelectroportionationenprocessing the immunogenisitio of hepatitis Cvirerusinstral 3/4 ADNAbyincrustationDNAuptake, protexpression, infection, and annefiltratino CD3+ cells, JIMMunal, 2007.179(7): pages 4741-53 ]. In addition, studies have shown that the use of a common immunogen can increase the breadth of the cellular immune response compared to the native antigen alone [ Yan., J. et al, enhanced cellular immune response and enhanced ByannenneeneeeeredHIV-1 subtype BYb-basenensensense, 2007.15(2): pages 411-21; rolland, M.et al, ReconstructionandFunctionofancestracter-of-treehumanimodimentcyvirustype1proteins. JVirol,2007.81(16): 8507-14 ].

DNA vaccines encoding HCV NS3 and NS4 are disclosed in Lang, K.A. et al Vaccine, Vol.26, No. 49, pp.6225-6231 (11 months 2008).

Thus, there remains a need for effective vaccines against HCV. Moreover, there remains a need for effective methods of treating individuals infected with HCV.

Summary of The Invention

Aspects of the invention include nucleic acid molecules comprising a coding sequence encoding one or more proteins selected from the group comprising: a) SEQ ID NO. 2; protein which is 298% homologous with SEQ ID NO; or an immunogenic fragment of SEQ ID NO. 2; b) SEQ ID NO. 4; a protein homologous to SEQ ID NO: 498%; or an immunogenic fragment of SEQ ID NO. 4; c) 6 is SEQ ID NO; 698% homologous protein to seq id no; or an immunogenic fragment of SEQ ID NO. 6. In some embodiments, the nucleic acid molecule may lack the coding sequence encoding the IgE leader sequence of seq id No. 9. Preferably, the nucleic acid molecule may be one or more sequences selected from the group comprising: a) SEQ ID NO. 1; or a coding sequence which is 198% homologous to SEQ ID NO; b) SEQ ID NO. 3; or a coding sequence which is 398% homologous to SEQ ID NO; or c) SEQ ID NO. 5; or a coding sequence which is 598 percent homologous with SEQ ID NO. In some embodiments, these nucleic acid molecules lack the coding sequence for the IgE leader sequence having the sequence of seq id No. 7 or seq id No. 8.

In addition, aspects are disclosed that include methods of treating a subject diagnosed with HCV, comprising administering to the subject a nucleic acid molecule described herein.

In another aspect, a protein selected from the group consisting of: a) SEQ ID NO. 2; protein which is 298% homologous with SEQ ID NO; or an immunogenic fragment of SEQ ID NO. 2; b) SEQ ID NO. 4; a protein homologous to SEQ ID NO: 498%; or an immunogenic fragment of SEQ ID NO. 4; or c) SEQ ID NO 6; 698% homologous protein to seq id no; or an immunogenic fragment of SEQ ID NO. 6. In some embodiments, the proteins described herein may lack the IgE leader sequence having the sequence of seq id No. 9.

Further described herein are methods of treating a subject diagnosed with HCV, comprising administering a protein described herein.

In addition, described herein are pharmaceutical compositions comprising a nucleic acid molecule provided herein and a pharmaceutically acceptable excipient. In addition, pharmaceutical compositions are described comprising a protein provided herein and a pharmaceutically acceptable excipient.

Brief Description of Drawings

FIG. 1: dose response of pConNS4B, pConNS5A, and pConNS 5B. Animals were immunized with 5 μ g, 12.5 μ g or 25 μ g of pConNS4B, pConNS5A and pConNS5B (n ═ 5). Animals received a total of two intramuscular immunizations followed by electroporation, each immunization being provided two weeks apart. Animals were sacrificed one week after the last immunization, after which splenocytes were isolated and analyzed separately. The response of each individual animal was determined by using an IFN- γ ELISpot assay, from which the optimal dose of each construct was determined.

FIG. 2: flow cytometric analysis of IFN-. gamma. + T cell responses of isolated splenocytes. Splenocytes were isolated from each animal (n ═ 5) and analyzed individually for NS4B-, NS5A-, or NS 5B-specific T cell responses. Splenocytes were stimulated with R10 (negative control) or NS4B, NS5A, or NS5B peptide pools ex vivo for 5 hours. After incubation, cells were stained intracellularly for IFN- γ and analyzed using flow cytometry. The immune specific response was reported as the percentage of IFN- γ + T cells in the peptide-stimulated group minus the percentage of IFN- γ + T cells in the R10-stimulated group. The figure shows representative animals in each group. The values shown are the average response of five individual animals from the untreated group and the immunized group. Significance was determined by student t-test (. p <0.05,. p <0.005 and. p < 0.0005).

FIG. 3: graphical representation of the percent immunospecific IFN-. gamma. + T cell responses of isolated splenocytes. Values are reported as a) average CD4+ IFN- γ + T cell response percentage and B) average CD8+ IFN- γ + T cell response percentage for each animal (n-5) from untreated and immunized groups. Significance was determined by student t-test (. p <0.05,. p <0.005 and. p < 0.0005).

FIG. 4: graphical representation of the percent immunospecific IFN- γ + T cell responses of isolated hepatic lymphocytes. Values are reported as the following mean (± SE) for each animal from untreated and immunized groups (n ═ 5): A) percent CD4+ IFN-. gamma. + T cell response and B) percent CD8+ IFN-. gamma. + T cell response. Significance was determined by student t-test (. p <0.05,. p <0.005 and. p < 0.0005).

FIG. 5: flow cytometric analysis of the percentage of IFN- γ + T cell responses of lymphocytes isolated from spleen, resting liver and transfected liver. Lymphocytes were isolated from each animal (n ═ 5) and analyzed individually for NS4B-, NS5A-, or NS 5B-specific T cell responses. The isolated lymphocytes were stained intracellularly for IFN- γ and analyzed using flow cytometry. The figure shows representative animals in each group. The values shown are the mean responses (± SE) of five individual animals from the untreated and immunized groups. Significance was determined by student t-test (. p <0.05,. p <0.005 and. p < 0.0005).

FIG. 6: graphical representation of the percentage of IFN- γ + T cell responses of lymphocytes isolated from spleen, resting liver and transfected liver. Values are reported as the following average percentage (± SE) per animal from untreated and immunized groups (n ═ 5): A) CD4+ IFN- γ + T cell response to pConNS 4B; B) CD4+ IFN- γ + T cell response to pConNS 5A; C) CD4+ IFN- γ + T cell response to pConNS 5B; D) CD8+ IFN- γ + T cell response to pConNS 4B; E) CD8+ IFN- γ + T cell response to pConNS 5A; F) CD8+ IFN-. gamma. + T cell response to pCONNS 5B. Significance was determined by student t-test (. p <0.05,. p <0.005 and. p < 0.0005).

FIG. 7: graphs of MFI ratios expressed by NS4B, NS5A, or NS5B normalized to DAPI. For each group, three images were captured for each animal (n-5). The MFI value (red) for NS4B, NS5A or NS5B was calculated for each image and normalized to the MFI value (blue) for DAPI. The values shown are the mean responses (± SE) of five individual animals from the untreated and immunized groups. Significance was determined by student t-test (. p <0.05,. p <0.005 and. p < 0.0005).

Figure 8 shows the following plasmid maps: figure 8A expression construct pConNS4B _ pVAX1, including the consensus antigen NS 4B; figure 8B expression construct pConNS5A _ pVAX1, including consensus antigen NS 5A; and figure 8C expression construct pConNS5B _ pVAX1, including the consensus antigen NS 5B.

Detailed description of the preferred embodiments

As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a nucleic acid molecule will hybridize to another nucleic acid molecule, but not to other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Typically, stringent conditions are selected to be about 5 ℃ below the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration): upon equilibration at this temperature, 50% of the probes complementary to the target sequence hybridize to the target sequence. Since the target sequence is usually present in excess, 50% of the probes are occupied at equilibrium at the Tm. Generally, stringent conditions are those in which: wherein the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion (or other salt) at pH7.0 to 8.3, and the temperature is at least about 30 ℃ for shorter probes, primers or oligonucleotides (e.g., 10 to 50 nucleotides) and at least about 60 ℃ for longer probes, primers or oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Nucleotide and amino acid sequence homology can be determined using FASTA, BLAST and GappedBLAST (Altschul et al, nuc. acids sres.,1997,25,3389, which are incorporated herein by reference in their entirety) as well as PAUP 4.0b10 software (d.l. swofford, sinauuerasates, Massachusetts). The "percent similarity" was calculated using PAUP 4.0b10 software (d.l. swofford, sinauuerassiates, Massachusetts). The average similarity of consensus sequences is calculated compared to all sequences in the phylogenetic tree.

Briefly, the BLAST algorithm, representing the basic local alignment search tool (BasiclocalAlignmentSearchTool), is suitable for determining sequence similarity (Altschul et al, J.Mol.biol.,1990,215, 403-. The software for performing BLAST analysis is publicly available through the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /). The algorithm involves first identifying high-scoring sequence pairings (HSPs) by identifying short strings of length W in the query sequence that either match or satisfy some positive numerical threshold score T when aligned with a string of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. As long as the cumulative sequence alignment score can be increased, string hits extend along each sequence in both directions. Extension of string hits in each direction terminates when the following is encountered: 1) the cumulative sequence alignment score decreases by an amount X from its maximum value obtained; 2) a cumulative score of zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) reaching the end of either sequence. Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses the following default values: word length (W) is 11, BLOSUM62 score matrix (see Henikoff et al proc. natl. acad. sci. usa,1992,89, 10915-. The BLAST algorithm (Karlin et al, Proc. Natl. Acad. Sci. USA,1993,90,5873-5787, incorporated herein by reference in its entirety) and GappedBLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which two nucleotide sequences may be matched by chance. For example, a test nucleic acid is considered similar to another nucleic acid if the smallest sum probability in a comparison of one nucleic acid to another is less than about 1, preferably less than about 0.1, more preferably less than 0.01, and most preferably less than about 0.001.

As used herein, the term "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in the cells of the individual to which the nucleic acid molecule is administered.

As used herein, the term "expressible form" refers to a genetic construct containing the necessary regulatory elements operably linked to a coding sequence encoding a protein, such that the coding sequence will be expressed when present in the cells of an individual.

Improved vaccines derived from a multi-step approach to design different DNA vaccines that can enhance cellular immune responses, including cytotoxic and IFN- γ and HCV-specific T cell responses specifically targeted against multiple conserved regions within the virus are disclosed. Modified consensus sequences were generated, including, for example, DNA vaccines that included the consensus antigens NS4B, NS5A, and NS 5B. Also disclosed are genetic modifications, including codon optimization, RNA optimization, and high-potency immunoglobulin leader sequence addition.

Improved HCV vaccines are based on proteins and genetic constructs encoding proteins with epitopes that make them particularly effective as immunogens that can be used to induce anti-HCV.

Described in some embodiments are nucleic acid molecules comprising a coding sequence encoding one or more proteins selected from the group consisting of: a) SEQ ID NO. 2; protein which is 298% homologous with SEQ ID NO; or an immunogenic fragment of SEQ ID NO. 2; b) SEQ ID NO. 4; or a protein that is 498% homologous to SEQ ID NO; or an immunogenic fragment of SEQ ID NO. 4; c) 6 is SEQ ID NO; 698% homologous protein to seq id no; or an immunogenic fragment of SEQ ID NO. 6. In some embodiments, the nucleic acid molecule may lack the coding sequence encoding the IgE leader sequence of seq id No. 9. Preferably, the nucleic acid molecule may be one or more sequences selected from the group comprising: a) SEQ ID NO. 1; or a coding sequence which is 198% homologous to SEQ ID NO; b) SEQ ID NO. 3; or a coding sequence which is 398% homologous to SEQ ID NO; or c) SEQ ID NO. 5; or a coding sequence which is 598 percent homologous with SEQ ID NO. In some embodiments, these nucleic acid molecules lack the coding sequence for the IgE leader sequence having the sequence of seq id No. 7 or seq id No. 8.

Thus, vaccines can induce a therapeutic or prophylactic immune response. In some embodiments, the means of delivering the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, an attenuated vaccine, or an inactivated vaccine. In some embodiments, the vaccine comprises a combination selected from the group consisting of: one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more immunogen-containing compositions, one or more attenuated vaccines, and one or more inactivated vaccines.

According to some embodiments, the vaccine is delivered to an individual to modulate the activity of the individual's immune system and thereby enhance the immune response against HCV. When a nucleic acid molecule encoding a protein is taken up by the cells of an individual, the nucleotide sequence is expressed in the cells and the protein is thereby delivered to the individual. Providing: methods of delivering the coding sequence of a protein on a nucleic acid molecule such as a plasmid (as part of a recombinant vaccine and as part of an attenuated vaccine, as an isolated protein or protein portion of a vector).

In another aspect, a protein selected from the group consisting of: a) SEQ ID NO. 2; protein which is 298% homologous with SEQ ID NO; or an immunogenic fragment of SEQ ID NO. 2; b) SEQ ID NO. 4; or a protein that is 498% homologous to SEQ ID NO; or an immunogenic fragment of SEQ ID NO. 4; or c) SEQ ID NO 6; 698% homologous protein to seq id no; or an immunogenic fragment of SEQ ID NO. 6. In some embodiments, the proteins described herein may lack the IgE leader sequence having the sequence of seq id No. 9.

In addition, aspects are disclosed that include methods of treating a subject diagnosed with HCV, comprising administering to the subject a nucleic acid molecule described herein.

Further described herein are methods of treating a subject diagnosed with HCV, comprising administering a protein described herein.

In addition, described herein are pharmaceutical compositions comprising a nucleic acid molecule provided herein and a pharmaceutically acceptable excipient. In addition, pharmaceutical compositions are described comprising a protein provided herein and a pharmaceutically acceptable excipient.

Compositions and methods for prophylactically and/or therapeutically immunizing individuals against HCV are provided. Nucleic acid molecule compositions for delivery comprising a nucleotide sequence encoding an immunogen are operably linked to regulatory elements. The composition may comprise: a plasmid encoding an immunogen, a recombinant vaccine comprising a nucleotide sequence encoding an immunogen, a live attenuated pathogen encoding a protein of the invention and/or comprising a protein of the invention, an inactivated pathogen comprising a protein of the invention, or a composition comprising a protein of the invention, such as a liposome or subunit vaccine. The invention also relates to injectable pharmaceutical compositions comprising the compositions.

SEQ ID NO. 1 comprises the nucleotide sequence of the HCV genotype 1a consensus immunogen encoding the HCV protein NS 4B. Seq id No. 1 further comprises the IgE leader sequence linked to the nucleotide sequence of the HCV genotype 1a consensus immunogen encoding HCV protein NS4B, together with additional 5' upstream sequences of the IgE leader sequence. SEQ ID NO. 2 contains the amino acid sequence of the HCV genotype 1a consensus immunogen for HCV protein NS 4B. SEQ ID NO. 2 further comprises the IgE leader sequence linked to a consensus immunogen sequence. The IgE leader sequence is located N-terminal to consensus sequence NS4B and is seq id No. 9 and can be encoded by seq id No. 8.

The common antigens and vaccines made therefrom described herein may include or have had the IgE leader sequence removed.

In some embodiments, the vaccine preferably comprises seq id No. 2 or a nucleic acid molecule encoding same. In some embodiments, the vaccine preferably comprises seq id No. 1. The vaccine preferably comprises the IgE leader sequence of seq id No. 9 or a nucleic acid sequence encoding the same.

The homologous sequence of SEQ ID NO. 1 may comprise 90 or more nucleotides. In some embodiments, the fragment of seq id No. 1 can comprise 180 or more nucleotides; in some embodiments, 270 or more nucleotides may be included; in some embodiments, 360 or more nucleotides may be included; in some embodiments, 450 or more nucleotides may be included; in some embodiments, 540 or more nucleotides may be included; in some embodiments, 630 or more nucleotides may be included; in some embodiments, 720 or more nucleotides may be included; in some embodiments, 810 or more nucleotides may be included; in some embodiments, and in some embodiments, 870 or more nucleotides may be included. In some embodiments, the fragment of SEQ ID NO. 1 may comprise the coding sequence of the IgE leader sequence. In some embodiments, the homologous sequence of seq id No. 1 does not comprise the coding sequence of the IgE leader sequence. Preferably, the homologous sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to seq id No. 1, and more preferably has 98% or 99% homology. In some embodiments, immunogenic fragments of seq id No. 1 are described, as well as fragments preferably having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology and more preferably 98% or 99% homology to seq id No. 1.

The homologous sequence of SEQ ID NO. 2 may comprise 30 or more amino acids. In some embodiments, the fragment of seq id No. 2 can comprise 60 or more amino acids; in some embodiments, 90 or more amino acids may be included; in some embodiments, 120 or more amino acids may be included; in some embodiments, 150 or more amino acids may be included; in some embodiments, 180 or more amino acids may be included; in some embodiments, 210 or more amino acids may be included; in some embodiments, 240 or more amino acids may be included; and in some embodiments may comprise 270 or more amino acids. Preferably, the homologous sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to seq id No. 2, and more preferably has 98% or 99% homology. In some embodiments, immunogenic fragments of seq id No. 2 are described, as well as fragments preferably having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, and more preferably 98% or 99% homology to seq id No. 2.

SEQ ID NO. 3 comprises the nucleotide sequence of the HCV genotype 1a consensus immunogen encoding the HCV protein NS 5A. SEQ ID NO. 4 contains the amino acid sequence of the HCV genotype 1a consensus immunogen for HCV protein NS 5A. Seq id No. 3 further comprises the IgE leader sequence linked to the nucleotide sequence of the HCV genotype 1a consensus immunogen encoding HCV protein NS5A, together with additional 5' upstream sequences of the IgE leader sequence. SEQ ID NO. 4 contains the amino acid sequence of the HCV genotype 1a consensus immunogen for HCV protein NS 5A. SEQ ID NO. 4 further comprises the IgE leader sequence linked to the consensus immunogen sequence NS 5A. The IgE leader sequence is located N-terminal to consensus sequence NS5A and is seq id No. 9 and can be encoded by seq id No. 7.

The homologous sequence of SEQ ID NO. 3 may comprise 90 or more nucleotides. In some embodiments, the fragment of seq id No. 3 can comprise 180 or more nucleotides; in some embodiments, 270 or more nucleotides may be included; in some embodiments, 360 or more nucleotides may be included; in some embodiments, 450 or more nucleotides may be included; in some embodiments, 540 or more nucleotides may be included; in some embodiments, 630 or more nucleotides may be included; in some embodiments, 720 or more nucleotides may be included; in some embodiments, 810 or more nucleotides may be included; in some embodiments, 900 or more nucleotides may be included; in some embodiments, 990 or more nucleotides may be included; in some embodiments, 1080 or more nucleotides may be included; in some embodiments, 1170 or more nucleotides may be included; in some embodiments, 1260 or more nucleotides may be included; in some embodiments, 1350 nucleotides or more may be included; and in some embodiments may comprise 1430 or more nucleotides. Preferably, the homologous sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to seq id No. 3, and more preferably has 98% or 99% homology. In some embodiments, immunogenic fragments of seq id No. 3 are described, as well as fragments preferably having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, and more preferably 98% or 99% homology to seq id No. 3.

The homologous sequence of SEQ ID NO. 4 may comprise 30 or more amino acids. In some embodiments, the fragment of seq id No. 4 can comprise 60 or more amino acids; in some embodiments, 90 or more amino acids may be included; in some embodiments, 120 or more amino acids may be included; in some embodiments, 150 or more amino acids may be included; in some embodiments, 180 or more amino acids may be included; in some embodiments, 210 or more amino acids may be included; in some embodiments, 240 or more amino acids may be included; may comprise 270 or more amino acids; in some embodiments, 300 or more amino acids may be included; may comprise 330 or more amino acids; in some embodiments, 360 or more amino acids may be included; may comprise 390 or more amino acids; in some embodiments, 420 or more amino acids may be included; in some embodiments, 450 or more amino acids may be included; and in some embodiments may comprise 470 or more amino acids. Preferably, the homologous sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to seq id No. 4, and more preferably has 98% or 99% homology. In some embodiments, immunogenic fragments of seq id No. 4 are described, as well as fragments preferably having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, and more preferably 98% or 99% homology to seq id No. 4.

SEQ ID NO. 5 comprises the nucleotide sequence of the HCV genotype 1a consensus immunogen encoding the HCV protein NS 5B. SEQ ID NO 6 comprises the amino acid sequence of the HCV genotype 1a consensus immunogen for HCV protein NS 5B. Seq id No. 5 further comprises the IgE leader sequence linked to the nucleotide sequence of the HCV genotype 1a consensus immunogen encoding HCV protein NS5B, together with additional 5' upstream sequences of the IgE leader sequence. SEQ ID NO 6 comprises the amino acid sequence of the HCV genotype 1a consensus immunogen for HCV protein NS 5B. SEQ ID NO 6 further comprises the IgE leader sequence linked to the consensus immunogen sequence NS 5B. The IgE leader sequence is located N-terminal to consensus sequence NS5B and is seq id No. 9 and can be encoded by seq id No. 8.

The homologous sequence of SEQ ID NO. 5 may comprise 90 or more nucleotides. In some embodiments, the fragment of seq id No. 5 can comprise 180 or more nucleotides; in some embodiments, 270 or more nucleotides may be included; in some embodiments, 360 or more nucleotides may be included; in some embodiments, 450 or more nucleotides may be included; in some embodiments, 540 or more nucleotides may be included; in some embodiments, 630 or more nucleotides may be included; in some embodiments, 720 or more nucleotides may be included; in some embodiments, 810 or more nucleotides may be included; in some embodiments, 900 or more nucleotides may be included; in some embodiments, 990 or more nucleotides may be included; in some embodiments, 1080 or more nucleotides may be included; in some embodiments, 1170 or more nucleotides may be included; in some embodiments, 1260 or more nucleotides may be included; in some embodiments, 1350 nucleotides or more may be included; in some embodiments, 1440 or more nucleotides may be included; in some embodiments, 1530 or more nucleotides may be included; in some embodiments, 1620 nucleotides or more may be included; in some embodiments, 1710 or more nucleotides may be included; and in some embodiments may comprise 1800 or more nucleotides. Preferably, the homologous sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to seq id No. 5, and more preferably has 98% or 99% homology. In some embodiments, immunogenic fragments of seq id No. 5 are described, as well as fragments preferably having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, and more preferably 98% or 99% homology to seq id No. 5.

The homologous sequence of SEQ ID NO. 6 may comprise 30 or more amino acids. In some embodiments, the fragment of seq id No. 6 can comprise 60 or more amino acids; in some embodiments, 90 or more amino acids may be included; in some embodiments, 120 or more amino acids may be included; in some embodiments, 150 or more amino acids may be included; in some embodiments, 180 or more amino acids may be included; in some embodiments, 210 or more amino acids may be included; in some embodiments, 240 or more amino acids may be included; may comprise 270 or more amino acids; in some embodiments, 300 or more amino acids may be included; may comprise 330 or more amino acids; in some embodiments, 360 or more amino acids may be included; may comprise 390 or more amino acids; in some embodiments, 420 or more amino acids may be included; in some embodiments, 450 or more amino acids may be included; in some embodiments, 480 or more amino acids may be included; in some embodiments, 510 or more amino acids may be included; in some embodiments, 540 or more amino acids may be included; in some embodiments, 570 or more amino acids may be included; and in some embodiments may comprise 600 or more amino acids. Preferably, the homologous sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to seq id No. 6, and more preferably has 98% or 99% homology. In some embodiments, immunogenic fragments of seq id No. 6 are described, as well as fragments preferably having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, and more preferably 98% or 99% homology to seq id No. 6.

According to some embodiments, the method of inducing an immune response in an individual against an immunogen comprises administering to the individual the amino acid sequence of HCV protein NS4B, NS5A, or NS5B, a functional fragment thereof, or an expressible coding sequence thereof, or a combination of the foregoing, of the HCV genotype 1a consensus immunogen. Some embodiments include an isolated nucleic acid molecule encoding an amino acid sequence of an HCV genotype 1a consensus immunogen for HCV protein NS4B, NS5A, or NS5B, or a fragment thereof. Some embodiments include recombinant vaccines that encode the amino acid sequence of an HCV genotype 1a consensus immunogen for HCV protein NS4B, NS5A, or NS5B, or fragments thereof. Some embodiments include subunit vaccines comprising the amino acid sequence of an HCV genotype 1a consensus immunogen for HCV protein NS4B, NS5A, or NS5B, or fragments thereof. Some embodiments include live attenuated and/or inactivated vaccines comprising the amino acid sequence of the HCV genotype 1a consensus immunogen for HCV protein NS4B, NS5A, or NS 5B.

The improved vaccines comprise proteins and gene constructs encoding proteins with epitopes that make them particularly effective as immunogens for inducing anti-HCV immune responses, particularly HCV-specific T cell immunity in the liver. Thus, vaccines can be provided to induce a therapeutic or prophylactic immune response. In some embodiments, the means of delivering the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, an attenuated vaccine, or an inactivated vaccine. In some embodiments, the vaccine comprises a combination selected from the group consisting of: one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more immunogen-containing compositions, one or more attenuated vaccines, and one or more inactivated vaccines.

According to some embodiments of the invention, the vaccine is delivered to an individual to modulate the activity of the individual's immune system and thereby enhance the immune response. When a nucleic acid molecule encoding a protein is taken up by the cells of an individual, the nucleotide sequence is expressed in the cells and the protein is thereby delivered to the individual. Aspects of the invention provide methods of delivering the coding sequence of a protein on a nucleic acid molecule such as a plasmid (as part of a recombinant vaccine and as part of an attenuated vaccine, as an isolated protein or protein portion of a vector).

According to some aspects of the invention, compositions and methods are provided for prophylactically and/or therapeutically immunizing an individual.

DNA vaccines are described in U.S. patent nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594, and in the priority applications cited herein, all of which are incorporated herein by reference. In addition to the delivery protocols described in these applications, alternative methods of delivering DNA are also described in U.S. patent nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference.

The present invention relates to improved attenuated live vaccines, improved inactivated vaccines, and improved vaccines that use recombinant vectors to deliver foreign genes encoding antigens, as well as subunit vaccines and glycoprotein vaccines. Examples of attenuated live vaccines, those that use recombinant vectors to deliver foreign antigens, subunit vaccines, and glycoprotein vaccines are described in U.S. patent nos.: 4,510,245; 4,797,368; 4,722,848; 4,790,987, respectively; 4,920,209, respectively; 5,017,487, respectively; 5,077,044, respectively; 5,110,587; 5,112,749, respectively; 5,174,993; 5,223,424, respectively; 5,225,336, respectively; 5,240,703, respectively; 5,242,829, respectively; 5,294,441, respectively; 5,294,548, respectively; 5,310,668, respectively; 5,387,744, respectively; 5,389,368, respectively; 5,424,065, respectively; 5,451,499, respectively; 5,453,364, respectively; 5,462,734, respectively; 5,470,734, respectively; 5,474,935, respectively; 5,482,713, respectively; 5,591,439, respectively; 5,643,579, respectively; 5,650,309, respectively; 5,698,202, respectively; 5,955,088, respectively; 6,034,298; 6,042,836, respectively; 6,156,319 and 6,589,529, both of which are incorporated herein by reference.

When taken up by a cell, the gene construct may remain present in the cell as a functional extrachromosomal molecule and/or integrated into the chromosomal DNA of the cell. The DNA may be introduced into the cell where it remains as separate genetic material in the form of one or more plasmids. Alternatively, linear DNA that can be integrated into a chromosome can be introduced into the cell. When introducing DNA into a cell, an agent may be added that promotes integration of the DNA into the chromosome. DNA sequences suitable for facilitating integration may also be included in the DNA molecule. Alternatively, RNA can be administered to the cells. It is also contemplated to provide the genetic construct in the form of a linear minichromosome, including a centromere, a telomere, and an origin of replication. The genetic construct may be maintained as part of the genetic material in a vector of attenuated live microorganisms or recombinant microorganisms that survive in the cell. The genetic construct may be part of the genome of a recombinant viral vaccine, wherein the genetic material is integrated into the chromosome of the cell or remains extrachromosomal. The genetic construct comprises regulatory elements necessary for gene expression of the nucleic acid molecule. The element comprises: a promoter, a start codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of sequences encoding target proteins or immunomodulatory proteins. These elements need to be operably linked to a sequence encoding the desired protein, and the regulatory elements need to be operable in the individual to whom they are administered.

The start codon and stop codon are generally considered to be part of the nucleotide sequence encoding the desired protein. However, these elements need to be functional in the individual to which the gene construct is administered. The initiation codon and the stop codon must be in frame with the coding sequence.

The promoter and polyadenylation signal used must be functional within the cells of the individual.

Examples of promoters suitable for use in practicing the present invention, particularly in the manufacture of genetic vaccines for human use, include, but are not limited to: promoters from simian virus 40(SV40), Mouse Mammary Tumor Virus (MMTV) promoter, human immunodeficiency virus (MV) such as BIV Long Terminal Repeat (LTR) promoter, moloney virus, ALV, Cytomegalovirus (CMV) such as CMV immediate early promoter, EB virus (EBV), Rous Sarcoma Virus (RSV), and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein.

Examples of polyadenylation signals suitable for use in the practice of the present invention, particularly in the manufacture of human genetic vaccines, include, but are not limited to: SV40 polyadenylation signal and LTR polyadenylation signal. Specifically, the SV40 polyadenylation signal in the pCEP4 plasmid (Invitrogen, san DiegoCA) was used, which was designated as the SV40 polyadenylation signal.

In addition to the regulatory elements required for expression of the DNA, other elements may also be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including, but not limited to: human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.

Genetic constructs having mammalian origins of replication can be provided to maintain the construct extrachromosomally and to produce multiple copies of the construct in the cell. Plasmids pVAX1, pCEP4 and pREP4 from Invitrogen (SanDiego, Calif.) contain the EB virus origin of replication and the nuclear antigen EBNA-1 coding region, which produces high copies of episomal replication without integration.

In some preferred embodiments involving immunological applications, nucleic acid molecules are delivered that comprise a nucleotide sequence encoding a protein of the invention, and additionally comprise a gene for a protein that further enhances the immune response against such target protein. Examples of such genes are: those genes encoding other cytokines and lymphokines, such as alpha-interferon, gamma-interferon, Platelet Derived Growth Factor (PDGF), TNF alpha, TNF beta, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and IL-15 (IL-15 including deletion of the signal sequence and optionally including the signal peptide from IgE). Other genes that may be useful include those encoding: MCP-1, MIP-1 alpha, MIP-1P, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, P150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutated forms of IL-18, CD40, CD40L, vascular growth factors, IL-7, nerve growth factors, vascular endothelial growth factors, Fas, TNF receptors, Flt, Apo-1, P55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase, FOJSp-1, FO-1, ICE-7, PECAM, ICAM-1, ICAM-3, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAPK, SAP-1, JNK, interferon-responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANKLIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof

Additional elements may be added which serve as targets for cell destruction if it is desired for any reason to eliminate the cell receiving the gene construct. An expressible form of the herpes virus thymidine kinase (tk) gene may be included in the gene construct. The drug ganciclovir can be administered to an individual and will cause selective killing of any tk-producing cells, thereby providing a means to selectively destroy cells with the genetic construct.

To maximize protein production, regulatory sequences can be selected that are well suited for gene expression in the cell to which the construct is administered. Furthermore, codons may be selected that are transcribed most efficiently in the cell. One skilled in the art can prepare DNA constructs that are functional in cells.

In some embodiments, a genetic construct may be provided wherein the coding sequence for a protein described herein is linked to an IgE signal peptide. In some embodiments, the proteins described herein are linked to an IgE signal peptide.

In some embodiments where a protein is used, for example using well known techniques, one skilled in the art can use well known techniques to prepare and isolate the protein of the invention. In some embodiments where a protein is used, for example, one skilled in the art can use well known techniques to insert a DNA molecule encoding a protein of the invention into a commercially available expression vector used in well known expression systems. For example, the commercially available plasmid pSE420(Invitrogen, san Diego, Calif.) can be used to make proteins in E.coli. A commercially available plasmid pYES2(Invitrogen, san Diego, Calif.) can be used, for example, for preparation in a Saccharomyces cerevisiae strain of yeast. Commercially available MAXBAC^TMThe complete baculovirus expression system (Invitrogen, san diego, Calif) can be used, for example, for preparation in insect cells. Commercially available plasmids pcDNAI or pcDNA3(Invitrogen, san Diego, Calif.) can be used, for example, for preparation in mammalian cells, such as Chinese hamster ovary cells. One skilled in the art can use these commercial expression vectors and systems or other vectors and systems to prepare proteins by conventional techniques and readily available starting materials. (see, e.g., Sambrook et al, molecular cloning laboratory ManualSecond edition, coldspring harborpress (1989), which is incorporated herein by reference). Thus, the desired protein can be produced in prokaryotic and eukaryotic systems, thereby producing a range of processed forms of the protein.

One skilled in the art can use other commercially available expression vectors and systems or use well known methods and readily available starting materials to prepare the vectors. Expression systems containing the required control sequences (such as promoters and polyadenylation signals, and preferably enhancers) are readily available for a variety of hosts and are known in the art. See, for example, Sambrook et al, molecular cloning laboratory Manual, second edition, ColdSpringg harborPress (1989). The genetic construct includes a protein-encoding sequence operably linked to a promoter that is functional in the cell line into which the construct is transfected. Examples of constitutive promoters include promoters from cytomegalovirus or SV 40. Examples of inducible promoters include the mouse mammary leukemia virus or metallothionein promoter. One skilled in the art can readily prepare a gene construct suitable for transfecting cells with a DNA encoding the protein of the present invention from readily available starting materials. Compatible hosts are transformed with an expression vector comprising DNA encoding the protein, and then the host is cultured and maintained under conditions in which expression of the foreign DNA occurs.

The prepared protein is recovered from the culture by lysing the cells or from a medium appropriate and known to the person skilled in the art. The person skilled in the art can isolate proteins prepared using such expression systems using well known techniques. The method of purifying a protein from a natural source using an antibody that specifically binds to a specific protein as described above can be equally applied to the purification of a protein prepared by a recombinant DNA method.

In addition to producing proteins by recombinant techniques, an automated peptide synthesizer may be used to produce isolated, substantially pure proteins. Such techniques are well known to those skilled in the art and are applicable to derivatives having substitutions not provided in the preparation of DNA-encoded proteins.

Nucleic acid molecules can be delivered using any of several well-known techniques, including DNA injection (also known as DNA vaccination), recombinant vectors, such as recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccinia.

Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular and oral as well as topical, transdermal, administration by inhalation or suppository, or administration to mucosal tissues such as by lavage of vaginal, rectal, urethral, buccal and sublingual tissues. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. The gene constructs may be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, needleless injection devices, or "microprojectile bombardment biolistics".

Examples of electroporation devices and electroporation methods that are preferred for facilitating the delivery of DNA vaccines include those described in the following patents: U.S. Pat. No. 7,245,963(Draghia-Akli et al), U.S. Pat. publication 2005/0052630 (filed by Smith et al), the entire contents of which are incorporated herein by reference. Also preferred are electroporation devices and electroporation methods for facilitating delivery of DNA vaccines provided in co-pending and commonly owned U.S. patent application serial No. 11/874072, filed on 17.10.2007 and claiming priority of the following applications under 35USC119 (e): U.S. provisional application serial No. 60/852,149 filed on day 10, 17, 2006 and U.S. provisional application serial No. 60/978,982 filed on day 10, 2007, which are both incorporated herein by reference in their entirety.

The following are examples of embodiments using electroporation techniques, and are discussed in more detail in the above-mentioned patent references: the electroporation device may be configured to deliver an energy pulse that produces a constant current similar to a preset current input by a user to a desired tissue of a mammal. The electroporation device includes an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate one or more of a variety of elements of an electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. The electroporation component may serve as one element of the electroporation device, and the other element is a separate element (or component) in communication with the electroporation component. In some embodiments, the electroporation component may serve as more than one element of the electroporation device, which may be in communication with other elements of the electroporation device independent of the electroporation component. The use of electroporation technology to deliver an improved HCV vaccine is not limited by the elements of electroporation devices that are present as part of one electromechanical or mechanical device, as the elements can serve as one device or separate elements in communication with each other. The electroporation component is capable of delivering an energy pulse that produces a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives an energy pulse from the electroporation component and delivers the energy pulse to a desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during the energy pulse and measures an electrical resistance in the desired tissue and communicates the electrical resistance to the electroporation component. A feedback mechanism may receive the measured resistance and may adjust the energy pulse delivered by the electroporation component to maintain a constant current.

In some embodiments, multiple electrodes may deliver the energy pulses in a distributed pattern. In some embodiments, the plurality of electrodes may deliver the energy pulses in a decentralized pattern through the control electrodes under a programmed sequence, and the programmed sequence is input to the electroporation component by a user. In some implementations, the programming sequence includes a plurality of pulses delivered sequentially, wherein each pulse of the plurality of pulses is delivered through at least two active electrodes (with one neutral electrode measuring resistance), and wherein subsequent pulses of the plurality of pulses are delivered through a different one of the at least two active electrodes (with one neutral electrode measuring resistance).

In some embodiments, the feedback mechanism is performed by hardware or software. Preferably, the feedback mechanism is performed by an analog closed loop circuit. Preferably, this feedback occurs every 50, 20, 10 or 1 μ s, but is preferably real-time feedback or instantaneous (i.e., it is substantially instantaneous as determined by available techniques for determining response time). In some embodiments, the neutral electrode measures the resistance in the desired tissue and informs a feedback mechanism of the resistance, and the feedback mechanism responds to the resistance and adjusts the energy pulse to maintain the constant current at a value similar to the preset current. In some embodiments, the feedback mechanism maintains a constant current continuously and instantaneously during the delivery of the energy pulse.

In some embodiments, the nucleic acid molecule is delivered to the cell in conjunction with administration of a polynucleotide functional enhancer or a gene vaccine facilitator. Polynucleotide functional enhancers are described in U.S. Ser. No. 5,593,972, 5,962,428 and International application Ser. No. PCT/US94/00899 filed 1/26 of 1994, all of which are incorporated herein by reference. Genetic vaccine promoters are described in U.S. serial No. 021,579 filed on 1/4 of 1994, which is incorporated herein by reference. The adjuvant administered in conjunction with the nucleic acid molecule may be administered as a mixture with the nucleic acid molecule or separately administered simultaneously with, before or after administration of the nucleic acid molecule. In addition, other agents that may be used as transfection and/or complexation and/or inflammation agents and that may be co-administered with GVF include: growth factors, cytokines and lymphokines, such as a-interferon, gamma-interferon, GM-CSF, Platelet Derived Growth Factor (PDGF), TNF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15, as well as fibroblast growth factor, surfactants such as immunostimulatory complexes (OMISCS), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A (WL)), muramyl peptides, quinone analogs, and vesicles such as triacontene and squalene, and hyaluronic acid may also be used in conjunction with the gene construct for administration. In some embodiments, immunomodulatory proteins can be used as GVFs. In some embodiments, nucleic acid molecules are provided in combination with PLGs to enhance delivery/uptake.

The pharmaceutical composition according to the invention comprises about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition according to the invention comprises from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA.

The pharmaceutical compositions according to the invention are formulated according to the mode of administration used. In case the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particle-free. Preferably, isotonic formulations are used. Typically, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

According to some embodiments of the invention, there is provided a method of inducing an immune response. The vaccine may be a protein-based, live attenuated vaccine, a cellular vaccine, a recombinant vaccine, or a nucleic acid or DNA vaccine. In some embodiments, a method of inducing an immune response against an immunogen (including a method of inducing a mucosal immune response) in an individual comprises administering to the individual one or more of: a combination of a CTACK protein, a TECK protein, a MEC protein and functional fragments thereof, or expressible coding sequences thereof, with an isolated nucleic acid molecule encoding a protein of the invention, and/or a recombinant vaccine encoding a protein of the invention, and/or a subunit vaccine comprising a protein of the invention, and/or a live attenuated vaccine and/or an inactivated vaccine. One or more of CTACK protein, TECK protein, MEC protein, and functional fragments thereof may be administered before, simultaneously with, or after: an isolated nucleic acid molecule encoding an immunogen, and/or a recombinant vaccine encoding an immunogen, and/or a subunit vaccine comprising an immunogen, and/or a live attenuated vaccine and/or an inactivated vaccine. In some embodiments, an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of: CTACK, TECK, MEC, and functional fragments thereof.

The invention is further illustrated in the following examples. It should be understood that this example, while indicating embodiments of the invention, is given by way of illustration only. From the above discussion and this example, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

U.S. patents, U.S. applications, and cited documents cited throughout this disclosure are each incorporated by reference herein in their entirety.

Examples

Example 1

Design and expression of pConNS4B, pConNS5A, and pConNS5B

The HCV genotype 1a consensus sequences for HCV proteins NS4B, NS5A and NS5B were generated from 170 different sequences obtained from the losamomos national laboratory HCV sequence database. Several modifications were then made to the consensus constructs in order to enhance their expression and detection, including the addition of leader sequence IgE at the HA-tags at the C-and N-termini of each construct. In addition, GeneOptimizer was used by codon and RNA optimization^TM(GENEART, Germany) Each construct was further modified and subcloned into the clinical expression vector pVAX is under the control of CMV promoter. The final constructs were named pConNS4B, pConNS5A, and pConNS5B (plasmid maps are shown in fig. 8A-8C).

Protein expression for each construct was determined by transient transfection of human RD myocytes with each individual construct. Using Lipofectamine^TM(Invitrogen) RD myocytes were transiently transfected with pConNS4B, pConNS5A, and pConNS5B according to the manufacturer's instructions. 48 hours after transfection, cells were fixed and permeabilized. The expression of each protein was detected using anti-HA polyclonal rabbit antibody (Invitrogen) followed by Cy 3-coupled goat anti-rabbit secondary antibody (Invitrogen).

Cells were observed using a confocal microscope and at 250 x magnification (images not shown). Showing that all three constructs were expressed, pConNS4B showed the highest number of transfected cells and pConNS5B showed the lowest number of transfected cells. Transfection with the blank vector pVax was used as a control.

Example 2

Immunization of C57BL/6 mice with pConNS4B, pConNS5A, and pConNS5B induced a strong cellular immune response

Once the expression of the construct was determined, C57BL/6 mice were immunized to determine the immunogenicity of the construct. Female C57BL/6 mice, 6 to 8 weeks old, were purchased from jackson laboratories and maintained according to the guidelines of the national institutes of health and university of pennsylvania laboratory animal care and use committee (IACUC). Animals were divided into three different feeding groups for each individual construct, with five animals per group. Animals were immunized intramuscularly with 5. mu.g, 12.5. mu.g or 25. mu.g of pConNS4B, pConNS5A or pConNS5B, followed by electroporation.

Use CELLECTRA^TMAn adaptive constant current electroporation device and electrode array (inovoio pharmaceuticals, inc., BlueBell, PA) were used for electroporation.

Animals received a total of two immunizations, two weeks apart, and were sacrificed one week after the second immunization. The immunogenicity of the constructs was determined using the IFN-. gamma.ELISpot assay.

Mouse IFN-. gamma.ELISpot assays were performed as previously described in Yan, J.et al, enhanced cellular research and infection of ByaneeneeredHIV-1 subtype Btye sensuss-basedenvelope DNAvaine. MolTher,2007.15(2): pages 411-21. Splenocytes were stimulated with a pool of 15mer peptides overlapping 8 amino acids and covering the length of each construct. Peptides were synthesized by Genscript (Piscataway, NJ), resuspended in DMSO and pooled at a concentration of 2. mu.g/ml/peptide. Splenocytes were plated at a concentration of 200,000 cells per well. The results were adjusted and plotted as the average number of Spot Forming Units (SFU) per 1x10^6 splenocytes. The results are visible in figure 1.

The immunogenicity of the construct is closely related to the expression level of the construct as determined by immunofluorescence. Although all constructs were strongly immunogenic, the response to pConNS4B was maximal and the response to pConNS5B was minimal. Optimal dosage: pConnS4B was 12.5 μ g (1687. + -. 237SFU/10^6 splenocytes); pConnS5A was 5 μ g (1091. + -. 111SFU/10^6 splenocytes); and pConnS5B was 12.5. mu.g (736. + -. 136SFU/10^6 splenocytes).

Once the feed for each construct was determined, a more detailed analysis of the cellular immune response induced by each construct was performed. Animals were immunized and grouped as described previously. After sacrifice, spleens were separated and crushed individually using a Stomacher device. Splenocytes were filtered through a 40 μ M cell filter and treated with ACK lysis buffer (Biosource) for 5 minutes to clear RBCs. Splenocytes were resuspended in complete medium (RPMI1640 with 2 mM/LL-glutamate supplemented with 10% heat-inactivated FBS, 1X antibiotic/antifungal, and 55 μ M/L β -mercaptoethanol). Cell numbers were determined using a hemocytometer.

To determine the relative contribution rate of CD8+ and CD4+ T cell responses to each construct, splenocytes were stained intracellularly for IFN- γ and observed using flow cytometry, fig. 2. The results of intracellular cytokine staining are closely related to those previously seen using the IFN-. gamma.ELISpot assay. The response was maximal for pConNS4B, while pConNS5B was minimally immunogenic. The IFN- γ responses to pConNS4B and pConNS5A were mostly generated by CD8+ T cells, but CD4+ T cells specific for each construct were also recognized. Interestingly, the IFN- γ response to pConnS5B was mostly CD4+ T cell mediated, with only a few IFN- γ + CD8+ T cells recognized. The average percentages of IFN- γ + CD4+ T cells for pConNS4B, pConNS5A, and pConNS5B were 0.50% ± 0.11%, 0.27% ± 0.06%, and 0.32% ± 0.11%, respectively, fig. 3A. The average percentages of IFN- γ + CD8+ T cells for pConNS4B and pConNS5A were 3.29% ± 1.33% and 0.68% ± 0.22%, respectively, fig. 3B.

Example 3

Immunity-induced NS4B-, NS 5A-and NS 5B-specific T cells were detected in the liver after intramuscular immunization

Mice were immunized as previously described in example 1 above. One week after the last immunization, animals were sacrificed. After sacrifice, the liver was isolated and separately comminuted using a Stomacher machine. The resulting mixture was filtered and treated with 10ml of ack lysis buffer (Bioscience) for 5 minutes to clear RBCs. The mixture was pelleted and hepatocytes were separated from lymphocytes by using a 35% percoll gradient. The precipitated lymphocytes were resuspended in complete medium. No difference was observed when experiments were performed with or without liver perfusion.

T cells were isolated from each liver and stimulated with overlapping peptides corresponding to each individual construct. Immune-induced HCV-specific T cells are identified by IFN- γ expression detected by intracellular cytokine staining and flow cytometry. Each animal was analyzed separately. Interestingly, HCV-specific T cells were recognized in the livers of all immunized mice. CD4+ and CD8+ T cell responses were detected in the liver of mice immunized with pConNS4B and pConNS5A, whereas only CD4+ T cell responses were detected in mice immunized with pConNS 5B. The dominant T cell responses detected in the liver were identical to those recognized in the spleen. Mice immunized with pConNS4B and pConNS5A had a strong CD8+ T cell response in the liver, while mice immunized with pConNS5B showed a major CD4+ T cell response and a minor CD8+ T cell response. CD4+ T cell responses to pConNS4B, pConNS5A, and pConNS5B were 0.29% ± 0.07%, 0.41% ± 0.09%, and 0.41% ± 0.06%, respectively, fig. 4A. CD8+ T cell responses to pConNS4B, pConNS5A, and pConNS5B were 3.73% ± 0.73%, 2.28% ± 0.68%, and 0.06% ± 0.02%, respectively, fig. 4B.

Example 4

3.4 hepatic cell liver-specific expression of HCV antigens results in increased IFN- γ production and clearance of transfected hepatocytes

We next sought to determine whether liver-specific expression of NS4B, NS5A or NS5B proteins would activate HCV-specific T cells detected in the liver. To induce liver-specific expression of NS4B, NS5A, and NS5B, hepatocytes of immunized mice were transfected by administering tail vein injections of pCons 4B, pCons 5A, or pCons 5B as previously described in Ahlen, G.et al, Invivoceranceofaphthotis Cviral construction 3/4A-expressohepatocystes-primatocytiscs.JInFectDis, 2005.192(12): pages 2112-6. The liver was transfected for 48 hours, after which the liver was harvested and hepatic lymphocytes were isolated as described in example 3 above. As mentioned previously, immune-induced HCV-specific T cells are recognized by IFN- γ secretion detected via intracellular cytokine staining and flow cytometry.

Intracellular cytokine staining

Splenocytes were resuspended in complete medium at a concentration of 1x10^6 cells/100. mu.l and plated in round bottom 96-well plates. Splenocytes were stimulated with 100 μ l of: 1)2 μ g/ml pConNS4B, pConNS5A, or pConNS5B overlapping peptide; 2)1 μ g/ml staphylococcal enterotoxin B (positive control; Sigma-Aldrich, st.louis, MO) or 3) 0.1% dimethylsulfoxide (negative control), all diluted in complete medium, supplemented with GolgiStop and golgiplug (bdbioscience). Splenocytes were stimulated at 37C for a total of 5 hours, after which the cells were washed three times with PBS and stained to determine viability. Surface markers of splenocytes were stained extracellularly for 30 min at 4C for anti-CD 4, CD 8. The splenocytes were then permeabilized and washed using the bdkyfox/Cytoperm solution kit (BDBioscience) and then stained intracellularly with anti-IFN- γ and CD3 for 45 minutes at 4C. After staining, splenocytes were fixed with 1% paraformaldehyde and stored at 4C until analysis. For each animal, the specific function was reported as a function of the percentage of the peptide-stimulated group minus the percentage of the 0.1% dimethylsulfoxide-stimulated group (negative control).

Flow cytometry reagents

The following directly conjugated antibodies were used: anti-mouse CD 3-allophycocyanin cyanine dye 7(APC-Cy7) [ clone 145-C11], anti-mouse CD 4-Fluorescein Isothiocyanate (FITC) [ clone H129.19], anti-mouse CD 8-polymethacrylic chlorophyll protein 5.5(percp5.5) [ clone 53-6.7], anti-mouse IFN- γ -phycoerythrin cyanine dye 7(PE-Cy7) [ clone XMG1.2] (all from BDBiosciences, san jose, CA). An aqueous live/dead fixable dead cell staining kit (molecular probes, Eugene, OR) was used according to the manufacturer's protocol to identify live cells.

Samples were collected on a LSRII flow cytometer (BDBiosciences, FranklinLakes, NJ). Bdcomp heads (bdbiosciences) and a single fluorescent dye were used for compensation. Data were analyzed using FlowJo software version 8.7.1 for Mac (TreeStar, Ashland, OR).

After tail vein injection, a large increase in the percentage of CD4+ and CD8+ HCV-specific T cells was seen in all three immunization groups compared to the percentage of HCV-specific T cells detected in the spleen and resulting liver, fig. 5. For mice immunized with pConNS4B, pConNS5A, and pConNS5B, the percentages of CD4+ HCV-specific T cells were 2.27% ± 0.70%, 2.55% ± 0.70%, and 1.22% ± 0.22%, respectively, fig. 6A. For mice immunized with pConNS4B, pConNS5A, and pConNS5B, the percentage of CD8+ HCV-specific T cells was 9.46% ± 1.53%, 6.98% ± 0.48%, and 0.477% ± 0.16%, respectively, fig. 6B. The greatest fold increase was seen for the CD4+ T cell response as determined by the percentage of HCV-specific IFN- γ + T cells in the liver before and after tail vein injection. The fold increase in intrahepatic CD4+ T cell responses in mice immunized with pConNS4B, pConNS5A, and pConNS5B was approximately 8-fold, 6-fold, and 3-fold, respectively. Although the CD8+ T cell response was still a dominant response in the liver before and after tail vein injection, the observed fold increase in CD8+ T cell response was slightly smaller compared to the CD4+ T cell response. The fold increase in intrahepatic CD8+ T cell responses in mice immunized with pConNS4B, pConNS5A, and pConNS5B was approximately 3-fold, and 8-fold, respectively.

After assessing the intrahepatic HCV-specific IFN- γ responses produced by each construct, studies were performed to determine whether immunization also produced intrahepatic cytotoxic HCV-specific T cells. One lobe of liver was obtained from each animal of each group and stained for determining hepatocyte expression of NS4B, NS5A or NS 5B. Cytotoxicity of the intrahepatic T cell response resulting from immunization with each construct was assessed by the ability of each immunized animal to clear NS4B, NS5A, or NS 5B-expressing hepatocytes after transfection when compared to the transfected immune untreated control group. A representative confocal image of this staining was observed for each group (image not shown).

Confocal microscopy

The livers were dissected and the specimens were fixed in 2% paraformaldehyde, followed by overnight cryopreservation in 30% sucrose. The specimens were immersed in Tissue-tek oct (bayer corporation, Pittsburgh, PA) and flash frozen in 2-methylbutane over dry ice nitrogen. Tissue sections (6 μm) mounted on Superfrost plus glass slides (Fisherscientific, Pittsburgh, Pa.) were stained and kept at 80 ℃ until use. Prior to immunofluorescent staining, the slides were brought to room temperature and washed three times in Phosphate Buffered Saline (PBS), 10 minutes each, and blocked with PBS containing 10% of normal serum of the species in which the second reagent was produced and 0.1% Triton. The first reagent was applied to the sections and the sections were incubated for 1 hour at room temperature or overnight at 4 ℃. Sections were washed three times in PBS for 10 minutes each, with the second reagent applied for 30 minutes at room temperature if necessary. The sections were washed again three times in PBS for 10 minutes each. Coverslips were mounted with ProlongGod mounting media (Invitrogen, Carlsbad, Calif.) and slides were kept at 4 ℃ in the dark until study and photographed. All dyeings were carried out in a moist environment. The antibody used was obtained from Invitrogen, or a competitor company that made the antibody. All images were obtained using a zeiss axioviert 100 inverted confocal microscope and the fluorescence intensity was analyzed and quantified using ImageJ software (NIH, Rockville, MD).

Clearance of each group of transfected hepatocytes was quantified by Mean Fluorescence Intensity (MFI) of NS4B, NS5A, or NS5B expression normalized by the number of hepatocytes present in each field as measured by MFI as nuclear DAPI staining, fig. 9. A large reduction in the number of transfected hepatocytes was seen in the animal immunization group for all three constructs compared to the untreated control group. Transfected hepatocytes expression was reduced by approximately 9-fold, 3-fold, and 2-fold in animals immunized with pConNS4B, pConNS5A, or pConNS5B compared to untreated controls. The observed clearance in each immunization group correlated closely with the HCV-specific CD8+ T cell response detected in the transfected liver. The greatest clearance was observed in animals immunized with pConNS4B, while the least clearance was observed in animals immunized with pConNS 5B.

The results provided show that HCV-specific T cells induced by systemic immunity revert back into the liver in the absence of liver-specific expression of cognate antigens, resulting in the formation of a large intrahepatic HCV-specific T cell pool. These T cells still function well within the liver, suggesting that their recruitment into the quiescent liver may additionally serve as part of the persistence of immune surveillance and may prove to be an important mechanism by which the liver protects against infection. To support this, this liver-localized HCV-specific T cell population is able to rapidly induce IFN- γ expression and clear transfected hepatocytes in response to liver-specific expression of HCV antigens. Since it has been previously reported that no T cell infiltration was observed until 72 hours after liver transfection (Ahlen et al, supra), rapid clearance of HCV-transfected hepatocytes appears likely to be dependent on the liver-localized HCV-specific T cell population present in the liver prior to transfection. In addition, as seen in animals immunized with pConNS5B, even a relatively small percentage of the vaccine-specific response (as measured by IFN- γ production) was sufficient to induce a large 2-fold reduction in intrahepatic transfected hepatocytes, suggesting that a small percentage of the vaccine-specific response, as measured in the periphery, has the ability to play a large role in the liver.

Liver-induced T cell tolerance can be disrupted by systemic immunity, and effective liver-specific immunity can be achieved by exploiting the ability of the liver to recover and sequester antigen-specific T cells under quiescent conditions. This unique property of the liver can be exploited to enhance HCV-specific responses in patients already infected with virus and to generate a pool of HCV-specific T cells within the liver of untreated individuals that have the ability to respond and migrate rapidly following infection with the first signal. Taken together, the findings indicate that antigen-specific T cell-to-liver recruitment, together with their effector functions retained within the liver, can play an important and previously unrecognized role in the process of immune surveillance, which can be exploited in future T cell-based HCV vaccines.

Claims (12)

1. A nucleic acid molecule consisting of: a coding sequence encoding one or more proteins selected from the group consisting of:

a)SEQIDNO:2；

b)SEQIDNO:4；

or

c)SEQIDNO:6。

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule lacks the coding sequence for the IgE leader sequence consisting of the sequence of seq id No. 9.

3. The nucleic acid molecule of claim 1, consisting of: one or more sequences selected from the group consisting of:

a)SEQIDNO:1；

b)SEQIDNO:3；

or

c)SEQIDNO:5。

4. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule lacks the coding sequence for the IgE leader sequence, wherein the lacking coding sequence is SEQ ID NO. 7 or SEQ ID NO. 8.

5. A plasmid comprising the nucleic acid molecule of any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid molecule of any one of claims 1 to 4, wherein the sequence encoding the one or more proteins is operably linked to regulatory elements.

7. Use of the nucleic acid molecule of any one of claims 1 to 4, the plasmid of claim 5, or the expression vector of claim 6 in the manufacture of a medicament for the treatment of a subject diagnosed with HCV.

8. A protein selected from the group consisting of:

a)SEQIDNO:2；

b)SEQIDNO:4；

or

c)SEQIDNO:6。

9. The protein according to claim 8, wherein the protein lacks the IgE leader sequence consisting of the sequence of seq id No. 9.

10. Use of a protein according to any one of claims 8 to 9 in the manufacture of a medicament for the therapeutic treatment of a subject diagnosed with HCV.

11. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 4, the plasmid of claim 5 or the expression vector of claim 6 and a pharmaceutically acceptable excipient.

12. A pharmaceutical composition comprising the protein of any one of claims 8 to 9 and a pharmaceutically acceptable excipient.