HK1192456B - Nucleic acid molecule encoding hepatitis b virus core protein and vaccine comprising the same - Google Patents

Description

Nucleic acid molecules encoding hepatitis b virus core protein and vaccines containing the same

Technical Field

The present invention relates to nucleic acid sequences encoding Hepatitis B Virus (HBV) core protein and fragments thereof; to Hepatitis B Virus (HBV) core protein and fragments thereof, to improved HBV vaccines, to improved methods for inducing an immune response against HBV, and to improved methods for prophylactically and/or therapeutically immunizing an individual against HBV.

Background

This application claims priority to U.S. provisional patent application No. 61/442,162, filed on 11/2/2011, which is incorporated herein by reference.

Hepatitis b is a common infection prevalent worldwide, leading to the development of cirrhosis, liver failure, and hepatocellular carcinoma. A significant number of hepatitis cases have not been reported due to the asymptomatic nature of the disease. Nevertheless, about 3.5 million cases of chronic hepatitis B are reported each year. The majority of hepatitis infected people are in underdeveloped or developing countries.

The viruses are classified into four major serotypes (adr, adw, ayr, ayw) based on the antigenic epitopes present on their envelope proteins. HBV exists in at least eight genotypes (A-H) according to changes in genomic sequence. Alternative genotypes of HBV have a prevalent geographical distribution.

Table 1 is the geographical distribution of HBV genotypes.

TABLE 1 geographical distribution of HBV

Journal of medical virology (j.med.virol), DOI 10.1002jmv

The HBV genome is a circular DNA molecule that is predominantly double-stranded but has a single-stranded region created by one strand that is longer than the other. The double-stranded region results from the hybridization of one strand of a shorter strand of about 3020 nucleotides to a longer strand of about 3320 nucleotides. The single stranded region on the longer non-hybridizing nucleotide is associated with HBV DNA polymerase. Both HBV genomic DNA and HBVDNA polymerase are contained within a nucleoprotein shell formed by multiple HBV core protein (HBcAg) molecules. The HBV core protein is enveloped by HBV surface proteins (HBsAgs) and lipid molecules.

The HBV genome contains four Open Reading Frames (ORFs): 1) ORF encoding HBVDNA polymerase, 2) ORF with two start codons, wherein the sequence linked to the second start codon encodes the core protein and the sequence comprising the other upstream start codon encodes a sequence called pre-C; 3) an ORF with three start codons, one of which encodes a surface protein (gp27), one including an upstream start codon encoding a sequence referred to as pre-S2(gp36), and the other including a start codon further upstream of the sequence referred to as pre-S1(gp 42); and 4) an ORF encoding HBxAg, a less well understood protein of function.

Prophylactic vaccines and treatments for HBV infection involve injection of subviral particles purified from the plasma of chronic carriers or produced as recombinant proteins in stably transfected eukaryotic cell lines. The subviral particles are viral proteins and these vaccines are generally referred to as subunit vaccines. HBV proteins are administered to an individual and become targets of the individual's immune system. In an uninfected individual, an immune response against the subunit vaccine protects the uninfected individual from HBV infection. In infected individuals, the immune response induced by the vaccine may have a therapeutic effect.

International virology (Intervirology)2001.44:98-114, including Kissali F.V (Chisari F.V.), American journal of Patholol (Am J Pathol.), 2000.156:1117-1132 and Papas P (Pumpeus P.), disclose HBV genome structures. Dinen P. (Deny P.) and f. zhouli (f. zuoli), pathology and biology (Pathologie biologic) 2010, 8 months, 58(4): 24587 discuss the diagnosis and treatment of hepatitis b virus. Macke M.L (Michel M.L.) and p.tiollais (p.tiollais), pathology and biology 2010, 8 months, 58(4): 28895 discuss hepatitis b vaccines and their protective efficacy and therapeutic potential. PCT publication W02004026899 discloses the use of immunogens comprising polypeptide sequences having the amino acid sequence of HBV. PCT published application W02008093976 discloses HBV coding sequences, proteins and vaccines including vaccines comprising recombinant full length HBV surface antigens and HBV core antigens. The entire HBV surface antigen is composed of three types of surface proteins (L protein, M protein and S protein). PCT published application W02009130588 discloses HBV coding sequences, proteins and vaccines comprising nucleic acid encoding a hepatitis b virus core antigen, said nucleic acid being codon optimized for expression in humans. PCT publication W02010127115 discloses the use of recombinant vectors to deliver HBV sequences.

Available HBV vaccines have shown some efficacy but are expensive to produce. In addition, plasma-derived subunit vaccines also have concerns regarding safety. Several vaccine approaches have been investigated, including those based on recombinant live vectors, synthetic peptides, and DNA vaccines comprising codon-optimized coding sequences for HBV proteins. These other approaches have heretofore had varying limited efficacy. Furthermore, some HBV vaccines have demonstrated positive efficacy in some geographical regions and limited efficacy in other regions due to genomic differences.

The direct administration of nucleic acid sequences for vaccination against animal and human diseases has been investigated and much effort has been focused on efficient and effective means of nucleic acid delivery in order to generate the necessary expression of the desired antigen to elicit an immunogenic response and ultimately the success of this technology.

DNA vaccines allow endogenous antigen synthesis, which induces CD8+ histocompatible complex class I-restricted cytotoxic T lymphocytes, which are rarely obtained 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. In addition, DNA vaccines appear to be very stable and simple to produce. Furthermore, a broader cellular immune response can be induced by combining strategies such as codon optimization, RNA optimization and addition of immunoglobulin leaders.

DNA vaccines are safe, stable, easy to produce, and well tolerated in humans, with preclinical experiments indicating little evidence of plasmid integration [ Martin T. (Martin, T.) and the like, plasmid DNA malaria vaccines: possibility of genomic integration after intramuscular injection. (Plasmid DNA molecular vaccine: the genetic integration after intramucosal injection.) human Gene therapy (Hum Gene Ther), 1999.10(5): pages 759-68; nikes W.W, (Nichols, W.W), etc., with possible DNA vaccines integrated into the host cell genome. (Potential DNA vaccine integration in host cell genome.) academic annual newspaper of New York academy of sciences (Ann N Y Acad Sci), 1995.772:30-9 pages ]. Furthermore, DNA vaccines are well suited for repeated administration due to the fact that the efficacy of the vaccine is not affected by pre-existing antibody titers to the vector [ chantergo M. (Chattergoon, M.), j. New era of vaccines and immunotherapy. (Genetic immunization: a new era in vaccines and immunoherapeutics.) the American society for laboratory and biology Union J (FASEB J), 1997.11(10):753-63 ]. However, one major obstacle to clinical adoption of DNA vaccines is the reduction in immunogenicity of the platform when moved to larger animals [ Liu M.A. (Liu, M.A.) and j.b. emer (j.b. ulmer), human clinical trials of plasmid DNA vaccines. (Human clinical trials of plasmid DNA vaccines.) genetic progress (Adv Genet), 2005.55: pages 25-40 ].

Recent technological advances in the engineering of 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 [ anderle S. (Andre, S.) and the like, enhanced immune responses elicited by DNA vaccination with synthetic gpl20 sequences with optimized codon usage. (incorporated immune response infected by DNA vaccination with an antigenic gpl20 sequence with an optimized codon usage.) J virology (J Virol), 1998.72(2):1497-503 pages; daimi L. (Demi, L.), etc., and the multiple effects of codon usage optimization on the expression and immunogenicity of DNA candidate vaccines encoding human immunodeficiency virus type 1 Gag protein. (Multiple effects of coding optimization on expression and immunogenicity of DNA coding catalysis the human immunogenic virus type 1 Gag protein.) virology journal 2001.75(22): 10991-; leydi D.J. (Laddy, D.J.), etc., novel consensus-based DNA vaccines are immunogenic against avian influenza. (immunological of novel consensus DNA Vaccine influenza Vaccine) (Vaccine), 2007.25(16) p 2984-9; frelin L, etc., codon optimization and mRNA amplification effectively enhanced the immunogenicity of the hepatitis C virus non-structural 3/4A gene. (Codon optimization and mRNA amplification efficacy enhancement genes and Gene therapy (Gene Ther), 2004.11(6): pages 522-33 ], and recently developed techniques in plasmid delivery systems such as electroporation [ blunt-tailed L.A. (Hirao, L.A.) and the like, intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and efficacy in pigs and rhesus monkeys. (Intradermal/subceutaneous immunization by electroporation plasmid delivery and containment in pigs and rhesus macaques.) vaccine 2008.26(3) page 440-8; lackay a. (Luckay, a.), et al, plasmid DNA vaccine design and the effect of in vivo electroporation on vaccine-specific immune responses generated in rhesus monkeys. (Effect of plasmid DNA vaccine design and in vivo electrophoresis on the killing vaccine-specific immunological responses in rhesus macaques.) J.Virol. 2007.81(10) pages 5257-69; allen G (Ahlen, G.), etc., in vivo electroporation enhances the immunogenicity of hepatitis c virus non-structural 3/4a DNA through increased local DNA uptake, protein expression, inflammation, and CD3+ T cell infiltration. (In vivo electrophoresis of immunology of hepatitis C virus not structural 3/4A DNA by incubation DNA update, protein expression, and enrichment of CD3+ cells.) journal of immunology (J Immunol), 2007.179(7): pages 4741-53 ]. In vivo electroporation techniques have been used in human clinical trials to deliver anticancer drugs such as bleomycin and have been used in a large number of animal species in a number of preclinical studies. In addition, studies have shown that the use of shared immunogens can increase the breadth of cellular immune responses [ strictly J. (Yan, J.) ] and the like, as compared to native antigens alone, enhanced cellular immune responses elicited by engineered HIV-1 subtype B consensus-based envelope DNA vaccines. (Enhanced cellular immunity stressed by engineered HIV-1 subtype B consensus-based envelope DNA vaccine.) cell therapy (MolTher), 2007.15(2): pages 411-21; rollan. M. (Rolland, M.), et al, reconstruction and function of human immunodeficiency virus type 1 proteins at the center of the evolutionary system of ancestry. (Reconstruction and function of center-of-tree great immunity virus type 1 proteins.) J.Virol, 2007.81(16): pages 8507-14 ].

There remains a need for nucleic acid constructs encoding HBV antigens and compositions suitable for inducing an immune response against HBV. There remains a need for an effective vaccine against HBV that is economical and effective. There remains a need for effective vaccines that increase the level of neutralizing antibodies and elicit T cell components. There remains a need for effective vaccines against HBV, including those effective against HBV strains with a broad genotype range, and preferably universal vaccines that are effective on a global scale.

Summary of The Invention

One aspect of the present invention includes a vaccine suitable for inducing an immune response against HBV. The development of HBV immunotherapeutic vaccines with broad efficacy against multiple genotypes can be provided using therapeutic DNA vaccines against HBV infection based on targeting the ubiquitous preserved HBV core-specific antigen. The use of common HBV immunogens induces a broader cellular immune response and may be useful in minimizing sequence dissimilarity between different virus strains.

Provided herein are proteins selected from the group consisting of: a protein comprising SEQ ID NO 2; a protein 295% homologous to SEQ id no; 2, fragment of SEQ ID NO; a protein 95% homologous to the fragment of SEQ ID NO 2; 4, a protein having 495% homology to SEQ ID NO; 4, a fragment of SEQ ID NO; a protein 95% homologous to the fragment of SEQ ID NO. 4; 6, a protein which is 695% homologous to SEQ ID NO; a fragment of SEQ ID NO 6; and a protein 95% homologous to the fragment of SEQ ID NO 6.

Also provided are nucleic acid molecules comprising sequences 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, SEQ ID NO; a nucleic acid sequence which is 195% homologous to SEQ ID NO; 1, fragment of SEQ ID NO; a nucleic acid sequence 95% homologous to the fragment of SEQ ID NO. 1; SEQ ID NO. 3; a nucleic acid sequence which is 395% homologous to SEQ ID NO; a fragment of SEQ ID NO 3; a nucleic acid sequence 95% homologous to the fragment of SEQ ID NO. 3; 5, SEQ ID NO; a nucleic acid sequence which is 595% homologous to SEQ ID NO; a fragment of SEQ ID NO 5; and a nucleic acid sequence 95% homologous to the fragment of SEQ ID NO. 5.

Some aspects of the invention provide methods of inducing an immune response against a core antigen from one or more HBV genotypes, the method comprising the steps of: such nucleic acid molecules and/or compositions are administered to an individual.

Further aspects of the invention provide methods of protecting an individual from HBV infection. These methods include the steps of: administering to the individual a prophylactically effective amount of a nucleic acid molecule or composition comprising such a nucleic acid sequence; wherein the nucleic acid sequence is expressed in cells of the individual and induces a protective immune response against the protein encoded by the nucleic acid sequence.

In some aspects of the invention, methods are provided for treating an individual already infected with HBV. These methods include the steps of: administering to the individual a therapeutically effective amount of such nucleic acid molecules and/or compositions.

Aspects of the invention further relate to vaccines comprising a protein or a nucleic acid encoding a protein selected from the group consisting of: a protein comprising SEQ ID NO 2, a protein 295% homologous to SEQ ID NO; 2, fragment of SEQ ID NO; a protein 95% homologous to the fragment of SEQ ID NO 2; 4, a protein that is 495% homologous to SEQ ID NO; 4, a fragment of SEQ ID NO; a protein 95% homologous to the fragment of SEQ ID NO. 4; 6, a protein which is 695% homologous to SEQ ID NO; a fragment of SEQ ID NO 6; and a protein 95% homologous to the fragment of SEQ ID NO 6. The vaccine may further comprise an adjuvant protein or a nucleic acid sequence encoding an adjuvant protein. In some embodiments, the adjuvant is IL-12, IL-15, IL-28 or RANTES.

A vaccine comprising a nucleic acid molecule may comprise a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: 1, SEQ ID NO; a nucleic acid sequence which is 195% homologous to SEQ ID NO; 1, fragment of SEQ ID NO; a nucleic acid sequence 95% homologous to the fragment of SEQ ID NO. 1; 3, SEQ ID NO; a nucleic acid sequence which is 395% homologous to SEQ ID NO; a fragment of SEQ ID NO. 3; a nucleic acid sequence 95% homologous to the fragment of SEQ ID NO. 3; 5, SEQ ID NO; a nucleic acid sequence which is 595% homologous to SEQ ID NO; a fragment of SEQ ID NO 5; and a nucleic acid sequence 95% homologous to the fragment of SEQ ID NO. 5. The vaccine may further comprise a nucleic acid sequence encoding an adjuvant protein. In some embodiments, the adjuvant is IL-12, IL-15, IL-28 or RANTES.

Brief Description of Drawings

FIG. 1 is a schematic diagram showing the structure of HBV genome consisting of four overlapping ORFs.

FIGS. 2A and 2B show results from pM Core expression experiments. Figure 3A shows results from an in vitro translation protocol. Fig. 3B shows the results of western blotting.

FIGS. 3A and 3B show the amount of increased IFN- γ secretion in CD8+ and CD4+ T cells from the spleen of C57BL/6 mice vaccinated with pM-Core.

FIGS. 4A and 4B show the amount of increased TNF- α secretion in CD8+ and CD4+ T cells from the spleen of C57BL/6 mice vaccinated with pM-Core.

FIGS. 5A and 5B show the amount of increased CD 107a secretion in CD8+ and CD4+ T cells from the spleen of C57BL/6 mice vaccinated with pM-Core.

FIGS. 6A and 6B show interferon- γ T cell responses in the liver from C57BL/6 mice vaccinated with pM-Core.

FIGS. 7A and 7B show the TNF-. alpha.T cell response in the liver from C57BL/6 mice vaccinated with pM-Core.

Figure 8 shows data from ELISPOT assays.

Figure 9 shows data from experiments using CSFE labeled cells to compare the in vivo removal of peptide-treated target cells from CD 8T cells in vaccinated and unvaccinated animals.

FIG. 10 shows a comparison of the proliferation percentage of CD3+ CD4+ cells and CD3+ CD8+ treated with pVax vector (control) or with plasmid pMCore expressing HBV M-core.

FIGS. 11A and 11B show a comparison of anti-HBV core antibody in serially diluted sera from animals treated with pVax vector (control) or with plasmid pMCore expressing HBVM-core.

FIG. 12 shows the percent of TNF-a and IFN-g in spleen and liver cells from CD4+ and CD8 +.

Figure 13 shows data from experiments in which the level of ALT in serum was measured to determine whether clearance induced by immunized mice affected the liver.

Detailed description of the invention

1. And (4) defining.

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 in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

To the extent that numerical ranges are recited herein, each intervening number between equal degrees of precision is explicitly recited. For example, for the range of 6-9, the numbers 7 and 8 are encompassed in addition to 6 and 9, and for the range of 6.0-7.0, 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 encompassed.

a. Adjuvant

As used herein, "adjuvant" means any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigen encoded by the DNA plasmids and encoding nucleic acid sequences described below.

b. Antibodies

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

c. Coding sequence

"coding sequence" or "coding nucleic acid" as used herein means a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in the cells of the subject or mammal to which the nucleic acid is administered.

d. Complementary body

"complement" or "complementary" as used herein means that a nucleic acid can refer to Watson-Crick (e.g., A-T/U and C-G) or Hustein (Hoogsteen) base pairing between nucleotides or nucleotide analogs of the nucleic acid molecule.

e. Consensus or consensus sequences

As used herein, "consensus" or "consensus sequence" means a polypeptide sequence of multiple subtypes based on a queue of specific HBV antigens for analysis. Nucleic acid sequences encoding the consensus polypeptide sequence may be prepared. Vaccines comprising proteins comprising consensus sequences and/or nucleic acid molecules encoding these proteins can be used to induce broad immunity against multiple subtypes or serotypes of a particular HBV antigen.

f. Electroporation

"electroporation," "electro-permeabilization," or "electrokinetic enhancement" ("EP") as used interchangeably herein means the use of transmembrane electric field pulses to induce microscopic pathways (pores) in a biological membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to flow from one side of the cell membrane to the other.

g. Fragments

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

By "fragment" or "immunogenic fragment" with respect to a polypeptide sequence is meant a polypeptide capable of eliciting an immune response in a mammal that is cross-reactive with a full-length wild-type strain HBV antigen. Fragments of a consensus protein may 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 may comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more of the consensus protein.

h. Gene construct

The term "genetic construct" as used herein refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence comprises an initiation signal and a termination signal operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. The term "expression form" as used herein refers to a genetic construct containing the necessary regulatory elements operably linked to a coding sequence encoding a protein such that the coding sequence will be expressed when present in the cells of the individual.

i. Are identical to each other

In the case of two or more nucleic acid or polypeptide sequences, "identical" or "identity" as used herein means that the sequences have a specified percentage of identical residues in a specified region. The percentage may be calculated by: optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions of the identical residue in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. Where two sequences are of different lengths or the alignment produces one or more staggered ends and the specified regions of comparison include only a single sequence, the residues of the single sequence are included in the denominator of the calculation rather than in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

j. Immune response

As used herein, "immune response" means activation of the host's immune system (e.g., the mammalian immune system) in response to the introduction of an antigen, such as an HBV consensus antigen. The immune response may be in the form of a cellular response or a humoral response or both.

k. Nucleic acids

As used herein, a "nucleic acid" or "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The description of single strands also defines the sequence of the complementary strand. Thus, nucleic acids also encompass the complementary strand of the single strand described. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acids also encompass substantially the same nucleic acids and their complements. Single strands provide probes that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids also encompass probes that hybridize under stringent hybridization conditions.

The nucleic acid may be single-stranded or double-stranded or may contain portions of both double-stranded or single-stranded sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acid may be obtained by chemical synthesis methods or by recombinant methods.

Is operatively connected to

"operably linked" as used herein means that expression of a gene is under the control of a promoter to which it is spatially linked. Under its control, the promoter may be positioned 5 '(upstream) or 3' (downstream) of the gene. The distance between the promoter and the gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, this change in distance can be adjusted without loss of promoter function.

m. promoter

As used herein, "promoter" means a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. The promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or alter spatial and/or temporal expression thereof. A promoter may also contain distal enhancer or repressor elements, which can be located as much as several thousand pairs of base pairs from the start of transcription. Promoters may be obtained from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate expression of a gene component either substantially or differentially with respect to the cell, tissue or organ in which expression occurs or with respect to the developmental stage at which expression occurs or in response to an external stimulus such as a physiological stress, pathogen, metal ion or inducer. Representative examples of promoters include the phage T7 promoter, the phage T3 promoter, the SP6 promoter, the lactose operon-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 CMVIE promoter.

n. signal peptide

"Signal peptide" and "leader sequence" are used interchangeably herein and refer to amino acid sequences that can be attached to the amino terminus of HBV proteins described herein. The signal peptide/leader sequence is generally indicative of the position of the protein. The signal peptide/leader sequence used herein preferably facilitates secretion of the protein from the cell in which it is produced. The signal peptide/leader sequence is often cleaved from the remainder of the protein, which is often referred to as the mature protein after secretion from the cell. The signal peptide/leader sequence is linked to the N-terminus of the protein. As mentioned herein with respect to linking a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of the nucleic acid sequence, rather than encoding the protein without the signal peptide coding sequence. Thus, for example, SEQ ID NO. 4 is SEQ ID NO. 2 with the signal peptide/leader sequence attached to the N-terminus of SEQ ID NO. 2, i.e., SEQ ID NO. 4 is a protein comprising a signal peptide attached to the N-terminus of SEQ ID NO. 2. The first residue "Xaa" in SEQ ID NO:2 is typically methionine in the absence of the signal peptide. However, the protein comprising the signal peptide linked to SEQ ID NO. 2 is as in SEQ ID NO. 4 with the substitution of the residue linking the signal peptide to the protein for the residue 1 methionine at Xaa. Thus, the N-terminal residue of SEQ ID NO. 2 can be any amino acid, but it is methionine if it is encoded by the starting sequence. The attachment of the signal peptide/leader sequence at the N-terminus of SEQ ID NO. 2 generally eliminates the N-terminal methionine. As used herein, it is intended that SEQ ID NO 4 comprises SEQ ID NO 2 with the signal peptide/leader sequence attached at the N-terminus of SEQ ID NO 2, although the N stretch Xaa residues of SEQ ID NO 2 have been removed. Similarly, the coding sequence of SEQ ID NO. 4 includes the coding sequence of SEQ ID NO. 2 and the coding sequence of the signal peptide/leader sequence linked to the 5' end of the coding sequence encoding SEQ ID NO. 2. The initiation codon may be "nnn" in the coding sequence of SEQ ID NO. 2, but is eliminated when the coding sequence of the signal peptide/leader sequence is ligated to the 5' end of the coding sequence encoding SEQ ID NO. 2. As used herein, it is intended that the coding sequence of SEQ ID NO. 4 comprises the coding sequence of SEQ ID NO. 2 and the coding sequence of the signal peptide/leader sequence linked 5' to the coding sequence of SEQ ID NO. 2 where nnn occurs. Thus, for example, it is desirable that SEQ ID NO 3 comprises the coding sequence of SEQ ID NO 1 and the signal peptide/leader sequence attached to the 5' end of SEQ ID NO 1 in place of nnn. In some embodiments, nnn is the start codon at the 5' end of SEQ ID NO 1.

Stringent hybridization conditions

As used herein, "stringent hybridization conditions" means conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize 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 may be selected to be more than the thermodynamic melting point (T) of a particular sequence at a defined ionic strength pH_m) About 5-10 deg.c lower. The T is_mMay 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 (at T, due to the presence of excess target sequence)_mNext, 50% of the probes are occupied in the equilibrium state). Stringent conditions may be those in which the salt concentration is less than about 1.0M sodium ion, such as about 0.01-1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes (e.g., about 10-50 nucleotides) and at least about 60 ℃ for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For a selected or specific hybridization, the positive signal can be at least 2 to 10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃ or 5 XSSC, 1% SDS, incubated at 65 ℃ washed with 0.2 XSSC and 0.1% SDS at 65 ℃.

Is substantially complementary to

"substantially complementary" as used herein means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 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, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that two sequences hybridize under stringent hybridization conditions.

q. essentially the same

"substantially identical" as used herein means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acid regions at least 60%, 65%, 70%, 95%, 97%, 98% or 99% identical, or, in the case of nucleic acids, if the first and second sequences are substantially complementary, so are the first and second sequences, within 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, 85, 90, 95, 80,85, 540 or more nucleotides or amino acid regions.

Subtype or serotype

"subtype" or "serotype": as used interchangeably herein and with HBV is meant a genetic variant of HBV such that one subtype is recognized by the immune system and separated from a different subtype.

s. variants

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

"variants" in the case of peptides or polypeptides differ in amino acid sequence by insertion, deletion or conservative substitution of amino acids, but retain at least one biological activity. Variant also means a protein having substantially the same amino acid sequence as a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., the replacement of an amino acid with a different amino acid of similar characteristics (e.g., hydrophilicity, extent and distribution of charged regions) are believed in the art to typically involve minor changes. As understood in the art, these minor changes may be identified in part by considering the hydropathic index of amino acids. Kate (Kyte), et al, J.Mol.biol., 157:105-132 (1982). The hydropathic index of the amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the case of peptides allows the calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101 is incorporated by reference herein in its entirety. As is understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity (e.g., immunogenicity). Substitutions may be made with amino acids having hydrophilicity values within ± 2 of each other. Both the hydropathic index and the hydropathic value of an amino acid are affected by the specific side chain of the amino acid. Consistent with the observations, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of these amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

t. vector

"vector" as used herein means a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extra-chromosomal vector, and is preferably a DNA plasmid.

HBV core antigen

HBV core antigen represents an important target for cross-presentation 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 immune-mediated viral clearance.

Table 2 shows the similarity between genotypes of core antigen with shared HBV core protein from HBV-A, HBV-B, HBV-C, HBV-D and HBV-E genotypes, which is called "HBV-M-core" in the figure. For some embodiments, HBV M core constructs are designed to have increased homology to a broad range of HBV core targets. Similarity between genotypes of core antigens with designed M-core constructs-increased homology to a wide range of HBV core targets. All genotypes should be represented by the universal immunotherapeutic vaccines for HBV.

TABLE 2

Provided herein are antigens capable of eliciting an immune response in a mammal against one or more HBV serotypes. The antigens may comprise core protein epitopes that make them particularly effective as immunogens against which an anti-HBV immune response can be induced. The HBV antigen may include a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof.

A consensus HBV core protein (SEQ ID NO:2) is provided. An amino acid sequence is generated that contains the IgE leader sequence at the N-terminus of the HBV core protein consensus sequence. Thus, proteins having an IgE leader (SEQ ID NO:7) linked to a consensus HBV core protein (SEQ ID NO:2) are also provided, so as to provide the IgE leader-consensus HBV core protein (SEQ ID NO: 4). Some embodiments provided further include an HA tag (SEQ ID NO:8) attached to the C-terminus of the HBV core protein consensus sequence. Thus, there is provided an HBV core protein consensus protein (SEQ ID NO:6) comprising an IgE leader (SEQ ID NO:7) linked to the HBV core protein consensus sequence (SEQ ID NO:2) and an HA tag (SEQ ID NO:8) linked to the C-terminus of the HBV core protein consensus sequence.

The protein may be homologous to SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequences herein. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequences herein. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequences herein. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequences herein. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequences herein.

In some embodiments, the protein is free of a leader sequence. In some embodiments, the protein lacks an IgE leader sequence. Fragments of a consensus protein 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 consensus protein. Immunogenic fragments of SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 may be provided. An immunogenic fragment 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 SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6. In some embodiments, the fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, i.e., an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to the immunogenic fragments of SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6 can 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 proteins that are homologous to S SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 695%. Some embodiments relate to immunogenic fragments that are 96% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 97% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 98% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 99% homologous to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, i.e., an IgE leader sequence.

3. Gene sequences, constructs and plasmids

Nucleic acid sequences encoding SEQ ID NO 2,4 and 6 and homologous proteins, immunogenic fragments and immunogenic fragments of homologous proteins can be routinely generated. Thus, nucleic acid molecules encoding immunogenic proteins having up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence can be provided. Likewise, nucleic acid sequences encoding the immunogenic fragments described herein and immunogenic fragments of proteins homologous to the proteins described herein are also provided.

Producing a nucleic acid molecule encoding the consensus amino acid sequence. The vaccine may comprise one or more nucleic acid sequences encoding one or more consensus forms of immunogenic proteins selected from the group of sequences generated to optimize stability and expression in humans. A nucleic acid sequence (SEQ ID NO:1) encoding HBV core protein consensus protein (SEQ ID NO:2), a nucleic acid sequence (SEQ ID NO:3) encoding IgE leader-HBV core protein consensus protein (SEQ ID NO:4), and a nucleic acid sequence (SEQ ID NO:5) encoding IgE leader-HBV core protein consensus protein-HA tag (SEQ ID NO: 6). Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 96% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding 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.

In some embodiments, the nucleic acid sequence is free of coding sequences that encode a leader sequence. In some embodiments, the nucleic acid sequence is free of coding sequences that encode an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5. 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% 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% homologous to the fragments of SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5. In some embodiments, a fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, a fragment lacks a coding sequence that encodes a leader sequence. In some embodiments, the fragment does not encode a leader sequence, i.e., the coding sequence for the IgE leader sequence.

Provided herein are genetic constructs that can comprise a nucleic acid sequence encoding an HBV core antigen disclosed herein, including a consensus protein sequence, a sequence homologous to the consensus protein sequence, a fragment of the consensus protein sequence, and a sequence homologous to a fragment of the consensus protein sequence. The genetic construct may be present as a functional extrachromosomal molecule. The genetic construct may be a linear minichromosome comprising a centromere, telomere or plasmid or cosmid.

The genetic construct may also be part of the genome of a recombinant viral vector, including recombinant adenovirus, recombinant adenovirus associated virus, and recombinant vaccinia. The genetic construct may be part of the genetic material in a recombinant microbial vector in an attenuated live microorganism or live in a cell.

The genetic construct may comprise regulatory elements for gene expression of the coding sequence of the nucleic acid. The regulatory element may be a promoter, enhancer, start codon, stop codon or polyadenylation signal.

The nucleic acid sequence may constitute a genetic construct which may be a vector. The vector is capable of expressing an antigen in cells of a mammal in an amount effective to elicit an immune response in the mammal. The vector may be recombinant. The vector may comprise a heterologous nucleic acid encoding an antigen. The vector may be a plasmid. The vector may be suitable for transfecting cells with nucleic acid encoding an antigen, the transformed host cells being cultured and maintained under conditions in which expression of the antigen occurs.

The coding sequence can be optimized for stability and high levels of expression. In some cases, the codons are selected to reduce the formation of RNA secondary structures, such as those due to intramolecular bonds.

The vector may comprise a heterologous nucleic acid encoding an antigen, and may further comprise a start codon that may be upstream of the antigen encoding sequence and a stop codon that may be downstream of the antigen encoding sequence. The initiation codon and the stop codon can be in frame with the antigen coding sequence. The vector further comprises a promoter operably linked to the antigen coding sequence. The promoter operably linked to the antigen-encoding sequence may be a promoter from simian virus 40(SV40), mouse mammary virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) promoter such as the Bovine Immunodeficiency Virus (BIV) Long Terminal Repeat (LTR) promoter, Moloney (Moloney) virus promoter, Avian Leukemia Virus (ALV) promoter, Cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr Virus (EBV) promoter, or Rous Sarcoma Virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human heme, 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 these promoters are described in U.S. patent application publication No. US20040175727, the entire contents of which are incorporated herein.

The vector may further comprise a polyadenylation signal, which may be downstream of the HBV core protein coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β -globin polyadenylation signal. The SV40 polyadenylation signal can be a polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, CA).

The vector may also comprise an enhancer upstream of the consensus HBV core protein coding sequence. The enhancer is necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV or EBV. Enhancer of polynucleotide function is described in U.S. Pat. Nos. 5,593,972, 5,962,428 and WO94/016737, each of which is incorporated herein by reference in its entirety.

The vector may also comprise a mammalian origin of replication, in order to maintain the vector extrachromosomally and produce multiple copies of the vector in the cell. The vector may be pVAX1, pCEP4, or pREP4 from invitrogen (san diego, ca), which may contain the replication origin of epstein-barr virus and the nuclear antigen EBNA-1 coding region, which may produce high copy episomal replication without integration. The vector may be a pVAX1 or a pVAX1 variant, such as a variant plasmid described herein, with a variation. 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 base 137-724. The T7 promoter/initiation site was located at base 664-683. The multiple cloning site is located at bases 696-811. The bovine GH polyadenylation signal is at base 829-1053. The Kanamycin (Kanamycin) resistance gene is at base 1226-containing 2020. The pUC origin is at base 2320-2993.

Based on the sequence of pVAX1 available from invitrogen, the following mutations were found in the sequence of pVAX1 used as the backbone of plasmids 1-6 described herein:

the backbone of the vector may be pAV 0242. The vector may be a replication-defective adenovirus 5 (Ad5) vector.

The vector may also comprise regulatory sequences which may be well suited for gene expression in a mammalian or human cell to which the vector is administered. The consensus HBV coding sequence may comprise codons that may allow for more efficient transcription of the coding sequence in a host cell.

The vector may be pSE420 (invitrogen, san diego, california), which is useful for the production of proteins in e. The vector may be pYES2 (Invitrogen, san Diego, Calif.) which can be used to produce proteins in a Saccharomyces cerevisiae strain of yeast (Saccharomyces cerevisiae strains). The vector may also have a MAXBAC^TMThe complete baculovirus expression system (Invitrogen, san Diego, Calif.) can be used to produce proteins in insect cells. The vector may also be pcDNA I or pcDNA3 (Invitrogen, san Diego, Calif.) which can be used to produce proteins in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The vector may be an expression vector or system for producing a protein by conventional techniques and readily available starting materials, including Sambrook (Sambrook) et al, Molecular Cloning and Laboratory Manual, second edition, Cold Spring gallery Laboratory (Cold Spring Harbor) (1989), which are incorporated herein by reference in their entirety.

4. Pharmaceutical composition

Provided herein are pharmaceutical compositions according to the invention comprising from about 1 nanogram to about 10mg of DNA. In some embodiments, the pharmaceutical composition according to the invention comprises between: 1) at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 nanograms or at least 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, 235, 240, 245, 250, 255, 260, 265, 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, 440, 460, 605, 455, 470, 610, 445, 610, 475, 440, 470, 610, 475, 440, 480, 220, 475, 220, 230, 240, 250, 255, 260, 265, 270, 275, 280, 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, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms or 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 10mg or more; and 2) up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 nanograms, or up to and including 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, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 440, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 495, 370, 375, 380, 385, 405, 410, 395, 400, 405, 410, 415, 450, 470, 455, 470, 500, 475, 445, 470, 200, 205, 215, 220, 225, 230, 235, 240, 245, 250, 255, 265, 240, 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, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms, or up to and including 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.5, or 10 mg. In some embodiments, a pharmaceutical composition according to the invention comprises about 5 nanograms to about 10mg of DNA. In some embodiments, a pharmaceutical composition according to the invention comprises from about 25 nanograms to about 5 milligrams of DNA. In some embodiments, the pharmaceutical composition contains from about 50 nanograms to about 1 milligram of DNA in some embodiments, the pharmaceutical composition contains from about 0.1 micrograms to about 500 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 1 microgram to about 350 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 5 micrograms to about 250 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 10 micrograms to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 15 micrograms to about 150 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 20 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 75 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 30 micrograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 35 micrograms to about 40 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 100 micrograms to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 35 nanograms to about 45 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 micrograms to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 microgram to about 350 microgram of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 micrograms to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 100 micrograms to about 200 micrograms of DNA.

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

Preferably, the pharmaceutical composition is a vaccine, and more preferably a DNA vaccine.

Provided herein are vaccines that are capable of generating an immune response in a mammal against one or more genotypes of HBV. The vaccine may comprise a genetic construct as discussed above.

Although not bound by scientific theory, vaccines can be used to elicit an immune response (humoral, cellular, or both) that is broadly directed against one or more genotypes of HBV. The vaccine may comprise a coding sequence of a consensus HBV core protein sequence (SEQ ID NO: 2); an IgE leader linked to the consensus HBV core protein sequence (SEQ ID NO: 4); and an IgE leader linked to a consensus HBV core protein linked to the HA tag sequence (SEQ ID NO: 6). The vaccine may comprise a specific coding sequence of a consensus HBV core protein sequence (SEQ ID NO:2) such as (SEQ ID NO: 1); the IgE leader sequence as shown (SEQ ID NO:3) linked to the consensus HBV core protein sequence (SEQ ID NO: 4); and the IgE leader linked to the consensus HBV core protein linked to the HA tag sequence (SEQ ID NO:6) as shown (SEQ ID NO: 5).

Some alternative embodiments include those comprising nucleic acid sequences encoding: an immunogenic fragment of a consensus HBV core protein, one or more proteins homologous to the consensus HBV core protein, and an immunogenic fragment of one or more proteins homologous to the consensus HBV core protein.

Some embodiments provide methods of generating an immune response against HBV core protein, the method comprising administering to an individual one or more compositions described herein. Some embodiments provide methods of prophylactically vaccinating an individual against HBV infection, the method comprising administering one or more compositions described herein. Some embodiments provide methods of therapeutically vaccinating an individual infected with HBV, comprising administering one or more compositions described herein. Diagnosis of HBV infection can be routinely accomplished prior to administration.

The vaccine may be a DNA vaccine. The DNA vaccine may comprise a plurality of identical or different plasmids comprising a nucleic acid sequence encoding a consensus HBV core protein.

DNA vaccines are disclosed in U.S. patent nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are all incorporated herein by reference. The DNA vaccine may further comprise an element or agent that prevents integration into the chromosome. The vaccine may be RNA of HBV core protein. The RNA vaccine can be introduced into a cell.

The vaccine may be a recombinant vaccine comprising a genetic construct or antigen as described above. The vaccine may further comprise one or more consensus HBV core proteins in the form of one or more protein subunits, one or more killed viral particles comprising one or more consensus HBV core proteins, or attenuated viral particles comprising one or more consensus HBV core proteins. The attenuated vaccines can be live attenuated vaccines, killed vaccines or vaccines that use recombinant vectors to deliver foreign genes encoding one or more consensus HBV core proteins, as well as subunit vaccines and glycoprotein vaccines. Examples of attenuated live vaccines, those that use recombinant vectors to deliver foreign antigens, subunit vaccines, and glycoprotein vaccines are described in U.S. patent nos.: 4,510,245; 4,797,368; 4,722,848; 4,790,987, respectively; 4,920,209, respectively; 5,017,487, respectively; 5,077,044, respectively; 5,110,587; 5,112,749, respectively; 5,174,993; 5,223,424, respectively; 5,225,336, respectively; 5,240,703, respectively; 5,242,829, respectively; 5,294,441, respectively; 5,294,548, respectively; 5,310,668, respectively; 5,387,744, respectively; 5,389,368, respectively; 5,424,065, respectively; 5,451,499, respectively; 5,453,364, respectively; 5,462,734, respectively; 5,470,734, respectively; 5,474,935, respectively; 5,482,713, respectively; 5,591,439, respectively; 5,643,579, respectively; 5,650,309, respectively; 5,698,202, respectively; 5,955,088, respectively; 6,034,298; 6,042,836, respectively; 6,156,319 and 6,589,529, each of which is incorporated herein by reference.

The vaccine may comprise vectors and/or proteins directed against multiple HBV genotypes from multiple specific regions of the world. The vaccines provided are useful for inducing immune responses, including therapeutic or prophylactic immune responses. Antibodies and/or killer T cells can be generated against the common HBV core protein and also widely across multiple genotypes of HBV viruses. Such antibodies and cells can be isolated.

The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule that acts as a vehicle, adjuvant, carrier or diluent. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surfactants such as Immune Stimulating Complexes (ISCOMS), Freunds incomplete adjuvant, LPS analogs including monophosphoryl ester a, muramyl peptides, benzoquinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitating agents.

The transfection facilitating agent is a polyanion, polycation (including poly-L-glutamic acid (LGS)), or lipid. 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 also include surfactants such as Immune Stimulating Complexes (ISCOMS), freunds incomplete adjuvant, LPS analogs (including monophosphoryl ester a), muramyl peptides, benzoquinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be used to be administered with the genetic construct. In some embodiments, the DNA vector vaccine may further include transfection-facilitating agents such as lipids, liposomes (including lecithin liposomes or other liposomes known in the art (such as DNA-liposome mixtures (see, e.g., W09324640))), calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection-facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation (including poly-L-glutamic acid (LGS)), or lipid. The concentration of the transfection agent in the vaccine is less than 4mg/ml, less than 2mg/ml, less than 1mg/ml, less than 0.750mg/ml, less than 0.500mg/ml, less than 0.250mg/ml, less than 0.100mg/ml, less than 0.050mg/ml or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be an adjuvant. The adjuvant may be other genes expressed in alternative plasmids or delivered as proteins in combination with the above plasmids in vaccines. The adjuvant may be selected from the group consisting of: alpha-interferon (IFN-. alpha.), beta. -interferon (IFN-. beta.), gamma-interferon, platelet-derived growth factor (PDGF), TNF. alpha., TNF. beta., GM-CSF, Epidermal Growth Factor (EGF), cutaneous T cell attracting chemokine (CTACK), epithelial thymus-expressing chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 lacking a signal sequence and optionally including a signal peptide from IgE). The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

Other genes that may be suitable adjuvants include those encoding: MCP-1, MIP-1a, 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, mutant forms of IL-18, CD40, CD40L, vascular growth factors, fibroblast growth factors, IL-7, nerve growth factors, vascular endothelial growth factors, Fas, TNF receptors, Flt, ApoApo-1, P55, WSL-1, DR3, AIMP, Apo-3, LARD, NGRF, DR4, DR5, KILLER, TRAIL-46R 35, TRICK2, TRISPL 6, caspase-1, FO-1, FOICE-3, FO-1, FORD-1, FO-1, FOSP-7, FORCE-1, and so, Ap-1, Ap-2, p38, p65Re1, MyD88, IRAK, TRAF6, IkB, inactivated NIK, SAP K, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

5. Method of delivery

Provided herein are methods for delivering pharmaceutical formulations, preferably vaccines, for providing genetic constructs and proteins of HBV core protein comprising epitopes that make them particularly effective immunogens against which an immune response to HBV viral infection can be induced. Methods of delivering the vaccine or vaccination may be provided to induce a therapeutic and/or prophylactic immune response. The vaccination process can generate immune responses in mammals against a variety of HBV genotypes. The vaccine can be delivered to an individual in order to modulate the activity of the mammalian immune system and enhance the immune response. The delivery of the vaccine may be transfection of HA antigens as nucleic acid molecules that are expressed in the cells and delivered to the surface of the cells where the immune system recognizes and induces cellular, humoral, or both cellular and humoral immunity. Delivery of the vaccine can be used to induce or elicit an immune response in a mammal against a variety of HBV viruses by administering the vaccine as discussed herein to the mammal.

After delivery of the vaccine to the mammal and thus the vector into the cells of the mammal, the transfected cells will express and secrete the consensus HBV core protein. These secreted proteins or synthetic antigens will be recognized by the immune system as foreign substances, which will perform an immune response that may include: generating antibodies to the antigen and T cell responses specific for the antigen. In some embodiments, a mammal vaccinated with a vaccine as discussed herein will have a primed immune system, and when challenged with an HBV strain, the primed immune system will allow rapid clearance of subsequent HBV viruses through humoral immunity, cellular immunity, or both. The vaccine can be delivered to an individual to modulate the activity of the immune system throughout the individual, thereby enhancing the immune response.

The vaccine can be delivered in the form of a DNA vaccine and methods of delivering DNA vaccines are described in U.S. patent nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entirety.

The vaccine can be administered to a mammal to elicit an immune response in the mammal. The mammal may be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, buffalo, bovidae, deer, hedgehog, elephant, camel, alpaca, mouse, rat or chicken, and is preferably a human, cow, pig or chicken.

a. Combination therapy

The pharmaceutical composition, preferably the vaccine described herein, may be administered with a protein or a genome encoding an adjuvant, which may include: alpha-interferon (IFN-. alpha.), beta. -interferon (IFN-. beta.), gamma-interferon, IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNF. alpha., TNF. beta., GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MCP-1, MIP-1a, 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, mutant forms of IL-18, CD40, CD40L, vascular growth factors, fibroblast growth factors, IL-7, nerve growth factors, vascular endothelial growth factors, Fas, TNF receptors, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase, Fos, c-jun, Sp-1, TAP-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactivated NIK, SAP K, SAP-1, JNK, interferon-responsive genes, NFkB, Bax, TRAIL, REC, TRAIL 24, DRC 24, NKR 5, NKR 24, NKR 8642, NKG C, NKG-2, NKG-8653, NKG-8427, NKG-7, NKG-C, NKG-7, NKG-8653, or functional fragments thereof.

b. Route of administration

The vaccine may be administered by different routes including oral, parenteral, sublingual, transdermal, rectal, mucosal, topical, via inhalation, via buccal administration, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasally and intraarticular or combinations thereof. For veterinary use, the combination may be administered as a suitably acceptable formulation according to standard veterinary practice. Veterinarians can readily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The vaccine may be administered by conventional syringes, needleless injection devices, "particle bombardment guns", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or ultrasound.

The vector of the vaccine can be delivered to mammals by several well-known techniques including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome-mediated nanoparticle-facilitated recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. The HBV antigens may be delivered via DNA injection and along with electroporation in vivo.

c. Electroporation

Administration of a vaccine via electroporation of its plasmids can be accomplished using electroporation devices configured to administer the vaccine as desiredThe mammalian tissue delivers a pulse energy effective to form reversible pores in the cell membrane, and preferably the pulse energy is a constant current similar to a preset current input by a 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 controller, a current waveform generator, an impedance meter, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. Electroporation may be carried out using an in vivo electroporation device such as CELLECTRAEP systems (Inovio Pharmaceuticals, Inc.), Blue Bell, Penny Fania (PA)) or Elgen electroporator (Novo Pharmaceuticals, Inc.) facilitate plasmid transfection of cells.

Examples of electroporation devices and electroporation methods that can facilitate delivery of the DNA vaccines of the present invention include those described in U.S. patent No. 7,245,963 to delagi-okley (Draghia-Akli), et al, U.S. patent publication 2005/0052630 filed by Smith (Smith), et al, the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that may be used to facilitate delivery of DNA vaccines include those provided in co-pending and commonly owned U.S. patent publication serial No. 11/874072 filed on day 10, 17, 2007, which discloses the benefit of U.S. 35, clause (e) of the U.S. code of code 35, U.S. provisional application serial No. 60/852,149 filed on day 10, 17, 2006, and U.S. provisional application serial No. 60/978,982 filed on day 10, 2007, all of which are incorporated herein in their entirety.

U.S. Pat. No. 7,245,963 to delarginia-octocryl et al describes modular electrode systems and their use for facilitating the introduction of biomolecules into cells of a selected tissue in a body or plant. The modular electrode system may include a plurality of needle electrodes; hypodermic needles; an electrical connector providing conductive connections from the programmable constant current pulse controller to the plurality of pin electrodes; and a power source. An operator may grasp a plurality of needle electrodes secured to a support structure and insert them firmly into selected tissue in a body or plant. The biomolecules are then delivered into the selected tissue via a hypodermic needle. A programmable constant current pulse controller is activated and constant current electrical pulses are applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of biomolecules into the cell between the plurality of electrodes. U.S. Pat. No. 7,245,963 is incorporated herein by reference in its entirety.

U.S. patent publication 2005/0052630 to smith et al describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of a selected tissue in a body or plant. The electroporation devices include electrodynamics devices ("EKD devices") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between an array of electrodes based on user control and input of pulse parameters and allows storage and retrieval of current waveform data. The electroporation apparatus further comprises a replaceable electrode disc with an array of needle electrodes, a central injection channel for injection needles and a movable guide disc. U.S. patent publication 2005/0052630 is incorporated herein by reference in its entirety.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963 and U.S. patent publication 2005/0052630 may be adapted for deep penetration not only into tissues such as muscles, but also into other tissues or organs. Due to the configuration of the electrode array, the injection needle (to deliver the selected biomolecules) is also fully inserted into the target organ, and the injection is applied perpendicularly onto the target tissue within the area pre-delineated by the electrodes. The electrodes described in U.S. Pat. No. 7,245,963 and U.S. patent publication 2005/005263 are preferably 20mm long and 21 gauge.

Further, contemplated in some embodiments are a combined electroporation device and uses thereof, with the electroporation device described in the following patents: U.S. patent 5,273,525, 28/1993, U.S. patent 6,110,161, 8/29/2000, 6,261,281, 17/7/2001, 6,958,060, 10/25/2005, and 6,939,862, 6/9/2005. Further, patents covering the subject matter provided in U.S. patent 6,697,669 issued on 2/24/2004 and U.S. patent 7,328,064 issued on 2/5/2008 relating to methods of injecting DNA relating to the use of any of a variety of devices to deliver DNA are encompassed herein. The entire contents of the above patents are incorporated herein by reference.

d. Method for preparing vaccine

Provided herein are methods for preparing DNA plasmids comprising the DNA vaccines discussed herein. The DNA plasmid, after the final subcloning step into a mammalian expression plasmid, can be used to inoculate a cell culture in a large scale fermentor using methods known in the art.

DNA plasmids for use with the EP apparatus of the present invention can be formulated or manufactured using a combination of known apparatus and techniques, but preferably they are manufactured using optimized plasmid manufacturing techniques described in U.S. published application No. 20090004716 filed on 23/5/2007. In some examples, the DNA plasmids used in these studies can be formulated at a concentration of greater than or equal to 10 mg/mL. Manufacturing techniques also include or incorporate various devices and protocols commonly known to those of ordinary skill in the art, including those described in U.S. serial No. 60/939792, the licensed patents issued on 7/3/2007, and those described in U.S. patent No. 7,238,522. The above-referenced applications and patents, U.S. serial No. 60/939,792 and U.S. patent No. 7,238,522, respectively, are incorporated herein in their entirety.

Examples

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

The consensus HBV core protein, also known as HBV modified or M-core construct, was designed from epitope sequences from HBV genotypes A, B, C, D and E. HBV core protein sequences from these genotypes are selected for inclusion in the construction of a consensus core that induces immunity against a wide range of genotypes, providing a universal vaccine for HBV. In some embodiments, the modification of the M-core construct comprises the addition of an IgE leader sequence. In some embodiments, the M-core protein is encoded using codon optimization and RNA optimization for enhanced expression.

The nucleic acid sequence encoding the M-core sequence with the IgE leader and the HA tag (SEQ ID NO:5) was cloned into the expression vector pVAX to produce the construct pM-core. In vitro expression testing was done using pM constructs and pVAX and was used as a control. Results showing positive expression are depicted in the gel images shown in fig. 2A and 2B.

The C57BL/6 transgenic mice were divided into two groups of 4 mice each and immunized three times at a biweekly interval with 20. mu.g of DNA using electroporation (group 1-pVAX vector control; group 2 pM-core). Mice were immunized on days 0, 14, 28 and sacrificed on day 35. Spleen, liver and serum were harvested from sacrificed animals.

In vivo studies of the C57BL/6 mouse strain showed increased amounts of tumor necrosis factor (TNF-. alpha.), interferon gamma T-cells (IFN-. gamma.) and CD 107a secretion in CD8 and CD 4T cells obtained from spleen. FIGS. 3A and 3B show increased amounts of IFN- γ secretion from spleen derived CD8+ and CD4+ T cells from C57BL/6 mice vaccinated with pM-core. FIGS. 4A and 4B show increased amounts of TNF- α secretion in CD8+ and CD4+ T cells from spleen of C57BL/6 mice vaccinated with pM-Core. FIGS. 5A and 5B show increased amounts of CD 107a secretion in CD8+ and CD4+ T cells from spleen of C57BL/6 mice vaccinated with pM-Core.

Migration of HBV-specific T-cells to the liver was also demonstrated in animals administered with the pM-Core DNA vaccine. Targeting HBV core antigen-specific T cells with high frequency and effector function to the liver is an important goal in the development of HBV immunotherapy. After immunization, animals were sacrificed and their livers removed, and migration of HBV-specific effector T cells to the liver was determined. The results show that the pM-Core vaccine drives effector T cells into the liver in vivo. Fig. 6A and 6B show interferon- γ T cell liver responses, and fig. 7A and 7B show tumor necrosis factor- α liver immune responses and enhanced responses resulting from vaccination with pM-Core.

The M-core consensus immunogen encoded by the pM-core DNA construct drives a strongly balanced CD4+/CD8+ T cell immune response. T cells induced after HBV infection flow into the liver at high frequency and exhibit the correct effector phenotype for immune clearance.

FIG. 8 shows the cellular immune response induced by pM-Core using enzyme-linked immunosorbent spot (ELISPOT) assay. Splenocytes were stimulated with two pools of 15-mer peptides spanning the entire pMCore length and having an 8 amino acid overlap. IFN-gamma capture antibody (R) from 96 wells inoculated with 200,000 splenocytes in R10 medium&D System (R)&D system)) and 5% CO at 37 ℃₂In the presence of a specific peptide pool overnight. Cells were washed away and plates were incubated with biotinylated anti-mouse IFN-. gamma.detection antibody (R)&System D) was incubated overnight. Streptavidin-alkaline phosphatase and 5-bromo-4-chloro-3' -indolyl phosphate p-toluidine salt and nitrotetrazolium blue chloride were subsequently used for the growth spots. Use automationThe ELISPOT reader (CTL Limited) counts the spots. As shown in figure 8, immunization with pMCore induced a strong cellular immune response. The data show that the dominant epitope is biased towards peptide set 2. The average HBcAg-specific IFN- γ T cell response was about 2000(± 210) SFU per million splenocytes.

In vivo cytotoxicity assay studies were performed using carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling coupled with flow cytometry. We evaluated cell division in cells of a cell population. Splenocytes were isolated from the initial experimental mice and divided into two populations. One population of CFSE highly labeled is pulsed with the relevant peptide (e.g., HBV core peptide). Another population of CFSE low-labeled was pulsed with an irrelevant peptide (e.g., HCV NS3 peptide). Labeled, peptide-treated cells were combined and used in an inheritance transfer experiment for flow analysis. The combined population of treated, labeled target cells was administered to two groups of mice, a control group and an immunization group. Splenocytes were isolated from each group of mice and samples were run on a flow cytometer. The number of CFSEs is measured. Typically, in such experiments, two peaks are formed, the first being an irrelevant peptide; the second is the immunopeptide in the peak indicating the larger CFSE.

Figure 9 shows that CD 8T cells induced by vaccination can specifically eliminate target cells in vivo. These results show that samples of spleen and liver from mice from the initial experiment contained nearly equal amounts of cells in the peaks of the irrelevant and relevant peptides, while these results clearly show that in the immunization group, the peaks of cells derived from those pulsed with relevant peptides were significantly smaller than the irrelevant peptides. These data show that target cells treated with HBV peptide are specifically removed in mice immunized with HBV vaccine, but not in non-immunized mice. Any removal of target cells treated with irrelevant peptides, if all occurred, was identical in mice immunized with HBV vaccine and in non-immunized mice and was significantly less than removal of target cells treated with HBV peptide.

Figure 10 shows data collected from T cell proliferation assays labeled with CFSE. The percentage of proliferation of CD3+ CD4+ cells and CD3+ CD8+ treated with pVax vector (control) or with plasmid pMCore expressing HBV M-core was compared. Briefly, isolated splenocytes were stained with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) Cell Tracer Kit (Cell Tracer Kit) (Invitrogen corporation) according to the manufacturer's instructions. Stained cells were washed three times with saline and stimulated with pMCore-specific overlapping peptide. Cells were incubated at 37 ℃ for 96 hours. After 48 hours, 50% of the medium was removed and replaced with fresh R10. On day 4, cells were harvested and stained with CD3, CD4, and CD 8-specific monoclonal antibody (BDPharmingen). Cells were fixed with PBS containing 1% Paraformaldehyde (PFA) and obtained on a flow cytometer (FACScalibur) (Becton Dickinson). Data was analyzed using the FlowJo program. The CFSE low and CFSE moderate populations are considered proliferating cells. As shown in figure 10, CD3+ CD8+ T cells isolated from spleen proliferated more compared to CD3+ CD4+ T cells.

Fig. 11A and 11B are ELISA data showing a comparison of anti-HBV core antibodies in serial diluted sera from animals treated with either the pVax vector (control) or with the HBVM-core expressing plasmid pMCore. Briefly, high binding ELISA plates (Corning Corp. (Costar), Corning (Coming), New York (NY)) were coated with 1 μ g/ml HBcAg protein in PBS, left at 4 ℃ for 24h, and then washed with PBS-Tween and blocked with 1% BSA in PBS at room temperature for 2 h. Serial diluted serum samples were added to the wells and incubated for 1h at room temperature. After washing, bound serum antibodies were revealed by HRP-labeled goat anti-mouse IgG (fig. 11A) or IgA (fig. 11B). Peroxidase-conjugated abs were detected using tetramethylbenzidine (Sigma-Aldrich) as substrate and OD at 450nm was measured with a multiple scanning ELISA plate reader. Antigen-specific humoral responses were observed in sera collected from immunized mice.

FIG. 12 shows the percent of TNF- α and IFN- γ in spleen and liver cells from CD4+ and CD8 +.

HBcAg was used to transiently transfect mouse liver by hydrodynamic injection in the absence of a small animal model of HBV. The immunized mouse liver was transfected with pMCore or HCV NS 3/4A. Immunohistochemical staining three days after transfection revealed clearance of HBcAg-transfected hepatocytes compared to NS 3/4A-transfected hepatocytes. ALT levels in serum were measured to ensure that clearance induced by immunized mice did not cause any liver damage. The results in figure 13 show that the clearance induced by the immunized mice did not cause any liver damage.

Claims (20)

1. A nucleic acid molecule encoding a sequence of a consensus HBV core protein that induces an immune response in an individual, said protein being: 2,4 or 6.

2. The nucleic acid molecule of claim 1, further comprising a nucleic acid sequence encoding a signal peptide linked to the N-terminus of the protein.

3. The nucleic acid molecule of claim 1, which encodes the protein of SEQ ID NO 2.

4. The nucleic acid molecule of claim 1, wherein the nucleotide sequence is SEQ ID NO 1.

5. The nucleic acid molecule of claim 1, further comprising a nucleic acid sequence encoding a signal peptide linked to the 5' end of the protein sequence.

6. The nucleic acid molecule of claim 1, wherein the nucleotide sequence is one or more nucleotide sequences selected from the group consisting of: 1, SEQ ID NO; 3, SEQ ID NO; and SEQ ID NO 5.

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

8. A plasmid comprising the nucleic acid molecule of claim 1.

9. An expression vector comprising the nucleic acid molecule of claim 1 operably linked to a regulatory element.

10. Use of a nucleic acid molecule according to claim 1 for the preparation of a medicament for inducing an immune response against an HBV antigen.

11. Use of a nucleic acid molecule according to claim 1 for the preparation of a medicament for protecting an individual against HBV infection.

12. Use of a nucleic acid molecule according to claim 1 for the preparation of a medicament for protecting an individual diagnosed with HBV infection.

13. A protein of SEQ ID NO. 2.

14. A protein of SEQ ID NO. 4.

15. A protein of SEQ ID NO 6.

16. Use of a protein according to any one of claims 13 to 15 for the preparation of a medicament for inducing an immune response against an HBV antigen.

17. Use of a protein according to any one of claims 13 to 15 for the preparation of a medicament for protecting an individual against HBV infection.

18. Use of a protein according to any one of claims 13 to 15 for the preparation of a medicament for the protection of an individual who has been diagnosed with HBV infection.

19. A vaccine suitable for generating an immune response against HBV in a subject, the vaccine comprising:

the nucleic acid molecule of claim 1, and

an adjuvant molecule.

20. The vaccine of claim 19, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.