Nucleic acid molecule encoding hepatitis b virus core protein and surface antigen protein and vaccine comprising the same

Abstract

To provide an effective vaccine against hepatitis B virus (HBV).SOLUTION: There are provided herein nucleic acid sequences encoding hepatitis B virus (HBV) core proteins, surface antigen proteins, fragments and combinations thereof, as well as genetic constructs / vectors and vaccines that express the protein sequences. These vaccines are able to induce an immune response peripherally and in the liver by recruiting both cellular and humoral agents. Also there are provided methods for prophylactically and / or therapeutically immunizing individuals against HBV. The combination vaccine may also be used for particular design vaccines for particular levels of immune responses to HBV challenge.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to nucleic acid sequences encoding Hepatitis B Virus (HBV) core proteins, surface antigen proteins, fragments and combinations thereof, Hepatitis B Virus (HBV) core proteins, surface antigen proteins, fragments and combinations thereof, improved HBV vaccines, improved methods for inducing an immune response against HBV, and improved methods for prophylactically and / or therapeutically immunizing individuals against HBV. [Background technology]

[0002] Hepatitis B is a common infectious disease that is endemic worldwide and leads to the development of cirrhosis, liver failure, and hepatocellular carcinoma. A significant number of hepatitis cases go unreported due to the asymptomatic nature of the disease. Nevertheless, approximately 350 million cases of chronic hepatitis B are reported annually. The majority of the hepatitis-infected population are from underdeveloped or developing countries.

[0003] The virus is classified into four main serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on its envelope protein. According to variations in the genome sequence, there are at least eight genotypes (A-H) of HBV. The alternative genotypes of HBV have endemic geographical distributions.

[0004] Table 1 shows the geographic distribution of HBV genotypes. [Table 1]

[0005] The HBV genome is a circular DNA molecule that is primarily double-stranded, but has a single-stranded region resulting from one strand that is longer than the other. The double-stranded region results from hybridization of a short strand of about 3020 nucleotides long with a long strand of about 3320 nucleotides long. The single-stranded region on the long strand of unhybridized nucleotides is associated with HBV DNA polymerase. Both HBV genomic DNA and HBV DNA polymerase are contained within a nucleocapsid formed by multiple HBV core protein (HBcAg) molecules. The HBV core protein is covered by HBV surface protein or antigen (HBsAg) and lipid molecules.

[0006] The HBV genome contains four open reading frames (ORFs) that code for seven different proteins: 1) an ORF that codes for the HBV DNA polymerase, 2) an ORF with two start codons (the sequence linked to the second start codon codes for the core protein, and the sequence containing an additional upstream start codon codes for a sequence designated pre-C), 3) an ORF with three start codons (one that codes for a surface protein (S protein, gp27), one that contains an upstream start codon that codes for a sequence designated pre-S2 (gp36), and one that contains an even more upstream start codon that codes for a sequence designated pre-S1 (gp42), and 4) an ORF that codes for HBxAg, a protein whose function is not well understood (Figure 1). These include three surface proteins or antigens (HBsAg): preS1, preS2, and major S.

[0007] The characteristics of HBsAg are illustrated in Figure 2. The epitope of HBsAg responsible for the expression of subtype specificity is located in a region of the HBV surface antigen molecule that includes two external loops (i.e., amino acids 110-180 of the S protein), making HBV strains antigenically diverse. The same region contains an unknown number of epitopes, which define "a" determinant of HBsAg that is common to all known HBV wild-type strains.

[0008] Prophylactic vaccines and treatments for HBV infection involve the injection of subviral particles purified from the plasma of chronic carriers or produced as recombinant proteins in stably transfected eukaryotic cell lines. Subviral particles are viral proteins, and such vaccines are often called subunit vaccines. HBV proteins are administered to an individual and targeted by the individual's immune system. In uninfected individuals, the immune response to the subunit vaccine protects the uninfected individual from HBV infection. In infected individuals, the immune response induced by the vaccine can have a therapeutic effect.

[0009] Chisari FV, Am J Pathol., 2000.156:1117-1132 and Pumpeus P. et al. Intervirology 2001.44:98-114 disclose the HBV genome structure. Deny P. and F. Zoulim, Pathologie Biologie 2010, Aug,58(4):245 53 discuss Hepatitis B virus diagnosis and treatment. Michel ML and P. Tiollais, Pathologie Biologie 2010, Aug,58(4):288 95 discuss Hepatitis B vaccines and their protective efficacy and therapeutic potential. PCT Publication No. 2004026899 discloses the use of immunogens containing polypeptide sequences having HBV amino acid sequences. PCT Published Application No. 2008093976 discloses vaccines including HBV coding sequences, proteins, and vaccines including recombinant full-length HBV surface antigen and HBV core antigen. HBV surface antigen is generally composed of three surface proteins (L protein, M protein, and S protein). PCT Published Application No. 2009130588 discloses vaccines including HBV coding sequences, proteins, and nucleic acids encoding Hepatitis B virus core antigen that are codon optimized for expression in humans. PCT Publication No. 2010127115 discloses delivery of HBV sequences using recombinant vectors.

[0010] Available HBV vaccines show some efficacy but are expensive to produce. In addition, plasma-derived subunit vaccines also have safety concerns. Several vaccine approaches have been explored, including recombinant live vectors, synthetic peptides, and DNA vaccines containing codon-optimized coding sequences of HBV proteins. These other approaches have had varying degrees of limited efficacy so far. In addition, due to genomic differences, some HBV vaccines have shown positive efficacy in some geographic regions and limited efficacy in others.

[0011] Currently available HBsAg-based vaccines derived from yeast transfected with DNA encoding the S protein (e.g., ENGERIX-B available from SmithKline Biologicals, Belgium, and RECOMBIVAX / HB-VAX II available from Merck & Co., USA) do not induce a response in approximately 5%-10% of individuals (C. Belloni, Immunogenicity of hepatitis B vaccine in term and preterm infants. Acta Paediatrica, 1998.87:p.336-338). Furthermore, the rate of non-response increases to 30% in older individuals, and immunity to HBV may decrease several years after vaccination. Also, multiple doses are used to achieve full protection. The safety of the HBsAg-based vaccine, ENGERIX-B, is a concern, as it triples the risk of central nervous system (CNS) inflammatory demyelination.

[0012] Other HBsAg-based vaccines derived from mammalian cells utilize pre-S1 and pre-S2 in addition to the S protein. The pre-S1 and pre-S2 antigens express highly immunogenic T and B cell epitopes, and one such vaccine induces an immune response in approximately 80% of non- or low-responders (Rendi-Wagner, P. et al., Comparative immunogenicity of PreS / S hepatitis B vaccine in non- and low responders to conventional vaccine. Vaccine, 2006.24:p.2781-9.).

[0013] Direct administration of nucleic acid sequences for vaccination against animal and human diseases has been investigated, and much effort has focused on effective and efficient means of nucleic acid delivery to produce the necessary expression of the desired antigen, result in an immune response, and ultimately lead to the success of this technology.

[0014] DNA vaccines allow for endogenous antigen synthesis, which induces class I-restricted cytotoxic T lymphocytes of the CD8+ histocompatibility complex, which is rarely achieved with subunit vaccines. In addition, antigen synthesis that occurs over a sustained period of time can help overcome low responsiveness and eliminate or reduce the need for booster injections. Furthermore, DNA vaccines appear to be very stable and easy to produce.

[0015] DNA vaccines are safe, stable, easily produced, and well tolerated in humans, and preclinical studies have shown little evidence of plasmid integration [Martin, T., et al., Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther, 1999.10(5):p.759-68; Nichols, WW, et al., Potential DNA vaccine integration into host cell genome. Ann NY Acad Sci, 1995.772:p.30-9]. In addition, DNA vaccines are well suited for repeated administration, since vaccine efficacy is not affected by preexisting antibody titers against the vector [Chattergoon, M., J. Boyer, and DB Weiner, Genetic immunization: a new era in vaccines and immune therapeutics. FASEB J, 1997.11(10):p.753-63]. However, one major obstacle in the clinical application of DNA vaccines is the diminished immunogenicity of the platform when moved to larger animals [Liu, M. Aand J. B. Elmer, Human clinical trials of plasmid DNA vaccines. Adv Genet, 2005. 55: p. 25-40].

[0016] Recent technological advances in engineering DNA vaccine immunogens have improved the expression and immunogenicity of DNA vaccines, such as codon optimization, RNA optimization, and the addition of immunoglobulin leader sequences [Andre,S.,et al.,Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage.J Virol,1998.72(2):p.1497-503; Deml,L.,et al.,Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein.J Virol,2001.75(22):p.10991-1001; Laddy,DJ,et al.,Immunogenicity of novel consensus-based DNA vaccines against avian influenza.Vaccine,2007.25(16):p.2984-9; Frelin,L.,et al.,Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3 / 4A gene.Gene Ther,2004.11(6):p.522-33], and recently developed techniques for plasmid delivery systems such as electroporation [Hirao,LA,et al.,Intradermal / subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques.Vaccine,2008.26(3):p.440-8, Luckay,A.,et al.,Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques.J Virol,2007.81(10):p.5257-69、Ahlen,G.,et al.,In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3 / 4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells.J Immunol,2007.179(7):p.4741-53]. In vivo electroporation techniques have been used to deliver anticancer drugs such as bleomycin in human clinical trials and in many animal species in many preclinical studies. In addition, studies suggest that the use of consensus immunogens can broaden the scope of cellular immune responses compared to native antigens alone [Yan, J., et al., Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ther, 2007. 15(2): p. 411-21, Rolland, M., et al., Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins. J Virol, 2007. 81(16): p. 8507-14].

[0017] Antibodies and cytotoxic T cells (CTLs) against different HBV antigens may be involved in the reduction of viral load and the clearance of HBV-infected cells from the liver. The major challenge of current candidate vaccines is the induction of both humoral and cellular immunity against multiple HBV antigen targets and against the major genotypes of HBV. There remains a need for nucleic acid constructs encoding HBV proteins and compositions useful for inducing immune responses against HBV. There remains a need for an effective vaccine against HBV that is economical and effective. There remains a need for an effective vaccine that increases neutralizing antibody levels and induces T cell components. There remains a need for an effective vaccine against HBV, including one that is effective against HBV strains with a wide range of genotypes, and preferably a universal vaccine that is effective worldwide. Summary of the Invention

[0018] The present invention is directed to a vaccine comprising a nucleic acid molecule encoding one or more proteins selected from the group consisting of (a) SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, (b) SEQ ID NO:10, SEQ ID NO:14, a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, and a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:14, and (c) SEQ ID NO:12, SEQ ID NO:16, a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:12, and a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:16. The nucleic acid molecule may comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15.

[0019] The present invention may also be directed to a vaccine comprising: (a) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; (b) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:10, SEQ ID NO:14, a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:10, and a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:14; and (c) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:12, and a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:16.

[0020] The vaccine may be a plasmid containing the nucleic acid described above. The nucleic acid molecule may be incorporated into a viral particle. The vaccine may further include an adjuvant molecule. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof, and in some embodiments, may be IL-12, IL-15, IL-28, or RANTES.

[0021] The present invention is also directed to a method of inducing an immune response against an HBV antigen comprising administering a vaccine of the present invention to a subject.

[0022] The present invention is also directed to a method of protecting a subject against HBV infection comprising administering to the subject a vaccine of the present invention.

[0023] The present invention is further directed to a method of protecting a subject diagnosed with HBV infection comprising administering to the subject a vaccine of the present invention.

[0024] The present invention further includes a vaccine that is useful for inducing an immune response against HBV.The development of an HBV immunotherapeutic vaccine with broad-spectrum efficacy against multiple genotypes can be achieved by using a therapeutic DNA vaccine for HBV infection based on targeting universally conserved HBV core-specific antigens.The use of consensus HBV immunogens can be useful for inducing a broader range of cellular immune responses and minimizing the degree of sequence dissimilarity between different virus strains.

[0025] Provided herein is a protein selected from the group consisting of a protein comprising SEQ ID NO:2, a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:2, a fragment of SEQ ID NO:2, a protein that is 95% identical to a fragment of SEQ ID NO:2, SEQ ID NO:4, a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:4, a fragment of SEQ ID NO:4, a protein that is 95% identical to a fragment of SEQ ID NO:4, SEQ ID NO:6, a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:6, a fragment of SEQ ID NO:6, and a protein that is 95% identical to a fragment of SEQ ID NO:6.

[0026] Provided herein is a protein selected from the group consisting of: (a) SEQ ID NO:2, (b) a protein that is 95% identical over the entire length of the amino acid sequence of the full-length sequence shown in SEQ ID NO:2, (c) an immunogenic fragment of SEQ ID NO:2 that comprises 20 or more amino acids of SEQ ID NO:2, and (d) an immunogenic fragment of a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:2 that comprises 20 or more amino acids.

[0027] Also provided is a nucleic acid molecule comprising a sequence encoding one or more of the protein molecules described above. In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO:1, a nucleic acid sequence 95% identical to the full length of the amino acid sequence of SEQ ID NO:1, a fragment of SEQ ID NO:1, a nucleic acid sequence 95% identical to a fragment of SEQ ID NO:1, SEQ ID NO:3, a nucleic acid sequence 95% identical to the full length of the amino acid sequence of SEQ ID NO:3, a fragment of SEQ ID NO:3, a nucleic acid sequence 95% identical to a fragment of SEQ ID NO:3, SEQ ID NO:5, a nucleic acid sequence 95% identical to the full length of the amino acid sequence of SEQ ID NO:5, a fragment of SEQ ID NO:5, and a nucleic acid sequence 95% identical to a fragment of SEQ ID NO:5.

[0028] Some embodiments of the present invention provide methods of inducing an immune response against HBV comprising administering to an individual an HBV core antigen and / or HBV surface antigen molecule and / or a composition.

[0029] A further aspect of the invention provides a method of protecting an individual from HBV infection comprising administering to said individual a prophylactically effective amount of a nucleic acid molecule comprising such a nucleic acid sequence or composition, wherein the nucleic acid sequence is expressed in cells of the individual and a protective immune response is induced against the protein encoded by the nucleic acid sequence.

[0030] In some embodiments of the present invention, methods are provided for treating an individual infected with HBV, comprising administering to said individual a therapeutically effective amount of such a nucleic acid molecule and / or composition.

[0031] In another aspect, the present invention provides a nucleic acid molecule comprising a coding sequence encoding one or more proteins selected from the group consisting of: (a) a protein comprising SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; (b) a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; (c) an immunogenic fragment of a protein comprising SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 that is at least 20 amino acids; and (d) an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 that is at least 20 amino acids.

[0032] The nucleic acid molecule may comprise one or more sequences selected from the group consisting of: (a) a nucleic acid sequence comprising SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; (b) a nucleic acid sequence that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; (c) a fragment thereof comprising a nucleic acid sequence encoding an immunogenic fragment comprising at least 20 amino acids encoded by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; and (d) a fragment thereof comprising a nucleic acid sequence encoding an immunogenic fragment comprising at least 20 amino acids of a protein that is 98% identical over the entire length of the amino acid sequence of the protein encoded by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. The nucleic acid molecule may be a plasmid. The nucleic acid molecule may be an expression vector, and the sequence encoding the one or more proteins is operably linked to a regulatory element. The nucleic acid molecule may be incorporated into a viral particle.

[0033] Some embodiments of the invention provide methods of inducing an immune response against an HBV antigen, comprising administering to a subject a nucleic acid molecule of the invention.

[0034] Some embodiments of the invention provide methods of protecting a subject from HBV infection comprising administering to the subject a nucleic acid molecule of the invention.

[0035] Some embodiments of the invention provide methods for protecting a subject diagnosed with HBV infection comprising administering to the subject a nucleic acid molecule of the invention.

[0036] In yet another aspect, the present invention provides a protein selected from the group consisting of: (a) SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; (b) a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; (c) an immunogenic fragment of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 comprising 20 or more amino acids; and (d) an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 comprising 20 or more amino acids.

[0037] Some aspects of the invention provide a vaccine useful for generating an immune response against HBV in a subject, comprising a nucleic acid molecule of the invention and an adjuvant molecule, which can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof, and in some embodiments can be IL-12, IL-15, IL-28, or RANTES. [Brief description of the drawings]

[0038] [Figure 1] 1 is a map showing the structure of the HBV genome, which is composed of four overlapping ORFs. [Diagram 2] Illustrates the structure and characteristics of the HBV surface antigen. [Diagram 3]Illustrates IgELS and intracellular protein cleavage sites in the consensus HBV surface antigen. [Figure 4] The long consensus HBV surface antigen (LHB) and the small consensus HBV surface antigen (SHB) are illustrated. [Diagram 5]

[0036] Figure 1 is a map of vector pGX1801 HepB-Mcore (SEQ ID NO: 17). [Figure 6]

[0046] Figure 1 is a map of vector pGX1802 HepB pLHBs-A (SEQ ID NO:18). [Figure 7]

[0046] Figure 1 is a map of vector pGX1803 HepB pLHBs-C (SEQ ID NO:19). [Figure 8]

[0046] Figure 2 is a map of vector pGX1804 HepB pSHBs-A (SEQ ID NO:20). [Figure 9]

[0046] Figure 2 is a map of vector pGX1805 HepB SHBs-C (SEQ ID NO:21). [Figure 10] FIG. 1 illustrates a phylogenetic analysis of the HBcAg consensus sequence compared to individual genotypes A, B, C, D, and E. [Figure 11A] Figure 11 shows the results of the pM Core expression experiment. Figure 11A shows the results of the in vitro translation protocol. Figure 11B shows the results of a Western blot. [Figure 11B] FIG. 11B shows the results of the Western blot. [Figure 12] FIG. 1 illustrates the plasmid map and sequence of pMCore. [Figure 13] A transcription / translation reaction using the pMCore plasmid is shown, in which the resulting MCore protein was immunoprecipitated with anti-HA monoclonal antibody and run on an SDS-PAGE gel. [Figure 14]Detection of MCore protein in transiently transfected cells using a primary monoclonal HA tag antibody followed by detection with a DyLight 594-conjugated anti-rabbit secondary antibody. Hoechst staining was also used to fluorescently label cell nuclei. MCore expression is mostly localized to the cytoplasm as indicated by staining outside the nucleus. [Figure 15] The immunization scheme is illustrated below: Four mice were immunized intramuscularly with 30 μg pMCore. [Figure 16] FIG. 1 shows the increased magnitude of IFN-γ secretion in CD8+ and CD4+ T cells from the spleens of Balb / C mice vaccinated with pM-Core. [Figure 17] FIG. 1 shows the increased magnitude of IFN-γ secretion in CD8+ and CD4+ T cells from the spleens of Balb / C mice vaccinated with pM-Core. [Figure 18] FIG. 1 shows the increased magnitude of TNF-α secretion in CD8+ and CD4+ T cells from the spleens of Balb / C mice vaccinated with pM-Core. [Figure 19] FIG. 1 shows the increased magnitude of TNF-α secretion in CD8+ and CD4+ T cells from the spleens of Balb / C mice vaccinated with pM-Core. [Figure 20] FIG. 1 shows the increased magnitude of CD107a secretion in CD8+ and CD4+ T cells from the spleens of Balb / C mice vaccinated with pM-Core. [Figure 21] FIG. 1 shows the increased magnitude of CD107a secretion in CD8+ and CD4+ T cells from the spleens of Balb / C mice vaccinated with pM-Core. [Figure 22] The mean percentage of HBcAg-specific CD4 or CD8 IFN-γ+, TNF-α+ secreting cells from Balb / c mice immunized with control pVAX or consensus core antigen pMCore is shown. [Figure 23]The mean percentage of HBcAg-specific CD4 or CD8 double positive producing cells from Balb / c mice immunized with control pVAX or consensus core antigen pMCore is shown. [Figure 24] 1 shows interferon-gamma T cell responses in livers from Balb / C mice vaccinated with pM-Core. [Diagram 25] 1 shows interferon-gamma T cell responses in livers from Balb / C mice vaccinated with pM-Core. [Figure 26] 1 shows tumor necrosis factor-α T cell responses in livers from Balb / C mice vaccinated with pM-Core. [Figure 27] 1 shows tumor necrosis factor-α T cell responses in livers from Balb / C mice vaccinated with pM-Core. [Figure 28] The percentage of HBcAg-specific CD4 and CD8 IFN-γ+, TNF-α+ producing cells in livers from Balb / c mice immunized with control pVAX or consensus core antigen pMCore is shown. [Figure 29] The mean percentage of HBcAg-specific CD4 or CD8 double positive producing cells in livers from Balb / c mice immunized with control pVAX or consensus core antigen pMCore is shown. [Diagram 30] Antigen-specific antibody producing splenocytes are shown. Values ​​are the mean ± standard error of the mean. Significance was determined by Student's t-test. [Diagram 31] Data from an ELISPOT assay are shown. [Diagram 32] The frequency of HBcAg-specific IFN-γ spot-forming units (SFU) per million splenocytes following stimulation of splenocytes from immunized mice is shown. [Diagram 33] Data are presented from an experiment using CSFE labeled cells to compare in vivo elimination of peptide-treated target cells by CD8 T cells in vaccinated and non-vaccinated animals. [Diagram 34]Showing in vivo specific killing. Two groups of mice immunized with either pVax (control) or pMCore received CFSE-labeled target cells (CFSElo pulsed with an irrelevant peptide or CFSEhi pulsed with an epitope-specific peptide) through the tail vein. CFSE-labeled cells were harvested and analyzed by FACS to quantify the percentage killing. [Diagram 35] A comparison of the proliferation rates of CD3+CD4+ and CD3+CD8+ cells treated with pVax vector (control) or with the plasmid pMCore expressing the HBV M core is shown. [Diagram 36] Percent proliferation of CD4 and CD8 T cells is shown. [Figure 37A] A comparison of anti-HBV core IgG and IgA in serial dilutions of sera from animals treated with pVax vector (control) or with the plasmid pMCore expressing HBV M core is shown. [Figure 37B] A comparison of anti-HBV core IgG and IgA in serial dilutions of sera from animals treated with pVax vector (control) or with the plasmid pMCore expressing HBV M core is shown. [Figure 38] Figure 1 shows HBcAg-specific humoral immune responses induced by pMCore in serum and splenocytes. Values ​​are means ± standard error of the mean. Significance was determined by Student's t-test. [Figure 39] Figure 1 shows HBcAg-specific humoral immune responses induced by pMCore in serum and splenocytes. Values ​​are means ± standard error of the mean. Significance was determined by Student's t-test. [Diagram 40] The percentages of TNF-α and IFN-γ from CD4+ and CD8+ spleen and liver cells are shown. [Diagram 41] 1 shows data from an experiment determining whether the clearance induced by immunized mice affected the liver by measuring ALT levels in serum. [Diagram 42]Immunostaining of liver sections taken 3 days after hydrodynamic injection of PBS, pMCore (HBcAg), or pNS3 / 4A (HCV NS3-4A) from naive mice or mice immunized with the indicated pMCore are shown. Clearance is much higher in pMCore-immunized livers compared to NS3 / 4a-transfected control livers. PMcore or NS3 / 4A expression was detected with anti-HA antibody (white / light grey cells). [Diagram 43] Shown three days after transfection, cells were analyzed for degranulation marker expression, CD107a, and IFN-γ+ expression following stimulation with HBcAg peptide. [Diagram 44] Serum ALT levels were measured 3, 6, and 12 days after transfection and show no evidence of elevation in relevant vaccinated animals. [Diagram 45] Shown are total cellular immune responses in monkeys immunized with small (i.e., pMCore, pSHb A, and pSHb C), long (i.e., pMCore, pLHb A, and pLHb C), and long + IL-12 (i.e., pMCore, pLHb A, pLHb C, and prlIL-12) vaccines. [Figure 46] 1 shows core-specific cellular immune responses in monkeys immunized with small (i.e., pMCore, pSHb A, and pSHb C), long (i.e., pMCore, pLHb A, and pLHb C), and long+IL-12 (i.e., pMCore, pLHb A, pLHb C, and prlIL-12) vaccines. [Figure 47] Shown are surface antigen A-specific cellular immune responses in monkeys immunized with small (i.e., pMCore, pSHb A, and pSHb C), long (i.e., pMCore, pLHb A, and pLHb C), and long + IL-12 (i.e., pMCore, pLHb A, pLHb C, and prlIL-12) vaccines. [Figure 48]ELISPOT assay data showing surface antigen C-specific cellular immune responses in monkeys immunized with small (i.e., pMCore, pSHb A, and pSHb C), long (i.e., pMCore, pLHb A, and pLHb C), and long + IL-12 (i.e., pMCore, pLHb A, pLHb C, and prlIL-12) vaccines. [Figure 49] Shown are total cellular immune responses in monkeys immunized with small (i.e., pMCore, pSHb A, and pSHb C), long (i.e., pMCore, pLHb A, and pLHb C), and long + IL-12 (i.e., pMCore, pLHb A, pLHb C, and prlIL-12) vaccines. [Figure 50] Comparison of anti-HBV antibodies in serial dilutions of sera from monkeys immunized with small (i.e., pMCore, pSHb A, and pSHb C), long (i.e., pMCore, pLHb A, and pLHb C), and long + IL-12 (i.e., pMCore, pLHb A, pLHb C, and prlIL-12) vaccines is shown. [Figure 51] A comparison of anti-HBV antibodies at serial dilutions from animals immunized with long + IL-12 (i.e., pMcore, pLHb A, pLHb C, and prlIL-12) vaccines is shown. [Figure 52A] HBV surface antigen DNA constructs and their expression are shown. (A) Phylogenetic analysis of HBs consensus sequences of genotypes A and C compared to native HBs. The consensus sequences are indicated by an asterisk. (B) Schematic diagram of HBs gene insertion into DNA plasmids. pLHBs and pSHBs represent the large and small HBs protein antigens. The preS1, preS2, and major S proteins are linked with an intracellular protein cleavage site. (C) Detection of pLHBs and pSHBs by intracellular staining of transfected RD cells with monoclonal antibodies and polyclonal mouse serum. [Figure 52B] Same as explanation for Figure 52A. [Figure 52C] Same as explanation for Figure 52A. [Figure 53A] Figure 1 shows that both pLHBs and SHBs induced strong antibody and T cell responses in Balb / c. Five Balb / c mice per group were immunized in three different experiments and their induction of both humoral and cellular responses was analyzed. (A) HBs-specific IgG responses measured one week after each immunization. Arrows indicate the immunization time points. (B) Total IFN-γ responses induced by each construct to synthetic preS1 / S2 and major S peptides. (C) Characterization of HBs-specific immunodominant epitopes by using individual HBs peptides in 16 and 12 matrix pools of LHBs and SHBs, respectively. This indicates an enhanced spread of cell-mediated immune responses. [Figure 53B] Same as explanation for Figure 53A. [Figure 53C] Same as explanation for Figure 53A. [Figure 54] Showing specific killing in vivo. Different groups of mice immunized with pVax (control) or one of the four HBs constructs received CFSE-labeled target cells (CFSElo pulsed with an irrelevant peptide or CFSEhi pulsed with an epitope-specific peptide) through the tail vein. CFSE-labeled cells were harvested and the percentage of killing was quantified using analysis by FACS (fluorescence activated cell sorting). [Figure 55A] (A) HBs-specific antibody responses from sera of mice immunized with the HBs / HBc vaccine cocktail at different immunization time points (arrows), showing that the HBc / HBs vaccine cocktail induces a strong immune response. (B) IFN-γ responses after ex vivo stimulation of splenocytes from mice immunized with the HBs / HBc cocktail. Immunized mice responded to both core and surface antigen peptides. [Figure 55B] Same as explanation for Figure 55A. [Figure 56A]Antigen-specific CD8 T cells induce antiviral cytokines. Intracellular staining of antiviral cytokines after ex vivo stimulation of splenocytes of BALB / c mice from different immunization groups (Table 3). Lymphocytes were stained with antibodies against CD3, CD4, CD8, IFN-γ, TNF-α, IL-2, and CD107a. The data are representative of 4 mice per group in 3 different experiments. [Figure 56B] Same as explanation for Figure 56A. [Figure 56C] Same as explanation for Figure 56A. [Figure 56D] Same as explanation for Figure 56A. [Figure 56E] Same as explanation for Figure 56A. [Fig. 56F] Same as explanation for Figure 56A. [Figure 57A] Immunogenicity in non-human primates is shown. Five rhesus macaques were immunized by intramuscular injection of pSHBs / pMCore, pLHBs / pMCore, or pLHBs / pMCore+pIL-12 (Table 4) followed by electroporation. The macaques received three immunizations. (A) High antibody titers against HBs were observed only after two immunizations. (B) IFN-γ responses were measured once before the first immunization and two weeks after each immunization. (C) Many positive HBs matrix peptide pools were observed in most monkeys from different groups confirming a broad T cell response in the immunized macaques. (D) Intracellular staining (IFN-γ, TNF-α, IL-2) of PBMCs after the last immunization shows HBs and HBc specific antiviral cytokine production in CD4 and CD8. [Figure 57B]Immunogenicity in non-human primates is shown. Five rhesus macaques were immunized by intramuscular injection of pSHBs / pMCore, pLHBs / pMCore, or pLHBs / pMCore+pIL-12 (Table 4) followed by electroporation. The macaques received three immunizations. (A) High antibody titers against HBs were observed only after two immunizations. (B) IFN-γ responses were measured once before the first immunization and two weeks after each immunization. (C) Many positive HBs matrix peptide pools were observed in most monkeys from different groups confirming a broad T cell response in the immunized macaques. (D) Intracellular staining (IFN-γ, TNF-α, IL-2) of PBMCs after the last immunization shows HBs and HBc specific antiviral cytokine production in CD4 and CD8. [Figure 57C] Immunogenicity in non-human primates is shown. Five rhesus macaques were immunized by intramuscular injection of pSHBs / pMCore, pLHBs / pMCore, or pLHBs / pMCore+pIL-12 (Table 4) followed by electroporation. The macaques received three immunizations. (A) High antibody titers against HBs were observed only after two immunizations. (B) IFN-γ responses were measured once before the first immunization and two weeks after each immunization. (C) Many positive HBs matrix peptide pools were observed in most monkeys from different groups confirming a broad T cell response in the immunized macaques. (D) Intracellular staining (IFN-γ, TNF-α, IL-2) of PBMCs after the last immunization shows HBs and HBc specific antiviral cytokine production in CD4 and CD8. [Fig. 57D]Immunogenicity in non-human primates is shown. Five rhesus macaques were immunized by intramuscular injection of pSHBs / pMCore, pLHBs / pMCore, or pLHBs / pMCore+pIL-12 (Table 4) followed by electroporation. The macaques received three immunizations. (A) High antibody titers against HBs were observed only after two immunizations. (B) IFN-γ responses were measured once before the first immunization and two weeks after each immunization. (C) Many positive HBs matrix peptide pools were observed in most monkeys from different groups confirming a broad T cell response in the immunized macaques. (D) Intracellular staining (IFN-γ, TNF-α, IL-2) of PBMCs after the last immunization shows HBs and HBc specific antiviral cytokine production in CD4 and CD8. [Figure 58] Figure 1 shows the safety of therapeutic HBV vaccine in rhesus macaques. Samples were analyzed before vaccination (pre), after the third vaccination (3rd), before the fourth vaccination (pre4th), and after the fourth vaccination (4th). Analytical error existed for alkaline phosphatase after the third vaccination. Normal ranges for all measurements are indicated by dashed lines. (A) Serum alkaline phosphatase. (B) Serum alanine aminotransferase (ALT) levels. (C) Serum aspartate aminotransferase (AST) levels. (D) Serum total bilirubin levels. (E) Serum creatinine levels. (F) Serum blood urea nitrogen levels. [Figure 59A] Sequence annotation for the four HBs plasmids is shown. Sequences for the IgE leader, intracellular protein cleavage site, and C-terminal HA tag are underlined. The four HBs plasmids are genotype A large surface protein (pLHBs-A) (A) and small surface protein (pSHBs-A) (B) and genotype C large surface protein (pLHBs-C) (C) and small surface protein (pSHBs-C) (D). [Figure 59B] Same as explanation for Figure 59A. [Figure 59C] Same as explanation for Figure 59A. [Fig. 59D] Same as explanation for Figure 59A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention relates to highly optimized, multivalent synthetic Hepatitis B virus (HBV) immunogens that induce both antibody and cell-mediated immunity against HBV surface (HBs) and HBV core (HBc) antigens. These plasmids encoding the most prevalent viral genotypes induce binding antibodies to HBs antigen and drive strong cell-mediated immunity (CMI) against both HBc and HBs antigen. The plasmids independently induced broad cross-reactive T cell responses and high titer antibody responses, as well as vaccine-induced cytotoxic activity. Plasmid cocktails containing both surface antigens induce strong broad cellular immune responses across multiple antigen epitopes, particularly broad cross-reactivity between the A and C genotypes of the virus. Optimized plasmid cocktails encoding HBs and HBc antigens induced strong humoral and cellular responses targeting the S antigen and drove a wide variety of multi-epitope responses involved in the wiping of infected hepatocytes.

[0040] Specifically, the present invention relates to a vaccine for Hepatitis B Virus (HBV), which can be used to protect individuals against HBV infection and provide a therapeutic effect to individuals diagnosed with HBV infection. Such a vaccine can also be used to induce an immune response to HBV antigens. The HBV vaccine of the present invention comprises one or more nucleic acid molecules. Specifically, the nucleic acid molecule encodes one or more consensus HBV core proteins. The consensus HBV core proteins are derived from the sequences of core proteins from HBV genotypes A, B, C, D, and E, and therefore the consensus HBV core proteins are unique. The nucleic acid molecule can also encode one or more immunogenic fragments of the consensus HBV core protein.

[0041] Furthermore, the nucleic acid molecule can also encode one or more consensus HBV surface antigens. The consensus HBV surface antigen is derived from the sequence of surface antigens from HBV genotype A isolates, thus the consensus HBV surface antigen is unique. The consensus HBV surface antigen can also be derived from the sequence of surface antigens from HBV genotype C isolates, resulting in a unique HBV surface antigen. The nucleic acid molecule can also encode one or more immunogenic fragments of the consensus HBV surface antigen.

[0042] The vaccine of the present invention may comprise any combination of nucleic acid molecules encoding consensus HBV core protein and / or consensus HBV surface antigens. The vaccine may also comprise any combination of nucleic acid molecules encoding immunogenic fragments of consensus HBV core protein and / or consensus HBV surface antigens.

[0043] These combinations of HBV core protein and HBV surface antigen surprisingly and unexpectedly induce differential immune response to HBV core protein and HBV surface antigen.In other words, the strength of immune response to HBV core protein and HBV surface antigen varies according to the specific combination administered to the subject.Therefore, any user of the vaccine of the present invention can design a vaccine that contains a specific combination, and induce desired immune response in the subject administered with such designed vaccine.Therefore, any user can tailor the vaccine of the present invention to control the level of immune response in the subject.

[0044] The vaccine of the present invention has broad applicability to multiple types of HBV due to the unique consensus sequences of the HBV core protein and HBV surface antigen.

[0045] Vaccines of the present invention can further comprise one or more of the nucleic acid molecules described above and one or more proteins encoded by such nucleic acid molecules. 1.Definition

[0046] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047] In reciting numerical ranges herein, each intervening number is expressly contemplated with the same degree of precision, for example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0048] "Adjuvant," as used herein, means any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigen encoded by the DNA plasmid and encoding nucleic acid sequence described hereinafter.

[0049] "Antibody" as used herein means an antibody of the IgG, IgM, IgA, IgD, or IgE class, or a fragment, fragment, or derivative thereof, including Fab, F(ab')2, Fd, as well as single chain antibodies, bispecific antibodies, diabodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0050] "Coding sequence" or "encoding nucleic acid" as used herein means a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that codes for a protein. The coding sequence can further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0051] "Complement" or "complementary" as used herein means a nucleic acid can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0052] "Consensus" or "consensus sequence" as used herein means a polypeptide sequence based on an analysis of an alignment of multiple subtypes of a particular HBV antigen, such as HBV core or surface antigen. Nucleic acid sequences encoding consensus polypeptide sequences can be prepared. Vaccines containing proteins containing consensus sequences and / or nucleic acid molecules encoding such proteins can be used to induce broad immunity against multiple subgroups or serogroups of a particular HBV antigen.

[0053] "Electroporation," "electropermeabilization," or "electro-kinetic enhancement" ("EP"), as used interchangeably herein, refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0054] "Fragment," as used herein with respect to a nucleic acid sequence, means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with a full-length wild-type strain HBV antigen. The fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding protein fragments described below.

[0055] "Fragment" or "immunogenic fragment" refers to a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with a full-length wild-type strain HBV antigen, with reference to the polypeptide sequence. A fragment of a consensus protein can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the consensus protein. In some embodiments, a fragment of a consensus protein can comprise at least 20 or more amino acids, at least 30 or more amino acids, at least 40 or more amino acids, at least 50 or more amino acids, at least 60 or more amino acids, at least 70 or more amino acids, at least 80 or more amino acids, at least 90 or more amino acids, at least 100 or more amino acids, at least 110 or more amino acids, at least 120 or more amino acids, at least 130 or more amino acids, at least 140 or more amino acids, at least 150 or more amino acids, at least 160 or more amino acids, at least 170 or more amino acids, at least 180 or more amino acids, at least 190 or more amino acids, at least 200 or more amino acids, at least 210 or more amino acids, at least 220 or more amino acids, at least 230 or more amino acids, or at least 240 or more amino acids of the consensus protein.

[0056] As used herein, the term "genetic construct" refers to a DNA or RNA molecule that contains a nucleotide sequence that codes for a protein. The coding sequence includes initiation and termination signals that are operably linked to regulatory elements, including promoters and polyadenylation signals that can direct expression in the cells of an individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that contains the necessary regulatory elements that are operably linked to the coding sequence that codes for a protein, such that the coding sequence is expressed when present in the cells of an individual.

[0057] As used herein, the term "homology" refers to the degree of complementarity. There may be partial or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to with the functional term "substantially homologous". When used in reference to a double-stranded nucleic acid sequence, such as a cDNA or gene clone, the term "substantially homologous" as used herein refers to a probe that can hybridize to a strand of a double-stranded nucleic acid sequence under low stringency conditions. When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" as used herein refers to a probe that can hybridize to (i.e., is the complement of) a template sequence of a single-stranded nucleic acid under low stringency conditions.

[0058] "Identical" or "identity", as used herein in the context of two or more nucleic acid or polypeptide sequences, means that the sequences have a certain percentage of residues that are the same over a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a particular region, determining the number of positions where identical residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the particular region, and multiplying the result by 100 to yield the percentage of sequence identity. If the two sequences are of different length or the alignment results in one or more sticky ends and a particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0059] "Immune response" as used herein means activation of a host's immune system, e.g., a mammalian immune system, in response to the introduction of an antigen, such as an HBV consensus antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0060] "Nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0061] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, where the nucleic acids can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0062] "Operably linked" as used herein means that the expression of a gene is under the control of a promoter that is spatially linked. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The spacing between the promoter and the gene can be approximately the same as the spacing between the promoter and the gene it controls in the gene from which the promoter is derived. As known in the art, variations in this spacing can be accommodated without loss of promoter function.

[0063] "Promoter" as used herein means a synthetic or naturally derived molecule that can confer, activate or enhance expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance its expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located up to several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can differentially regulate the expression of genetic components constitutively, or with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[0064] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked to the amino terminus of an HBV protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. As used herein, a signal peptide / leader sequence preferably facilitates secretion of a protein from the cell in which it is produced. A signal peptide / leader sequence is often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. A signal peptide / leader sequence is linked to the N-terminus of a protein.

[0065] "Stringent hybridization conditions," as used herein, refer to the conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions can be selected to be about 5-10°C lower than the thermal melting point (Tm) of a particular sequence at a defined pH of ionic strength. The Tm can be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (50% of the probes occupy equilibrium, since the target sequence is present in excess at the Tm). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion (or other salt), such as about 0.01-1.0 M sodium ion concentration at pH 7.0-8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5×SSC, and 1% SDS, incubation at 42° C., or 5×SSC, 1% SDS, incubation at 65° C. with a wash in 0.2×SSC and 0.1% SDS at 65° C.

[0066] "Substantially complementary," as used herein, means that a first sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 142, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2008, 2008, 2009, By "stringent hybridization" it is meant that the two sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 1, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0067] "Substantially identical," as used herein, means that the first and second sequences have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, With respect to nucleic acids, it means at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or where a first sequence is substantially complementary to the complement of a second sequence.

[0068] "Subtype" or "serotype" as used herein interchangeably with reference to HBV refer to genetic variants of HBV such that one subtype is recognized by the immune system separately from different subtypes.

[0069] "Variant," as used herein with respect to a nucleic acid, means (i) a portion or fragment of a reference nucleotide sequence, (ii) the complement of a reference nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0070] "Variants" refers to peptides or polypeptides that differ in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variants also refer to proteins with amino acid sequences that are substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with an amino acid that has a different property (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art, typically with minor modifications. These minor modifications can be identified, in part, by considering the hydropathic index of an amino acid as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic index can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in the retention of biological activity of a protein. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference. Substitution of amino acids with similar hydrophilicity values ​​results in peptides retaining biological activity, such as immunogenicity, as is understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are comparable in biological function depend on the relative similarity of amino acids, particularly the side chains of these amino acids, as evidenced by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0071] "Vector" as used herein means a nucleic acid sequence that contains an origin of replication.Vector can be a virus vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome.Vector can be a DNA or RNA vector.Vector can be a self-replicating extrachromosomal vector, and is preferably a DNA plasmid. 2. Vaccines

[0072] The present invention is directed to a hepatitis B vaccine. The hepatitis B vaccine (HBV) may comprise a nucleic acid encoding HBV core antigen, HBV surface antigen, or a combination thereof, HBV core antigen, HBV surface antigen, or a combination thereof, or (1) a nucleic acid encoding HBV core antigen and / or HBV surface antigen, and (2) a combination of HBV core antigen and / or HBV surface antigen, or (3) a combination thereof. The HBV core antigen may comprise a consensus protein derived from the amino acid sequence of core proteins from multiple HBV genotypes. Similarly, the HBV surface antigen may comprise a consensus protein derived from the amino acid sequence of surface antigens from multiple HBV genotypes. Such consensus HBV core protein and consensus HBV surface antigen are unique and have similarity with core and surface antigens, respectively, across multiple HBV genotypes. Thus, the vaccine of the present invention is broadly applicable to multiple types of HBV and useful for a large population. Furthermore, the vaccine of the present invention may be tailored to a specific nucleic acid encoding consensus HBV core protein, consensus HBV surface protein, or a combination thereof. In other words, the vaccines of the present invention can be designed to control the level or strength of the immune response in a subject against one or more HBV serotypes.

[0073] Vaccine can be DNA vaccine.DNA vaccine is disclosed in U.S. Patent No. 5,593,972, U.S. Patent No. 5,739,118, U.S. Patent No. 5,817,637, U.S. Patent No. 5,830,876, U.S. Patent No. 5,962,428, U.S. Patent No. 5,981,505, U.S. Patent No. 5,580,859, U.S. Patent No. 5,703,055 and U.S. Patent No. 5,676,594, which are fully incorporated herein by reference.DNA vaccine can further comprise an element or reagent that inhibits it from being integrated into chromosome.

[0074] The vaccine can be RNA of HBV core protein and / or HBV surface antigen protein. The RNA vaccine can be introduced into cells. a.HBV Core antigen

[0075] The vaccine of the present invention may comprise HBV core protein, which is a target for immune-mediated viral clearance by inducing 1) a cytotoxic T lymphocyte (CTL) response, 2) a T helper cell response, and / or 3) a B cell response, or preferably all of the above, for cross-presentation.

[0076] Table 2 shows the genotypic similarity of the core antigens of HBV-A, HBV-B, HBV-C, HBV-D, and HBV-E genotypes with the consensus HBV core protein, referred to in the table as "HBV-M Core". In some embodiments, HBV M Core constructs were designed to have increased homology to a broad range of HBV core targets. Genotypic similarity of Core Antigen with M-Core constructs designed to have increased homology to a broad range of HBV core targets. All genotypes should be represented in a universal immunotherapeutic vaccine for HBV. [Table 2]

[0077] The antigen may comprise a core protein epitope that makes it particularly effective as an immunogen against which an anti-HBV immune response can be induced. The HBV antigen may comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof.

[0078] HBV core antigen can comprise consensus protein.HBV consensus core antigen induces antigen-specific T cell and high titer antibody response both systemically and in liver.Therefore, vaccines that comprise HBV consensus core antigen provide protective immune response to liver.As a result, any user can design the vaccine of the present invention to comprise HBV consensus core antigen to provide immune system therapy and / or protection to liver.

[0079] In particular, in the immune response induced, HBV consensus core antigen stimulates spleen cells, specifically CD4+ cells and CD8+ cells, to secrete or produce the same amount of interferon-γ (INF-γ), but secrete or produce different amounts of tumor necrosis factor-α (TNF-α). Interestingly, the immune response induced is different in the spleen and liver. In the spleen, more CD8+ cells than CD4+ cells produce both INF-γ and TNF-α, but in the liver, the opposite occurs. That is, in the liver, more CD4+ cells than CD8+ cells produce both INF-γ and TNF-α. Furthermore, in the liver, more antigen-specific IgA is produced than IgG, and the protective immune response surprisingly and unexpectedly includes an antigen-specific CTL response that does not cause liver damage. As a result, the vaccine of the present invention containing an HBV consensus core antigen can be delivered into the periphery, establishing antigen specificity that targets the liver, wiping out or eliminating HBV-infected cells without causing liver damage or inflammation.

[0080] The nucleic acid sequences encoding the consensus HBV core protein are SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. SEQ ID NO:1 encodes the HBV consensus core protein. SEQ ID NO:3 encodes the HBV consensus core protein linked to the IgE leader. SEQ ID NO:5 encodes the HBV consensus core protein linked to the IgE leader and HA tag.

[0081] Some embodiments relate to nucleic acid sequences that code for proteins identical or homologous to HBV consensus proteins, immunogenic fragments of HBV consensus proteins, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that code for immunogenic proteins with up to 95% identity over the entire length of the amino acid sequence of the consensus sequence, up to 96% identity over the entire length of the amino acid sequence of the consensus sequence, up to 96% identity over the entire length of the amino acid sequence of the consensus sequence, up to 97% identity over the entire length of the amino acid sequence of the consensus sequence, up to 98% identity over the entire length of the amino acid sequence of the consensus sequence, and up to 99% identity over the entire length of the amino acid sequence of the consensus sequence. Similarly, nucleic acid sequences that code for immunogenic fragments as shown herein and immunogenic fragments of proteins identical to the proteins as shown herein are also provided.

[0082] Some embodiments relate to nucleic acid molecules that encode immunogenic proteins having 95% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules that encode immunogenic proteins having 96% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules that encode immunogenic proteins having 97% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules that encode immunogenic proteins having 98% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules that encode immunogenic proteins having 99% homology to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to a coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0083] Some embodiments relate to proteins having a specific percent identity over the entire length of the amino acid sequence of the full-length HBV core consensus protein, immunogenic fragments of the HBV core consensus protein, and nucleic acid sequences encoding immunogenic fragments of the HBV core consensus protein, such as up to 80% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 85% identity over the entire length of the amino acid sequence of the full-length consensus sequence, up to 90% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 91% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 92% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 93% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 94% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 95% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 96% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 97% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 98% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 99% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 100% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 101% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 102% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 103% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence Such nucleic acid molecules may be provided that encode immunogenic proteins having up to 94% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 95% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 96% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 97% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 98% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence, up to 99% identity over the entire length of the amino acid sequence of the full-length HBV core consensus sequence. Similarly, nucleic acid sequences are provided that encode immunogenic fragments as set forth herein and immunogenic fragments of proteins having similar percent identities as set forth above for the HBV core protein as set forth herein.

[0084] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 80% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 83% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 85% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 90% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 91% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 92% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 93% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 94% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 95% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 96% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 97% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein.Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 98% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 99% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV core protein coding sequence herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to a coding sequence of a consensus HBV core protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0085] In some embodiments, the nucleic acid sequence does not include a coding sequence that encodes a leader sequence.In some embodiments, the nucleic acid sequence does not include a coding sequence that encodes an IgE leader.

[0086] Some embodiments relate to fragments of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to fragments of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to fragments of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. The fragments can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to fragments of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. In some embodiments, the fragments include sequences encoding a leader sequence, such as, for example, an immunoglobulin leader, such as, for example, an IgE leader. In some embodiments, the fragments do not include coding sequences encoding a leader sequence. In some embodiments, the fragments do not include coding sequences encoding a leader sequence, such as, for example, an IgE leader.

[0087] Further, the amino acid sequence of the consensus HBV core protein is SEQ ID NO:2. The amino acid sequence of the consensus HBV core protein linked to the IgE leader is SEQ ID NO:4. In particular, in SEQ ID NO:4, the IgE leader is linked to the amino terminus of the consensus HBV core protein. The amino acid sequence of the consensus HBV core protein linked to the IgE leader and HA tag is SEQ ID NO:6. In SEQ ID NO:6, the IgE leader and HA tag are linked to the amino and carboxy termini of the consensus HBV core protein, respectively.

[0088] Some embodiments relate to proteins that are homologous to SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Some embodiments relate to immunogenic proteins that have 95% homology to the consensus protein sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins that have 96% homology to the consensus protein sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins that have 97% homology to the consensus protein sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins that have 98% homology to the consensus protein sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins that have 99% homology to the consensus protein sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0089] Some embodiments relate to a protein that is identical to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0090] In some embodiments, the protein does not include a leader sequence. In some embodiments, the protein does not include an IgE leader. A fragment of a consensus protein can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. Immunogenic fragments of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 may be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In some embodiments, the fragment comprises a leader sequence, such as, for example, an immunoglobulin leader, such as, for example, an IgE leader. In some embodiments, the fragment does not comprise a leader sequence. In some embodiments, the fragment does not comprise a leader sequence, such as, for example, an IgE leader.

[0091] Immunogenic fragments of proteins having amino acid sequence homology to immunogenic fragments of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 may be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% homologous to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, such as an immunoglobulin leader, e.g., an IgE leader. In some embodiments, the fragments do not include a leader sequence. In some embodiments, the fragments do not include a leader sequence, e.g., an IgE leader.

[0092] Immunogenic fragments of proteins having amino acid sequences identical to immunogenic fragments of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 may be provided. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over the entire length of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In some embodiments, the fragment comprises a leader sequence, such as an immunoglobulin leader, e.g., an IgE leader. In some embodiments, the fragment does not include a leader sequence. In some embodiments, the fragment does not include a leader sequence, such as, for example, an IgE leader.

[0093] With respect to linking a signal peptide or leader sequence to the N-terminus of a protein, as referred to herein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, which then encodes a protein that does not have a signal peptide coding sequence. Thus, for example, SEQ ID NO:4 is SEQ ID NO:2 with a signal peptide / leader sequence linked to the N-terminus of SEQ ID NO:2, i.e., SEQ ID NO:4 is a protein that includes a signal peptide linked to the N-terminus of SEQ ID NO:2. The first residue "Xaa" of SEQ ID NO:2 is typically a methionine if no signal peptide is present. However, a protein that includes a signal peptide linked to SEQ ID NO:2, such as SEQ ID NO:4, replaces the methionine at residue 1 at Xaa with a residue that links the signal peptide to the protein. Thus, the N-terminal residue of SEQ ID NO:2 can be anything, but if it is encoded by the start sequence, it is a methionine. The attachment of a signal peptide / leader sequence to the N-terminus of SEQ ID NO:2 typically excludes the N-terminal methionine. As used herein, SEQ ID NO:4 is intended to include SEQ ID NO:2 with a signal peptide / leader sequence linked to the N-terminus of SEQ ID NO:2, even though the N-terminal Xaa residue of SEQ ID NO:2 has been eliminated. Similarly, the coding sequence of SEQ ID NO:4 includes the coding sequence of SEQ ID NO:2 with a signal peptide / leader sequence coding sequence linked to the 5' end of the coding sequence encoding SEQ ID NO:2. The start codon may be "nnn" in the coding sequence of SEQ ID NO:2, but is eliminated when the signal peptide / leader sequence coding sequence is linked to the 5' end of the coding sequence encoding SEQ ID NO:2. As used herein, the coding sequence of SEQ ID NO:4 is intended to include the coding sequence of SEQ ID NO:2 with a signal peptide / leader sequence coding sequence linked to the 5' end of the coding sequence of SEQ ID NO:2 where nnn occurs. Thus, for example, SEQ ID NO:3 is intended to include SEQ ID NO:1 with a signal peptide / leader sequence coding sequence linked to the 5' end of SEQ ID NO:1 instead of nnn. In some embodiments, nnn is the start codon at the 5' end of SEQ ID NO:1. b.HBV surface antigen

[0094] The vaccine may comprise an HBV surface antigen. Provided herein is an HBV surface antigen that can induce an immune response against one or more HBV serotypes in a mammal. The surface antigen may comprise a surface protein epitope that is particularly effective as an immunogen that can induce an anti-HBV immune response. The HBV surface antigen may comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof.

[0095] HBV surface antigens may include consensus proteins. The consensus HBV surface antigen was generated from the sequences of surface antigens from primary isolates from either HBV genotype A or C. Specifically, the consensus HBV surface antigen includes the S protein or a combination of the S protein, pre-S2, and pre-S1 (Figures 3 and 4). An intracellular protein cleavage site was introduced into the consensus HBV surface antigen to provide proper protein folding and better CTL processing. The codon usage in the consensus HBV surface antigen was modified to reflect the codon bias of human genes. Additionally, regions of very high (e.g., greater than 80 percent) or very low (e.g., less than 30 percent) GC content, cis-acting motifs such as internal TATA boxes, repeat sequences, and structured sequences, were avoided. A Kozak sequence was introduced into the consensus HBV surface antigen to increase translation initiation, and an IgE leader sequence was added to increase protein expression.

[0096] Nucleic acid sequences encoding consensus HBV surface antigens are SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15. SEQ ID NO:9 is derived from genotype A and encodes a long consensus HBV surface antigen protein comprising S protein, pre-S2, and pre-S1 (LHBs-A). SEQ ID NO:11 is derived from genotype C and encodes a long consensus HBV surface antigen protein comprising S protein, pre-S2, and pre-S1 (LHBs-C). SEQ ID NO:13 is derived from genotype A and encodes a short consensus HBV surface antigen protein comprising S protein (SHBs-A). SEQ ID NO:15 is derived from genotype C and encodes a short consensus HBV surface antigen protein comprising S protein (SHBs-C).

[0097] Some embodiments relate to nucleic acid sequences encoding proteins identical to the entire amino acid sequence of the consensus HBV surface antigen, immunogenic fragments of the consensus HBV surface antigen, and immunogenic fragments of the identical proteins. Thus, nucleic acid molecules may be provided that encode immunogenic proteins having up to 95% identity over the entire amino acid sequence of the consensus HBV surface antigen sequence, up to 96% identity over the entire amino acid sequence of the consensus HBV surface antigen sequence, up to 96% identity over the entire amino acid sequence of the consensus HBV surface antigen sequence, up to 97% identity over the entire amino acid sequence of the consensus HBV surface antigen sequence, up to 98% identity over the entire amino acid sequence of the consensus HBV surface antigen sequence, and up to 99% identity over the entire amino acid sequence of the consensus HBV surface antigen sequence. Similarly, nucleic acid sequences are provided that encode immunogenic fragments as presented herein and immunogenic fragments of proteins identical to the entire amino acid sequence of the HBV surface antigen protein as presented herein.

[0098] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 95% identity over the entire length of the amino acid sequence of a nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 96% identity over the entire length of the amino acid sequence of a nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 97% identity over the entire length of the amino acid sequence of a nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 98% identity over the entire length of the amino acid sequence of a nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 99% identity over the entire length of the amino acid sequence of a nucleic acid HBV surface antigen coding sequence herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is identical over the entire length of the amino acid sequence of a consensus HBV surface antigen protein coding sequence disclosed herein includes a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the identical protein sequence disclosed herein.

[0099] Some embodiments relate to proteins having a particular percent identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus protein, immunogenic fragments of the HBV surface antigen consensus protein, and nucleic acid sequences encoding immunogenic fragments of the HBV surface antigen consensus protein, such as up to 80% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 85% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 90% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 91% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 92% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 93% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 94% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 95% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 96% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 97% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 98% identity over the entire length of the amino acid sequence of the full length HBV surface antigen consensus sequence, up to 9 ... Such nucleic acid molecules may be provided that encode immunogenic proteins having up to 94% identity over the entire length of the amino acid sequence of the full-length HBV surface antigen consensus sequence, up to 95% identity over the entire length of the amino acid sequence of the full-length HBV surface antigen consensus sequence, up to 96% identity over the entire length of the amino acid sequence of the full-length HBV surface antigen consensus sequence, up to 97% identity over the entire length of the amino acid sequence of the full-length HBV surface antigen consensus sequence, up to 98% identity over the entire length of the amino acid sequence of the full-length HBV surface antigen consensus sequence, up to 99% identity over the entire length of the amino acid sequence of the full-length HBV surface antigen consensus sequence. Similarly, nucleic acid sequences are provided that encode immunogenic fragments of the proteins as set forth herein and having similar percent identities as set forth above for the HBV surface antigen proteins as set forth herein.

[0100] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 80% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 83% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 85% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 90% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 91% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 92% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 93% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 94% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 95% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 96% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 97% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 98% identity over the entire length of the amino acid sequence of the full-length nucleic acid HBV surface antigen coding sequence herein.Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that have 99% identity over the entire length of the amino acid sequence of a full-length nucleic acid HBV surface antigen coding sequence herein. In some embodiments, a nucleic acid molecule having an encoded HBV surface antigen sequence disclosed herein that is identical over the entire length of the amino acid sequence of a consensus HBV surface antigen protein coding sequence disclosed herein includes a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence that encodes the identical protein sequence disclosed herein.

[0101] In some embodiments, the nucleic acid sequence does not include a coding sequence that encodes a leader sequence.In some embodiments, the nucleic acid sequence does not include a coding sequence that encodes an IgE leader.

[0102] Some embodiments relate to fragments of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15. The fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. The fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. The fragment may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. In some embodiments, the fragment includes a sequence encoding a leader sequence, such as an immunoglobulin leader, e.g., an IgE leader. In some embodiments, the fragment does not include a coding sequence encoding a leader sequence. In some embodiments, the fragment does not include a coding sequence encoding a leader sequence, such as an IgE leader.

[0103] Further, the amino acid sequences of the consensus HBV surface antigens, LHBs-A, LHBs-C, SHBs-A, and SHBs-C, are SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively.

[0104] The protein may be identical to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having 95% identity over the entire length of the amino acid sequence of the consensus protein sequence herein. Some embodiments relate to immunogenic proteins having 96% identity over the entire length of the amino acid sequence of the consensus protein sequence herein. Some embodiments relate to immunogenic proteins having 97% identity over the entire length of the amino acid sequence of the consensus protein sequence herein. Some embodiments relate to immunogenic proteins having 98% identity over the entire length of the amino acid sequence of the consensus protein sequence herein. Some embodiments relate to immunogenic proteins having 99% identity over the entire length of the amino acid sequence of the consensus protein sequence herein.

[0105] Some embodiments relate to proteins that are identical to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins that have an amino acid sequence that is 80% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins that have an amino acid sequence that is 85% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins that have an amino acid sequence that is 90% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins that have an amino acid sequence that is 91% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical over the entire length of the amino acid sequence of the full-length consensus amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

[0106] Fragments of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. Immunogenic fragments of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

[0107] Immunogenic fragments of proteins may be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16. Such immunogenic fragments may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.

[0108] Immunogenic fragments of proteins having amino acid sequences identical to immunogenic fragments of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 may be provided. Such immunogenic fragments may include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Some embodiments relate to immunogenic fragments having 96% identity to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% identity to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% identity to the immunogenic fragments of the consensus protein sequences herein.Some embodiments relate to immunogenic fragments having 99% identity to the immunogenic fragments of the consensus protein sequences herein. C. HBV core and surface antigen combination

[0109] The vaccine may comprise a combination of the HBV core and surface antigens described above. This combination of HBV antigens can induce an immune response against one or more HBV serotypes in a mammal. The vaccine may be designed or tailored to have a specific combination of HBV antigens, which provides the ability to control the level or strength of the immune response in a mammal.

[0110] The combinations were: (1) HBV-M-Core, LHBs-A, SHBs-A, LHBs-C, and SHBs-C; (2) HBV-M-Core, LHBs-A, SHBs-A, and LHBs-C; (3) HBV-M-Core, LHBs-A, and SHBs-A; (4) HBV-M-Core and LHBs-A; (5) HBV-M-Core, SHBs-A, LHBs-C, and SHBs-C; (6) HBV-M-Core , LHBs-C, and SHBs-C, (7) HBV-M-Core and SHBs-C, (8) HBV-M-Core, LHBs-A, LHBs-C, and SHBs-C, (9) HBV-M-core, LHBs-A, and SHBs-C, (10) HBV-M-Core and SHBs-C, (11) HBV-M-Core, LHBs-A, SHBs-A, and SHBs-C, (12) HBV-M-Core, LHBs-A, and SHBs-C, (13) HBV-M-Core, SHBs-A, and SHBs-C, (14) LHBs-A, SHBs-A, LHBs-C, and SHBs-C, (15) LHBs-A, SHBs-A, and LHBs-C, (16) LHBs-A and SHBs-A, (17) SHBs-A, LHBs-C, and SHBs-C, (18) LHBs-C and SHBs-C, (19) LHBs-A, LHBs-C, and S The nucleic acid sequence may include one or more nucleic acids encoding HBs-C, (20) LHBs-A and SHBs-C, (21) LHBs-A, SHBs-A, and SHBs-C, (22) LHBs-A and SHBs-C, (23) SHBs-A and SHBs-C, (24) HBV-M-Core and SHBs-A, (25) HBV-M-Core, LHBs-A, and LHBs-C, or (26) LHBs-A and LHBs-C.

[0111] An exemplary embodiment relates to a vaccine comprising one or more nucleic acids encoding HBV-M-Core, LHBs-A, and LHBs-C. Another exemplary embodiment relates to a vaccine comprising one or more nucleic acids encoding HBV-M-Core, SHBs-A, and SHBs-C. Yet another exemplary embodiment relates to a vaccine comprising one or more nucleic acids encoding HBV-M-Core, LHBs-A, and LHBs-C, and an adjuvant such as IL-12.

[0112] Combination vaccines also include one or more consensus HBV core proteins and / or HBV surface antigen proteins in the form of one or more protein subunits, one or more killed viral particles containing one or more consensus HBV core proteins and / or consensus HBV surface antigen proteins, or one or more attenuated viral particles containing one or more consensus HBV core proteins and / or HBV surface antigen proteins. Attenuated vaccines can be live attenuated vaccines, killed vaccines, and vaccines that use recombinant vectors to deliver foreign genes encoding one or more consensus HBV core proteins and / or HBV surface antigen proteins, as well as subunit and glycoprotein vaccines. Examples of live attenuated vaccines, those using recombinant vectors to deliver foreign antigens, subunit vaccines, and saccharide vaccines are described in U.S. Pat. Nos. 4,510,245, 4,797,368, 4,722,848, 4,790,987, 4,920,209, 5,017,487, 5,077,044, 5,110,587, 5,112,749, 5,174,993, 5,223,424, 5,225,336, 5,240,703, 5,242,829, 5,294,441, 5,294,548, 5,310,66 No. 8, No. 5,387,744, No. 5,389,368, No. 5,424,065, No. 5,451,499, No. 5,453,364, No. No. 5,462,734, No. 5,470,734, No. 5,474,935, No. 5,482,713, No. 5,591,439, No. 5,6 Nos. 43,579, 5,650,309, 5,698,202, 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is incorporated herein by reference. d. Vaccine Constructs and Plasmids

[0113] The vaccine may include a nucleic acid construct or plasmid encoding HBV core protein, HBV surface antigen, and HBV core protein / surface antigen combination. Provided herein is a genetic construct that can include a nucleic acid sequence encoding the disclosed HBV core antigen, including consensus protein sequences, sequences homologous to consensus protein sequences, fragments of consensus protein sequences, and sequences homologous to fragments of consensus protein sequences. Further provided herein is a genetic construct that can include a nucleic acid sequence encoding the disclosed HBV surface antigen, including consensus protein sequences, sequences homologous to consensus protein sequences, fragments of consensus protein sequences, and sequences homologous to fragments of consensus protein sequences. The genetic construct can be present in a cell as a functional extrachromosomal molecule. The genetic construct can be a centromere, a telomere, or a linear microchromosome, including a plasmid or cosmid.

[0114] The genetic construct may also be part of the genome of a recombinant viral vector, including recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant vaccines. The genetic construct may be part of the genetic material of a live attenuated microorganism or a recombinant microbial vector that lives intracellularly.

[0115] The genetic construct can include regulatory elements for gene expression of the coding sequence of the nucleic acid. The regulatory elements can be promoters, enhancers, initiation codons, stop codons, or polyadenylation signals.

[0116] The nucleic acid sequence may constitute a genetic construct that may be a vector. The vector may express the antigen in the cells of a mammal in an amount effective to elicit an immune response in the mammal. The vector may be recombinant. The vector may contain a heterologous nucleic acid encoding the antigen. The vector may be a plasmid. The vector may be useful for transfecting cells with a nucleic acid encoding the antigen, and the transformed host cells are cultured and maintained under conditions that result in expression of the antigen.

[0117] The coding sequence can be optimized for stability and high levels of expression. In some cases, codons are selected to reduce secondary structure formation in RNA, such as those formed by intramolecular bonds.

[0118] The vector can include a heterologous nucleic acid encoding an antigen, and can further include an initiation codon that can be upstream of the antigen coding sequence, and a stop codon that can be downstream of the antigen coding sequence. The initiation and stop codons can be in frame with the antigen coding sequence. The vector can also include a promoter operably linked to the antigen coding sequence. The promoter operably linked to the antigen coding sequence can be a human immunodeficiency virus (HIV) promoter, such as a Simian Virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a natural or synthetic muscle- or skin-specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. 20040175727, the contents of which are incorporated herein by reference in their entirety.

[0119] The vector can also include a polyadenylation signal that can be downstream of the HBV core protein coding sequence. The polyadenylation signal can be the SV40 polyadenylation signal, the LTR polyadenylation signal, the bovine growth hormone (bGH) polyadenylation signal, the human growth hormone (hGH) polyadenylation signal, or the human β-globulin polyadenylation signal. The SV40 polyadenylation signal can be the polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, CA).

[0120] The vector can also contain an enhancer upstream of the consensus HBV core protein coding sequence or the consensus HBV surface antigen protein coding sequence. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as one of CMV, HA, RSV, or EBV. Enhancement of polynucleotide function is described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94 / 016737, the contents of each of which are fully incorporated by reference.

[0121] The vector can also contain a mammalian origin of replication to maintain the vector extrachromosomal and produce multiple copies of the vector within a cell. The vector can be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which can contain an origin of replication for Epstein-Barr virus, and a nuclear antigen EBNA-1 coding region that can result in high copy episomal replication without integration. The vector can be pVAX1, or a pVax1 variant with changes such as the variant plasmids described herein. The variant pVax1 plasmid is a 2998 base pair variant of the backbone vector plasmid pVAX1 (Invitrogen, Carlsbad CA). The CMV promoter is located at bases 137-724. The T7 promoter / priming site is at bases 664-683. The multiple cloning site is at bases 696-811. The bovine GH polyadenylation signal is at bases 829-1053. The kanamycin resistance gene is at bases 1226 to 2020. The pUC origin is at bases 2320 to 2993.

[0122] Based on the sequence of pVAX1 available from Invitrogen, the following mutations were found in the sequence of pVAX1, which was used as the backbone for the set of plasmids 1-6 described herein:

[0123] C>G241 CMV promoter,

[0124] C>T1942 backbone, downstream of bovine growth hormone polyadenylation signal (bGHpolyA);

[0125] A>-2876 main chain, downstream of the kanamycin gene;

[0126] C>T3277 pUC origin of replication (origin of replication) high copy number mutant (see Nucleic Acid Research 1985),

[0127] G>C 3753, the very end of the pUC origin of replication upstream of the RNASeH site;

[0128] Base pairs 2, 3, and 4 are changed from ACT to CTG in the backbone upstream of the CMV promoter.

[0129] The vector backbone can be pAV0242. The vector can be a replication-deficient adenovirus type 5 (Ad5) vector.

[0130] The vector may also contain regulatory sequences that may be sufficient for gene expression in mammalian or human cells to which the vector is administered. The consensus HBV coding sequence may contain codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0131] The vector can be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). The vector can also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in Saccharomyces cerevisiae strains of yeast. The vector can also be the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The vector can also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. The vector can be an expression vector or system that produces proteins by routine techniques and readily available starting materials, including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated by reference in its entirety.

[0132] In some embodiments, the vector may comprise the nucleic acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. SEQ ID NO: 17 encodes the consensus HBV core protein, and SEQ ID NOs: 18-21 encode the consensus HBV surface antigen. Vector maps of SEQ ID NOs: 17-21 are shown in Figures 5-9, respectively. e. Pharmaceutical Composition of Vaccine

[0133] The vaccine may be in the form of a pharmaceutical composition. The pharmaceutical composition may comprise the vaccine.

[0134] The pharmaceutical composition may contain about 5 nanograms to about 10 mg of vaccine DNA. In some embodiments, the pharmaceutical composition according to the present invention contains about 25 nanograms to about 5 mg of vaccine DNA. In some embodiments, the pharmaceutical composition contains about 50 nanograms to about 1 mg of DNA. In some embodiments, the pharmaceutical composition contains about 0.1 to about 500 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 5 to about 250 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 10 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 15 to about 150 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 20 to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 25 to about 75 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 30 to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 35 to about 40 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 35 nanograms to about 45 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 compositions contain about 100 to about 200 micrograms of DNA.

[0135] In some embodiments, the pharmaceutical composition according to the present invention comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nanograms of DNA vaccine. 30, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 46 5, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800 , 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms of DNA vaccine. In some embodiments, the pharmaceutical composition can comprise at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg or more of DNA vaccine.

[0136] In other embodiments, the pharmaceutical composition can include up to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nanograms of DNA vaccine. In some embodiments, the pharmaceutical composition can include up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 35, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 47 0, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 80 In some embodiments, the pharmaceutical composition can comprise up to 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 1000 micrograms of DNA of the vaccine.

[0137] The pharmaceutical composition may further include other agents for formulation purposes according to the mode of administration used.When the pharmaceutical composition is an injectable pharmaceutical composition, it is sterile, pyrogen-free, and particle-free.Isotonic formulations are preferably used.Generally, additives for isotonicity 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, vasoconstrictors are added to the formulation.

[0138] The vaccine can further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a functional molecule as a vehicle, adjuvant, carrier, or diluent. The pharmaceutically acceptable excipient can be a transfection enhancer, which can include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection enhancers.

[0139] The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The transfection facilitating agent is poly-L-glutamic acid, and more preferably, the poly-L-glutamic acid is present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent may include detergents such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be used for administration with the gene construct. In some embodiments, the DNA vector vaccine may also include transfection facilitating agents such as liposomes, including lipids, lectin liposomes, or other liposomes known in the art such as DNA liposome mixtures (see, for example, International Publication No. WO 09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0140] The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be another gene that is expressed in an alternative plasmid or delivered as a protein combined with the above-mentioned plasmid in the vaccine. The adjuvant is selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell attack chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, D86 (including IL-15 that lacks the signal sequence from IgE and optionally includes a signal peptide). The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof. In an exemplary embodiment, the adjuvant is IL-12.

[0141] Other genes that may be useful adjuvants include MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutated forms of IL-18, CD40, CD40L, vascular proliferation Factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP These include those encoding K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof. f. Method of Vaccine Delivery

[0142] Provided herein are methods for delivering pharmaceutical formulations to provide genetic constructs and proteins of HBV core protein and / or HBV surface antigens that contain particularly effective immunogenic epitopes that can induce an immune response against HBV virus infection. Vaccine delivery or vaccination methods can be provided to induce therapeutic and / or prophylactic immune responses. Vaccination methods can generate immune responses in mammals against multiple HBV genotypes. Vaccines can be delivered to individuals to modulate the activity of the mammal's immune system and enhance the immune response. Vaccine delivery can be transfection of HA antigen as a nucleic acid molecule that is expressed in cells and delivered to the surface of cells where it is recognized by the immune system and induces a cellular, humoral, or cellular and humoral response. Vaccine delivery can be used to administer the vaccines discussed herein to mammals to induce and induce an immune response in mammals against multiple HBV viruses.

[0143] By delivering the vaccine to a mammal, and thus the vector to the cells of the mammal, the transfected cells express and secrete consensus HBV core protein and consensus HBV surface antigen. These secreted proteins or synthetic antigens are recognized as foreign by the immune system and initiate an immune response that may include antibodies made against the antigen and T cell responses specific to the antigen. In some instances, a mammal vaccinated with the vaccine discussed herein has a primed immune system, and when challenged with an HBV virus strain, the primed immune system allows for the subsequent rapid clearance of the HBV virus, whether humoral, cellular, or both. The vaccine can be delivered to an individual to modulate the activity of the individual's immune system, thereby enhancing the immune response.

[0144] Methods for delivering vaccine DNA are described in US Pat. Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entireties.

[0145] The vaccine can be administered to a mammal to induce an immune response in the mammal, which can be a human, a non-human primate, a cow, a pig, a sheep, a goat, an antelope, a bison, a water buffalo, a bovine, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, or a chicken, preferably a human, a cow, a pig, or a chicken. g. Delivery of Vaccines with Adjuvants

[0146] The pharmaceutical compositions, preferably vaccines described herein, can be administered in combination with a protein or gene-encoded adjuvant, including, but not limited to, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, L FA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt , Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, or TAP2, or functional fragments thereof. In an exemplary embodiment, the adjuvant is IL-12. h. Methods of generating an immune response using a vaccine

[0147] The vaccine can be used to generate an immune response, including a therapeutic or prophylactic immune response, in a mammal. The immune response can generate antibodies and / or killer T cells directed against HBV core antigen, HBV surface antigen, or a combination thereof. Such antibodies and T cells can be isolated.

[0148] Some embodiments provide a method of generating an immune response against HBV core protein, HBV surface antigen protein, and combinations thereof, comprising administering a vaccine to an individual. Some embodiments include a method of prophylactically vaccinating an individual against HBV infection, comprising administering a vaccine. Some embodiments provide a method of therapeutically vaccinating an individual infected with HBV, comprising administering a vaccine. Diagnosis of HBV infection before administering a vaccine can be routinely performed. i. Vaccine-based treatment methods

[0149] The vaccine can be used to generate immune responses in mammals that protect the liver. The immune response can generate antigen-specific CTL responses that do not cause liver damage or liver inflammation. In some embodiments, the vaccine can be delivered to the periphery to establish an antigen-specific immune response that targets the liver and can wipe out or eliminate HBV-infected cells without causing liver damage or liver inflammation. In some embodiments, the treatment includes delivery of a vaccine that includes an HBV consensus core antigen to the periphery to establish an antigen-specific immune response that targets the liver and can wipe out or eliminate HBV-infected cells without causing liver damage or liver inflammation. 3. Route of Administration

[0150] The vaccine or pharmaceutical composition can be administered by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or a combination thereof. For veterinary use, the composition can be administered in a formulation that is appropriately acceptable for normal veterinary practice. Veterinarians can easily determine the administration regimen and route of administration that is most suitable for a particular animal. The vaccine can be administered by traditional syringes, needleless injection devices, "microprojectile guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0151] Vaccine vectors can be delivered to mammals by several well-known techniques, including DNA injection with and without in vivo electroporation (also called DNA vaccination), liposome-mediated, nanoparticle-facilitated recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. HBV antigens can be delivered via DNA injection and in conjunction with in vivo electroporation. A electroporation

[0152] The vaccine or pharmaceutical composition may be administered by electroporation. Administration of the vaccine via electroporation may be accomplished using an electroporation device that may be configured to deliver a pulse of energy to mammalian tissue effective to form reversible pores in cell membranes, preferably the pulse of energy is a constant current similar to the current input preset by the user. The electroporation device may include an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include and incorporate one or more of the various elements of the electroporation device, including a control device, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory device component, a power source, and a power switch. Electroporation may be achieved by using an in vivo electroporation device, such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.), to facilitate transfection of cells with plasmids.

[0153] Examples of electroporation devices and electroporation methods that can enhance delivery of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al. and U.S. Patent Publication No. 2005 / 0052630 by Smith et al., the contents of which are incorporated herein by reference in their entireties. Other electroporation devices and electroporation methods that can be used to enhance delivery of DNA vaccines include those provided in co-pending and co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, which are incorporated herein by reference in their entireties.

[0154] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes modular electrode systems and their use for facilitating the introduction of biomolecules into cells of selected tissues in a body or plant. The modular electrode system can include a plurality of needle electrodes, a hypodermic needle, an electrical connector that provides conductive ink from a programmable constant current pulse controller to the plurality of needle electrodes, and a power source. An operator can grasp the plurality of needle electrodes loaded on a support structure and firmly insert them into a selected tissue of a body or plant. The biomolecules are then delivered through the hypodermic needle into the selected tissue. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse promotes the introduction of the biomolecules into cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0155] US Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device used to effectively facilitate the introduction of biomolecules into cells of selected tissues within a body or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between an electrode array based on user control and input of pulse parameters, and allows for storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of US Patent Publication No. 2005 / 0052630 are incorporated herein by reference.

[0156] The electrode array and method described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 can be adapted to penetrate deep into tissues such as muscle, as well as other tissues or organs. Due to the configuration of the electrode array, the injection needle (delivering the selected biomolecule) is also inserted completely into the target organ, and the injection is applied perpendicular to the target tissue in the area pre-delineated by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 005263 are preferably 20 mm in length and 21 in gauge.

[0157] In addition, there are electroporation devices described in the following patents, as contemplated in some embodiments incorporating electroporation devices and their use: U.S. Patent No. 5,273,525 issued December 28, 1993, U.S. Patent No. 6,110,161 issued August 29, 2000, U.S. Patent No. 6,261,281 issued July 17, 2001, and U.S. Patent No. 6,958,060 issued October 25, 2005, and U.S. Patent No. 6,939,862 issued September 6, 2005. Additionally, patents covering subject matter provided in U.S. Patent No. 6,697,669 issued February 24, 2004, which relates to the delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064 issued February 5, 2008, which describes a method of injecting DNA, are contemplated herein. The above patents are incorporated by reference in their entirety. 4. Methods for preparing the vaccine

[0158] Provided herein are methods for preparing DNA plasmids that contain the vaccines discussed herein, which, after a final subcloning step into a mammalian expression plasmid, can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0159] The DNA plasmids used in the EP devices of the present invention can be formulated and manufactured using a combination of known equipment and techniques, but preferably they are manufactured using the optimized plasmid manufacturing techniques described in U.S. Published Application No. 20090004716, filed May 23, 2007. In some instances, the DNA plasmids used in these studies can be formulated at concentrations of 10 mg / mL or greater. The manufacturing techniques also include or incorporate a variety of equipment and protocols generally known to those of skill in the art in addition to those described in U.S. Patent Application No. 60 / 939,792, including those described in covenant patent U.S. Patent No. 7,238,522, filed July 3, 2007. The above referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are each incorporated herein in their entirety. EXAMPLES

[0160] The present invention will be further described in the following examples. It should be understood that these examples, although showing preferred embodiments of the present invention, are presented for illustrative purposes only. From the above discussion and examples, those skilled in the art can ascertain the essential characteristics of the present invention, and can make various changes and modifications of the present invention to suit various applications and conditions without departing from the spirit and scope of the present invention. Therefore, in addition to those shown and described herein, various modifications of the present invention will be 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. Example 1 HBV core protein

[0161] A consensus HBV core protein, also referred to as HBV-modified MCore or M-core construct, was designed from epitope sequences of HBV genotypes A, B, C, D, and E. HBV core protein sequences from these genotypes were selected for inclusion in the construction of a consensus core that induces immunity against a wide range of genotypes and thus provides a universal vaccine for HBV. In some embodiments, the modification of the M core construct included the addition of an IgE leader sequence. In some embodiments, the M core protein was encoded using codon optimization and RNA optimization for enhanced expression. 1. Construction and Expression of Consensus Core Antigens

[0162] HBV genotypes A, B, C, D, and E Core consensus nucleotide sequences were constructed by generating consensus sequences of the core genes of each genotype, and then generating consensus sequences of all five genotype consensuses, thus avoiding bias toward severe contiguous genotypes. Furthermore, sequences were taken from different countries to avoid sampling bias toward severe contiguous genotypes. To develop the final HBcAg consensus sequence, the sequences were aligned using ClustalX software. As shown in Figure 10, the relative proximity of the multi-genotype consensus HBcAg sequence to all sample sequences from different genotypes was observed.

[0163] After generating the consensus sequence, some modifications were made to increase the antigen expression level from the plasmid. Specifically, a highly efficient IgE leader sequence and a C-terminal HA tag were added, and the construct was RNA-optimized and codon-optimized. This resulted in a nucleic acid sequence encoding the M-core sequence (SEQ ID NO: 5) with an IgE leader and HA Tag, which was digested with EcoRI and NotI and cloned into the expression vector pVAX (Invitrogen) under the control of the cytomegalovirus-mediated early promoter. The resulting construct was then named pMCore (SEQ ID NO: 17).

[0164] In vitro expression studies were completed with pMCore constructs and pVAX used as controls. Results showing positive expression are depicted in the gel images shown in Figures 11A and 11B.

[0165] In addition, HBcAg protein was expressed by transfecting pMCore DNA plasmid containing the Hepatitis B core gene (FIG. 12). 35 Expression of pMCore was detected using the TNT® Quick Coupled Expression of Transcription / Translation (Promega, Madison, WI) System containing S-methionine. The synthesized gene product was immunoprecipitated using an anti-HA monoclonal antibody targeting the encoded HA epitope, Clone HA-7 (Sigma-Aldrich). The immunoprecipitated proteins were electrophoresed on a 12% SDS-PAGE gel, followed by fixation and drying. Radioactive 35 Proteins synthesized upon incorporation of S were detected by autoradiography. In vitro translation assays on this lysate showed detectable HBcAg with the expected molecular weight of 28 kDa (Fig. 13).

[0166] Expression was further confirmed using anti-HA tagged monoclonal antibody by immunofluorescence assay. Rhabdomyosarcoma (RD) cell line was transfected with pMCore using TURBOFECT (Thermo Scientific) according to the manufacturer's guidelines. First, cells were fixed with 2% formaldehyde and then assayed for protein expression. Fixed cells were incubated with rabbit monoclonal HA tag (Invitrogen) diluted in "primary standard solution" (0.1% BSA, 0.2% saponin, 0.02% sodium azide) for 1 hour at room temperature. Subsequently, cells were incubated with DyLight 594 labeled anti-rabbit secondary antibody (Thermo Scientific) for 20 minutes at room temperature. Confocal imaging analysis was used to visualize HBcAg around the cytoplasm and nucleus of transfected RD cells, as shown in FIG. 14. Expression patterns confirmed that DNA plasmids carrying consensus core genes were highly expressed in different cells in vitro. Specifically, images were acquired using a Zeiss Axiovert 100 inverted confocal microscope, and flowering intensity was analyzed and quantified using Image J software (NIH, Rockville, MD). 2. Immunization of Mice

[0167] C57BL / 6 transgenic mice were divided into two groups of four mice each and used for three electroporation immunizations with 20 μg of DNA at biweekly intervals (group 1-pVAX vector control, group 2 pM-Core). Mice were immunized on days 0, 14, and 28 and sacrificed on day 35. Spleens, livers, and serum were collected from sacrificed animals.

[0168] Additionally, to assess the generation of T and B cell immune responses, Balb / c mice were immunized and measured for both responses in various peripheral tissues. As shown in the immunization scheme, mice received three intramuscular immunizations of 30 μg pMCore or pVax followed by electroporation (Figure 15). In particular, 6-8 week old female Balb / c mice were purchased from Jackson Laboratories. Animals were maintained in accordance with the Institutional Care and Use Committee (IACUC) guidelines of the National Institutes of Health and the University of Pennsylvania. For DNA immunization studies, eight animals were divided into two groups. Each animal in the immunization group received a total of three immunizations of 30 μg pMCore in the tibialis anterior (TA) muscle at 2-week intervals. Each immunization was accompanied by in vivo electroporation using a CELLECTRA adaptive constant current electroporation device (Inovio Pharmaceuticals, Blue Bell, PA). Two 0.2 Amp constant current square wave pulses were delivered through a triangular three-electrode array consisting of 26 gauge solid stainless steel electrodes inserted fully into the muscle, each pulse 52 ms long with a 1 s delay between pulses.

[0169] In vivo studies of Balb / C mouse strains showed increased magnitude of secretion of tumor necrosis factor (TNF-α), interferon gamma T cells (IFN-γ), and CD107a in CD8 and CD4 T cells from the spleen. Figures 16 and 17 show that vaccination of Balb / C mice with pM-Core increased the magnitude of IFN-γ secretion by CD8+ and CD4+ T cells from the spleen. Figures 18 and 19 show that vaccination of Balb / C mice with pM-Core increased the magnitude of TNF-α secretion by CD8+ and CD4+ T cells from the spleen. Figures 20 and 21 show that vaccination of Balb / C mice with pM-Core increased the magnitude of CD 107a secretion by CD8+ and CD4+ T cells from the spleen.

[0170] In further experiments, IFN-γ and TNF-α secretion in T cells was examined. To collect splenocytes for these further experiments, mice were sacrificed 1 week after the last immunization, and spleens were collected and placed in R10 medium (RPMI medium supplemented with 10% FBS and 1x Antibiotic-Antimycotic). Spleens were individually ground, strained through a 40μM cell strainer, and treated with 1mL of ACK lysis buffer for 5 minutes to lyse red blood cells. Splenocytes were resuspended in complete R10 medium and used for further immunological assays.

[0171] Spleen cell subsets were cultured at 10 7 The cells were resuspended in R10 medium at a concentration of 100 μL and seeded onto 96-well round-bottom plates. 100 μL of medium containing pMCore was pooled with peptide or 10 ng / ml PMA (Sigma, St. Louis, MO, USA) and 500 ng / ml ionomycin (Calbiochem, Novabiochem, La Jolla, CA, USA) mixture as a positive control or 0.1% dimethyl sulfoxide (Sigma, St. Louis, MO, USA) as a negative control. All wells contained 5 μL / mL of two protein transport inhibitors, brefeldin A (GolgiPlug) and monensin (Golgistop) (all from BD Bioscience). Cells were incubated at 37 °C, 5% CO 2 The cells were incubated in 500 mM NaCl for 5 h and stained with LIVE / DEAD Fixable Dead Cell Stain (Invitrogen) for 10 min at 37° C. Extracellular staining was performed using antibodies specific for mouse CD3, CD4, and CD8. Splenocytes were then permeabilized and washed using BD CYTOFIX / CYTOPERM and PERM / WASH (BD Bioscience), respectively.

[0172] Intracellular cytokine staining.

[0173] Intracellular cytokines were then stained with antibodies against mouse interferon-γ and tumor necrosis factor-α. Lymphocyte subsets were cultured at 10 7 The cells were resuspended in R10 medium at a concentration of 100 μL and seeded onto 96-well round-bottom plates. 100 μL of medium containing pMCore was pooled with peptide or 10 ng / ml PMA (Sigma, St. Louis, MO, USA) and 500 ng / ml ionomycin (Calbiochem, Novabiochem, La Jolla, CA, USA) mixture as a positive control or 0.1% dimethyl sulfoxide (Sigma, St. Louis, MO, USA) as a negative control. All wells contained 5 μL / mL of two protein transport inhibitors, brefeldin A (GolgiPlug) and monensin (Golgistop) (all from BD Bioscience). Cells were incubated at 37 °C, 5% CO 2 The cells were incubated in PBS for 5 hours and stained with LIVE / DEAD® Fixable Dead Cell Stain (Invitrogen) for 10 minutes at 37° C. Extracellular staining was performed using antibodies specific for mouse CD3, CD4, and CD8. Splenocytes were then permeabilized and washed using BD Cytofix / Cytoperm™ and Perm / Wash™ (BD Bioscience), respectively. Cells were then stained intracellularly with antibodies against interferon-γ and tumor necrosis factor-α, interleukin-2, and lysosome-associated membrane protein 1.

[0174] Conjugated anti-mouse antibodies were used during extracellular and intracellular staining, including CD3-Phycoerythrin / Cy7 (PE / Cy7), CD4-Peridinin chlorophyll protein (PerCP), CD8-Allophycocyanin (APC), IFN-γ-Alexa Fluor 700, TNF-α-Fluorescein isothiocyanate (FITC), and IL-2-Phycoerythryin cyanine (PE) (all from BD Biosciences, San Jose, CA).

[0175] The mean HBcAg-specific IFN-γ T cell response induced was potent at 2000 (±210) SFU per million splenocytes. Interestingly, intracellular staining of stimulated splenocytes was consistent with CD4 + and CD8 + Both CD4+ and CD8+ cells produced approximately the same amount of antigen-specific IFN-γ, 0.74 and 0.94, respectively. + and CD8 + Approximately 0.3% and 1.5% of cells were found to produce different levels of TNF-α (Figure 22). A similar trend was observed in cells that were double positive for both cytokines. In the spleen, double positive CD8 + Fewer than 0.2% of cells are double positive CD4 + Cells were present, averaging 0.7% (Figure 23).

[0176] HBV-specific T cell migration to the liver was also shown in animals administered pM-Core DNA vaccine. High frequency targeting of HBV core antigen-specific T cells and effector function to the liver is the goal of developing HBV immunotherapy. After immunization, animals were sacrificed, livers were removed, and HBV-specific effector T cell migration to the liver was determined. The results show that pM-Core vaccine drives effector T cells to the liver in vivo. Figures 24 and 25 show interferon-γ T cell liver response, and Figures 26 and 27 show tumor necrosis factor-α liver immune response, as well as the increased response resulting from vaccination with pM-Core.

[0177] The M-core consensus immunogen encoded by the pM-core DNA construct drives a potent and balanced CD4+ / CD8+ T-cell immune response. The induced T cells migrate to the liver at high frequency and display the correct effector phenotype for immune clearance after HBV infection, supporting the further development of this immunotherapeutic vaccine.

[0178] We also examined the cytokine producing capacity of intrahepatic antigen-specific T cells after DNA immunization. Each liver was perfused by injecting 1 mL of PBS directly into the hepatic vein of each mouse. Specifically, the liver was collected, pulverized, and resuspended in 5 mL of 44% isotonic Percoll. The mixture with 3 mL of 66% isotonic Percoll was based and centrifuged at 2000 rpm for 20 minutes for gradient separation. Lymphocytes were harvested, washed in 10 mL of R10, and treated with ACK lysis buffer, if necessary. Both CD4 and CD8 T cells isolated from liver produced IFN-γ and TNF-α when stimulated with HBcAg peptide in vitro (Figures 28 and 29). CD4 T cells showed a high percentage of dual producers, while CD8 showed little to no IFN-γ+TNF-α+ producers. Instead, the majority of CD8 T cells produced only IFN-γ or TNF-α. Abundant HBcAg-specific CD4 T cells were observed in the liver, which was opposite to that in the spleen. The HBcAg-specific CD4 T double positive percentage in the quiescent liver was comparable to that observed in the spleen. Moreover, peripheral CD8 T cells were confirmed to be better double producers than liver-resident CD8 T cells. The antibody-producing ability of liver-resident B cells from immunized mice was also observed. Interestingly, the liver, being a mucosal organ, produced higher antigen-specific IgA than IgG (Figure 30), an observation that has not been studied before.

[0179] Figure 31 shows the cellular immune response induced by pM-Core using an enzyme-linked immunosorbent spot (ELISPOT) assay. Splenocytes were stimulated with two pools of 15mer peptides spanning the full length of pMCore and overlapping with 8 amino acids. 200,000 splenocytes in R10 medium were plated on 96-well IFN-γ capture antibody (R&D system) coated plates and incubated at 5% CO in the presence of specific peptide pools. 2The cells were stimulated overnight at 37°C in 100% CO. The cells were washed and the plates were incubated overnight with biotinylated anti-mouse IFN-γ detection antibody (R&D system). Spots were developed using streptavidin-alkaline phosphatase, 5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt, and nitroblue tetrazolium chloride sequentially. Spots were counted using an automated ELISPOT reader (CTL Limited). As shown in Figure 31, immunization with pMCore could induce a strong cellular immune response. The average HBcAg-specific IFN-γ T cell response was about 2000 (±210) SFU per million splenocytes.

[0180] IFN-γ was further examined using an ELISPOT assay. Specifically, splenocytes were stimulated with two pools of 15mer peptides spanning the full length of HBcAg and overlapping with 8 amino acids. There were 33 total individual peptides, which were randomly pooled so that the first 17 peptides went into pool 1 and the last 16 into pool 2. IgELs and the HA tag were omitted to generate peptides as close to the native antigen as possible. 200,000 splenocytes in R10 medium were plated on 96-well IFN-γ capture antibody (R&D systems) coated plates and incubated in 5% CO in the presence of specific peptide pools. 2 The cells were stimulated overnight at 37°C in 5% CO. The cells were washed and the plates were incubated overnight with biotinylated anti-mouse IFN-γ detection antibody (R&D system). Spots were developed using streptavidin-alkaline phosphatase, 5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt, and nitroblue tetrazolium chloride sequentially. Spots were counted using an automated ELISPOT reader (CTL Limited). One week after the final immunization, it was confirmed that mice immunized with pMCore showed evidence of a strong HBcAg T cell response as confirmed by IFN-γ ELISPOT assay following ex vivo stimulation. Figure 32 showed that the major epitopes were biased towards peptide pool 2.

[0181] In an in vivo cytotoxicity assay, the study was carried out using carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling combined with flow cytometry. Cell division in cells of a cell population was evaluated. Splenocytes were isolated from naive mice and divided into two populations. One population, highly labeled with CFSE, was pulsed with a relevant peptide (e.g., HBV core peptide). The other population, low labeled with CFSE, was pulsed with an irrelevant peptide (e.g., HCV NS3 peptide). The labeled peptide-treated cells were combined and used in an adoptive transfer experiment in which flow analysis was performed. The combined groups of treated labeled target cells were administered to two groups of mice, a control group and an immunized group. Splenocytes were isolated from each group of mice and samples were subjected to flow cytometry. The amount of CFSE was measured. Typically, two peaks are formed in such experiments, the first being the irrelevant peptide and the second being the immunized peptide showing a larger CFSE in the peak.

[0182] Figure 33 shows that CD8 T cells induced by vaccination can specifically eliminate target cells in vivo. Results show that spleen and liver samples from untreated mice contained approximately equal amounts of cells in the peaks of irrelevant and relevant peptides, while results show that the peaks of cells induced from the immunized group pulsed with relevant peptides were significantly lower than those of irrelevant peptides. These data show that target cells treated with HBV peptides were specifically eliminated in mice immunized with HBV vaccines, but not in non-immunized mice. Elimination of target cells treated with irrelevant peptides, if any, was the same in mice immunized with HBV vaccines and non-immunized mice, and was significantly less than that of target cells treated with HBV peptides.

[0183] The ability of HBV-specific CD8 T cells induced after DNA immunization to specifically eliminate target cells in vivo was further examined. Human CTLs targeting core antigens are important in the acute clearance of HBV versus chronic infection. One week after the final immunization, four mice immunized with pVax or pMCore from each of the two groups were adoptively transferred with target splenocytes that had been pulsed with either HBcAg (relevant) or HCV-NS3 / 4A (irrelevant) peptides. Briefly, splenocytes from naive mice were stained with either 1 μM or 1 nM CFDA SE (invitrogen). Labeled splenocytes were then coated with the indicated peptides (1 μM) and 10 of each population were transferred to the HBV-specific CD8 T cells. 7 Cells were injected intravenously into naive or immunized mice. After 24 or 90 hours, cells from spleen and liver were isolated and analyzed by flow cytometry. Percent killing was calculated as follows: 100-([(% infected with relevant peptide pulsed / % infected with irrelevant peptide pulsed / % uninfected with irrelevant peptide pulsed] x 100). By gating on CFSE-labeled splenocytes to follow killing, it was observed that pMCore-vaccinated mice were able to induce strong specific killing of antigen-pulsed target cells, as shown in Figure 34. The average percent killing observed in the spleen was about 83%, while the average in the liver was 76%, indicating that vaccine-induced CTLs that migrated to and were retained in the liver were able to kill HBV peptide-pulsed target cells. This was the first study to show the induction of HBcAg-specific CTL responses in the liver by any method, and especially by systemic immunization. This data provides evidence that peripheral immunization can induce effector cells that can migrate to the liver and lyse target cells.

[0184] Figure 35 shows data collected from a T cell proliferation assay using CFSE labeling. The proliferation rates of CD3+CD4+ and CD3+CD8+ cells treated with pVax vector (control) or with the plasmid pMCore expressing HBV M core were compared. Briefly, isolated splenocytes were stained with Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) Cell Tracer Kit (Invitrogen) according to the manufacturer's instructions. Stained cells were washed three times with saline and stimulated with pMCore-specific overlapping peptides. Cells were incubated at 37°C for 96 hours. After 48 hours, 50% of the culture medium was removed and replaced with fresh R10. On day 4, cells were harvested and stained with CD3, CD4, and CD8 specific monoclonal antibodies (BD Pharmingen). Cells were fixed with PBS with 1% paraformaldehyde (PFA) and acquired on a FACScalibur (Becton Dickinson). Data was analyzed by using the FlowJo program. Low and medium CFSE populations were considered as proliferating cells. As shown in Figure 35, CD3+CD8+ T cells isolated from spleen proliferated more compared to CD3+CD4+ T cells.

[0185] Further experiments using T cell proliferation assays also used CFSE labeling. Specifically, isolated splenocytes were stained with Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) Cell Tracer Kit (Invitrogen) according to the manufacturer's instructions. The stained cells were washed three times with saline and seeded in a 96-well U-bottom plate in a total volume of 200 μL of medium containing HBcAg pool peptides at a concentration of 1 μg / mL. The cells were incubated at 37°C for 96 hours. After 48 hours, 50% of the culture medium was removed and replaced with fresh R10. As discussed above, the difference in cytokine production between both CD4 and CD8 T cells corresponded to their final proliferation capacity. Four days after stimulation with antigen-specific peptides, CD8 + T cells are CD4 +Cells proliferated more than two-fold higher than those of control subjects (FIG. 36), indicating a clear CD8 T cell bias in the response.

[0186] Figures 37A and 37B are ELISA data showing a comparison of anti-HBV core antibodies in serial dilutions of sera from animals treated with pVax vector (control) or with the plasmid pMCore expressing HBV M core. Briefly, high-binding ELISA plates (Costar, Corning, NY) were coated with 1 μg / ml HBcAg protein in PBS for 24 hours at 4°C, then washed with PBS-Tween and blocked with PBS containing 1% BSA for 2 hours at room temperature. Serially diluted serum samples were added to the wells and incubated for 1 hour at room temperature. After washing, bound serum Antibody was revealed with HRP-labeled goat anti-mouse IgG (Figure 37A) or IgA (Figure 37B). Peroxidase-conjugated Ab was detected using tetramethylbenzidine (Sigma-Aldrich) as substrate, and OD at 450 nm was measured in a Multiscan ELISA Plate Reader. Antigen-specific humoral responses in sera collected from immunized mice were observed.

[0187] To further explore the immune response induced in mice immunized with pMCore, antigen-specific IgG and IgA responses were analyzed by B cell ELISpot and in ELISA using splenocytes and sera collected after vaccination, respectively. Splenocytes were isolated and purified as described above. For ELISA, high-binding ELISA plates (Costar, Corning, NY) were coated with 1 μg / ml HBcAg protein in PBS for 24 hours at 4°C, then washed with 0.1% PBS-Tween, and then blocked with PBS containing 1% BSA for 2 hours at room temperature. Serially diluted serum samples were added to the wells and incubated for 1 hour at room temperature. After washing, bound serum antibodies were revealed with HRP-labeled goat anti-mouse IgA or IgG. Peroxidase-bound antibodies were detected using tetramethylbenzidine (Sigma-Aldrich) as substrate, and OD values ​​at 450 nm were measured on a Multiscan ELISA Plate Reader. High IgG and IgA titers were observed in the serum of immunized mice compared to control animals (Figure 38). B cell ELISpot (Figure 39) from immunized mice shows approximately 200 and 100 SFU of HBcAg-specific IgG and IgA per million cells, respectively. This illustrated the activation of the B cell compartment by immunization with pMCore. The synthetic HBcAg plasmid effectively induced antigen-specific cellular and humoral responses after three immunizations.

[0188] FIG. 40 shows the percentage of TNF-α and IFN-γ from CD4+ and CD8+ cells in the spleen and liver.

[0189] In the absence of a small animal model of HBV, HBcAg was used to transiently transfect the liver of mice via hydrodynamic injection. The liver of immunized mice was transfected with either pMCore or HCV NS3 / 4A. Immunohistochemical staining 3 days after transfection showed clearance of HBcAg-transfected hepatocytes compared to those transfected with NS3 / 4A. ALT levels in serum were measured to ensure that the clearance induced by immunized mice did not cause liver damage. The results in Figure 41 showed that the clearance induced by immunized mice did not cause liver damage.

[0190] Direct hydrodynamic injection was also used to transiently transfect mouse livers. Here, immunized or naive mouse livers were transfected with either pMCore or an irrelevant plasmid encoding a Hepatitis C antigen (HCV NS3 / 4A). Briefly, immunized mice were intravenously injected with 100 μg of plasmid in 2 mL (approximately 10% volume of mouse weight) of Ringers solution within 7 seconds to transiently transfect the liver. Plasmid expression or clearance was determined by staining the liver with an anti-HA monoclonal antibody. Immunohistochemical staining 3 days after transfection (Figure 42) showed clearance of HBcAg-transfected hepatocytes compared to livers of animals transfected with NS3 / 4A. CD8 T cells isolated from mice hydrodynamically injected with pMCore in Figure 43 showed high frequency of IFN-γ, a marker of degranulation, compared to livers of immunized animals transfected with an irrelevant plasmid. + CD107a + showed.

[0191] Since the clearance of pMCore-transfected hepatocytes appears to involve degranulation, it was believed that death would cause liver damage. To test whether immunized mice were able to clear the transfected hepatocytes without inducing significant liver damage, an assay was used that measures the activity of the enzyme alanine aminotransferase (ALT), as the enzyme levels rise in serum (Figure 44), indicating the presence of liver damage. Specifically, serum alanine aminotransferase (ALT) activity was measured using an absorbance-based assay (Stanbio Laboratory) on a BioTek Synergy 2 microplate reader. Results were reported in units per liter (U / L) and represent the amount of enzyme that oxidizes 1 μmol / L of NADH per minute. These studies showed that the specific clearance of HBcAg-transfected hepatocytes did not elevate ALT levels in transfected and immunized animals above the normal range of 5-30 U / L (Figure 44). Example 2 HBV surface protein 1.HBsAg DNA vaccine design

[0192] The use of consensus immunogens in the context of variable pathogens with highly variable antigen sequences may be more effective in inducing a more directed immune response to combat natural viral diversity compared to immunization with a single innate immunogen. 8-2 hepatitis genotype A large surface antigen sequences and 70 hepatitis genotype C large surface antigen sequences were taken from GenBank, and after performing multiple alignments, genotype-specific large and small surface antigen consensus sequences were obtained. ClusterX (version 1.8) software was used to generate the multiple alignments used to generate the consensus sequences.

[0193] Using consensus sequences encoding genotypes A or C, two of the most common genotypes worldwide, we generated DNA constructs encoding the HBV surface envelope proteins major S alone, or preS1 / S2 and major S (Figure 52A, Figures 59A-59D). As the amino-terminal domain of preS1 may assist in virus binding and entry, while the carboxyl terminus of preS2 may be involved in infectivity, we examined differences in the immune response elicited by constructs encoding full-length HBs or major S alone. The major S only construct was designated small HBs (SHBs) and the plasmid containing preS1 / S2 was designated large HBs (LHBs).

[0194] A consensus HBV surface protein was designed from epitope sequences from HBV genotype A primary isolates, such that the resulting consensus protein has 94.2 percent to 99.8 percent sequence identity with the surface protein of HBV genotype A primary isolates. Specifically, two types of such consensus proteins were designed (FIGS. 4 and 52B). One contains the S protein, also referred to as SHBs-A, in which SEQ ID NO: 13 is the nucleic acid sequence and SEQ ID NO: 14 is the amino acid sequence. The second contains the S protein, pre-S2, and pre-S1, also referred to as LHBs-A, in which SEQ ID NO: 9 is the nucleic acid sequence and SEQ ID NO: 10 is the amino acid sequence.

[0195] We also designed a consensus HBV surface protein from epitope sequences from primary isolates of HBV genotype C, such that the resulting consensus protein has 96.5 percent to 99.8 percent sequence identity with the surface protein of HBV genotype C primary isolates. Specifically, two types of such consensus proteins were designed (FIG. 4). One contains the S protein, also referred to as SHBs-C, in which SEQ ID NO: 15 is the nucleic acid sequence and SEQ ID NO: 16 is the amino acid sequence. The second contains the S protein, pre-S2, and pre-S1, also referred to as LHBs-C, in which SEQ ID NO: 11 is the nucleic acid sequence and SEQ ID NO: 12 is the amino acid sequence.

[0196] After generation of the consensus genotype A or C large and small surface antigen consensus sequences, codon optimization and RNA optimization were performed. For the above consensus HBV surface antigen, an intracellular protein cleavage site was introduced into the consensus HBV surface antigen to provide proper protein folding and better antigen processing (Figure 3). The codon usage in the consensus HBV surface antigen was modified to reflect the codon bias of human genes. In addition, regions of very high (e.g., greater than 80 percent) or very low (e.g., less than 30 percent) GC content, which are cis-acting motifs such as internal TATA boxes, repeat sequences, and structured sequences, were avoided. A Kozak sequence was introduced into the consensus HBV surface antigen to increase translation initiation. As previously described (Yan et al., Vaccine (2008) 26:5210-5215), a highly efficient IgE leader sequence was added upstream of the start codon to enhance and increase protein expression (see Figures 3 and 52B). The optimized gene was synthesized and the sequence was verified.

[0197] Consensus HBV surface antigens SHBs-A, LHBs-A, SHBs-C, and LHBs-C were cloned into expression vectors to generate pSHb A (also referred to as pSHb-A), pLHb A (also referred to as pLHb-A), pSHb C (also referred to as pSHb-C), and pLHb C (also referred to as pLHb-C) constructs, respectively (Figure 4). The synthesized genotype A large and small surface antigen genes were subcloned into the expression vector pGX0001 at the BamHI and XhoI sites, while the synthesized genotype C large and small surface antigen genes were subcloned into pGX0001 at the EcoRI and NotI sites. The generation of HBcAg plasmid (pMCore) was described in Example 1. 2. In Vitro Expression of Multiplex HBsAg DNA Vaccine

[0198] Rhabdomyosarcoma (RD) and human hepatoma (Hep G2) cell lines were transfected with pLHBs-A, pLHBs-C, pSHBs-A, and pSHBs-C using TurboFect™ (Thermo Scientific) according to the manufacturer's guidelines. For intracellular assays, cells were washed and fixed using a Cytofix / Cytoperm kit (BD PharMingen, San Diego, CA) according to their instructions. Following fixation, cells were stained with serum from immunized mice or Mouse monoclonal antibody to Hepatitis B Virus Surface (Abcam, Cambridge, MA). Cells were then stained with fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (BD Biosciences, San Jose, CA). Cells were washed and fixed with 2% paraformaldehyde. Stained and fixed cells were acquired on the LSR instrument using CellQuest software (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

[0199] Following construction and optimization of the four constructs, protein expression of each plasmid was confirmed by intracellular staining. RD or Hep G2 cells were transiently transfected with individual constructs or with empty pGX0001 vector as a control. After 48 hours, cells were stained intracellularly using anti-HBsAg polyclonal mouse serum or a commercially available mouse monoclonal antibody. Expression was confirmed using flow cytometry (Figure 52C). Elevated levels of HBs protein expression were observed in cells transfected with each construct compared to cells transfected with empty vector (pGX0001). Example 3 Immunogenicity of individual synthetic HBsAg DNA plasmids in a small animal model

[0200] Following expression studies, the ability of individual plasmids to induce antigen-specific humoral and cellular responses was evaluated in vivo. Female Balb / c mice aged 6–8 weeks were purchased from Jackson Laboratories. Animals were maintained in accordance with the Institutional Care and Use Committee (IACUC) guidelines of the National Institutes of Health and the University of Pennsylvania. Balb / c mice were immunized intramuscularly (IM) three times at 2-week intervals at weeks 0, 2, and 4 with 15 μg of plasmid expressing either the small or large S antigen or the pGX0001 control. Electroporation at the site of infection was performed immediately after each vaccination as previously described (Hirao et al., Vaccine (2008) 26:440-448).

[0201] For immunization, all DNA was diluted using nuclease-free water (Qiagen, Valencia, CA 91355). Each animal in the immunization group received a total of three intramuscular immunizations of 15 μg of the appropriate plasmid. For combination studies, each animal received a total of 45 μg of DNA in both tibialis anterior (TA) muscles. Following immunization, in vivo electroporation was performed using a CELLECTRA® adaptive constant current electroporation device (Inovio Pharmaceuticals, Blue Bell, PA). Two 0.2 Amp constant current square wave pulses were delivered through a triangular three-electrode array consisting of a 26-gauge solid stainless steel electrode inserted fully into the muscle. Each pulse was 52 ms long with a 1-second delay between pulses. Because older DNA vaccines have not been consistent in generating significant antibody responses, vaccine-induced humoral responses were first examined. 1. Antibody ELISA Assay

[0202] ELISA was performed to analyze antigen-specific IgG responses in sera collected from mice 7 days after each immunization using recombinant full-length HBsAg protein as the target antigen. High-binding ELISA plates (Costar, Corning, NY) were coated with 1 μg / ml of recombinant protein in PBS for 24 h at 4°C, then washed with 0.1% PBS-Tween and blocked with PBS containing 1% BSA for 2 h at room temperature. Serially diluted serum samples were added to the wells and incubated for 1 h at room temperature. After washing, bound IgG was detected by HRP-labeled goat anti-mouse IgG. Peroxidase-conjugated antibodies were detected using tetramethylbenzidine (Sigma-Aldrich) as a substrate, and OD values ​​at 450 nm were measured with a Multiscan ELISA Plate Reader.

[0203] Both pLHBs and pSHBs from either genotype were highly immunogenic. Further immunization boosted these responses with antibody responses, with the greatest boost two weeks after the third immunization (Figure 53A). In contrast, mice immunized with the pGX0001 control exhibited only background levels of IgG responses. Some differences in the magnitude of the IgG responses were observed in each group, but the progressive boost after each immunization was consistent within groups. 2. IFN-γ ELISPOT

[0204] The ability of pLHBs-A, pLHBs-C, pSHBs-A, and pSHBs-C constructs to induce cellular immune responses was examined by IFN-γ ELISPOT. Matching HepB consensus core, surface antigen A, and surface antigen C 15-mer peptides were synthesized by Genescript (Piscataway, NJ), resuspended in DMSO, and pooled at a final concentration of approximately 1 mg / mL for each peptide. Cellular responses were measured using IFN-γ ELISpot (MabTech, Sweden) according to the manufacturer's instructions. Samples were run in triplicate using R10 (RPMI 1640 containing L-glutamine and 1% penicillin / streptomycin with 10% heat-inactivated fetal bovine serum) and PMA / IM (PMA 0.1 μg / mL and ionomycin 0.5 μg / mL, Sigma Aldrich, St. Louis, MO) controls.

[0205] HBsAg-specific IFN-γ-secreting cells were analyzed in response to two pools of synthetic peptides developed from the consensus large HBV surface protein. The first peptide pool contained peptides spanning the preS1 and preS2 proteins, while the second peptide pool was made from peptides spanning the major S protein. All four consensus constructs were able to induce strong T cell responses, and cross-immunoreactivity was observed in the two genotypes Figure 53B. Taken together, the preS protein is almost as large as the major S protein, but there was an epitope bias in the cell-mediated immune (CMI) response to the major S protein. These results indicate that synthetic DNA immunogens encoding either the full-length or only the major S portion of the HBV surface antigen exhibit sufficient immunogenic diversity to target the major genotypes of HBV. The full-length constructs did not show significant differences in the induction of IgG against HBV native proteins over the shorter constructs. 3.HBV surface antigen (15-mer) peptide mapping

[0206] After confirmation of the magnitude of CMI, the breadth of cellular responses against genotype-specific targets was investigated. Therefore, a set of interferon-γ ELISpot assays was performed against different matrix peptide pools. Two sets of peptides spanning either the LBHs-A or LHBs-C proteins, each containing 15 amino acid residues overlapping by 8 amino acids, were synthesized by GenScript (Piscataway, NJ). To map immunodominant epitopes, the two sets of peptides were pooled into either 16 or 12 pools for the large or small HBsAg groups, respectively. IFN-γ ELISpot assays were performed as described above. These different sets of pooled peptides were used in matrix assays to map the epitopes of each construct. Responses of more than 50 SFU per million cells were considered positive.

[0207] These data illustrate the wide variety of epitope responses induced by these synthetic surface antigen plasmids in BALB / c mice. 12 out of 16 positive LHBs-A matrix pools were observed in mice immunized with pLHBs-A, and 8 out of 16 positive LHBs-C matrix pools in mice immunized with pLHBs-C. Small HBS plasmids showed higher fold responses in mice immunized with pSHBs-A, where 9 out of 12 SHBs-A matrix peptide pools corresponded, while mice immunized with pSHBs-C exhibited responses against all SHBs-C matrix pools (Figure 53C). These data indicate the high and broad cross-reactive cellular responses induced by these four HBs constructs. As a result, these genotype-specific consensus vaccines may be able to induce cross-reactive cellular responses against various primary surface antigen isolates that differ by up to 8% across these genotypes. Example 4 Cytotoxicity assays and antigen-specific CTLs in the liver and spleen of vaccinated mice

[0208] Antiviral effector CD8 T cells can induce cytotoxicity, a cellular activity to eliminate HBV-infected hepatocytes. The ability of the constructs to generate HBs-specific CTLs in the periphery that can also home to the liver of immunized mice was evaluated using an in vivo cytotoxicity assay. Splenocytes from naive mice were stained with either 1 μM (high) or 1 nM (low) CFDA SE (Invitrogen, Grand Island, NY, USA). The labeled splenocytes were then coated with the indicated peptides (relevant or irrelevant peptides) (1 μM) and 10 of each population were cultured. 7Cells were injected intravenously into naive or immunized mice. After 90 hours, cells were isolated from blood, spleen, or liver and analyzed by flow cytometry. Percent killing was calculated as follows: 100-([(% infected pulsed relevant peptide / % infected pulsed irrelevant peptide / % non-infected pulsed irrelevant peptide)] x 100).

[0209] Transfer of HBV-specific CTL to the liver is essential for intrahepatic clearance of infected cells. Antigen-specific clearance of adoptively transferred target splenocytes pulsed with HBsAg synthetic peptide was observed. The average percent killing of target cells in the spleen of mice immunized with SHBs construct was 39% and 48% for genotypes A and C, respectively. The average target cell clearance of LHBs construct was 35% for genotype A and 51% for genotype C. Clearance was strong, but not as strong as the response observed in mice immunized with core Ag plasmid alone, as described in Example 1, where 83% and 76% target clearance was recorded in the spleen and liver, respectively. The demonstration that CTLs generated against HBs can target antigen clearance in the liver encourages the combination of HBs and HBc to further expand cell capacity and limit escape. The percent elimination of target cells in the liver was 45% and 66% for SHBs-A and SHBs-C, and 60% and 74% in the LHBs-A and LHBs-C groups (Figure 54). The trafficking of vaccine-specific CTLs to the liver, an organ known to suppress T cell responses and induce tolerance, highlights the efficacy of the construct in inducing functional CMI. Example 5 Immunogenicity of HBsAg-HBcAg DNA cocktail

[0210] Immunization with each synthetic HBsAg plasmid elicited broadly cross-reactive immune responses, thus extending the response beyond surface antigens and further enhancing immunogenicity against a wide variety of antigens within the virus.We combined the HBcAg(pMCore)-encoding plasmid with the HBs plasmid to generate an HBs-HBc DNA vaccine cocktail for immunization and determined whether the cocktail would enhance the cellular response by elevating the number of immune epitopes that T cells recognize for clearance, since the core antigen of HBV is involved in viral clearance through the induction of HBcAg-specific CTL responses.

[0211] Five groups of mice were immunized with a vaccine cocktail of HBs antigen only or HBs and HBc antigen combined. Each mouse received two injections followed by electroporation in both TAs. Mice were immunized with either a cocktail containing only surface antigens (both genotypes) or a cocktail containing both surface antigens and consensus core antigen plasmid (pMCore) as described in Example 1 (see Table 3). Mice immunized with HBsAg only produced high IgG titers in their serum. Interestingly, similar titers were seen in the SHBs-pMCore group, but were weaker in the LHBs-pMCore cocktail group (Figure 55A). The rise in antigen target may cause some antibody inhibition. [Table 3]

[0212] To further explore the immune response elicited by mice immunized with HBs-HBc, the production of IFN-γ from each immunization group was evaluated. The average SFU per million splenocytes for the group without pMCore was about 689, 666 for genotype A peptide, and 1160 and 1312 for genotype C peptide, respectively. Moreover, the addition of the average pMCore increased the total SFU per million splenocytes by 400 spots for genotype C, doubling the response when splenocytes were stimulated with genotype A peptide (Figure 55B).

[0213] CD8 T cells are known to be the primary effector cells involved in HBV clearance. It was important to determine which T cell type was responsible for the observed IFN-γ production and to examine whether these T cells could induce multiple antiviral functions upon re-encounter by releasing other antiviral cytokines such as TNF-α and IL-2, as well as degranulation markers such as CD107a. Following ex vivo stimulation with the appropriate peptides, the ability of CD8 T cells from immunized mice to induce these cytokines and markers was assessed. Multicolor flow analysis revealed that in all groups, a significant proportion of CD8 T cells from each group were polyfunctional (i.e., produced multiple cytokines) activated CD8 T cells (Figure 56A-F). Antigen-specific induction of the lymphocyte degranulation marker CD107a was also observed in these CD8 T cells. CD8 T cells from the HBs-HBc cocktail group showed higher polyfunctional activity when stimulated with surface antigen peptides, confirming the synergistic effect of CD8 T cell antiviral cytokines such as IFN-γ, TNF-α, and IL-2, as well as the degranulation marker CD107a. Induction of CTLs through clearance from transferred antigen-pulsed cells was also observed. Although there was a decrease in the antibody response against HBs with respect to the cocktail containing the LHBs construct, the SHBs-pMCore vaccine cocktail maintained the humoral response detected in animals immunized only with SHBs. Example 6 Immunization of rhesus macaques with the cocktail induces strong cellular HBc-specific and HBs-specific immune responses as well as humoral HBs-specific immune responses

[0214] DNA vaccine efficacy of previous DNA constructs has been consistently weak in large animals and humans. The strong antibody and T cell responses elicited by the combination of pLHBs or pSHBs and pMCore in mice precedes the evaluation of the immunogenicity of these constructs in non-human primate models (NHPs) that more closely mimic the human immune response. This is an obstacle to vaccine immunogenicity, as the large size and outbred MHC population of NHPs typically results in reduced or absent immunogenicity compared to mouse models. In these studies, IL-12, a Th1-driving cytokine, was added and analyzed. IL-12 has been shown to drive CMI by stimulating and aiding in the expansion of CD8 T cells when added to immunizations as an adjuvant to enhance immune responses to different antigens from various pathogens. This adjuvant boosts the T cell response of DNA vaccines in mice, as well as in macaques and humans. 1. Vaccination

[0215] Fifteen Indian rhesus monkeys were housed at Bioqual (Rockville, MD) in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. The monkeys were placed into groups with each group containing five monkeys. See Table 4. [Table 4]

[0216] One group received a vaccine containing 1.0 mg of each of constructs pMCore, pSHb A, and pSHb C via intramuscular (IM) delivery (also known as in vivo electroporation). This group is referred to as the "small" group. A second group received a vaccine containing 1.0 mg of each of constructs pMCore, pLHb A, and pLHb C via IM delivery. This group is referred to as the "long" group. A third group received a vaccine containing 1.0 mg of each of constructs pMCore, pLHb A, and pLHb C, and 0.4 mg of prhIL-12 (a plasmid that expressed optimized rhesus IL-12) via IM delivery. This group is referred to as the "long + IL-12" or "pLHBs + 12" group. The construct prhIL-12 encodes the rhesus IL-12 protein. DNA was delivered to a single site in the quadriceps muscle, followed by in vivo EP using a constant current CELLECTRA® device (Inovio Pharmaceuticals, Blue Bell, PA) with 3 pulses at 0.5 A constant current, 52 ms pulse length and 1 second interval between pulses. Animals were vaccinated at weeks 0, 4, 12, and 30. 2. Sample Collection

[0217] Samples were collected from the monkeys prior to the 0 week immunization as well as after each immunization (i.e., after weeks 0, 4, and 12). Animals were bled 2 weeks after each immunization. Blood (20 mL at each time point) was collected into EDTA tubes and PBMCs were isolated using a standard Ficoll-Hypaque procedure with Accuspin tubes (Sigma-Aldrich). An additional 5 mL of blood was collected into clot tubes and an aliquot of serum was removed for analysis.

[0218] High titer antibody responses to HBsAg were observed after two immunizations with pSHBs / pMCore, but not after immunization with pLHBs / pMCore. This response was consistent with the mouse antibody data in Figure 55A. Responses to pLHBs / pMCore were boosted after the third immunization. IL-12 adjuvant enhanced the antibody titers of pLHBs / pMCore cocktail immunization as early as two immunizations (Figure 57A).

[0219] Cellular immune responses from PBMCs show little or no HBs or HBc specific responses after the first immunization. However, responses were observed after the second immunization and boosted after each subsequent immunization. After the third immunization, robust responses were observed in all groups, over 2000 SFU per million PBMCs, with the majority targeting against core antigens. The addition of IL-12 adjuvant enhanced the magnitude of T cell responses by 1000 SFU per million PBMCs compared to the pLHBs / pMCore cocktail alone (Figure 57B). To further define the breadth of the cellular response, a matrix pool of consensus core peptides was generated and the number of potential epitopes was determined. T cell responses spanned multiple peptide pools, with an average response to 12 peptide epitopes (Figure 57C). These responses are consistent with those previously seen in small animal models. These results now suggest that the cocktail is capable of inducing cellular responses that are both robust in magnitude and breadth, and that the magnitude of these responses can be further enhanced by the addition of IL-12.

[0220] Figures 45-48 show the cellular immune responses induced by the vaccines administered to the small, long, and long + IL-12 groups. The cellular immune responses were determined utilizing an enzyme-linked immunosorbent spot (ELISPOT) assay, as detailed above in Example 1. As shown in Figures 45-48, T cell responses were raised by each vaccination. However, HBV core antigen was more immunogenic than HBV surface antigen A and HBV surface antigen C. The data also show that the longer consensus HBV surface antigen (i.e., including S protein, pre-S2, and pre-S1) is as immunogenic as the smaller consensus HBV surface antigen (i.e., including S protein).

[0221] Similar to Figures 45-48, Figure 49 shows the cellular immune responses induced by vaccines administered to the small, long, and long+IL-12 groups as measured by ELISPOT assay, however, different pools of peptides covering core antigen, surface antigen A, and surface antigen C were tested. Again, T cell responses were boosted by each vaccination, the addition of IL-12 enhanced the cellular immune response, and the long and short consensus HBV surface antigens were similarly immunogenic.

[0222] Figure 50 shows ELISA data comparing anti-HBV antibody responses to small, long, and long + IL-12 groups. ELISA was performed as detailed above in Example 1. Antigen-specific humoral responses were observed in serum collected from immunized monkeys. Specifically, vaccines containing long consensus surface antigens (i.e., S protein, pre-S2, and pre-S1) had better antibody responses, which may be due to the additional epitopes present in the vaccine.

[0223] Figure 51 shows ELISA data comparing anti-HBV antibody responses to the long + IL-12 group before immunization (prevac) and at 0, 4, and 12 weeks after immunization (i.e., after the 1st, 2nd, and 3rd doses, respectively). ELISA was performed as described above in Example 1. The data in Figure 51 showed that two immunizations or doses were required for measurable antibody production. Furthermore, a significant increase in anti-HBV antibody responses was observed after the third immunization or dose. Example 7

[0224] Similar to the mouse experiments in Example 5, splenocytes, i.e., CD8 and CD4 T-cells, were isolated from the immunized rhesus macaques of Example 6 and the levels of INF-γ and TNF-α secretion were examined in response to administration of a combination of consensus core and consensus surface antigens. To examine the secretion of tumor necrosis factor, interferon gamma, and CD107a from these CD8 and CD4 T cells, the CD8 and CD4 T cells were studied using polyfunctional and phenotypic flow analysis to determine whether the CD8 and CD4 T cells of the immunized monkeys of Example 6 have similar characteristics to the mouse CD8 and CD4 T cells studied in Example 1.

[0225] To further clarify the function of the cellular immune response, intracellular staining for antiviral cytokines was performed following stimulation with core and surface antigens of genotypes A and C. Intracellular staining with antibodies against antiviral cytokines revealed that antigen-specific production of antiviral cytokines (IFN-γ, TNF-α, and IL-2) was induced in both CD4 and CD8 T cells of immunized monkeys. Figure 57D shows that following three immunizations, both HBV-specific CD4 and CD8 T cell responses were observed. Similar to the results seen in the IFN-γ ELISpot, responses to the core antigens were predominant. The addition of IL-12 enhanced the overall magnitude of the response, however, as expected, this effect was more evident for CD8 T cells. The induction of both CD4 and CD8 responses, averaging 1%, suggests the induction of a broad range of immune responses constituting both Th1 and Th2 immunity, strongly encouraging the advancement of this strategy in human trials.

[0226] Antibody and T cell responses against both HBs and HBc antigens were similar to those generated by mice in Example 5. These responses were even better when IL-12, a Th1 polarizing cytokine adjuvant, was added to the pLHBs-pMCore cocktail. The pLHBs-pMCore+IL-12 formulation appeared to be highly effective as a preferred combination. It is noteworthy that the formulation without vaccine resulted in an increase in parameters related to liver damage. Example 8 Analysis of Liver Function in Non-Human Primates Following Vaccination Using a Liver Blood Chemistry Panel

[0227] Because clinical studies of actively HBV-infected patients suggest that T cell responses may be associated with liver pathology during disease, a preliminary analysis of the overall safety of these multivalent vaccines on liver function was performed. To examine liver function, serum levels of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and total bilirubin were analyzed in vaccinated animals before immunization, after the third vaccination, and immediately before and after the fourth vaccination. The blood chemistry panel was performed by IDEXX Laboratories and included alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and total bilirubin. The results of this analysis are shown in Figure 58. The lack of elevation of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and total bilirubin in liver function tests before, during, and after the completion of the immunization period suggested that induction of an HBV-specific immune response did not induce significant damage to the liver of vaccinated animals.

[0228] Taken together, the data support that administration of this synthetic DNA vaccine cocktail encoding engineered HBV antigens in combination with specific electroporation drives robust and diverse cellular and humoral responses in both mice and NHPs while avoiding overall toxicity.

[0229] It is understood that the foregoing detailed description and the accompanying examples are merely illustrative and are not to be taken as limitations on the scope of the invention, which is defined solely by the appended claims and equivalents thereof.

[0230] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those relating to the chemical structures, substitutes, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

1. A vaccine comprising: (a) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:10, SEQ ID NO:14, a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:10, and a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:14; or (b) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:12, and a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:16; Including, vaccines.

2. 2. The vaccine of claim 1, further comprising: (c) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:

6.

3. (a) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:10, SEQ ID NO:14, a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:10, and a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:14; (b) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:12, and a protein that is 95% identical over the entire length of the amino acid sequence of the full-length amino acid sequence of SEQ ID NO:16; and (c) a nucleic acid molecule encoding one or more proteins selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; The vaccine of claim 1 , comprising:

4. 4. The vaccine of claim 3, wherein the nucleic acid molecule comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:

15.

5. The vaccine of claim 1 , wherein the nucleic acid molecule is a plasmid.

6. The vaccine of claim 1 , wherein the nucleic acid molecule is incorporated into a viral particle.

7. The vaccine of claim 1 further comprising an adjuvant molecule.

8. The vaccine of claim 7, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

9. A method for inducing an immune response against an HBV antigen, comprising administering the vaccine of claim 1 to a subject.

10. 13. A method of protecting a subject against HBV infection comprising administering to the subject the vaccine of claim 1.

11. 13. A method of protecting a subject diagnosed with HBV infection comprising administering to the subject the vaccine of claim 1.

12. A nucleic acid molecule comprising a coding sequence encoding one or more proteins selected from the group consisting of a protein comprising SEQ ID NO:2, a protein that is 95% identical over the entire length of its amino acid sequence to the full-length amino acid sequence of SEQ ID NO:2, an immunogenic fragment of a protein comprising SEQ ID NO:2 that is at least 20 amino acids, and an immunogenic fragment of a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:2 that is at least 20 amino acids.

13. The nucleic acid molecule of claim 12, further comprising a signal peptide linked to the N-terminus of the protein.

14. The nucleic acid molecule of claim 12, encoding one or more proteins selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:

6.

15. The nucleic acid molecule of claim 12, comprising one or more sequences selected from the group consisting of a nucleic acid sequence comprising SEQ ID NO:1, a nucleic acid sequence that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:1, a fragment thereof comprising a nucleic acid sequence that encodes an immunogenic fragment comprising at least 20 amino acids encoded by SEQ ID NO:1, and a fragment thereof comprising a nucleic acid sequence that encodes an immunogenic fragment comprising at least 20 amino acids of a protein that is 98% identical over the entire length of the amino acid sequence of the protein encoded by SEQ ID NO:

1.

16. The nucleic acid molecule of claim 12, further comprising a signal peptide linked to the 5' end of the nucleic acid sequence.

17. 13. The nucleic acid molecule of claim 12, comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:

5.

18. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule is a plasmid.

19. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule is an expression vector and the sequence encoding another protein is operably linked to a regulatory element.

20. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule is incorporated into a viral particle.

21. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule comprises a coding sequence encoding one or more proteins selected from the group consisting of an immunogenic fragment of a protein comprising SEQ ID NO:2, which is at least 60 amino acids, and an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:2, which is at least 60 amino acids.

22. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule comprises a coding sequence encoding one or more proteins selected from the group consisting of an immunogenic fragment of a protein comprising SEQ ID NO:2, which is at least 120 amino acids long, and an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:2, which is at least 120 amino acids long.

23. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule comprises a coding sequence encoding one or more proteins selected from the group consisting of an immunogenic fragment of a protein comprising SEQ ID NO:2, which is at least 180 amino acids, and an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:2, which is at least 180 amino acids.

24. A method of inducing an immune response against an HBV antigen, comprising administering to an individual the nucleic acid molecule of claim 12.

25. 13. A method of protecting an individual from HBV infection comprising administering to the individual a nucleic acid molecule according to claim 12.

26. 13. A method of protecting an individual diagnosed with HBV infection comprising administering to the individual the nucleic acid molecule of claim 12.

27. A protein, (a) SEQ ID NO:2, (b) a protein that is 95% identical over the entire length of the amino acid sequence of the full-length sequence set forth in SEQ ID NO:2; (c) an immunogenic fragment of SEQ ID NO:2 comprising 20 or more amino acids of SEQ ID NO:2; (d) a protein selected from the group consisting of immunogenic fragments of a protein that is 95% identical over the entire length of the amino acid sequence of SEQ ID NO:2 containing 20 or more amino acids.

28. 28. The protein of claim 27, wherein the protein is selected from the group consisting of an immunogenic fragment of a protein comprising SEQ ID NO: 2, which is at least 60 amino acids long, and an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO: 2, which is at least 60 amino acids long.

29. 28. The protein of claim 27, wherein the protein is selected from the group consisting of an immunogenic fragment of a protein comprising SEQ ID NO: 2, which is at least 120 amino acids long, and an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO: 2, which is at least 120 amino acids long.

30. 28. The protein of claim 27, wherein the protein is selected from the group consisting of an immunogenic fragment of a protein comprising SEQ ID NO: 2, which is at least 180 amino acids long, and an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO: 2, which is at least 180 amino acids long.

31. 28. A method of inducing an immune response against an HBV antigen, comprising administering to an individual the nucleic acid molecule of claim 27.

32. 28. A method of protecting an individual from HBV infection comprising administering to the individual the nucleic acid molecule of claim 27.

33. 28. A method of protecting an individual diagnosed with HBV infection comprising administering to the individual the nucleic acid molecule of claim 27.

34. 1. A nucleic acid molecule comprising: (a) a protein comprising SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; (b) a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; (c) an immunogenic fragment of the protein comprising at least 20 amino acids of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; and (d) an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 that is at least 20 amino acids; A nucleic acid molecule comprising a coding sequence encoding one or more proteins selected from the group consisting of:

35. (a) a nucleic acid sequence comprising SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; (b) a nucleic acid sequence that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; (c) a nucleic acid sequence encoding an immunogenic fragment comprising at least 20 amino acids encoded by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, and a fragment thereof comprising the nucleic acid sequence encoding the immunogenic fragment comprising at least 20 amino acids encoded by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; and (d) a nucleic acid sequence encoding an immunogenic fragment comprising at least 20 amino acids of a protein that is 98% identical over the entire length of the amino acid sequence of the protein encoded by SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15; 35. The nucleic acid molecule of claim 34, comprising one or more sequences selected from the group consisting of:

36. 35. The nucleic acid molecule of claim 34, wherein the nucleic acid molecule is a plasmid.

37. 35. The nucleic acid molecule of claim 34, wherein the nucleic acid molecule is an expression vector, and the sequence encoding the one or more proteins is operably linked to a regulatory element.

38. The nucleic acid molecule of claim 34, wherein the nucleic acid molecule is incorporated into a viral particle.

39. 35. A method of inducing an immune response against an HBV antigen, comprising administering to a subject the nucleic acid molecule of claim 34.

40. 35. A method of protecting a subject from HBV infection comprising administering to the subject a nucleic acid molecule according to claim 34.

41. 35. A method of protecting a subject diagnosed with HBV infection comprising administering to the subject a nucleic acid molecule according to claim 34.

42. A protein, (a) SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, (b) a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; (c) an immunogenic fragment of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, which comprises 20 or more amino acids; and (d) an immunogenic fragment of a protein that is 98% identical over the entire length of the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, comprising 20 or more amino acids; A protein selected from the group consisting of:

43. 1. A vaccine useful for generating an immune response to HBV in a subject, comprising: A nucleic acid molecule according to claim 34; and an adjuvant molecule.

44. 44. The vaccine of claim 43, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

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