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

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DESCRIPTION

Nucleic acid molecule that encodes the core protein of the hepatitis B virus and vaccine that comprises the same

Field of the invention

The present invention relates to nucleic acid sequences encoding Hepatitis B virus (HBV) core proteins, for improved vaccines against HBV and nucleic acids or vaccines of the invention for use in methods improved to induce immune responses against HBV, and for use in improved methods for prophylactically and / or therapeutically immunizing individuals against HBV. The disclosure relates to nucleic acid sequence fragments encoding hepatitis B virus (HBV) core proteins; to hepatitis B virus (HBV) core proteins and fragments thereof, improved methods for inducing immune responses against HBV, and improved methods for prophylactically and / or therapeutically immunizing individuals against HBV

Background of the invention

Hepatitis B is a common infection common throughout the world that leads to the development of cirrhosis, liver failure, and hepatocellular carcinoma. A significant number of hepatitis cases are not reported due to the asymptomatic nature of the disease. However, approximately 350 million cases of chronic hepatitis B are reported each year. The majority of the population infected with hepatitis is in underdeveloped or developing countries.

The virus is divided into four main serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on its envelope proteins. There are at least eight genotypes (HA) of HBV according to the variation of the genomic sequences. Alternative HBV genotypes have a predominant geographic distribution.

Table 1 the geographic distribution of HBV genotypes.

Table 1 - Geographic Distribution of HBV

<td>HBV genotype</td><td>HBV genosubtype</td><td>HBsAg subtype</td><td>Frequency</td><td>Main Geographic Distribution</td>

<td>TO</td><td>A2</td><td>adw2</td><td>high</td><td>Europe, North America, Australia</td>

<td></td><td>A1</td><td>aywl, adw2</td><td>high</td><td>Africa</td>

<td>B</td><td>B1 B2, B3</td><td>adw2</td><td>high</td><td>Far East</td>

<td></td><td>B4</td><td>ayw1</td><td>high</td><td>Far East</td>

<td></td><td>B2</td><td>adw3</td><td>Come down</td><td>Far East</td>

<td>C</td><td>C1, C2, C4</td><td>adr</td><td>high</td><td>Far East</td>

<td></td><td>C3</td><td>adrq-</td><td>high</td><td>New Guinea., Pacific</td>

<td></td><td>C1, C2</td><td>ayr</td><td>high</td><td>Far East</td>

<td></td><td>C1, C3</td><td>adw2</td><td>Come down</td><td>Far East</td>

<td></td><td>C4</td><td>ayw3</td><td>Come down</td><td>Far East, Pacific</td>

<td>D</td><td>D1, D3, D4</td><td>ayw2</td><td>high</td><td>Western Asia, Eastern Europe, Mediterranean</td>

<td></td><td>D2, D3</td><td>ayw3</td><td>high</td><td>world</td>

<td></td><td>Unidentified</td><td>adw3</td><td>Come down</td><td>Eastern Europe, Spain</td>

<td></td><td>D2</td><td>ayw4</td><td>Come down</td><td>Eastern Europe, Spain, United States</td>

<td>AND</td><td> -</td><td>ayw4</td><td>high</td><td>Africa</td>

<td>F</td><td>F1, F2</td><td>adw4q-</td><td>high</td><td>Latin America, Alaska, Pacific</td>

<td></td><td>F1, F2</td><td>ayw4</td><td>Come down</td><td>Latin America</td>

<td>G</td><td> -</td><td>adw2</td><td>Come down</td><td>Europe, North America</td>

<td>H</td><td> -</td><td>ayw4</td><td>Come down</td><td>Central America</td>

<td colspan="5">J. Med. Virol, DOI 10.1002jmv</td>

The HBV genome is a circular DNA molecule that is primarily double-stranded but has a single-stranded region that arises from one strand that is longer than the other. The double-stranded region arises from the hybridization of a shorter chain of approximately 3020 nucleotides with a longer chain of approximately 3320 nucleotides. The single-stranded region in the unhybridized nucleotides of the longest chain is associated with HBV DNA polymerase. HBV genomic DNA and HBV DNA polymerase are found within a nucleocapsid made up of multiple HBV core protein (HBcAg) molecules. The HBV core protein is enveloped by HBV surface protein (HBsAgs) and lipid molecules.

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The HBV genome contains four open reading frames (ORFs): 1) an ORF that encodes HBV DNA polymerase, 2) an ORF that has two start codons, where the sequence is attached to the second start codon encodes the core protein and the sequence including the additional start codon upstream encodes a sequence called pre-C; 3) an ORF that has three start codons, where one encodes the surface protein (gp27), one includes an upstream start codon that encodes a sequence called pre-S2 (gp36) and another that includes a start codon additional upstream encoding a sequence called pre-S 1 (gp42); and 4) an ORF encoding HBxAg, a protein whose function is less understood.

Prophylactic vaccines and therapies for HBV infection involve the injection of purified subviral particles from the plasma of chronic carriers, or subviral particles produced as recombinant proteins into stably transfected eukaryotic cell lines. Subviral particles are viral proteins and such vaccines are often referred to as subunit vaccines. The HBV proteins are delivered to an individual and become targets for the individual's immune system. In uninfected individuals, an immune response against subunit vaccines protects the uninfected individual from hBv infection. In infected individuals, the immune response induced by the vaccine may have therapeutic effects.

Chisari FV, Am J Pathol., 2000. 156: 1117-1132 and Pumpeus P. et al. Intervirology 2001.44: 98-114 disclose the genomic organization of HBV. Deny P. and F. Zoulim, Pathologie Biologie 2010, August, 58 (4): 245 53 discuss the diagnosis and treatment of hepatitis B virus. Michel ML and P. Tiollais, Pathologie Biologie 2010, August, 58 (4 ): 288 95 discuss hepatitis B vaccines and their protective efficacy and therapeutic potential. PCT publication WO2004026899 discloses the use of polypeptide sequences containing immunogens with HBV amino acid sequences. Published PCT application WO2008093976 discloses HBV coding sequences, proteins, and vaccines including a vaccine comprising a full-length recombinant HBV surface antigen and an HBV core antigen. All HBV surface antigen consists of three types of surface protein (protein L, protein M, and protein S). Published PCT application WO2009130588 discloses HBV coding sequences, proteins, and vaccines that include a nucleic acid that encodes a hepatitis B virus core antigen that is a codon optimized for expression in humans. PCT publication WO2010127115 discloses the delivery of HBV sequences using recombinant vectors.

Available HBV vaccines have shown some efficacy, but are expensive to produce. Additionally, plasma-derived subunit vaccines also have safety concerns. Various vaccine approaches have been explored, including those based on live recombinant vectors, synthetic peptides, and DNA vaccines comprising codon optimized coding sequences for HBV proteins. These other approaches have so far had variable limited efficacy. Additionally, due to genomic differences, some HBV vaccines have shown positive efficacy in some geographic areas and limited efficacy in other areas.

Direct administration of nucleic acid sequences to vaccinate against animal and human diseases has been studied, and much effort has been focused on effective and efficient means of nucleic acid delivery to produce the necessary expression of the desired antigens, resulting in a response immunogenicity and, ultimately, the success of this technique.

DNA vaccines allow the synthesis of endogenous antigens, which induces the histocompatible CD8 + complex, class I restricted cytotoxic T lymphocytes that are rarely obtained with subunit vaccines. Additionally, antigen synthesis that occurs over a sustained period can help overcome poor responsiveness and eliminate or reduce the need for booster injections. Furthermore, DNA vaccines appear to be very stable and easy to produce. Furthermore, broader cellular immune responses can be induced by combining strategies such as codon optimization, RNA optimization, and adding immunoglobulin leader sequences.

DNA vaccines are safe, stable, easily produced, and well tolerated in humans with preclinical trials indicating 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: pp. 30-9]. Furthermore, DNA vaccines are very suitable for repeat administration due to the fact that the efficacy of the vaccine is not influenced by pre-existing antibody titers to the vector [Chattergoon, M., J. Boyer and DB Weiner, Genetic immunization: a new era in vaccines and immune therapeutics. FASEB J, 1997. 11 (10): pp. 75363]. However, a major obstacle to the clinical adoption of DNA vaccines has been a decrease in the immunogenicity of the platform when it is moved to larger animals [Liu, MA and JB Ulmer, Human clinical trials of plasmid DNA vaccines. Adv Genet, 2005. 55: pp. 25-40].

Recent technological advances in the engineering of the immunogen of DNA vaccines have improved the expression and immunogenicity of DNA vaccines, since they have codon optimization, RNA optimization and 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.

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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): pp. 522-33], as well as recently developed technology in 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): pp. 4741-53]. The in vivo electroporation technique has been used in human clinical trials to deliver anticancer drugs, such as bleomycin, and in many preclinical studies in a large number of animal species. Furthermore, studies have suggested that the use of consensus immunogens may increase the amplitude of the cellular immune response compared to natural 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): pp. 8507-14].

Jessica Nystrom et al, The Journal of Infectious Diseases, vol. 201, No. 12, June 15, 2010 (2010-06-15), pages 1867-1879, discloses the enhancement of the ability of endogenous hepatitis B core antigen to prime cytotoxic T lymphocytes. Yi-Ping Xing et al., World J Gastroenterol., January 1, 2005 (2005-01-01), pages 45834586, discloses a new DNA vaccine based on the core gene of the hepatitis B virus that induces a response specific immune system in Balb / c mice. Nader Shahrokhi et al, Iranian Biomedical Journal, April 1, 2006, pages 61-68, discloses an immunogen to stimulate multivalent immunity against hepatitis B surface antigen (HBsAg) and hepatitis B core antigen ( HBcAg). US 2004/156863 discloses a method for treating chronic hepatitis B which comprises administering a stimulating amount of T lymphocytes from a vaccine to a patient. WO 00/26385 discloses recombinant nucleic acid molecules. WO 02/14478 discloses immogenic HBc chimeric particles having enhanced stability.

There remains a need for nucleic acid constructs encoding HBV antigens and for compositions useful for inducing immune responses against HBV. There remains a need for effective HBV vaccines that are inexpensive and effective. There remains a need for effective vaccines that increase neutralizing antibody levels and produce a T-lymphocyte component. There remains a need for effective HBV vaccines, including those that are effective against HBV strains that have a wide range of genotypes, and preferably, a universal vaccine that is globally effective.

Summary of the invention

The subject matter for which protection is sought is as defined in the claims.

The invention provides a nucleic acid molecule comprising a coding sequence that encodes a protein with SEQ ID NO: 2. The invention also provides a nucleic acid of the invention for use in a method of inducing an immune response against an antigen of HBV.

The invention also provides a nucleic acid of the invention for use in a method of protecting an individual from HBV infection. The invention also provides 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.

One aspect of the present invention includes vaccines useful for inducing an immune response against HBV. The development of a therapeutic HBV immune vaccine with broad efficacy against a multitude of genotypes can be provided using a therapeutic DNA vaccine for HBV infection based on the targeting of universally conserved HBV core specific antigens. The use of HBV consensus immunogens induces broader cellular immune responses and may be useful in minimizing the degree of sequence dissimilarity between different virus strains.

Proteins selected from the group consisting of: proteins comprising SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 are disclosed herein.

Also provided are nucleic acid molecules comprising sequences encoding one or more protein molecules set forth above. In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 3; and SEQ ID NO: 5.

Some aspects of the invention provide the nucleic acid molecules of the invention for use in inducing an immune response against the core antigen of one or more HBV genotypes.

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Additional aspects of the invention provide the nucleic acid molecules of the invention for use in methods of protecting an individual against HBV infection. The methods comprise the steps of: administering to said individual a prophylactically effective amount of a nucleic acid molecule comprising said nucleic acid sequence or compositions; wherein the nucleic acid sequence is expressed in cells of said individual and a protective immune response is induced against a protein encoded by said nucleic acid sequence.

In some aspects of the invention, nucleic acid molecules of the invention are provided for use in methods of treating an individual who has been infected with HBV. The methods comprise the steps of: administering to said individual a therapeutically effective amount of such nucleic acid molecules and / or composition.

Aspects of the invention further relate to vaccines comprising nucleic acids encoding proteins selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. The vaccine may additionally 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.

Vaccines comprising nucleic acid molecules may comprise nucleic acid molecules comprising nucleic acid sequences selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 3 and 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 the figures

Figure 1 is a map showing the organization of the HBV genome consisting of four overlapping ORFs. Figures 2A and 2B show results of pM-Core expression experiments. Figure 3A shows the results of the in vitro translation protocol. Figure 3B shows the results of a Western blot.

Figures 3A and 3B show the enhanced magnitude of IFN-γ secretion in CD8 + and CD4 + T cells from spleens of C57BL / 6 mice vaccinated with pM-Core.

Figures 4A and 4B show the enhanced magnitude of TNF-α secretion in CD8 + and CD4 + T lymphocytes from spleens of C57BL / 6 mice vaccinated with pM-Core.

Figures 5A and 5B show the enhanced magnitude of CD 107a secretion in CD8 + and CD4 + T cells from spleens of C57BL / 6 mice vaccinated with pM-Core.

Figures 6A and 6B show the response of interferon-gamma T cells in the liver of C57BL / 6 mice vaccinated with pM-Core.

Figures 7A and 7B show the response of tumor necrosis factor-α T cells in the liver of C57BL / 6 mice vaccinated with pM-Core.

Figure 8 shows ELISPOT assay data.

Figure 9 shows data from experiments using CSFE-labeled cells to compare the killing of peptide-treated target cells in vivo by CD8 T cells in vaccinated and unvaccinated animals.

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

Figures 11A and 11B show a comparison of HBV anticore antibody in serial dilution of sera from animals treated with the vector pVax (control) or with the plasmid pMCore expressing the M core of HBV.

Figure 12 shows the percentage of TNF-a and IFN-g of CD4 + and CD8 + cells of spleen and liver.

Figure 13 shows data from experiments to determine whether clearance induced by immunized mice had effects on the liver by measuring serum ALT levels.

Detailed description

1. Definitions.

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 appended claims, the singular forms a, an, and the or the include plural referents unless the context clearly indicates otherwise.

For recitation of number ranges herein, every number in between is explicitly contemplated with the same degree of precision. For example, for the interval 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the interval 6.0-7.0, the numbers 6.0, 6.1 are explicitly contemplated , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

to. Adjuvant

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Adjuvant as used herein, means any molecule added to the plasmid DNA vaccines described herein, to enhance the immunogenicity of the antigens encoded by the plasmid DNA and the nucleic acid sequences described in hereinafter in this document.

B. Antibody

Antibody as used herein means an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F (ab ') 2, Fd and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody can be an antibody isolated from the mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or mixtures thereof that show sufficient specificity for binding to a desired epitope or a sequence derived therefrom.

C. Coding Sequence

Coding sequence or coding nucleic acid as used herein means nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence that encodes a protein. The coding sequence may further include start and stop signals operably linked to regulatory elements including a promoter and a polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered.

d. Complement

Complement or complementary, as used herein, means a nucleic acid that can indicate a Watson-Crick (eg AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

and. Consensus or Consensus Sequence

Consensus or consensus sequence as used herein means a polypeptide sequence based on the analysis of an alignment of multiple subtypes of a particular HBV antigen. Nucleic acid sequences can be prepared that encode a consensus polypeptide sequence. Vaccines comprising proteins comprising consensus sequence and / or nucleic acid molecules encoding said proteins can be used to induce broad immunity against multiple subtypes or serotypes of a particular HBV antigen.

F. Electroporation

Electroporation, electropermeabilization, or electrokinetic potentiation (EP) as used interchangeably herein means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

g. Fragment

Fragment as used herein with respect to nucleic acid sequences 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 fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode the protein fragments set out below.

"Immunogenic fragment or fragment with respect to polypeptide sequences" means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with a full-length wild-type strain HBV antigen. Consensus protein fragments 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 minus 80%, at least 90%, or at least 95% of a consensus protein. In some embodiments, the consensus protein fragments 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 plus, 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 a consensus protein.

h. Genetic construction

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As used herein, the term "genetic construct" refers to DNA or RNA molecules that comprise a nucleotide sequence that encodes protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and a polyadenylation signal capable of directing expression in cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes a protein so that when present in the individual's cell, the coding sequence will be expressed. .

i. Identical

Identical or identity as used herein in the context of two or more nucleic acids or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same in a specific region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions where the identical residue occurs in both sequences to produce the number of matching positions, dividing the number of matching positions between the total number of positions in the specified region and multiplying the result by 100 to produce the percent sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only one sequence, single sequence residues are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be done manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

j. Immune response

Immune response as used herein means the activation of the immune system of a host, eg, that of a mammal, in response to the introduction of antigen, such as a HBV consensus antigen. The immune response can be in the form of a cellular or humoral response, or both.

k. Nucleic acid

Nucleic acid or oligonucleotide or polynucleotide as used herein means at least two nucleotides covalently linked to each other. The representation of a single chain also defines the sequence of the complementary chain. Thus, a nucleic acid also encompasses the complementary strand of a 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 complements thereof. 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.

Nucleic acids can be single-stranded or double-stranded, or they can contain portions of both double-stranded and single-stranded sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid can contain combinations of deoxyribo and ribonucleotides 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.

l. Operationally united

"Operatively linked" as used herein means that the expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be placed 5 '(upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the controlling gene in the gene from which the promoter is derived. As is known in the art, the variation in this distance can be satisfied without loss of promoter function.

m. Promoter

Promoter, as used herein, means a naturally derived or synthetic molecule that is capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or alter spatial expression and / or temporal expression thereof. A promoter can also comprise distal enhancer or repressor elements, which can be located up to several thousand base pairs from the transcriptional start site. A promoter can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue or organ where expression occurs or, with respect to the stage of development at which expression occurs, or in response to external stimuli such as

ES 2 806 263 T3 physiological stress, pathogens, metal ions or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, early promoter of SV40 or SV40 late promoter and CMV IE promoter.

n. Signal peptide

Signal peptide and leader sequence are used interchangeably herein and refer to an amino acid sequence that can be joined at the amino terminus of an HBV protein discussed herein. Signal peptides / leader sequences generally direct the localization of a protein. The signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the rest of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are attached at the N-terminus of the protein. As mentioned herein with respect to the binding of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of a protein that is encoded by the initiation codon of the nucleic acid sequence encoding the protein without signal peptide coding sequences. Thus, for example, SEQ ID NO: 4 is SEQ ID NO: 2 with the signal peptide / leader sequence attached at the N-terminus of SEQ ID NO: 2, that is, SEQ ID NO: 4 is a protein comprising a signal peptide linked to the N-terminus of SEQ ID NO: 2. The first residue in SEQ ID NO: 2, Xaa, is generally methionine when no signal peptide is present. However, proteins comprising signal peptides linked to SEQ ID NO: 2, such as SEQ ID NO: 4, replace the methionine 1 residue in Xaa with the residue that binds the signal peptide to the protein. Therefore, the N-terminal residue of SEQ ID NO: 2 can be anything, but if it is encoded by an initiation sequence it is methionine. The binding of the signal peptide / leader sequence at the N-terminus of SEQ ID NO: 2 generally removes the N-terminal methionine. As used herein, SEQ ID NO: 4 is intended to comprise SEQ ID NO: 2 with a signal peptide / leader sequence attached at the N-terminus of SEQ ID NO: 2 despite removal of the remainder. N-terminal Xaa of SEQ ID NO: 2. Similarly, the coding sequences for SEQ ID NO: 4 comprise coding sequences for SEQ ID NO: 2 with coding sequences for a signal peptide / leader sequence attached to the 5 'end of the coding sequences coding for SEQ ID NO: two. The initiation codon can be the nnn in the coding sequences for SEQ ID NO: 2, but it is eliminated when the coding sequences for a signal peptide / leader sequence are joined to the 5 'end of the coding sequences coding for SEQ ID NO : two. As used herein, the coding sequences for SEQ ID NO: 4 are intended to comprise sequences coding for SEQ ID NO: 2 with coding sequences for a signal peptide / leader sequence attached at the 5 'end of the sequence. encoding SEQ ID NO: 2 where nnn is produced. Thus, for example, SEQ ID NO: 3 is intended to comprise SEQ ID NO: 1 with coding sequences for a signal peptide / leader sequence attached at the 5 'end of SEQ ID NO: 1, rather than the nnn. In some embodiments, the nnn is a start codon at the 5 'end of SEQ ID NO: 1.

or. Rigorous Hybridization Conditions

Stringent hybridization conditions, as used herein, means conditions in which a first nucleic acid sequence (eg, probe) will hybridize to a second nucleic acid sequence (eg, 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 approximately 5-10 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm can be the temperature (under defined ionic strength, pH and nucleic concentration) at which 50% of the probes complementary to the target hybridize with the target sequence at equilibrium (since the target sequences are present in excess, in Tm , 50% of the probes are occupied in equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ions, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0. at 8.3 and the temperature is at least about 30 ° C for short probes (eg, about 10-50 nucleotides) and at least about 60 ° C for long probes (eg, greater than about 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents, such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubation at 42 ° C, or, 5x SSC, 1% SdS, incubation at 65 ° C, with wash at 0 , 2x SSC, and 0.1% SDS at 65 ° C.

p. Substantially Complementary

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 the two sequences hybridize under stringent hybridization conditions.

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q. Substantially Identical

Substantially identical as used herein means that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical 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 with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

r. Subtype or Serotype

Subtype or Serotype: As used herein, interchangeably, and in reference to HBV, means genetic variants of an HBV such that one subtype is recognized by an immune system from a different subtype.

s. Variant

"Variant 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 the reference nucleic acid, its complement, or a sequence substantially identical thereto.

Variant with respect to a peptide or polypeptide that differs in amino acid sequence by conservative amino acid insertion, deletion, or substitution, but retains at least one biological activity. "Variant" can also mean a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. A conservative amino acid substitution, that is, replacing an amino acid with a different amino acid of similar properties (eg, hydrophilicity, degree, and distribution of charged regions) is recognized in the art as generally involving a minor change. These minor changes can be identified, in part, by considering the hydropathic amino acid index, 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 a consideration 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 having hydropathic indices of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins that retain biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide allows calculation of the highest local mean hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. US Patent No. 4,554,101. Substitution of amino acids that have similar hydrophilicity values ​​can result in peptides that retain biological activity, eg, immunogenicity, as understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ± 2 of each other. Both the hypophobia index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Based on that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, and particularly on 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 that contains an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector and is preferably a DNA plasmid.

two. HBV core antigen

The HBV core protein represents an important target for immune-mediated viral clearance by inducing 1) cytotoxic T lymphocyte (CTL) responses, 2) T helper lymphocyte responses, and / or 3) B lymphocyte responses, or preferably all of those mentioned above, for cross presentation.

Table 2 shows the similarities between genotypes for the core antigen of the HBV-A, HBV-B, HBV-C, HBV-D and HBV-E genotypes with the HBV core consensus proteins, named in the table HBV- M-core. For some embodiments, the HBV core M construct was designed to have increased homologies for broad HBV core targets. Similarities between genotypes for core antigen with engineered M core construct: increased homologies for broad HBV core targets. All genotypes must be represented in a universal immunotherapeutic vaccine for HBV

ES 2 806 263 T3

Table 2

<td colspan="10">Percentage of Identity</td>

<td rowspan="8">Divergence</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1</td><td></td><td> 96,2</td><td> 96,2</td><td> 97,8</td><td> 95,6</td><td> 98,4</td><td></td><td>1 - HBV-A-ConCore</td>

<td> 2</td><td> 3,9</td><td></td><td> 100</td><td> 95,6</td><td> 93,4</td><td> 96,7</td><td></td><td>2 - HBV-B-ConCore</td>

<td> 3</td><td> 3,9</td><td> 0</td><td></td><td> 95,6</td><td> 93,4</td><td> 96,7</td><td></td><td>3 - HBV-C-ConCore</td>

<td> 4</td><td> 2,2</td><td> 4,5</td><td> 4,5</td><td></td><td> 97,8</td><td> 97,8</td><td></td><td>4 - HBV-D-ConCore</td>

<td> 5</td><td> 4,5</td><td> 6,9</td><td> 6,9</td><td> 2,2</td><td></td><td> 95,6</td><td></td><td>5 - HBV-E-ConCore</td>

<td> 6</td><td> 1,7</td><td> 3,4</td><td> 3,4</td><td> 2,2</td><td> 4,5</td><td></td><td></td><td>6 - HBV-M-Core</td>

<td></td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td></td><td></td>

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

A consensus HBV core protein is provided (SEQ ID NO: 2). An amino acid sequence was generated comprising the IgE leader at the N-terminus of the HBV core protein consensus sequences. Thus, a protein with an IgE leader (SEQ ID NO: 7) bound to the consensus HBV core protein (SEQ iD NO: 2) is also provided to provide a consensus hBv core protein-IgE leader. (SEQ ID NO: 4). Some embodiments provided also comprise an HA tag (SEQ ID NO: 8) attached at the C-terminus of the HBV core protein consensus sequence. Accordingly, a HBV core protein consensus protein (SEQ ID NO: 6) is provided 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) attached to the C-terminus of the HBV core protein consensus sequences.

Proteins can be homologous to SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Disclosed herein are immunogenic proteins that have 95% homology to the consensus protein sequences herein. Disclosed herein are immunogenic proteins that have 96% homology to the consensus protein sequences herein. Disclosed herein are immunogenic proteins that have 97% homology to the consensus protein sequences herein. Disclosed herein are immunogenic proteins that have 98% homology to the consensus protein sequences herein. Disclosed herein are immunogenic proteins that have 99% homology to the consensus protein sequences herein.

In some embodiments, the protein is free of a leader sequence. In some embodiments, the protein is free from the IgE leader.

The consensus protein fragments disclosed herein 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 consensus protein. Immunogenic fragments of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 are disclosed. Immunogenic 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 the 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. As disclosed herein, the fragments include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. As disclosed herein, the fragments are free of a leader sequence. As disclosed herein, the fragments are free of a leader sequence, the IgE leader.

Immunogenic protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 are disclosed. Said immunogenic 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 the 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 proteins that are 95% homologous to SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Disclosed herein are immunogenic fragments that have 96% homology to the immunogenic fragments of consensus protein sequences of the

ES 2 806 263 T3 present document. Disclosed herein are immunogenic fragments that have 97% homology to immunogenic fragments of consensus protein sequences herein. Disclosed herein are immunogenic fragments that have 98% homology to immunogenic fragments of consensus protein sequences herein. Disclosed herein are immunogenic fragments that have 99% homology to immunogenic fragments of consensus protein sequences herein. As disclosed herein, the fragments can include a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. As disclosed herein, the fragments can be free of a leader sequence. As disclosed herein, the fragments can be free of a leader sequence, the IgE leader.

3. Genetic Sequences, Constructs and Plasmids

Nucleic acid sequences encoding SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, as well as homologous proteins, immunogenic fragments, and immunogenic fragments of homologous proteins can be generated in the usual manner. Thus, nucleic acid molecules can be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% 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%. Also provided are nucleic acid sequences that encode the immunogenic fragments set forth herein and the immunogenic fragments of the protein homologous to the proteins set forth herein.

Nucleic acid molecules encoding the consensus amino acid sequences were generated. Vaccines may comprise one or more nucleic acid sequences encoding one or more of the consensus versions of immunogenic proteins selected from this group of sequences generated to optimize stability and expression in humans. Nucleic acid sequence (SEQ ID NO: 1) encoding the HBV core protein consensus protein (SEQ ID NO: 2), nucleic acid sequence (SEQ ID NO: 3) encoding the IgE-protein leader HBV core protein consensus (SEQ ID NO: 4), and the nucleic acid sequence (SEQ ID NO: 5) encoding the HBV core protein consensus IgE-leader-HA tag (SEQ ID NO: 6). Disclosed herein are nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences herein. Disclosed herein are nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences herein. Disclosed herein are nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences herein. Disclosed herein are nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences herein. Disclosed herein are nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences herein. As disclosed herein, nucleic acid molecules with coding sequences disclosed herein that are homologous to a disclosed consensus protein coding sequence may include sequences that encode an IgE leader sequence attached to the 5 'end of the coding sequence that encodes the homologous protein sequences disclosed herein.

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

The disclosure also provides fragments of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5. Fragments can 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 less 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 can 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. Fragments can include sequences that encode a leader sequence, such as for example an immunoglobulin leader, such as the IgE leader. It is disclosed herein that the fragments may be free of coding sequences that encode a leader sequence. It is disclosed herein that the fragments may be free of coding sequences that encode a leader sequence, the IgE leader.

Provided herein are genetic constructs that may comprise a nucleic acid sequence encoding the HBV core antigen disclosed herein, including consensus protein sequences, sequences homologous to consensus protein sequences, fragments of protein sequences consensus and sequences homologous to fragments of consensus protein sequences. The genetic construct can be present in the cell as a functional extrachromosomal molecule. The

ES 2 806 263 T3 genetic construct can be a linear minichromosome including centromere, telomeres or plasmids or cosmids.

The genetic construct can also be part of a recombinant viral vector genome, including recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. The genetic construct can be part of the genetic material in live attenuated microorganisms or recombinant microbial vectors that live in cells.

Genetic constructs can comprise regulatory elements for gene expression of nucleic acid coding sequences. Regulatory elements can be a promoter, enhancer, start codon, stop codon, or polyadenylation signal.

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

Coding sequences can be optimized for stability and high levels of expression. In some cases, codons are selected to reduce the formation of RNA secondary structure, such as that formed due to intramolecular binding.

The vector may comprise heterologous nucleic acid encoding an antigen and may further comprise an initiation codon, which may be upstream of the antigen coding sequence, and a stop codon, which may be downstream of the antigen coding sequence. The start and stop codons can be in phase with the coding sequence. The vector may also comprise a promoter that is operably linked to the antigen coding sequence. The promoter operably linked to the antigen coding sequence may be a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus ( HIV) such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV, a cytomegalovirus (CMV) promoter such as the cMv immediate early promoter, the Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter , for its acronym in English). 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 can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in United States Patent Application Publication No. US20040175727.

The vector may also comprise a polyadenylation signal, which may be downstream of the HBV core protein coding sequence. The polyadenylation signal can be SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, for its acronym in English) and polyadenylation signal of human β-globin. The SV40 polyadenylation signal can be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).

The vector may also comprise an enhancer upstream of the consensus HBV core protein coding sequence. The enhancer may be required for expression of the DNA. The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as that of CMV, HA, RSV, or EBV. Enhancers of polynucleotide function are described in US Patent Nos. 5,593,972, 5,962,428, and WO94 / 016737.

The vector may also comprise a mammalian origin of replication to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The vector can be pVAXI, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which can comprise the Epstein Barr virus origin of replication and the coding region for the EBNA-1 nuclear antigen, which can produce high copy episomal replication without integration. The vector can be pVAX1 or a variant pVax1 with changes such as the variant plasmid described herein. The plasmid variant pVax1 is a 2998 base pair variant of the backbone of plasmid vector pVAXI (Invitrogen, Carlsbad CA). The CMV promoter is located at bases 137724. The T7 promoter / priming site is at bases 664-683. Multiple cloning sites are at bases 696-811. The bovine GH polyadenylation signal is at bases 829-1053. The kanamycin resistance gene is at bases 1226-2020. The pUC origin is at bases 2320-2993.

Based on the sequence of pVAX1 available from Invitrogen, the following mutations were found in the sequence of pVAX1 that was used as the backbone for plasmids 1-6 discussed herein:

ES 2 806 263 T3

C&gt; G 241 in the CMV promoter

C&gt; T 1942 in the main chain, downstream of the bovine growth hormone polyadenylation signal (bGHpoliA)

A&gt; - 2876 on the main chain, downstream of the Kanamycin gene

C&gt; T 3277 high copy number mutation in the pUC origin of replication (Ori) (see Nucleic Acid Research 1985)

G&gt; C 3753 at the end of the pUC Ori upstream of the RNASeH site

Base pairs 2, 3 and 4 are changed from ACT to CTG on the main chain, upstream of the CMV promoter.

The backbone of the vector can be pAV0242. The vector may be a replication-defective adenovirus type 5 (Ad5) vector.

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

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 yeast strains. The vector can also be from the MAXBAC ™ full-length 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 vectors or expression systems for producing proteins by standard techniques and readily available starting materials, including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Edition, Cold Spring Harbor (1989).

Four. Pharmaceutical compositions

Provided herein are pharmaceutical compositions in accordance with the present invention comprising from about 1 nanogram to about 10 mg of DNA. In some embodiments, the pharmaceutical compositions according to the present invention comprise from 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, 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, 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, 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 10 mg 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 inclusive, 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, 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, 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 o 1000 micrograms, or up to and including 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. In some embodiments, the pharmaceutical compositions according to the present invention comprise about 5 nanograms to about 10 mg of DNA. In some embodiments, the pharmaceutical compositions according to the present invention comprise from about 25 nanograms to about 5 mg of DNA. In some embodiments, the pharmaceutical compositions contain about 50 nanograms to about 1 mg of DNA. In some embodiments, the pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 1 to about 350 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 5 to about 250 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 10 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 15 to about 150 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 20 to about 100 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 25 to

ES 2 806 263 T3 approximately 75 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 30 to about 50 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 35 to about 40 micrograms of DNA. In some embodiments, the pharmaceutical compositions contain about 100 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical compositions comprise about 35 nanograms to about 45 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 100 to about 200 micrograms of DNA.

The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free, and particle-free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin or albumin. In some embodiments, an agent for vasoconstriction is added to the formulation.

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

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

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

Some alternative embodiments include those that comprise nucleic acid sequences encoding immunogenic fragments of the consensus HBV core protein, one or more proteins homologous to the HBV consensus core protein, and immunogenic fragments of one or more proteins homologous to the HBV protein. nucleus of the HBV consensus.

Some embodiments provide the compositions described herein for use in methods of generating immune responses against HBV core proteins that comprise administering to an individual one or more compositions described herein. Some embodiments provide the compositions described herein for use in methods of prophylactic vaccination of an individual against HBV infection comprising administering one or more compositions described herein. Some embodiments provide the compositions described herein for use in methods of therapeutic vaccination of an individual who has been infected with HBV which comprises administering one or more compositions described herein. Diagnosis of HBV infection prior to administration can be made routinely.

The vaccine can be a DNA vaccine. The DNA vaccine may comprise a plurality of the same or different plasmids comprising nucleic acid sequences encoding the 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. The DNA vaccine may further comprise elements or reagents that inhibit its integration into the chromosome. The vaccine can be an RNA from the HBV core protein. The RNA vaccine can be introduced into the cell.

ES 2 806 263 T3

The vaccine can be a recombinant vaccine comprising the genetic construct or antigen described above. The vaccine may also comprise one or more consensus HBV core proteins in the form of one or more protein subunits, one or more inactivated viral particles comprising one or more consensus HBV core proteins, or one or more attenuated virus particles comprising one or more core proteins of the consensus HBV. The attenuated vaccine can be live attenuated vaccines, inactivated vaccines, and vaccines that use recombinant vectors to deliver foreign genes encoding one or more consensus HBV core proteins and also subunit and glycoprotein vaccines. Examples of live attenuated vaccines, those that use recombinant vectors to deliver foreign antigens, subunit vaccines, and glycoprotein vaccines are described in US Pat. No. 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; 5.310.668; 5.387.744; 5.389.368; 5.424.065; 5.451.499; 5.453.364; 5.462.734;

5.470.734; 5.474.935; 5.482.713; 5.591.439; 5.643.579; 5.650.309; 5.698.202; 5.955.088; 6.034.298; 6.042.836;

6,156,319 and 6,589,529.

The vaccine may comprise vectors and / or proteins targeting multiple HBV genotypes from multiple particular regions in the world. The vaccine provided can be used to induce immune responses, including therapeutic or prophylactic immune responses. Antibodies and / or cytotoxic T lymphocytes targeting the consensus HBV core protein and also generally across multiple HBV virus genotypes can be generated. Said antibodies and cells can be isolated.

The vaccine may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable excipient can be functional molecules such as carriers, adjuvants, carriers, or diluents. The pharmaceutically acceptable excipient is an agent that facilitates transfection, which may include surfactants, such as immunostimulating complexes (ISCOMS), incomplete Freund's adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalane and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other agents that facilitate transfection.

The agent that facilitates transfection is a polyanion, polycation, including poly-L-glutamate (LGS), or a lipid. The agent that facilitates transfection is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent can also include surfactants such as immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and can also be use hyaluronic acid administered in conjunction with the genetic build. In some embodiments, the DNA vector vaccines may also include an agent that facilitates transfection such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, such as a DNA-liposome mixture (see, for example, WO9324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other agents that facilitate transfection. Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or a lipid. 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. ml, less than 0.100 mg / ml, less than 0.050 mg / ml or less than 0.010 mg / ml.

The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be other genes that are expressed on an alternative plasmid or supplied as proteins in combination with the above plasmid in the vaccine. The adjuvant can be selected from the group consisting of: interferon α (IFN-α), interferon β (IFN-β), interferon γ, platelet-derived growth factor (PDGF), TNFa, TNFe, GM-CSF, epidermal growth factor (EGF), T-cell attractant chemokine (CTACK), thymus-expressed epithelial chemokine (TeCK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, mHc, CD80, CD86, including IL-15, which has the signal sequence deleted and including optionally the IgE signal peptide. The adjuvant can be IL12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFa, TNFe, 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 useful adjuvants include those that encode: 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, mutant forms of IL-18, CD40, CD40L, factor 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, genes response to interferon, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LINKING, Ox40, Ox40 LINKING, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E , TAP1, TAP2 and functional fragments thereof.

5. Supply Methods

Provided herein is the supply of pharmaceutical formulations, preferably

ES 2 806 263 T3 vaccines, to provide genetic constructs and proteins of the HBV core protein comprising epitopes that make them particularly effective immunogens against which an immune response to HBV viral infections can be induced. Delivery of the vaccine, or vaccination, can be provided to induce a therapeutic and / or prophylactic immune response. The vaccination process can generate an immune response in the mammal against a plurality of HBV genotypes. The vaccine can be administered to an individual to modulate the activity of the mammalian immune system and enhance the immune response. Administration of the vaccine can be the transfection of the HA antigen as a nucleic acid molecule that is expressed in the cell and delivered to the cell surface upon which the immune system recognizes and induces a cellular, humoral, or cellular response and humoral. The vaccine delivery can be used to induce or elicit an immune response in mammals against a plurality of HBV viruses by administering the vaccine to mammals as discussed herein.

Upon delivery of the vaccine to the mammal, and following 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 as foreign by the immune system, which will generate an immune response that may include: antibodies raised against the antigens, and the T lymphocyte response specifically against the antigen. In some examples, a mammal vaccinated with the vaccines discussed herein will have a primed immune system and when challenged with a viral strain of HBV, the primed immune system will allow rapid clearance of subsequent HBV viruses, either through of humoral or cellular response, or both. The vaccine may be for delivery to an individual to modulate the activity of the individual's immune system, thereby enhancing the immune response.

The vaccine can be for delivery in the form of a DNA vaccine and methods for delivering the DNA vaccines are described in US Patent Nos. 4,945,050 and 5,036,006.

The vaccine can be for administration to a mammal to elicit an immune response in a mammal. The mammal can be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bovine, deer, hedgehog, elephant, llama, alpaca, mouse, rat or chicken, and preferably human. cow, pig or chicken.

to. Combination Treatments

The pharmaceutical compositions, preferably the vaccines described herein, may be for administration in combination with proteins or genes encoding adjuvants, which may include: interferon α (IFN-α), interferon β (IFN-β), interferon γ, 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, 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 factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL1, 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 LINKAGE, Ox40, Ox40 LINKAGE, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, or TAP2, or functional fragments thereof.

b. Administration routes

The vaccine can be for administration by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, through inhalation, through buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal and intraarticular or combinations thereof. For veterinary use, the composition may be for administration as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can easily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The vaccine can be for administration by traditional syringes, needleless injection devices, gene guns for microprojectile bombardment, or other physical methods such as electroporation (EP), the hydrodynamic method, or ultrasound.

The vaccine vector can be delivered to the mammal by various well-known technologies, including DNA injection (also known as DNA vaccine) with and without in vivo electroporation, liposome-mediated, particle-facilitated, recombinant vectors such as Recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant vaccinia. The HBV antigen can be for delivery via DNA injection and in conjunction with in vivo electroporation.

c. Electroporation

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Administration of the vaccine through electroporation of the vaccine plasmids can be accomplished using electroporation devices that can be configured to deliver a desired tissue of a mammal with a pulse of energy effective to cause reversible pores to form in the membranes. cell phones, and preferably the energy pulse is a constant current similar to a current input preset by a user. The electroporation device may comprise an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate one or more of the various elements of electroporation devices, including: a controller, a current waveform generator, an impedance evaluator, a waveform recorder, an input element, a status report element, a communication port, a memory component, a power supply, and a power switch. Electroporation can be accomplished using an in vivo electroporation device, for example the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.) to facilitate transfection of cells. by the plasmid.

Examples of preferred electroporation devices and electroporation methods that can facilitate administration of the DNA vaccines of the present invention include those described in US Patent No. 7,245,963 to Draghia-Akli, et al., Pub. US Patent 2005/0052630 filed by Smith, et al. Other electroporation devices and electroporation methods that can be used to facilitate the delivery of DNA vaccines include those provided co-pending with and from the same owner as the present US patent application. US, Serial number US2008091135.

US Patent No. 7,245,963 to Draghia-Akli, et al. describes modular electrode systems and their use to facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. Modular electrode systems can comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector providing a conductive link from a programmable constant current pulse controller to the plurality of needle electrodes; and a power supply. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue on 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 the constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecule into the cell between the plurality of electrodes.

United States Patent Publication 2005/0052630 filed by Smith, et al. describes an electroporation device that can be used to effectively facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. The electroporation device comprises an electrokinetic device (EKD device) the operation of which is specified by an embedded computer program or software. The EKD device produces a series of programmable constant current pulse patterns between the electrodes in an array, based on user control and input of pulse parameters, and allows data acquisition and storage in the form of current wave. The electroporation device also comprises a replaceable electrode disc having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disc.

The electrode arrays and methods described in US Patent No. 7,245,963 and US Patent Publication 2005/0052630 are 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 biomolecule of choice) is also fully inserted into the target organ and the injection is delivered perpendicular to the target, in the area pre-aligned by the electrodes. The electrodes described in US Patent No. 7,245,963 and US Patent Publication 2005/005263 are preferably 20mm long and 21 gauge.

Additionally, contemplated in some embodiments that incorporate electroporation devices and uses thereof, there are electroporation devices that are described in the following patents: US Patent 5,273,525 issued December 28, 1993, US Patents 6,110,161 issued August 29, 2000, 6,261,281 issued July 17, 2001, and 6,958,060 issued October 25, 2005, and US Patent 6,939,862 issued on September 6, 2005. Also contemplated herein are patents that cover the subject matter provided in U.S. Patent 6,697,669 issued February 24, 2004, which relates to the delivery of DNA using any of a variety of devices, and the US Patent 7,328,064 issued February 5, 2008, which refers to the DNA injection method.

d. Vaccine Preparation Method

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

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DNA plasmids for use with the EP devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but are preferably manufactured using an optimized plasmid manufacturing technique described in an application published in United States No. 20090004716, which was filed on May 23, 2007. In some examples, the DNA plasmids used in these studies can be formulated at concentrations greater than or equal to 10 mg / ml. Manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those skilled in the art, in addition to those described in US serial number 60/939792, including those described in a licensed patent, Patent United States No. 7,238,522, which was filed on July 3, 2007.

Example

The present invention is further illustrated in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are provided by way of illustration only. Based on the foregoing discussion and the examples, one skilled in the art can determine the essential features of the present invention and, without departing from the scope of the present invention, can make various changes and modifications of the invention to adapt it to various uses and conditions. Thus, thanks to the foregoing description, various modifications of the invention will be apparent to those skilled in the art, beyond those shown and described herein. The subject matter for which protection is requested is as established in the claims.

A consensus HBV core protein, also known as a modified HBV or M core construct, was designed from epitope sequences of HBV A genotypes. B, C, D, and E. The HBV core protein sequences of these genotypes were selected for inclusion in a consensus core construct that would induce immunity against a wide range of genotypes, thus providing a universal vaccine for HBV. In some embodiments, modifications to the M core construct included the addition of an IgE leader sequence. In some embodiments, the M core protein is encoded using codon optimization and RNA optimization to enhance expression.

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

The C57BL / 6 transgenic mice were separated into two groups of four mice each and using electroporation immunized three times with 20 µg of DNA at fortnightly intervals (group 1 - pVAX vector control; group 2 pM-core). Mice were immunized on Day 0, Day 14, Day 28 and sacrificed on Day 35. The spleens, liver and sera of the sacrificed animals were harvested.

In vivo studies of C57BL / 6 mouse strains indicate an enhancement in the extent of secretion of tumor necrosis factor (TNF-α), interferon gamma (IFN-γ) in T lymphocytes and CD107a in CD8 and T lymphocytes CD4 taken from the spleen. Figures 3A and 3B show that vaccination of C57BL / 6 mice with pM-Core enhanced the magnitude of IFN-γ secretion in spleen CD8 + and CD4 + T lymphocytes. Figures 4A and 4B show that vaccination of C57BL / 6 mice with pM-Core enhanced the magnitude of TNF-α secretion from CD8 + and CD4 + T lymphocytes from spleens. Figures 5A and 5B show that vaccination of C57BL / 6 mice with pM-Core enhanced the magnitude of CD 107a secretion in spleen CD8 + and CD4 + T lymphocytes.

Migration of HBV-specific T lymphocytes to the liver was also demonstrated in animals administered 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 for the development of immune therapy against HBV. After immunization, the animals were sacrificed and their livers were removed and the migration of HBV-specific effector T lymphocytes to the liver was determined. The results show that the pM-Core vaccine carries effector T cells to the liver in vivo. Figures 6A and 6B show the hepatic response of interferon-γ T cells, Figures 7A and 7B show the hepatic immune response of Tumor Necrosis Factor α and the elevated response resulting from vaccination with pM-Core.

The M core consensus immunogen encoded by the pM-core DNA construct drives strongly balanced CD4 + / CD8 + T cell immune responses. Induced T cells circulate to the liver at high frequency and display the correct effector phenotype for immune clearance after HBV infection.

Figure 8 shows cellular immune responses induced by pM-Core using an enzyme-linked immunosorbent spot assay (ELISPOT). Splenocytes were stimulated with two 15-mer peptide pools spanning the full length of pMCore and overlapping by 8 amino acids. 200,000 splenocytes were plated in R10 medium in a 96-well plate coated with IFN-γ capture antibody (R&amp;D system) and stimulated overnight in the presence of a group of specific peptides at 37 ° C in CO2 at 5 %. Cells were washed and plates were incubated overnight with biotinylated mouse anti-IFN-γ detection antibody (R&amp;D system). Streptavidin-alkaline phosphatase and p-toluidine 5-bromo-4-chloro-3'-indolyl phosphate salt