Compositions and methods for treating and preventing hepatitis B and hepatitis D

Abstract

Disclosed herein are immunogenic compositions or immunogenic combination products comprised of genetically modified Hepatitis B virus (HBV) and / or Hepatitis D virus (HDV) nucleic acids, genes, peptides, or proteins for use in inducing an immune response against Hepatitis B virus (HBV) and / or Hepatitis D virus (HDV) infection. Also disclosed are methods of using the immunogenic compositions or immunogenic combination products of the disclosure in a subject to induce an immune response against HBV and / or HDV, comprising administering the composition or combination product in a nucleic acid prime / polypeptide boost regimen.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 966970, filed on 28 January 2020, which is expressly incorporated herein by reference in its entirety.

[0002] Sequence listing reference This application was filed together with an electronic sequence listing. This sequence listing was created with the filename SVF005SeqListing.TXT, last modified on January 26, 2021, and has a file size of 141,377 bytes. The information contained in this electronic sequence listing is expressly incorporated herein by reference in its entirety.

[0003] field Aspects of this disclosure relate, as a whole, to immunogenic compositions or immunogenic combination products comprising genetically modified HBV and HDV nucleic acids, genes, peptides or proteins used to induce an immune response to hepatitis B virus (HBV) infection and / or hepatitis D virus (HDV) infection. This immune response includes, is essentially derived from, or comprises activated immune cells that produce neutralizing antibodies against HBV and / or HDV, as well as activated immune cells against HBV and / or HDV, such as T cells and B cells. The disclosure also relates, as a whole, to a method of using the above-mentioned immunogenic compositions or immunogenic combination products in a subject to induce an immune response to HBV and / or HDV, comprising administering the compositions or combination products in a homogeneous or heterogeneous inoculation method comprising a priming with nucleic acids and / or polypeptides and a boosting with nucleic acids and / or polypeptides.

[0004] background Hepatitis is a disease characterized by swelling and inflammation of the liver. This disease is generally caused by viruses, and currently five types (A, B, C, D, and E) are known. Hepatitis B infection can be acute or chronic, and severe chronic infection can lead to chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. The hepatitis B virus (HBV) has an incomplete double-stranded circular DNA genome. Once translocated to the host nucleus, the genome is transcribed into four viral mRNA molecules by the host's RNA polymerase. These mRNA molecules are then translated into viral proteins such as capsid proteins and surface antigens, or reverse transcriptase generates a large DNA genome. The hepatitis D virus (HDV) is a virus that replicates by co-infection or co-infection with HBV. The circular single-stranded RNA of HDV is amplified by the host's RNA polymerase, but this RNA contains only one hepatitis D antigen (HDAg) gene. In co-infection with HBV and HDV, naked HDV is packaged in an envelope containing the HBV surface antigen, and the RNA genome coated with the HDAg protein is surrounded by this envelope. Since HDV does not encode its own receptor-binding protein, the uptake of the HBV surface antigen is essential for HDV infection. Co-infection with hepatitis D can lead to even more serious complications, increasing the risk of liver failure, cirrhosis, and liver cancer. There is currently a need for effective immunogenic compositions and vaccines to acquire immunity that prevents infection with both hepatitis B and hepatitis D. [Overview of the Initiative] [Means for solving the problem]

[0005] This disclosure relates, as a whole, to the use of recombinant nucleic acids, recombinant DNA, recombinant RNA, recombinant proteins, recombinant polypeptides, or recombinant plutides containing HBV antigens and / or HDV antigens to induce an immune response, antibody production, immunoprotection, or immunity against HBV or HDV infection. In some embodiments, the recombinant nucleic acids, recombinant DNA, recombinant RNA, recombinant proteins, recombinant polypeptides, or recombinant plutides containing HBV antigens and / or HDV antigens are used in DNA prime / protein boost composition inoculation methods. In some embodiments, the DNA prime / protein boost composition inoculation methods yield a more potent immune response, antibody production, immunoprotection, or immunity against HBV or HDV infection than DNA alone, protein alone, or organism-based immunogenic compositions.

[0006] Chronic hepatitis B and D virus (HBV / HDV) infections can cause cancer. Current HBV treatments using nucleoside analogs (NAs) must be continued for life and, while they can reduce the risk of cancer, they cannot eliminate it. One characteristic of chronic hepatitis B is dysfunction of the HBV-specific T cell response. In some embodiments, immunotherapy is provided that is induced by normal naive T cells specific to the HDV antigen (HDAg). This immunotherapy can block HBV entry without the need for HBV-specific T cells by priming PreS1-specific T cells and PreS1 antibodies. In some embodiments, we evaluated whether it is possible to induce PreS1 antibodies and HBV-specific and HDV-specific T cells in vitro and in vivo by various combinations of PreS1 sequences and / or HDAg sequences. In some embodiments, the HBV neutralizing activity of PreS1-specific mouse and rabbit antibodies was evaluated in cell culture systems, and the HBV neutralizing activity of rabbit anti-PreS1 was investigated using mice in which hepatocytes were replaced with human hepatocytes. In some embodiments, adoptive transfer of PreS1 antibodies prevented or mitigated challenge-induced HBV infection in humanized mice.

[0007] In some embodiments, the nucleic acid composition or polypeptide composition comprises a sequence, gene, or polypeptide of HBV, HDV, PreS1, or HDAg. In some embodiments, PreS1 is PreS1 A or PreS1 B. In some embodiments, HDAg is a genotype 1A strain of HDAg (1A), a genotype 1B strain of HDAg (1B), a genotype 2A strain of HDAg (2A), or a genotype 2B strain of HDAg (2B). In some embodiments, the composition further comprises an autocatalytic peptide cleavage site. In some embodiments, the autocatalytic peptide cleavage site is a P2A autocatalytic peptide cleavage site. In some embodiments, the PreS1 component and the HDAg component are grouped together in the composition. In some embodiments, the PreS1 is located downstream of or immediately downstream of the HDAg sequence. In some embodiments, each group comprising the PreS1 and the HDAg is separated by an autocatalytic peptide cleavage site. In some embodiments, each group composed of PreS1 and HDAg is separated by a P2A autocatalytic peptide cleavage site.

[0008] In some embodiments, the nucleic acid composition is a plasmid, virus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC). In some embodiments, the nucleic acid composition is circular or linear. In some embodiments, the nucleic acid composition is produced in a biological system, which may include, but is not limited to, mammalian cells, human cells, bacterial cells, Escherichia coli, yeast, Saccharomyces cerevisiae, or other suitable biological systems. In some embodiments, the HBV and / or HDV nucleic acid or gene is contained in a cassette containing the elements necessary for the nucleic acid or gene to be transcribed and translated in the biological system.

[0009] In some embodiments, the polypeptide composition is properly folded or properly denatured. In some embodiments, the polypeptide composition is produced in biological systems such as mammalian expression systems, bacterial expression systems, yeast expression systems, insect expression systems, or cell-free recombinant expression systems, but is not limited to the following. In some embodiments, the polypeptide composition is produced in mammalian cells, human cells, primary cells, immortalized cells, cancer cells, stem cells, fibroblasts, human fetal kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, bacteria, Escherichia coli, yeast, Saccharomyces cerevisiae, Pichia pastris, insect cells, Spodoptera fulgiperda Sf9 cells, or Spodoptera fulgiperda Sf21 cells, or in cell-free systems. In some embodiments, the polypeptide composition is purified using techniques known in the art, including but not limited to extraction, freeze-thaw, homogenization, permeation, centrifugation, density gradient centrifugation, ultracentrifugation, precipitation, SDS-PAGE, native PAGE, size exclusion chromatography, liquid chromatography, gas chromatography, hydrophobic interaction chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, affinity chromatography, immunoaffinity chromatography, metal-bonded chromatography, nickel column chromatography, epitope tag purification, or lyophilization.

[0010] In some embodiments, the nucleic acid composition or the polypeptide composition is administered to animals such as humans, mice, rats, rabbits, cats, dogs, horses, cattle, pigs, sheep, monkeys, primates, or chickens, but is not limited to those mentioned above. In some embodiments, the administration interval of the nucleic acid composition or the polypeptide composition is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, or any two of these times as upper and lower limits. In some embodiments, the nucleic acid composition is administered before the polypeptide composition. In some embodiments, the polypeptide composition is administered before the nucleic acid composition.

[0011] In some embodiments, the dose of the nucleic acid composition or polypeptide composition is 1 ng, 10 ng, 100 ng, 1000 ng, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1000 μg, 1 mg, 10 mg, 100 mg, or 1000 mg, or any two of these amounts as upper and lower limits. In some embodiments, the nucleic acid composition or polypeptide composition is administered together with an additive. In some embodiments, the nucleic acid composition or polypeptide composition is administered together with an adjuvant. In some embodiments, the nucleic acid composition is administered by in vivo electroporation.

[0012] In some embodiments, the immunogenicity of the nucleic acid composition or polypeptide composition is evaluated by measuring interferon-γ (IFNγ)-producing immune cells using techniques known in the art, such as ELISpot; measuring the titer of IgG antibodies specific to HBV, HDV, HBV protein, HBV nucleic acid, HDV protein, HDV nucleic acid, PreS1, or HDAg; or measuring the neutralizing activity in vitro or in vivo of serum or purified antibodies obtained from immunized animals.

[0013] In some embodiments, administration of the nucleic acid composition or polypeptide composition provides transient, sustained, or permanent protection against HBV or HDV infection. In some embodiments, the transient, sustained, or permanent protection against HBV or HDV infection by administration of the composition is superior to that obtained by administration of other immunogenic compositions. In some embodiments, administration of the nucleic acid composition or polypeptide composition is carried out in combination with antiviral therapy. In some embodiments, administration of the nucleic acid composition or polypeptide composition to provide transient, sustained, or permanent protection against HBV or HDV infection is effective in humans. In some embodiments, the nucleic acid composition or polypeptide composition is used as a vaccine against HBV or HDV.

[0014] Preferred embodiments of the present invention relate to the following numbered embodiments.

[0015] 1. (a) A nucleic acid comprising at least one nucleic acid sequence encoding hepatitis D antigen (HDAg) and at least one nucleic acid sequence encoding PreS1; and (b) Polypeptide comprising at least one HDAg polypeptide sequence and at least one PreS1 polypeptide sequence Immunogenic compositions or immunogenic combination products containing [the specified substance].

[0016] 2. The immunogenic composition or immunogenic combination product according to embodiment 1, wherein at least one nucleic acid sequence encoding the HDAg comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or any combination thereof.

[0017] 3. The immunogenic composition or immunogenic combination product according to embodiment 1 or 2, wherein at least one nucleic acid sequence encoding the PreS1 comprises SEQ ID NO: 9 or SEQ ID NO: 10 or both.

[0018] 4. The immunogenic composition or immunogenic combination product according to any one of embodiments 1 to 3, wherein in the nucleic acid, each HDAg nucleic acid sequence is in a configuration grouped with the PreS1 nucleic acid sequence, and in each group, the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence.

[0019] 5. The immunogenic composition or immunogenic combination product according to embodiment 4, further comprising at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site, and each group composed of the HDAg nucleic acid sequence and the PreS1 nucleic acid sequence is separated by at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site.

[0020] 6. The immunogenic composition or immunogenic combination product according to embodiment 5, wherein at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of 2A (P2A) derived from swine teschovirus type 1, the nucleic acid sequence of 2A (F2A) derived from foot-and-mouth disease virus, the nucleic acid sequence of 2A (E2A) derived from equine rhinitis A virus (ERAV), and the nucleic acid sequence of 2A (T2A) derived from Thosea asigna virus, and the encoded autocatalytic peptide cleavage site may contain a GSG (glycine-serine-glycine) motif at its N-terminus.

[0021] 7. The immunogenic composition or immunogenic combination product according to Embodiment 5 or 6, wherein at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site comprises SEQ ID NO: 13.

[0022] 8. An immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 7, wherein the nucleic acid is codon-optimized for human expression.

[0023] 9. The immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 8, wherein the nucleic acid comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with SEQ ID NOs. 15-24, 35, or 36.

[0024] 10. An immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 9, wherein the nucleic acid comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with SEQ ID NO: 18, SEQ ID NO: 35, or SEQ ID NO: 36.

[0025] 11. The immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 10, wherein the at least one HDAg polypeptide comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or any combination thereof.

[0026] 12. The immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 11, wherein the at least one PreS1 polypeptide sequence comprises SEQ ID NO: 11 or SEQ ID NO: 12 or both.

[0027] 13. The immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 12, wherein the at least one PreS1 polypeptide sequence is located downstream of the at least one HDAg polypeptide sequence.

[0028] 14. The immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 13, wherein the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with the sequence of SEQ ID NOs. 25 to 34 or 37.

[0029] 15. The immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 14, wherein the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with the sequence of SEQ ID NO: 29, 31, 32, or 37.

[0030] 16. An immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 15, wherein the polypeptide is recombinantly expressed.

[0031] 17. The immunogenic composition or immunogenic combination product according to Embodiment 16, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0032] 18. An immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 17, further comprising an adjuvant.

[0033] 19. The immunogenic composition or immunogenic combination product according to Embodiment 18, wherein the adjuvant is Aram, QS-21, or MF59, or any combination thereof.

[0034] 20. An immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 19, wherein the nucleic acid comprises DNA.

[0035] 21. An immunogenic composition or immunogenic combination product according to any one of Embodiments 1 to 20, wherein the nucleic acid is provided in the form of a recombinant vector.

[0036] 22. A method for inducing an immune response in a subject using an immunogenic composition or immunogenic combination product described in any one of Embodiments 1 to 21, Administering at least one prime dose containing the nucleic acid described in any one of the preceding embodiments; and Administer to the subject at least one boost dose containing the polypeptide described in any one of the preceding embodiments. A method that includes this.

[0037] 23. The method according to Embodiment 22, wherein the at least one boost dose further comprises an adjuvant.

[0038] 24. The method according to Embodiment 23, wherein the adjuvant is Aram, QS-21, or MF59, or any combination thereof.

[0039] 25. The method according to any one of embodiments 22 to 24, wherein the at least one boost dose is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 24 days, 36 days, 48 ​​days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, or 48 weeks after the administration of the at least one prime dose, or within a time range with any two of these time points as upper and lower limits, for example, within 1 to 48 days or within 1 to 48 weeks.

[0040] 26. The method according to any one of Embodiments 22 to 25, wherein the administration is enteral, oral, nasal, parenteral, subcutaneous, intramuscular, intradermal, or intravenous, or any combination thereof.

[0041] 27. The method according to any one of embodiments 22 to 26, wherein the administration is performed in combination with antiviral therapy.

[0042] 28. The method according to Embodiment 27, wherein the antiviral therapy comprises the administration of entecavir, tenofovir, lamivudine, adefovir, terbivudine, emtricitabine, interferon α, pegylated interferon α, or interferon α-2b, or any combination thereof.

[0043] 29. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D,

[0044] (a) a nucleic acid comprising at least one nucleic acid sequence encoding hepatitis D antigen (HDAg) and at least one nucleic acid sequence encoding PreS1; and

[0045] (b) Polypeptide comprising at least one HDAg polypeptide sequence and at least one PreS1 polypeptide sequence Immunogenic compositions or immunogenic combination products containing [the specified substance].

[0046] 30. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D, as described in Embodiment 29, wherein at least one nucleic acid sequence encoding HDAg comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or any combination thereof.

[0047] 31. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D, according to Embodiment 29 or 30, wherein at least one nucleic acid sequence encoding PreS1 comprises SEQ ID NO: 9 or SEQ ID NO: 10 or both.

[0048] 32. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of Embodiments 29 to 31, wherein in the nucleic acid, each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and in each group, the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence.

[0049] 33. The immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to Embodiment 32, wherein the immunogenic composition or immunogenic combination product further comprises at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site, and each group consisting of an HDAg nucleic acid sequence and a PreS1 nucleic acid sequence is separated by at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site.

[0050] 34. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D, according to Embodiment 33, wherein at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site comprises a nucleic acid sequence selected from the group consisting of a nucleic acid sequence of 2A (P2A) derived from porcine rhinitis virus type 1, a nucleic acid sequence of 2A (F2A) derived from foot-and-mouth disease virus, a nucleic acid sequence of 2A (E2A) derived from equine rhinitis A virus (ERAV), and a nucleic acid sequence of 2A (T2A) derived from Thosea asigna virus, and the encoded autocatalytic peptide cleavage site may contain a GSG (glycine-serine-glycine) motif at its N-terminus.

[0051] 35. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to Embodiment 33 or 34, wherein at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site comprises SEQ ID NO: 13.

[0052] 36. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 35, wherein the nucleic acid is codon-optimized for human expression.

[0053] 37. An immunogenic composition or immunogenic combination product for use in the treatment or suppression of hepatitis B or hepatitis D according to any one of Embodiments 29 to 36, wherein the nucleic acid comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with SEQ ID NOs. 15 to 24, 35, or 36.

[0054] 38. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 37, wherein the nucleic acid comprises SEQ ID NO: 18, SEQ ID NO: 35, or SEQ ID NO: 36.

[0055] 39. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 38, wherein the at least one HDAg polypeptide comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or any combination thereof.

[0056] 40. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 39, wherein the at least one PreS1 polypeptide sequence comprises SEQ ID NO: 11 or SEQ ID NO: 12 or both.

[0057] 41. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 40, wherein the at least one PreS1 polypeptide sequence is located downstream of the at least one HDAg polypeptide sequence.

[0058] 42. An immunogenic composition or immunogenic combination product for use in the treatment or suppression of hepatitis B or hepatitis D according to any one of Embodiments 29 to 41, wherein the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with the sequence of SEQ ID NOs. 25 to 34 or 37.

[0059] 43. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of Embodiments 29 to 42, wherein the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with the sequence of SEQ ID NO: 29, 31, 32, or 37.

[0060] 44. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 43, wherein the polypeptide is recombinantly expressed.

[0061] 45. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D, as described in Embodiment 44, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0062] 46. ​​An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 45, further comprising an adjuvant.

[0063] 47. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D, as described in Embodiment 46, wherein the adjuvant is Aram, QS-21, or MF59, or any combination thereof.

[0064] 48. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 47, wherein the nucleic acid comprises DNA.

[0065] 49. An immunogenic composition or immunogenic combination product for use in the treatment of hepatitis B or hepatitis D according to any one of embodiments 29 to 48, wherein the nucleic acid is provided in the form of a recombinant vector. [Brief explanation of the drawing]

[0066] Further features and variations beyond those described above can be easily understood from the following description of the drawings and exemplary embodiments. The following drawings show representative embodiments and do not limit the scope of the present invention.

[0067] [Figure 1A-1B] These are nucleic acid constructs or polypeptide constructs containing HBV antigen and / or HDV antigen used herein. Ten constructs are presented: Delta-1 (Δ-1, D1), Delta-2 (Δ-2, D2), Delta-3 (Δ-3, D3), Delta-4 (Δ-4, D4), Delta-5 (Δ-5, D5), Delta-6 (Δ-6, D6), Delta-7 (Δ-7, D7), Delta-8 (Δ-8, D8), Delta-9 (Δ-9, D9), and Delta-10 (Δ-10, D10) (Figure 1A). Western blotting confirmed that these ten polypeptide constructs were properly expressed (Figure 1B). GFP was used as a control for Western blotting.

[0068] [Figure 2] This is a construct used in the DNA prime / protein boost composition inoculation method. The DNA composition contains a Δ-4 nucleic acid sequence, and the protein composition contains a Δ-7 or Δ-8 polypeptide sequence, or a polypeptide sequence fused with Δ-7 and Δ-8.

[0069] [Figure 3A-3E]This study shows the results of quantifying interferon-gamma (IFNγ) formation spots per 10⁶ cells, an indicator of T lymphocyte activation, by performing an ELISpot assay on a population of leukocytes purified from the serum of mice immunized with an HBV / HDV DNA composition, after exposure to various HBV or HDV antigens. The antigens used were PreS1 A (SEQ ID NO: 11), PreS1 A (SEQ ID NO: 12), HDAg genotype 1 A (SEQ ID NO: 5, "HDAg gtp 1A-pool 1" and "HDAg gtp 1A-pool 2"), HDAg genotype 1 B (SEQ ID NO: 6, "HDAg gtp 1B-pool 3" and "HDAg gtp 1B-pool 4"), HDAg genotype 2 A (SEQ ID NO: 7, "HDAg gtp 2C-pool 5" and "HDAg gtp 2C-pool 6"), or HDAg genotype 2 B (SEQ ID NO: 8, "HDAg gtp 2D-pool 7" and "HDAg gtp 2D-pool 6") This is a purified polypeptide containing "8"). Six weeks after initial immunization, mice were euthanized, and pooled spleen cells from each group were stimulated for 48 hours with HDV peptide pools 1-8 corresponding to genotype 1 (pools 1-4) and genotype 2 (pools 5-8). Pools 1 and 2 of genotype 1 represent sequence / isopropyl A, and pools 3 and 4 represent sequence / isopropyl B. Similarly, pools 5 and 6 of genotype 2 represent sequence / isopropyl C, and pools 7 and 8 of genotype 2 represent sequence / isopropyl D. Each pool contains 20 or 221 peptides (pools 1 and 5) consisting of 15 amino acids with 10 overlapping amino acids. Concanavalin A ("ConA") was used as a positive control, and two ovalbumin peptides ("OVA Th" and "OVA Th") were used as negative controls. CTLs and growth medium ("culture medium") were used. Experiments were conducted with N=3 in each peptide stimulation group. The bar graph shows the mean number and standard error of IFNγ spot-forming cells (SFCs) per 106 cells. The cutoff was set at 100 SFCs / 106 spleen cells. The concentrations of each antigen are as indicated in the figure.

[0070] [Figure 4A-4C]This shows the quantitative results of anti-PreS1 IgG antibody titers in the serum of mice immunized with HBV / HDV DNA compositions. Using constructs Δ-1 to Δ-10, the presence or absence of IgG antibody production against the PreS1A consensus sequence and the PreS1B consensus sequence in mice (5 mice / group) was investigated. Figures 4A and 4B show the reactivity to the amino acid region of PreS1 from 2 to 48. Figure 4C shows the cross-reactivity to HBV genotypes A1, A2, B, B2, C, D1, E1, and F.

[0071] [Figure 5A-5C] The results show the quantification of interferon-gamma (IFNγ) formation spots per 106 cells by performing an ELISpot assay on leukocyte populations purified from the serum of C57BL / 6 mice immunized with a Δ-4 DNA composition, HLA-A2 transgenic (Tg) HHD mice, or unsensitized C57BL / 6 mice, after exposure to various HBV or HDV antigens or peptides. The antigens used were purified polypeptides containing PreS1 A (SEQ ID NO: 11) or PreS1 A (SEQ ID NO: 12), pools containing HDAg genotype 1 A (SEQ ID NO: 5) or 1 B (SEQ ID NO: 6) ("gtp 1-pool 1", "gtp 1-pool 2", "gtp 1-pool 3", "gtp 1-pool 4"), pools containing HDAg genotype 2 A (SEQ ID NO: 7) or 2 B (SEQ ID NO: 8) ("gtp 2-pool B1", "gtp 2-pool B2", "gtp 2-pool B3", "gtp 2-pool B4"), an HDAg peptide fragment pool containing peptides KLEDDNPWL, KLEEENPWL, and FPVDILFPA ("pep-3-pool"), and the individual HDAg peptides KLEDDNPWL, KLEEENPWL, and FPVDILFPA. Concanavalin A ("ConA") was used as a positive control, and two types of ovalbumin peptides ("OVA Th" and "OVA CTL") and growth medium ("Culture Medium") were used as negative controls. The concentrations of each antigen are shown in the figure.

[0072] [Figure 6A-6C] This shows the quantitative results of anti-PreS1 IgG antibody titers in New Zealand white rabbits immunized with Δ-3 DNA composition or Δ-4 DNA composition. ELISA assays for PreS1A consensus peptide and PreS1B consensus peptide were performed using serum collected from New Zealand white rabbits (Figure 6B). Cross-reactivity to HBV genotypes A1, A2, B, B2, C, D1, E1, and F was also investigated using antiserum from these vaccinated New Zealand white rabbits (Figure 6C). The bar graph shows the mean endpoint anti-PreS1 antibody titer in each group, defined as the highest dilution ratio in which the OD value at 405 nm was 3 times higher than that of serum from non-immunized animals at the same dilution ratio. Serum was initially diluted 1:60, and then serially diluted 6-fold in series for antibody titer measurement.

[0073] [Figure 6D] This figure shows the reactivity of antiserum from D-4 vaccinated rabbits to different HBV genotypes of PreS1. The reactivity (OD405nm) to HBV genotypes D1, F, A1, C, A2, B, B2, and E1 was examined using ELISA with antiserum from 6-week-old rabbits vaccinated with D-4. For each HBV genotype, 20-amino acid PreS1 peptides with 10-amino acid overlaps (i.e., peptides containing amino acid regions 2-21, 12-31, 22-41, or 32-48 of PreS1) were used. The highest reactivity rate (%) was observed in the amino acid regions 22-41 and 32-48 of genotype D1, followed by the same amino acid regions of genotypes C, E1, and A1. This result suggests that the neutralizing epitope is primarily localized in these regions.

[0074] [Figure 7A-7C]The ELISpot assay was performed on leukocyte populations purified from serum of C57BL / 6 mice immunized with Δ-4 DNA alone, Δ-7 protein alone, or Δ-4 DNA / Δ-8 protein prime / boost compositions, and the number of interferon-gamma (IFNγ)-forming spots per 106 cells was quantified. The antigens used were purified polypeptides containing PreS1 A (SEQ ID NO: 11) or PreS1 A (SEQ ID NO: 12), pools containing HDAg genotype 1 A (SEQ ID NO: 5) or 1 B (SEQ ID NO: 6) ("gtp 1-pool 1", "gtp 1-pool 2", "gtp 1-pool 3", "gtp 1-pool 4"), and pools containing HDAg genotype 2 A (SEQ ID NO: 7) or 2 B (SEQ ID NO: 8) ("gtp 2-pool 5", "gtp 2-pool 6", "gtp 2-pool 7", "gtp 2-pool 8"). Concanavalin A ("ConA") was used as a positive control, and two types of ovalbumin peptides ("OVA Th" and "OVA CTL"), DMSO, and growth medium ("Culture Medium") were used as negative controls. The concentrations of each antigen are shown in the figure.

[0075] [Figures 8A-8C] This shows the quantitative results of anti-PreS1 IgG antibody titers in C57BL / 6 mice immunized with HBV / HDV DNA alone, protein alone, or a DNA prime / protein boost composition.

[0076] [Figure 9] This shows the quantitative results of anti-PreS1 IgG antibody titers in rabbits immunized with HBV / HDV DNA alone, protein alone, or a DNA prime / protein boost composition.

[0077] [Figure 10A-10B]This figure shows the protective effect against HBV infection one, two, three, four, six, and eight weeks after the initial vaccination, based on HBV antibody titers at each time point. Each line represents an individual mouse (Figure 10A). Two negative control mice (gray lines) were administered IgG from non-immunized mice, and three mice (red lines) were administered D4 PreS1 IgG. One mouse in the PreS1 IgG group died at week 4, so only measurements at weeks 1, 2, and 3 are available for this mouse. No significant differences were observed between the groups in serum levels of alanine transferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin (Figure 10B).

[0078] [Figure 11A-11D] This document presents the results of evaluating D-7 and D-8 peptide mixtures using different adjuvants (administered to mice at a dose of 10 μg each). The adjuvants used in the study were QS-21, MF59, and Aram. A control group received intramuscular administration of the D-4 DNA composition via electroporation. Figure 11A shows the administration schedule and exemplary endpoint antibody titers for the various adjuvants used in the study. Figure 11B shows the response rate (%) of individual mice under each condition, measured by ELISA. The X-axis (1, 3, 10, 30, 0) corresponds to the ID number of each mouse. Figure 11C shows IFNγ activation of spleen cells by HBV PreS1 consensus peptide and HDV antigen consensus peptide, as evaluated by ELISpot. Figure 11D shows the endpoint PreS1 antibody titers against PreS1A peptide and PreS1B peptide.

[0079] [Figures 12A-12D]The results show the comparison between D-7 and D-8 peptide mixture alone, D-7+D-8 fusion peptide alone, and D-4 DNA prime and D-7 and D-8 peptide mixture boost, compared to D-4 DNA alone and unsensitized controls. Figure 12A shows IFNγ activation of spleen cells by HBV PreS1 consensus peptide and HDV antigen consensus peptide, as assessed by ELISpot. Figure 12B shows antibody titers against PreS1A assessed 2 weeks after the first dose. Figure 12C shows antibody titers against PreS1A assessed 2 weeks after the second dose. Figure 12D shows antibody titers against PreS1B assessed 2 weeks after the second dose. The legends for Figures 12B-12D correspond to the ID numbers of individual mice. [Modes for carrying out the invention]

[0080] Despite the availability of preventive vaccines and antiviral therapies, chronic hepatitis B virus (HBV) infection currently affects more than 250 million people worldwide. Each year, one million chronic HBV carriers die from HBV-related liver complications, such as cirrhosis and ultimately hepatocellular carcinoma (HCC). Hepatitis D virus (HDV) is an RNA satellite virus of HBV that "steals" the HBV surface antigen (HBsAg) to replicate. Between 15 and 25 million HBV carriers worldwide are co-infected with HDV, and co-infection accelerates disease progression. Until now, there have been no effective and functional treatments for chronic HBV or HDV infections. Currently, the standard treatment for HBV involves the use of nucleoside analogs (NAs) that inhibit the reverse transcriptase (RT) function of HBV polymerase. This prevents viral maturation by inhibiting the synthesis of incomplete double-stranded DNA within the capsid. In other words, NAs can only suppress viral replication during treatment. This is because inhibiting RT does not affect the production and release of proteins including HBsAg, nor the synthesis of covalent closed circular DNA, which is the main cause of HBV persistence. While lifelong NA treatment reduces the risk of HCC, it cannot eliminate it. Currently, a schedule of at least one year of pegylated interferon (IFN)α administration is recommended for the treatment of chronic HDV, but sustained responses are rare. The combined effects of pegylated IFNα and NA on HDV and HBV have been shown to be limited.

[0081] HBV evades the host immune response through several means. It maintains the persistent presence of HBV proteins, causing T cell dysfunction. Cells infected with HBV overproduce subviral HBsAg particles, mainly small HBsAg (SHBsAg), diverting the neutralizing antibody population to SHBsAg and thus preventing attack by neutralizing antibodies. This allows viral particles with densely packed middle HBsAg (MHBsAg) proteins containing S and PreS2, and large HBsAg (LHBsAg) proteins containing S, PreS2, and PreS1 on their surface, to survive. Additionally, the PreS1 domain is present on the Na of HBV on hepatocytes. + - It is important to note that it is involved in binding to the taurocholic acid cotransport polypeptide (NTCP) receptor. Therefore, to target infectious HBV particles and prevent new infections in other hepatocytes, it is necessary to produce antibodies against the virus's PreS1 domain.

[0082] As disclosed herein, chimeric genes containing PreS1 and large HDV antigen (HDAg) in various combinations were created to construct immunotherapies targeting both HBV and HDV infections, inducing the production of PreS1 antibodies and HBV and HDV-specific T cells (Figure 1A). The advantage of linking PreS1 to HDAg is that in patients with HBV monoinfection, HDAg functions as a heterologous T cell epitope carrier. These HDAg-specific T cells can block viral entry without HBV-specific T cells by assisting the sustained endogenous production of PreS1 antibodies. In fact, the proportion of HBV monoinfections among HBV carriers exceeds 90%, and in such patients, the heterologous antigen HDAg primes healthy naive T cells, which then assist in priming the HBV-specific response. Furthermore, in such patients, HDAg-specific T cells and PreS1 antibodies are likely to prevent HDV co-infection. Therefore, to induce both neutralizing antibodies and T cells, we used gene-based immunization methods that have been shown to activate the immune response against HBV. Overall, we believe that by complementing this viral entry prevention strategy with maturation inhibitors and capsid formation inhibitors currently under development, it will be possible to achieve sustained complete remission against HBD infection and / or HDV infection.

[0083] The embodiments described herein relate to immunogenic compositions or immunogenic combination products comprising genetically modified HBV and HDV nucleic acids, genes, peptides, or proteins used to induce an immune response to hepatitis B virus (HBV) infection or hepatitis D virus (HDV) infection. The use of chimeric genes and chimeric proteins containing HBV and HDV nucleic acids, genes, peptides, or proteins has been characterized, for example, in WO2017 / 132332, which is expressly incorporated herein by reference in its entirety.

[0084] In the following detailed description, the present invention will be described with reference to the accompanying drawings which constitute part of this specification. Unless otherwise stated, similar symbols in the drawings generally indicate similar components. The embodiments described in the detailed description, drawings and claims are illustrative and not limiting the present invention in any way. Other embodiments and other modifications are also possible, as long as they do not depart from the spirit or scope of the subject matter shown herein. It will be readily apparent that the aspects of this disclosure outlined herein and shown in the drawings can be arranged, replaced, combined, separated and designed in a variety of configurations, any such aspects which are expressly encompassed herein.

[0085] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Unless otherwise specified, all patent documents, patent applications, published patent applications, and other publications cited herein as references are expressly incorporated by reference in their entirety. If a term has multiple definitions, unless otherwise specified, the definition in the section containing the term shall prevail.

[0086] In this specification, the articles "a" and "an" are used to indicate one or more (e.g., at least one) things that are the grammatical objects of these articles. For example, "an element" means one or more constituent elements.

[0087] In this specification, the term “about” means that a particular quantity, level, number, frequency, percentage, dimension, size, volume, weight, or length varies by 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a baseline quantity, level, number, frequency, percentage, dimension, size, volume, weight, or length.

[0088] Throughout this specification, unless otherwise stated, the terms “includes” and “contains” mean that they include the processes or components or groups of processes or components described herein, but do not exclude other processes or components or groups of processes or components.

[0089] "Consists of" means that it includes only those listed before it. Therefore, the term "consists of" means that the components listed before it are necessary or essential, and other components are not included. "Substantially consists of" means that it includes the components listed before it, and also includes other components that do not interfere with or contribute to the activity or action described in relation to the disclosure of those components. Therefore, the term "substantially consists of" means that the components listed before it are necessary or essential, but other components are optional and may or may not be included depending on whether they substantially affect the activity or action of the components listed before it.

[0090] In implementing this disclosure, unless otherwise stated, conventional molecular biological methods and recombinant DNA techniques known in the art are used, most of which are described below for explanatory purposes. Such techniques are described in detail in the literature. For example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000);DNA Cloning: A Practical Approach, vol. 1 & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984);Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004);Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986);Freshney, RI (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th See Ed. Hoboken NJ, John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd Edition 2005).

[0091] In this specification, the term “purity” of a given substance, compound, or material means the actual abundance of that substance, compound, or material relative to the expected abundance. For example, the purity of a substance, compound, or material may be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and may include decimal values ​​between these numbers. Purity may be affected by undesirable impurities, which include, but are not limited to, by-products, isomers, enantiomers, decomposition products, solvents, carriers, vehicles, or contaminants, or any combination thereof. Purity can be measured using a variety of techniques, including, but are not limited to, chromatography, liquid chromatography, gas chromatography, spectroscopy, ultraviolet-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric or titration, or any combination thereof.

[0092] In this specification, the terms “function” and “functional” mean biological function, enzymatic function, or therapeutic function.

[0093] As used herein, the terms “effective dose” or “effective dosage” mean an amount sufficient to obtain the desired result. Therefore, the “effective dose” or “effective dosage” will vary depending on the ingredients used and the desired result. However, if the desired effect is clear, determining the effective dose is within the scope of the skill of those skilled in the art.

[0094] Typically, the "error bars" in a figure indicate the standard error of the mean value.

[0095] In this specification, the terms "formulation" and "composition" are interchangeable terms meaning a substance composition administered to a subject.

[0096] In this specification, “isolated” means material from which components normally present in its natural state have been substantially or essentially removed. For example, “isolated cells” as used herein include cells purified from the surrounding environment or organism in which they existed in their natural state, cells recovered from a subject or culture, for example, cells substantially free of in vivo or in vitro material.

[0097] In this specification, the term “subject” has the general meaning understood in light of this specification and means an animal that is the subject of treatment, suppression, or improvement, observation, or experimentation. “Animal” has the general meaning understood in light of this specification and includes, for example, cold-blooded vertebrates, warm-blooded vertebrates, and invertebrates, specifically fish, mollusks, reptiles, etc., but particularly mammals. “Mammal” has the general meaning understood in light of this specification and includes, for example, but not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, and primates such as monkeys, chimpanzees, and apes, but particularly humans. In some embodiments, the subject is a human.

[0098] Some embodiments disclosed herein relate to the selection of subjects or patients requiring the present invention. In some embodiments, patients requiring treatment for a viral infection are selected. In some embodiments, patients who have previously received treatment for a viral infection are selected. In some embodiments, patients who have previously received treatment for being at risk of a viral infection are selected. In some embodiments, patients with a recurrent viral infection are selected. In some embodiments, patients who have developed resistance to treatment for a viral infection are selected. In some embodiments, patients having any combination of the above selection criteria are selected.

[0099] In this specification, the terms “to treat,” “treatment,” “therapeutic,” or “therapy” have their general meanings as understood in light of this specification and do not necessarily mean complete cure or elimination of a disease or condition. Also, in this specification (and as is well known in the art), the terms “to treat” or “therapy” mean an approach to obtain a beneficial or desired outcome for a condition in question, such as a clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reduction or alleviation of one or more symptoms or conditions, reduction in the severity of the disease, stabilization of the disease condition (i.e., no worsening), prevention of disease transmission or spread, delay or reduction in the rate of disease progression, alleviation or reduction of the condition, reduction in disease recurrence, and remission, and may be partial or total, and may be detectable or undetectable. In this specification, “to treat” and “therapy” also include prophylactic therapy. A therapeutic method comprises the step of administering a therapeutically effective amount of an active agent to a subject. This administration step may consist of a single dose or may include a series of multiple doses. The compositions of the present invention are administered to a subject in an amount and for a duration sufficient to treat the patient. The duration of treatment varies depending on various factors, including the severity of the condition, the patient's age and genetic profile, the concentration of the active drug, the activity of the composition used for treatment, or a combination of these. Furthermore, it is well understood that the effective dose of the drug used for treatment or prevention may be increased or decreased during the implementation of a particular treatment or prevention plan. Dosage changes may be revealed by standard diagnostic assays known in the art. In some cases, long-term administration may be necessary.

[0100] In this specification, the term “suppress” has the general meaning understood in light of this specification and may mean reducing or preventing a viral infection. The degree of reduction is expressed as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any two of these values ​​as upper and lower limits. In this specification, the term “delay” has the general meaning understood in light of this specification and means slowing down the progression of an event, such as a viral infection, or shifting the timing of the event's occurrence later than expected. The degree of delay is expressed as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any two of these values ​​as upper and lower limits. The terms “suppress” and “delay” do not necessarily mean 100% suppression or delay, and may refer to partial suppression or delay.

[0101] In this specification, the term “immunogenic composition” means a substance or mixture of substances intended to induce an immune response when administered to a host, including but not limited to antigens, epitopes, nucleic acids, peptides, polypeptides, proteins, polysaccharides, lipids, haptens, toxoids, inactivated organisms, or attenuated organisms, or any combination thereof. Immune responses include innate and adaptive immune responses, the latter establishing persistent immunological memory through cells such as memory T cells and memory B cells. Antibodies produced during the initial immune response to an immunogenic composition are generated when challenged by the same antigen, epitope, nucleic acid, peptide, polypeptide, protein, polysaccharide, lipid, hapten, toxoid, inactivated organism, or attenuated organism, or by a living organism or pathogen expressing the same antigen, epitope, nucleic acid, peptide, polypeptide, protein, polysaccharide, lipid, hapten, or toxoid, or by any combination thereof. Thus, immunogenic compositions function as vaccines against specific pathogens. The immunogenic composition may contain one or more adjuvants to activate the immune response and enhance the protective immune effect.

[0102] In this specification, the term “combination product” means a collection of two or more separate compounds, substances, materials, or compositions that can be used together to achieve a common function. In some embodiments, a combination product comprises at least one nucleic acid composition and at least one polypeptide composition, which, when used together, can be administered to a host to induce an immune response, and in some cases can induce a more potent immune response than when only one of the compositions is administered.

[0103] In this specification, the terms “nucleic acid” or “nucleic acid molecule” mean polynucleotides, including, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of natural nucleotide monomers (such as DNA or RNA), analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications to the sugar moiety and / or the pyrimidine base moiety or purine base moiety. Modifications to the sugar moiety include, for example, substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Furthermore, the entire sugar moiety may be substituted with a structure that is stereochemically or electronically similar, such as aza sugars and carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Similar bonds to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoranilothioate bonds, phosphoranilidate bonds, and phosphoramidate bonds. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids." Peptide nucleic acids contain native or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded. The term "oligonucleotide" is used synonymously with "nucleic acid" and refers to double-stranded or single-stranded DNA or RNA.Nucleic acids may be contained in nucleic acid vectors or nucleic acid constructs (e.g., plasmids, viruses, bacteriophages, cosmids, fosmids, phagemids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or human artificial chromosomes (HACs)) that can amplify and / or express nucleic acids in various biological systems. Typically, nucleic acid vectors or nucleic acid constructs also include, but are not limited to, elements such as promoters, enhancers, terminators, inducers, ribosome binding sites, translation start sites, start codons, stop codons, polyadenylation signals, replication start sites, cloning sites, multicloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, target sequences, peptide purification tags or accessory genes, or any combination thereof.

[0104] A nucleic acid or nucleic acid molecule may contain one or more sequences encoding multiple different peptides, polypeptides, or proteins. These one or more sequences may be linked adjacently within a single nucleic acid or nucleic acid molecule, or they may be linked with another nucleic acid in between. Examples of these other nucleic acids include linkers, repeat sequences, or restriction enzyme sites, or sequences of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or 300 nucleotides in length, or sequences of length within a range where any two of these lengths are the upper and lower limits. In this specification, the term “downstream” with respect to nucleic acids means, if the nucleic acid is double-stranded, a sequence located at the 3' end (back) of a given sequence on the strand containing the coding sequence (sense strand). In this specification, the term “upstream” with respect to nucleic acids means, if the nucleic acid is double-stranded, a sequence located at the 5' end (forward) of a given sequence on the strand containing the coding sequence (sense strand). In this specification, the term “grouped” with respect to nucleic acids means two or more sequences that are in close proximity to each other, for example, two or more sequences that are directly linked or linked with another nucleic acid in between.Examples of these other nucleic acids include linkers, repeat sequences, or restriction enzyme sites, or sequences of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or 300 nucleotides in length, or sequences of length within a range where any two of these lengths are the upper and lower limits. Furthermore, the nucleic acid sequences sandwiched between them typically do not encode functional or catalytic polypeptides, proteins, or protein domains.

[0105] In this specification, the term “codon optimization” in relation to nucleic acids means performing codon substitutions in nucleic acids to promote or maximize translation in a specific species host without altering the polypeptide sequence, based on species-specific codon usage bias and the relative utilization of each aminoacyl-tRNA in the cytoplasm of target cells. Codon optimization and codon optimization techniques are known in the art. Programs containing codon optimization algorithms are well known to those skilled in the art. Examples of such programs include algorithms such as OptimumGene and GeneGPS®. Furthermore, synthetic codon-optimized sequences are commercially available, for example, from Integrated DNA Technologies and other DNA sequencing service providers. Those skilled in the art will understand that gene expression levels are determined by many factors, including promoter sequences and regulatory elements. As seen in many bacteria, small subsets of codons are recognized by tRNA species, which can lead to translational selection and become a significant limit on protein expression. In this regard, many synthetic genes can be designed to increase the expression levels of their proteins.

[0106] The nucleic acids described herein include nucleic acid bases. Basic bases, in other words, standard bases, natural bases, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleic acid bases include, but are not limited to, purines, pyrimidines, modified nucleic acid bases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescently labeled bases, or biotin-labeled bases.

[0107] In this specification, "peptide," "polypeptide," and "protein" refer to macromolecules composed of multiple amino acids linked by peptide bonds. Numerous functions of peptides, polypeptides, and proteins are known in the art, including, but are not limited to, enzyme, structural, transport, defense, hormone, or signal transduction functions. Peptides, polypeptides, and proteins are often biologically produced by ribosome complexes using nucleic acids as templates, but are not limited to this method and can also be produced by chemical synthesis. Mutations can be introduced into peptides, polypeptides, or proteins by genetically modifying the template nucleic acid, and these mutations include substitution, deletion, cleavage, addition, duplication, or fusion of two or more peptides, polypeptides, or proteins. When two or more peptides, polypeptides, or proteins are fused, they may be linked adjacently within a single molecule or with other amino acids in between. Other amino acids include, for example, linkers, repeat sequences, epitopes, or tags, or sequences of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 nucleotides in length, or sequences of length within a range where any two of these lengths are the upper and lower limits.

[0108] In some embodiments, the nucleic acid sequences or peptide sequences presented herein and used in the examples are functional in various biological systems, including, but are not limited to, human, mouse, rabbit, Escherichia coli, yeast, and mammalian cells. In another embodiment, nucleic acid sequences or peptide sequences having numerical similarity to the nucleic acid sequences or peptide sequences presented herein and used in the examples in terms of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any two of these numbers as upper and lower limits, can also be used in biological systems without affecting their function. In this specification, the term “similarity” means that the nucleic acid sequence or peptide sequence has the same sequence of bases or amino acids as a whole as the template nucleic acid sequence or peptide sequence, despite certain changes such as substitutions, deletions, repetitions, or insertions. In some embodiments, two nucleic acid sequences having similarity of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% can encode the same polypeptide by containing different codons that translate to the same amino acid.

[0109] In this specification, the term “recombinantly expressed” means that the protein is produced in an optimized or adapted biological system. Such systems offer advantages compared to protein expression in a native host. These advantages include, but are not limited to, high expression (overexpression), ease of purification, ease of transformation, inducibility, low cost, or protein stability. In some embodiments, the protein is expressed in a mammalian expression system, a bacterial expression system, a yeast expression system, an insect expression system, or a cell-free recombinant expression system. Each system has its advantages and disadvantages. For example, while bacterial expression systems are highly optimized for overexpression, misfolding and aggregation of the resulting protein can occur. Also, yeast systems are useful when post-translational modification is required, and insect and mammalian systems are useful when proper RNA splicing, which occurs in higher organisms, is required. In some embodiments, Δ-7, Δ-8, and other recombinant polypeptides are generated in mammalian cells, human cells, primary cells, immortalized cells, cancer cells, stem cells, fibroblasts, human embryonic kidney (HEK) 293 cells, Chinese hamster ovary (CHO) cells, bacteria, Escherichia coli, yeast, Saccharomyces cerevisiae, Pichia pastris, insect cells, Spodoptera fulgiperda Sf9 cells, or Spodoptera fulgiperda Sf21 cells, or in a cell-free system and subsequently purified. In some embodiments, the expression gene, expression vector, or expression construct is introduced into the recombinant expression system in the form of a plasmid, bacteriophage, virus, adeno-associated virus (AAV), baculovirus, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC). For a detailed description of recombinant expression systems, see Gomes et al., "An Overview of Heterologous Expression Host Systems for Production of Recombinant Proteins" (2016) Adv. Anim. Vet. Sci. 4(7): 346-356. This document is expressly incorporated herein by reference in its entirety.

[0110] In this specification, the term “HDAg” means the gene or protein of the hepatitis D antigen. HDAg exists in small (24 kDa) and large (27 kDa, consisting of 213 amino acid residues excluding the start methionine) isoforms, both of which are translated from the same open reading frame of the HDV genome. Deamination of adenosine at the stop codon UAG, which is the 196th codon of the coding sequence, allows translation to continue and generates the large isoform. Unless otherwise stated, the embodiments described herein include the large isoform of HDAg. In some embodiments, the HDAg sequence includes at least one of four different HDAg strain sequences: “HDAg genotype 1 A,” “HDAg genotype 1 B,” “HDAg genotype 2 A,” and “HDAg genotype 2 B.” In some embodiments, the nucleic acid sequence encoding at least one HDAg polypeptide includes the nucleic acid sequence of HDAg genotype 1A (SEQ ID NO: 1), HDAg genotype 1B (SEQ ID NO: 2), HDAg genotype 2A (SEQ ID NO: 3), or HDAg genotype 2B (SEQ ID NO: 4). In some embodiments, the polypeptide comprising at least one HDAg polypeptide includes the polypeptide sequence of HDAg genotype 1A (SEQ ID NO: 5), HDAg genotype 1B (SEQ ID NO: 6), HDAg genotype 2A (SEQ ID NO: 7), or HDAg genotype 2B (SEQ ID NO: 8).

[0111] In this specification, the term “PreS1” means one segment of the N-terminal domain of the large HBV surface antigen (HBsAg). The 47-amino acid length PreS1 segment located in the 108-119 amino acid length N-terminal domain of large HBsAg is effective in inducing an immune response and high anti-PreS1 / anti-HBV antibody titers in mammalian models. In some embodiments, the PreS1 sequence comprises at least one of two different PreS1 consensus sequences, “PreS1 A” and / or “PreS1 B”. In some embodiments, the nucleic acid sequence encoding at least one PreS1 polypeptide comprises the nucleic acid sequence of PreS1 A (SEQ ID NO: 9) or PreS1 B (SEQ ID NO: 10). In some embodiments, the polypeptide comprising at least one PreS1 polypeptide comprises the polypeptide sequence of PreS1 A (SEQ ID NO: 11) or PreS1 B (SEQ ID NO: 12).

[0112] In some embodiments, the HBV PreS1 A consensus sequence and PreS1 B consensus sequence are either the PreS1 sequence of a known HBV genotype or a sequence similar to the PreS1 of a known genotype. It is widely known that HBV has 10 genotypes (genotypes A, B, C, D, E, F, G, H, I, J), and the genome sequence differs by up to approximately 8% between genotypes. Of these, there are further genotype subtypes that differ by up to approximately 4% to 8% in genome sequence. HBV genotypes include, but are not limited to, A1, A2, A3, A4, A5, A6, A7, B2, B3, B4, B5, B6, B7, B9, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, D1, D2, D3, D4, D5, D6, D7, F1, F2, F2a, F3, or F4. For a detailed explanation of HBV genotypes, see Sunbul, "Hepatitis B virus genotypes: Global distribution and clinical importance" (2014) World. J. Gastroenterology. 20(18): 5427-5434. This document is expressly incorporated herein by reference in its entirety.

[0113] In this specification, the terms "autocatalytic peptide cleavage site" or "2A peptide" refer to a peptide sequence configured such that the peptide bond between two constituent amino acids of the peptide sequence is cleaved, thereby separating the two proteins flanking the sequence. This cleavage is thought to be due to a ribosome "skip" in peptide bond formation between proline and glycine at the C-terminus of the 2A peptide sequence. To date, four types of autocatalytic peptide cleavage site sequences have been identified: 2A from foot-and-mouth disease virus (F2A), 2A from equine rhinitis A virus (ERAV) (E2A), 2A from porcine rhinitis virus type 1 (P2A), and 2A from Thosea asigna virus (T2A), and these are widely used in biomedical research. In some embodiments, the nucleic acid sequence (SEQ ID NO: 13) and polypeptide sequence (SEQ ID NO: 14) of the P2A autocatalytic peptide cleavage site are used.

[0114] In this specification, the term "HBeAg" refers to one of the HBV antigenic proteins, specifically the antigenic protein located between the HBV nucleocapsid core and lipid envelope. Since HBeAg produced in the host is secreted into the serum, it is a suitable marker for HBV infection activity. In vitro measurement of HBeAg secretion in cell culture models is used to evaluate the effects of biological or pharmaceutical compounds and compositions on HBV infectivity.

[0115] The term “additive” has the general meaning as understood herein and means any other substance, compound, or material contained in an immunogenic composition or vaccine. Additives having the desired properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Furthermore, additives may also be residues or contaminants from the manufacturing process of the immunogenic composition or vaccine, such as serum, albumin, ovalbumin, antibiotics, inactivators, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell fragments, nucleic acids, peptides, amino acids, or growth medium components, or any combination thereof. The amount of the additive contained in the immunogenic composition or vaccine may be within the range of 0%w / w, 0.1%w / w, 0.2%w / w, 0.3%w / w, 0.4%w / w, 0.5%w / w, 0.6%w / w, 0.7%w / w, 0.8%w / w, 0.9%w / w, 1%w / w, 2%w / w, 3%w / w, 4%w / w, 5%w / w, 6%w / w, 7%w / w, 8%w / w, 9%w / w, 10%w / w, 20%w / w, 30%w / w, 40%w / w, 50%w / w, 60%w / w, 70%w / w, 80%w / w, 90%w / w, 95%w / w, 100%w / w, or any two of these weight percentage values ​​as upper and lower limits.

[0116] In this specification, “adjuvant” means a substance, compound, or material that activates the immune response and enhances the protective immune effect, and is administered in combination with an antigen, epitope, or composition as an immunogen. Adjuvants enhance the immune response by enabling the continuous release of antigens, upregulation of cytokines and chemokines, recruitment of cells to the administration site, enhancement of antigen uptake and presentation by antigen-presenting cells, or activation of antigen-presenting cells and inflammasomes. Commonly used adjuvants include, but are not limited to, alum, aluminum salts, aluminum sulfate, aluminum hydroxide, aluminum phosphate, calcium hydroxide phosphate, potassium aluminum sulfate, oils, mineral oils, paraffin oils, oil-in-water emulsions, detergents, MF59 (registered trademark), squalene, AS03, α-tocopherol, polysorbate 80, AS04, monophosphoryl lipid A, virosoms, nucleic acids, polyinosinate-polycytidylic acid, saponins, QS-21, proteins, flagellin, cytokines, chemokines, IL-1, IL-2, IL-12, IL-15, IL-21, imidazoquinoline, CpG oligonucleotides, lipids, phospholipids, dioleoylphosphatidylcholine (DOPC), trehalose dimycolic acid, peptidoglycans, bacterial extracts, lipopolysaccharides, or Freund's adjuvants, or any combination thereof.

[0117] In this specification, the terms “prime” and “boost” refer to immunogenic compositions used separately in prime-boost heterologous immunization. Immunization or vaccines generally require multiple administrations of the same immunogenic composition to induce and establish immunity against a target pathogen in the host. Thus, compared to homogenous inoculation, where the same composition is provided for all administrations, prime-boost heterologous inoculation may be more effective in establishing robust immunity to certain pathogens, such as HBV and HDV, resulting in increased antibody levels and improved elimination or resistance. In prime-boost heterologous inoculation, at least one prime dose containing one type of immunogenic composition is administered first. After at least one prime dose has been administered, at least one boost dose containing another type of immunogenic composition is administered. The administration of the at least one boost dose is performed at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 24 days, 36 days, 48 ​​days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, or 48 weeks after the administration of the at least one prime dose, or within a time range with any two of these time points as upper and lower limits, for example, within 1 to 48 days or within 1 to 48 weeks. In some embodiments, the prime dose comprises nucleic acids (e.g., DNA or RNA) encoding one or more antigens or epitopes, and the boost dose comprises a polypeptide containing one or more antigens or epitopes. In the host, the prime nucleic acid is translated in vivo to induce an immune response, followed by a stronger response to the boost polypeptide. In some embodiments, the prime nucleic acid comprises a sequence encoding at least one HDAg polypeptide, a sequence encoding at least one PreS1 peptide, and a sequence encoding at least one autocatalytic peptide cleavage site. In some embodiments, the boost polypeptide comprises at least one HDAg polypeptide and at least one PreS1 polypeptide.

[0118] In some embodiments, when a prime nucleic acid and boost polypeptide containing HBV and HDV components are administered to experimental organisms, the anti-HDAg antibody titer, anti-PreS1 antibody titer, anti-HBV antibody titer, or anti-HDV antibody titer, as measured by techniques known in the art such as ELISA, is increased by 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 50x, 100x, 150x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 5000x, 10000x, 100000x, or 1000000x, or any two of these multipliers as upper and lower limits, compared to organisms immunized with nucleic acid alone or polypeptide alone, or non-immunized control organisms. In some embodiments, when experimental organisms were administered a prime nucleic acid and boost polypeptide containing HBV and HDV components, compared to serum obtained from organisms immunized with nucleic acid alone or polypeptide alone, or from non-immunized control organisms, in In vitro serums that more effectively neutralize HBV or HDV infectivity are obtained, specifically serums that reduce the incidence of infection by a factor of 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, or any two of these factors as upper and lower limits.In some embodiments, when experimental organisms were administered a prime nucleic acid and boost polypeptide containing HBV and HDV components, the number of interferon-gamma (IFNγ) positive cells (e.g., T cells) was 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, and 60 times higher compared to organisms immunized with nucleic acid alone or polypeptide alone, or to non-immunized control organisms. It increases by a multiplier of 1 / 2, 70 / 2, 80 / 2, 90 / 2, 100 / 2, 110 / 2, 120 / 2, 130 / 2, 140 / 2, 150 / 2, 200 / 250 / 2, 300 / 2, 350 / 2, 400 / 2, 450 / 2, 500 / 2, 550 / 2, 600 / 2, 650 / 2, 700 / 2, 750 / 2, 800 / 2, 850 / 2, 900 / 2, 950 / 2, 1000 / 2, 5000 / 2, or 10000 / 2, or any two of these multipliers as upper and lower limits.

[0119] In some embodiments, the immunogenic composition or immunogenic combination product is administered with an adjuvant. In some embodiments, the administration of the immunogenic composition or immunogenic combination product is enteral, oral, nasal, parenteral, subcutaneous, intramuscular, intradermal, or intravenous, or any combination thereof. In some embodiments, the immunogenic composition or immunogenic combination product is administered in combination with an antiviral therapeutic compound known to be effective against HBV or HDV, such as, but not limited to, entecavir, tenofovir, lamivudine, adefovir, terbivudine, emtricitabine, interferon α, pegylated interferon α, or interferon α-2b, or any combination thereof.

[0120] In this specification, the terms “in vivo electroporation,” “electroporation,” and “EP” mean the introduction of genes, nucleic acids, DNA, RNA, proteins, or vectors into the cells of a living tissue or organism using electric current by techniques known in the art. Electroporation can be used as an alternative to other gene transfer methods such as viral (transduction), lipofection, gene gun, microinjection, vesicle fusion, or chemical transformation. Electroporation reduces the risk of immunogenicity, harmful integration into the cell genome, and mutagenesis. DNA vectors, such as plasmids, translocate to the cell nucleus, where the constituent genes are transcribed and translated. In some embodiments, genes, nucleic acids, DNA, RNA, proteins, or vectors are delivered to a target tissue or organism by subcutaneous, intramuscular, or intradermal injection. An electroporator then emits short electric pulses from electrodes placed inside or near the injected sample. In this specification, “im / EP” means the delivery of a sample intramuscularly (im) by in vivo electroporation.

[0121] In this specification, "uPA" + / + The term "-SCID" refers to an immunodeficient mouse model used in the study of liver diseases, including hepatitis virus infections. This mouse is called Prkdc. scid This mouse is homozygous for [specific gene] and lacks functional T lymphocytes and B lymphocytes. Furthermore, overexpression of urokinase-type plasminogen activator (uPA) leads to severe hepatotoxicity and liver failure during development. Transplanting human liver tissue into this mouse and allowing it to engraft provides an ideal model for studying human liver diseases. uPA + / +For a detailed description of the SCID mouse, see Meuleman et al., "The human liver-uPA-SCID mouse: A model for the evaluation of antiviral compounds against HBV and HCV" ((2008) Antiviral Research 80(3): 231-238). This document is expressly incorporated herein by reference in its entirety.

[0122] In this specification, "%w / w" or "%wt / wt" has the general meaning understood in light of this specification, and represents the ratio of the weight of the component or agent to the total weight of the composition of the present invention multiplied by 100. In this specification, "%v / v" or "%vol / vol" has the general meaning understood in light of this specification, and represents the ratio of the liquid volume of the compound, substance, component or agent to the total liquid volume of the composition of the present invention multiplied by 100.

[0123] To illustrate various embodiments of the present invention, this specification generally discloses the invention in positive terms. The present invention also includes embodiments in which all or part of the subject matter of the invention, such as substances or materials, process and conditions of a method, or protocol or procedure, is excluded.

[0124] Immunogenic compositions and immunogenic combination products This specification discloses immunogenic compositions or immunogenic combination products. In some embodiments, the immunogenic compositions or immunogenic combination products of the present invention are intended to induce an immunogenic response to a specific antigen. In some embodiments, the immunogenic compositions or immunogenic combination products include (a) a nucleic acid comprising at least one nucleic acid sequence encoding hepatitis D antigen (HDAg) and at least one nucleic acid sequence encoding PreS1; and (b) a polypeptide comprising at least one HDAg polypeptide sequence and at least one PreS1 polypeptide sequence. In some embodiments, the at least one nucleic acid sequence encoding HDAg includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or any combination thereof. In some embodiments, the at least one nucleic acid sequence encoding PreS1 includes SEQ ID NO: 9 or SEQ ID NO: 10, or both. In some embodiments, the nucleic acid is configured such that each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and in each group, the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence. In some embodiments, the immunogenic composition or immunogenic combination product further comprises at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site, wherein each group consisting of the HDAg nucleic acid sequence and the PreS1 nucleic acid sequence is separated by at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site. In some embodiments, the at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of 2A (P2A) derived from porcine rhinitis virus type 1, the nucleic acid sequence of 2A (F2A) derived from foot-and-mouth disease virus, the nucleic acid sequence of 2A (E2A) derived from equine rhinitis A virus (ERAV), and the nucleic acid sequence of 2A (T2A) derived from Thosea asigna virus, wherein the encoded autocatalytic peptide cleavage site may contain a GSG (glycine-serine-glycine) motif at its N-terminus. In some embodiments, the at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site includes SEQ ID NO: 13.In some embodiments, the nucleic acid is codon-optimized for human expression. In some embodiments, the nucleic acid includes a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with SEQ ID NOs. 15-24, 35, or 36. 36. 4. In some embodiments, the nucleic acid includes a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with SEQ ID NOs. 35, or 36. 4. In some embodiments, the at least one HDAg polypeptide includes SEQ ID NOs. 5, 6, 7, or 8, or any combination thereof. 5. In some embodiments, the at least one PreS1 polypeptide sequence includes SEQ ID NOs. 11 or 12, or both. 6. In some embodiments, the at least one PreS1 polypeptide sequence is located downstream of the at least one HDAg polypeptide sequence. In some embodiments, the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the sequence of SEQ ID NOs. 25-34 or 37. In some embodiments, the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the sequence of SEQ ID NOs. 29, 31, 32, or 37. In some embodiments, the polypeptide is recombinantly expressed. In some embodiments, the polypeptide is recombinantly expressed in mammalian, bacterial, yeast, insect, or cell-free systems. In some embodiments, the immunogenic composition or immunogenic combination product further comprises an adjuvant. In some embodiments, the adjuvant is Aram, QS-21, or MF59, or any combination thereof. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid is provided in the form of a recombinant vector.

[0125] Furthermore, this specification discloses methods for inducing an immune response in a subject using an immunogenic composition or immunogenic combination product. In some embodiments, the immunogenic composition or immunogenic combination product is any of the immunogenic compositions or immunogenic combination products disclosed herein. In some embodiments, the method comprises administering at least one prime dose containing a nucleic acid disclosed herein to the subject; and administering at least one boost dose containing a polypeptide disclosed herein to the subject. In some embodiments, the at least one boost dose further comprises an adjuvant. In some embodiments, the adjuvant is Aram, QS-21, or MF59, or any combination thereof. In some embodiments, the at least one boost dose is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 24 days, 36 days, 48 ​​days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, or 48 weeks after the administration of the at least one prime dose, or within a time range with any two of these time points as upper and lower limits, for example, within 1 to 48 days or within 1 to 48 weeks. In some embodiments, the administration is enteral, oral, nasal, parenteral, subcutaneous, intramuscular, intradermal, or intravenous, or any combination thereof. In some embodiments, the administration is carried out in combination with antiviral therapy. In some embodiments, the antiviral therapy includes the administration of entecavir, tenofovir, lamivudine, adefovir, terbivudine, emtricitabine, interferon α, pegylated interferon α, or interferon α-2b, or any combination thereof.

[0126] Furthermore, this specification discloses immunogenic compositions or immunogenic combination products for use in the treatment or suppression of hepatitis B or hepatitis D. In some embodiments, the immunogenic composition or immunogenic combination product is any of the immunogenic compositions or immunogenic combination products disclosed herein. In some embodiments, the immunogenic composition or immunogenic combination product comprises (a) a nucleic acid comprising at least one nucleic acid sequence encoding hepatitis D antigen (HDAg) and at least one nucleic acid sequence encoding PreS1; and (b) a polypeptide comprising at least one HDAg polypeptide sequence and at least one PreS1 polypeptide sequence. In some embodiments, the at least one nucleic acid sequence encoding HDAg comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the at least one nucleic acid sequence encoding PreS1 comprises SEQ ID NO: 9 or SEQ ID NO: 10 or both. In some embodiments, the nucleic acid is configured such that each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and in each group, the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence. In some embodiments, the immunogenic composition or immunogenic combination product further comprises at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site, wherein each group consisting of the HDAg nucleic acid sequence and the PreS1 nucleic acid sequence is separated by at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site. In some embodiments, the at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences from porcine rhinitis virus type 1 2A (P2A), nucleic acid sequences from foot-and-mouth disease virus 2A (F2A), nucleic acid sequences from equine rhinitis A virus (ERAV) 2A (E2A), and nucleic acid sequences from Thosea asigna virus 2A (T2A), wherein the encoded autocatalytic peptide cleavage site may contain a GSG (glycine-serine-glycine) motif at its N-terminus.In some embodiments, the at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site includes SEQ ID NO: 13. In some embodiments, the nucleic acid is codon-optimized for human expression. In some embodiments, the nucleic acid includes a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with SEQ ID NOs: 15-24, 35, or 36. In some embodiments, the nucleic acid includes a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology with SEQ ID NO: 18, SEQ ID NO: 35, or SEQ ID NO: 36. In some embodiments, the at least one HDAg polypeptide includes SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or any combination thereof. In some embodiments, the at least one PreS1 polypeptide sequence includes SEQ ID NO: 11 or SEQ ID NO: 12, or both. In some embodiments, the at least one PreS1 polypeptide sequence is located downstream of the at least one HDAg polypeptide sequence. In some embodiments, the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the sequence of SEQ ID NOs. 25-34 or 37. In some embodiments, the polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the sequence of SEQ ID NOs. 29, 31, 32, or 37. In some embodiments, the polypeptide is recombinantly expressed. In some embodiments, the polypeptide is recombinantly expressed in mammalian, bacterial, yeast, insect, or cell-free systems. In some embodiments, the immunogenic composition or immunogenic combination product further comprises an adjuvant. In some embodiments, the adjuvant is Aram, QS-21, or MF59, or any combination thereof. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid is provided in the form of a recombinant vector. [Examples]

[0127] The following embodiments further disclose some aspects of the embodiments of the present invention described above, but these embodiments do not limit the scope of the disclosure of the present invention. Those skilled in the art will understand that many other embodiments, as described herein and in the claims, are also within the scope of the present invention.

[0128] Example 1: Method animal Female C57BL / 6(H-2 b The mice used were obtained from Charles River Laboratories. Human leukocyte antigen A2 (HLA-A2) transgenic HHD mice were bred in-house. All mice were 8-10 weeks old at the start of the experiment, and were kept under standard conditions. uPA with humanized liver + / + -SCID mice were bred and reared by the researchers. New Zealand white rabbits were purchased from a supplier.

[0129] DNA plasmid In this study, plasmids encoding L-HDAg genotypes 1 and 2 and the HBsAg PreS1 domain (amino acid region 2-48) were used as fusion constructs combining various HDAg and PreS1 sequences. P2A cleavage sites were inserted into the fusion constructs as needed. The HDAg sequences for genotypes 1 and 2 were obtained from four clinical isolates: US-2, CB, 7 / 18 / 83, and TW2476, respectively. Each gene was cloned into the pVAX1 backbone (Invitrogen, Carlsbad, California) using restriction sites with EcoRI and HindIII. The plasmids were grown in TOP10 E. coli (Life Technologies, Carlsbad, California) and purified for in vivo injection using the Qiagen Endofree DNA Purification Kit (Qiagen) according to the manufacturer's instructions. Appropriate gene size was confirmed using restriction enzyme digests with EcoRI and HindIII (Fast Digest, Thermo Fisher Scientific).

[0130] Western blot Western blotting was performed using methods generally known in the art. Each pVAX1 D1-D10 DNA plasmid and a pVAX1 containing the reporter gene GFP as a control were transfected into HeLa cells using Lipofectamine® 3000 transfection reagent (Thermo Fisher Scientific). For protein detection, D4 vaccinated rabbit serum diluted 1:1000 (primary antibody) and goat anti-rabbit immunoglobulin HRP 0.25 g / L diluted 1:4000 (DAKO) (secondary antibody) were used. For chemiluminescence detection, Pierce® ECL Plus Western Blotting Substrate was used, and images were collected using the Gel Doc XR+ System (Biorad).

[0131] peptide 168 HDAg peptides consisting of 15 amino acids overlapping by 10 amino acids each were purchased from Sigma-Aldrich (St. Louis, Missouri). These 168 peptides were divided into 8 pools containing 20 or 21 peptides each. Four pools (Pool 1 1-21 , Pool 2 22-42 , Pool 3 43-63 , Pool 4 64-84 ) correspond to genotype 1 of sequences A, B, C, and D, and the remaining 4 pools (Pool 1 1-21 , Pool 2 22-42 , Pool 3 43-63 , Pool 4 64-84 ) correspond to genotype 2 of sequences A, B, C, and D. Each sequence represents a different clinical isolate.

[0132] Two consensus sequences of PreS1 HBsAg consisting of 47 amino acids (PreS1A and PreS1B), and a group of PreS1 peptides consisting of 20 amino acids overlapping by 10 amino acids each, for each of the HBV genotypes (subtypes) A1, A2, B, B2, C, D1, E1, F, were purchased from Sigma-Aldrich (St. Louis, Missouri). All peptides passed quality inspection (Sigma-Aldrich PEPscreen® Directory) and had a purity value exceeding 70%. Ovalbumin peptide OVA 257-264 CTL (SIINFEKL (SEQ ID NO: 38)) and OVA 323-339 Th (ISQAVHAAHAEINEAGR (SEQ ID NO: 39)) were used as negative control peptides, and concanavalin A (ConA) purchased from Sigma-Aldrich (St. Louis, Missouri) was used as a positive control at a final concentration of 0.5 μg / μL.

[0133] Immunization protocols for evaluating the immunogenicity of HBV / HDV plasmids in mice and rabbits To evaluate the in vivo immunogenicity of the construct, mice and rabbits were immunized using essentially the same method as described above, boosted with a one-month interval, euthanized two weeks later, and their spleens and blood were collected. Briefly, 50 μg of plasmid DNA dissolved in 50 μL of sterile PBS was intramuscularly (im) injected into the tibialis anterior muscle (TA muscle) of female C57BL / 6 mice (5 mice / group) using a standard needle (27G), followed by in vivo electroporation (EP) using a Cliniporator 2 (IGEA, Calpi, Italy). For in vivo electroporation, a pattern of stimulation with a 1 ms 600 V / cm pulse followed by a 400 ms 60 V / cm pulse was used to promote DNA uptake. Before vaccine injection, mice were administered analgesics, and they were maintained under isoflurane anesthesia during vaccination. In the rabbit study, New Zealand white rabbits were divided into groups of two and immunized with either 300 μg of D3 DNA vaccine or D4 DNA vaccine. The vaccine was dissolved in 300 μL of sterile PBS and injected intramuscularly into the right TA muscle, followed by in vivo epiphysis testing.

[0134] Detection of IFNγ-producing T cells by enzyme-linked immunosorbent spot (ELISpot) Two weeks after the final vaccination, pooled spleen cells from each immunized group of mice (5 mice / group) were stimulated with peptides for 48 hours, and the ability of the vaccine to induce HBV / HDV-specific T cells was investigated by measuring IFNγ secretion using a commercially available ELISpot assay (Mabtech, Nakkastrand, Sweden) in a manner known in the art.

[0135] Antibody detection by ELISA Detection of mouse and rabbit IgG against PreS1 consensus peptide and 20-amino acid overlap peptide (10 μg / mL) was performed using protocols known in the art. Antibody titers were determined as the serum dilution of the endpoint, where the OD value at 405 nm was at least twice the OD value of the negative control (serum of an unimmunized animal or control animal) at the same dilution.

[0136] HBV neutralization assay using a humanized liver uPA-SCID mouse model HepG2-NTCP-A3 is a cell line selected from clones of previously reported human NTCP-expressing HepG2 cells. This cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U / mL penicillin, and 50 μg / mL streptomycin. 2.5% DMSO was added to the medium during and after HBV inoculation to promote HBV infection and replication. The HBV virus stock used for infection was prepared from HepAD38 cells by PEG precipitation as described. Cell cultures were collected 3–6 days after infection, diluted 1:5 with PBS, and HBeAg was quantified by ELISA using a commercially available antibody.

[0137] statistical analysis Data analysis was performed using GraphPad Prism V.5 and V.8 software and Microsoft Excel V.16.13.1.

[0138] Example 2: HBV / HDV immunogenic construct The use of recombinant HBV / HDV polypeptide constructs has been shown, for example, in WO2017 / 132332, to be effective in inducing antibody production and immunoprotection against these two hepatitis viruses, HBV and HDV. WO2017 / 132332 is incorporated herein by reference in its entirety. These recombinant polypeptide constructs are constructed by combining HDAg selected from four different HDV genotypes (HDAg genotype 1A, HDAg genotype 1B, HDAg genotype 2A, and HDAg genotype 2B), PreS1 selected from two genotype consensus sequences (PreS1A and PreS1B), and one or more P2A autocatalytic peptide cleavage sites. Schematic diagrams of 11 recombinant constructs are shown in Figures 1A and 2, and their respective DNA and polypeptide sequences, indicated by their sequence numbers, are listed in Table 1 where disclosed herein. Western blotting confirmed that polypeptides were properly expressed from the recombinant constructs Δ-1 to Δ-10 (Figure 1B). [Table 1]

[0139] Example 3: Induction of immunogenic response in mice by HBV / HDV DNA composition Until now, immunogenic compositions and vaccines have consisted of whole organisms or antigen proteins. However, in recent years, it has become clear that administering DNA to living tissues in vivo, and then transcribing and translating antigen proteins from that DNA, is also highly effective in eliciting an immune response. Such DNA immunogenic compositions are being studied as potential vaccine candidates for various diseases.

[0140] Mice were administered a DNA construct composition twice, and their immunity to HBV and HDV antigens was examined two weeks after the second administration. Leukocytes were purified from whole blood of mice and incubated with purified polypeptide antigens such as PreS1 A, PreS1 B, and HDAg genotypes 1 A, 1 B, 2 A, and 2 B. Concanavalin A ("ConA") was used as a positive control, and two types of ovalbumin peptides ("OVA Th" and "OVA CTL") were used as negative controls, both incubated with leukocytes. The population frequency of interferon-gamma (IFNγ) producing cells that appeared upon antigen exposure was examined using enzyme-linked immunosorbent spot (ELISpot). Briefly, leukocytes were incubated with antigens in wells coated with IFNγ antibody. Subsequently, the leukocytes were removed, and biotinylated IFNγ antibody, alkaline phosphatase-crosslinked streptavidin, and alkaline phosphatase substrate colorimetric reagents were sequentially added to the wells. Thorough washing was performed before adding each reagent. Subsequently, the plates were dried, and the remaining stained spots corresponding to IFNγ-secreting cells were counted under a microscope. Cell counts for each mouse against various peptide antigens were then measured. 6 The number of IFNγ spot-forming cells per cell is shown in Figures 3A (Δ-1 and Δ-2), 3B (Δ-3 and Δ-4), 3C (Δ-5 and Δ-6), 3D (Δ-7 and Δ-8), and 3E (Δ-9 and Δ-10).

[0141] The reactivity to PreS1A consensus peptide and PreS1B consensus peptide (amino acid region 2-48) was investigated using antiserum. Furthermore, cross-reactivity to HBV genotypes A1, A2, B, B2, C, D1, E1, and F was examined using a 20-amino acid PreS1 peptide pool with antiserum. Immunogenic compositions containing Δ-1, Δ-2, Δ-3, Δ-4, Δ-7, or Δ-8 showed potent immunogenicity against any of the HBV PreS1 antigens (Figures 4A and 4B). Δ-3 and Δ-4 were found to be effective in mice. 4Antibody titers exceeding Δ-4 were induced, followed by Δ-1, Δ-2, Δ-7, and Δ-8 in that order. Importantly, the antisera from mice immunized with Δ-4 and Δ-7 showed effective cross-reactivity among all HBV genotypes tested (Figure 4C). The immune response to HDAg peptide was generally greater than that of the ovalbumin control group, although there was some variability likely due to differences in genotype sequences. It should be noted that the HDV T cell response was slightly weaker in the Δ-3 and Δ-4 groups compared to constructs containing only HDAg (Δ-5, Δ-6, Δ-9, Δ-10), which is thought to be due to competition for epitope recognition through simultaneous priming of PreS1-specific T cells. The above findings demonstrate that active immunity induces functional T cells against PreS1 antigen and HDAg antigen, suggesting that for immunotherapies that deliver broad-spectrum functions, it is necessary to include both HDV genotypes 1 and 2 to reliably induce specific T cells.

[0142] Similar experiments were conducted using HLA-A2-restricted T cells purified from HLA-A2 transgenic HHD mice. IFNγ ELISpot was performed on healthy C57BL / 6 mice (Figure 5A) and HLA-A2 HHD mice (Figure 5B) introduced with a Δ-4-containing composition via electroporation, and on unsensitized HLA-A2 HHD mice (Figure 5C) as a control. The results confirmed immunogenicity in transgenic mice, suggesting that this DNA composition may be effective when administered to humans.

[0143] Example 4: Induction of immunogenic response in rabbits by HBV / HDV DNA composition The same experiment as in Example 3 was performed in rabbits (Oryctolagus cuniculus). New Zealand white rabbits were intramuscularly injected with saline solution containing 900 μg of a DNA composition containing Δ-3 or Δ-4, followed by electroporation. Administration was performed at weeks 0 and 4. After immunization, the anti-PreS1 antibody titer in rabbit serum against the DNA composition containing Δ-3 or Δ-4 was measured, and a stronger effect was observed with Δ-4 (>10). 3The results were shown (Figures 6A and 6B). Furthermore, cross-reactivity with HBV genotypes A1, A2, B, B2, C, D1, E1, and F was investigated using rabbit antiserum with a 20-amino acid PreS1 peptide pool (Figure 6C). The specificity of rabbit D4 antiserum was further investigated using a PreS1 peptide group consisting of 20 amino acids from each HBV genotype A1, A2, B, B2, C, D1, E1, and F (Figure 6D). Epitope mapping of PreS1 revealed high reactivity to the epitope located in the amino acid region 22-48 of genotype D1, followed by high reactivity in the order of genotypes C, E1, and A1. This overlaps with the NTCP binding site and also overlaps with known epitopes recognized by neutralizing antibodies.

[0144] Table 2 summarizes the immunogenic effects of 10 different DNA immunogenic compositions. The DNA composition containing Δ-4 yielded the highest anti-PreS1 / anti-HBV antibody titers in both mice and rabbits. In subsequent examples, this DNA composition was used for prime / boost immunization. Furthermore, Δ-4 exhibits the broadest reactivity to different HBV genotypes. "nd" indicates low or undetectable antibody activity. "n / a" indicates that the experiment was not performed. [Table 2]

[0145] Example 5: Improvement of immunogenicity response in mice using DNA prime / protein boost inoculation with HBV / HDV constructs To induce adaptive immunization against HBV and / or HDV in vivo and induce antibody production, DNA-priming / protein-boosting immunization was performed using a DNA composition containing Δ-4 (SEQ ID NO: 18) and a polypeptide composition containing Δ-7 (SEQ ID NO: 31) or Δ-8 (SEQ ID NO: 32) (Figure 2).

[0146] C57BL / 6 mice were immunized with (1) a DNA composition containing Δ-4 (50 μg DNA administered three times consecutively), (2) a polypeptide composition containing Δ-7 (20 μg protein + adjuvant administered three times consecutively), or (3) a polypeptide composition containing Δ-8 following a DNA composition containing Δ-4 (50 μg DNA administered twice, followed by two administrations of 20 μg protein + alum). After administration of each composition, leukocytes purified from the mice were examined using ELISpot (in the same manner as described in Examples 1 and 2) to determine whether IFNγ was produced in response to HBV and HDV antigens. Mice administered with (1) showed a response to hepatitis antigens similar to that in Example 3 and Figure 3B (Figure 7A), while mice administered with the DNA prime / protein boost composition of (3) showed a relatively strong immune cell response overall (Figure 7C). Because Δ-8 contains the HDAg genotype 2 polypeptide sequence, a particularly strong immune response was observed against the HDAg genotype 2 antigen (Figure 7C, gtp 2-pool 5, gtp 2-pool 6, gtp 2-pool 7, and gtp 2-pool 8). Conversely, the inoculation method using only the protein in (2) with the Δ-7 polypeptide could not induce a similarly effective immune response against both HBV and HDV antigens (Figure 7B). This indicates that the DNA prime / protein boost inoculation method is effective in inducing a stronger immunogenic response against specific pathogens such as HBV and HDV than conventional protein or organism-based compositions.

[0147] Other combinations of the DNA prime / protein boost inoculation method were also evaluated in mice. Mice were immunized with (1) a DNA-only composition containing Δ-4 ("D4"), (2) a protein-only composition containing Δ-7 ("D7-D7"), a protein-only composition containing Δ-8 ("D8-D8"), a protein-only composition containing Δ-9 ("D9-D9"), or a protein-only composition containing Δ-10 ("D10-D10"), or (3) a DNA-protein composition containing Δ-4 DNA and Δ-7 protein ("D4-D7"), a DNA-protein composition containing Δ-4 DNA and Δ-8 protein ("D4-D8"), a DNA-protein composition containing Δ-4 DNA and Δ-9 protein ("D4-D9"), or a DNA-protein composition containing Δ-4 DNA and Δ-10 protein ("D4-D10"), and then the anti-PreS1 IgG antibody titers of the mice were measured. Each composition was administered a total of three times, at week 0, week 4, and week 8. Administration consisted of either 50 μg DNA im / EP or 20 μg protein + alum. For DNA-protein composition (3), the first administration was 50 μg of DNA im / EP at week 0, and the second and third administrations were 20 μg of protein + alum at weeks 4 and 8, respectively. Serum anti-PreS1 IgG antibody titers were examined 2 weeks (Figure 8A), 6 weeks (Figure 8B), and 10 weeks (Figure 8C) after the first administration (i.e., 2 weeks after each administration). Excellent anti-PreS1 antibody titers were obtained after completion of the administration schedule with DNA prime / protein boost compositions D4-D7.

[0148] Example 6: Improvement of immunogenicity response in rabbits using DNA prime / protein boost inoculation with HBV / HDV constructs New Zealand white rabbits were immunized with (1) a DNA-only composition containing Δ-4, (2) a protein-only composition containing Δ-4, or (3) a DNA prime / protein boost composition containing Δ-4 DNA and Δ-4 protein. Each composition was administered a total of four times at weeks 0, 4, 8, and 12. Administration was either 900 μg DNA im / EP or 300 μg protein + Alum. For DNA-protein composition (3), the first dose was 900 μg DNA im / EP at week 0, followed by 300 μg protein + Alum at week 4, week 8, and week 12. Serum anti-PreS1 IgG antibody titers were measured at weeks 0, 2, 10, and 14 (i.e., 2 weeks after each administration) (Figure 9). The DNA prime / protein boost composition (3) yielded generally higher antibody titers compared to the DNA-only composition (1) and the protein-only composition (2). Furthermore, antibody production was induced more rapidly than with the protein-only composition, and strong antibody production was induced by the second week.

[0149] Example 7: Adoptive transfer of serum or purified IgG from immunized animals protects humanized mice from HBV and HDV challenge. The ability of D4-induced antibodies to neutralize HBV infection in vivo is described above as human liver chimeric uPA + / + -This was investigated using the SCID mouse model. Total IgG was purified from D4 immunized rabbits and non-immunized rabbits, and uPA was created by replacing hepatocytes with human hepatocytes. + / + -SCID mice were injected, and an HBV challenge was performed 3 days later. In all challenged mice, the development of viremia was prevented by PreS1 IgG antibodies induced on D4, and the peak of viremia was significantly delayed (Figure 10A). Of the three challenged mice, infection was prevented in one (weeks 1-3), and the other two had HBV serum levels of 10 by the 1-month screening. 4 The levels were kept below IU / ml and remained lower than the control levels until the follow-up 8 weeks later. In control mice administered with IgG from unsensitized rabbits, serum HBV DNA concentrations were 10% in all cases. 8The levels reached over IU / ml. No significant differences were observed between groups regarding serum levels of alanine transferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin (Figure 10B). Therefore, it was found that passive immunization with a single dose of D4-specific PreS1 IgG antibody can prevent or significantly delay in vivo HBV infection in mice in which hepatocytes have been replaced with human hepatocytes (Table 3). The presence of high levels of subviral particles SHBsAg in the inoculation indicates that the antibody is indeed resistant to SHBsAg inhibition, which is an important finding. PreS1 antibodies present at the time of inoculation or during the first few weeks can inhibit infection itself or the initial infection cycle, and suppress the number of infected hepatocytes. As a result, viral spread can be suppressed, and the peak of viremia can be delayed. [Table 3]

[0150] Example 8: Challenge using HBV / HDV peptide constructs with various adjuvants The mixture of D-7 and D-8 peptides was evaluated using various adjuvants. C57BL / 6J mice were administered 20 μg of the mixture of D-7 and D-8 peptides (10 μg each of D-7 and D-8) twice, at week 0 and week 3 (Figure 11A). Peripheral blood was collected at week 2 (between the two administrations), and endpoint antibody titers were measured by ELISA as a measure of reactivity to HBV and HDV (Figures 11A and 11B). Splenocytes were collected at week 5, and reactivity to HBV and HDV was examined using ELISpot (Figures 11C and 11D). The peptide composition was administered subcutaneously with various adjuvants including QS-21, MF59, and Aram. Unsensitized mice and mice administered intramuscularly with D-4 DNA plasmid by electroporation were used as controls. IFNγ ELISpot was performed using the HDAg peptide pool, PreS1A peptide, and PreS1B peptide, with OVA peptide and concanavalin A as controls, in the same manner as described above (Figures 11C and 11D). Compositions administered with QS-21 adjuvant showed higher HDAg reactivity compared to those administered with other adjuvants. The study was conducted with 5 mice in each group.

[0151] Example 9: Comparison of exemplary HBV / HDV DNA constructs and / or peptide constructs A comparative immunogenicity study was conducted for 1) a mixture consisting only of D-7 peptide and D-8 peptide, 2) D-7 / D-8 fusion peptide alone, and 3) D-4 DNA prime / D-7 peptide and D-8 peptide mixture boost. D-4 DNA alone and unsensitized conditions were used as controls. 20 μg of D-7 / D-8 fusion protein or 10 μg each of D-7 peptide and D-8 peptide were mixed with QS-21 adjuvant and administered subcutaneously to the base of the tail of mice in 100 μL volumes. Two doses were administered at week 0 and week 4. For the D-4 DNA control, 50 μg was dissolved in 50 μL of PBS and administered intramuscularly by electroporation. At week 6 (after the second dose), T cell responses to PreS1 and HDV antigen genotypes 1 and 2 were measured using IFNγ ELISpot (Figure 12A). Furthermore, antibody titers against PreS1A consensus peptide (Figures 12B and 12C) and PreS1B consensus peptide (Figure 12D) were examined at week 2 (after the first dose) and week 6 (after the second dose). It was confirmed that the reactivity of HBV and HDV was highest under DNA prime / peptide boost conditions.

[0152] Example 10: Human clinical trial to verify the efficacy of an immunotherapy consisting of DNA or protein priming / DNA or protein boosting against HBV and / or HDV. The following examples illustrate embodiments relating to the use of immunogenic compositions or immunogenic combination products, which may contain nucleic acid components and polypeptide components, for use in treating or preventing viral infections caused by viruses such as HBV and HDV.

[0153] The DNA prime / protein boost composition described in Example 5 is administered to human patients by enteral, oral, nasal, parenteral, subcutaneous, intramuscular, intradermal, or intravenous administration. The human patients may be those currently infected with HBV and / or HDV, those who have been infected with HBV and / or HDV in the past, those at risk of becoming infected with HBV and / or HDV, or those who are not infected with HBV and / or HDV.

[0154] The DNA prime dose is administered initially as 1 ng, 10 ng, 100 ng, 1000 ng, 1 μg, 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1000 μg, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, or any two of these amounts as upper and lower limits, or the dose that yields the optimal effect in humans. After the initial DNA Prime dose is administered, one, two, three, four, or five additional DNA Prime doses may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 24 days, 36 days, 48 ​​days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or within a range of any two of these times, for example, within 1 to 48 days or 1 to 48 weeks. After administering the DNA prime dose, the protein boost dose is initially administered in the following amounts: 1 ng, 10 ng, 100 ng, 1000 ng, 1 μg, 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1000 μg, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, or any two of these amounts as upper and lower limits, or the amount that yields the optimal effect in humans. The initial protein boost dose is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 24 days, 36 days, 48 ​​days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or any two of these times as upper and lower limits.After the initial protein boost dose is administered, one, two, three, four, or five additional protein boost doses may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 24 days, 36 days, 48 ​​days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or any two of these times as upper and lower limits.

[0155] In patients, monitor whether a response to HBV and / or HDV is obtained, for example, whether serum production of anti-HBV antibodies, anti-HDV antibodies, anti-PreS1 antibodies, and anti-HDAg antibodies, rapid activation of T cells and other immune cells upon exposure to HBV antigens and / or HDV antigens, and whether protection against future HBV and / or HDV infections is induced.

[0156] In patients currently infected with HBV and / or HDV, those with a history of HBV and / or HDV infection, and those at risk of HBV and / or HDV infection, DNA prime / protein booster compositions may be administered in conjunction with antiviral therapy. Antiviral therapies shown to be effective against HBV or HDV include, but are not limited to, entecavir, tenofovir, lamivudine, adefovir, terbivudine, emtricitabine, interferon α, pegylated interferon α, or interferon α-2b, or any combination thereof. Patients should be monitored for side effects such as dizziness, nausea, diarrhea, depression, insomnia, headache, itching, rash, fever, and other side effects known to occur with the antiviral agent used.

[0157] In at least some of the embodiments described above, if technically feasible, one or more components used in one embodiment may be substituted for components of another embodiment. Those skilled in the art will understand that various omissions, additions, and modifications other than those described herein may be made to the methods and structures described above, as long as they do not deviate from the scope of the subject matter described in the claims. Any such modifications and changes fall within the scope of the subject matter as defined by the appended claims.

[0158] With regard to substantially all use of plural and / or singular terms herein, a person skilled in the art can interpret plural as singular and / or singular as plural, depending on the context and / or use. For clarity, such various singular and plural substitutions may be specified herein.

[0159] As those skilled in the art will readily understand, the terminology used herein, particularly in the attached claims (e.g., the text of the attached claims), is generally intended to be "open" throughout (for example, the term "includes" is to be interpreted as "includes, but not limited to," the term "have" is to be interpreted as "have at least," and the term "includes" is to be interpreted as "includes, but not limited to," etc.). Furthermore, those skilled in the art will understand that if a particular number is intended in the subject matter of a claim, such an intention is explicitly stated in the claim, and if it is not stated, such an intention does not exist. To illustrate with a specific example, in the attached claims, for instance, prefixes such as "at least one" or "one or more" are sometimes used in the subject matter of the claim. However, even when such a preamble is used, a particular claim containing a statement using the indefinite article "a or an" is not limited to embodiments containing only that statement, even if a single claim contains a preamble such as "one or more" or "at least one" and the indefinite article "a or an" (for example, "a and / or an" should be interpreted as meaning "at least one" or "one or more"). The same applies to the statements in claims using the definite article. Furthermore, even when a specific number is explicitly stated in the statement of a claim, it will be understood by those skilled in the art that the stated number means the minimum number (for example, the statement "two" without modifiers means "at least two" or "two or more").Furthermore, when idiomatic expressions such as "at least one of A, B, and C" are used, they are usually intended to be interpreted in a way that a person skilled in the art would understand (for example, "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C). Also, when idiomatic expressions such as "at least one of A, B, or C" are used, they are usually intended to be interpreted in a way that a person skilled in the art would understand (for example, "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C). Furthermore, it will be understood by those skilled in the art that disjunctive words and / or disjunctive phrases used to present two or more options, whether in the specification, claims, or drawings, are intended to imply the possibility of including one of the terms listed as options, either of those terms, or both of those terms. For example, the expression "A or B" is understood to include the possibilities of "A" or "B," or "A and B."

[0160] Furthermore, if any feature or aspect of this disclosure is described in Markush form, it will be understood by those skilled in the art that this disclosure is also described in any individual element or subgroup of elements in Markush form.

[0161] A person skilled in the art will understand that all ranges described herein for any purpose, such as providing detailed explanations, encompass all conceivable subranges and combinations thereof. It will be readily apparent that each of these ranges is sufficiently described and capable of being divided into at least two, three, four, five, ten, or the like. For example, each of the ranges described herein can be easily divided into three, such as upper, middle, and lower, but is not limited to these divisions. Furthermore, a person skilled in the art will understand that the terms "less than or equal to," "at least," "greater than," and "less than" include the stated numerical values ​​and, as mentioned above, also represent ranges that can be divided into subranges. Moreover, a person skilled in the art will understand that each range described herein includes individual elements. Therefore, for example, a group having 1 to 3 elements represents a group having 1 element, a group having 2 elements, or a group having 3 elements. Similarly, a group having 1 to 5 elements represents a group having 1 element, a group having 2 elements, a group having 3 elements, a group having 4 elements, or a group having 5 elements, and so on.

[0162] While various aspects and embodiments have been disclosed in this specification, those skilled in the art will readily understand that other aspects and embodiments are also possible. The various aspects and embodiments disclosed in this specification are for illustrative purposes only and do not limit the invention in any way. The scope and gist of the invention are defined by the following claims.

[0163] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and academic literature, are incorporated herein by reference in their entirety and constitute part of this Specified. If any cited document, patent, or patent application conflicts with any disclosure herein, the provisions of this Specified shall prevail and / or take precedence over such conflict.

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Claims

1. (a) a nucleic acid comprising at least one nucleic acid sequence encoding hepatitis D antigen (HDAg) and at least one nucleic acid sequence encoding PreS1; and (b) Polypeptide comprising at least one HDAg polypeptide sequence and at least one PreS1 polypeptide sequence An immunogenic combination product including, An immunogenic combination product administered to a subject as an initial prime dose containing (a) nucleic acid, followed by one or more boost doses containing (b) polypeptide.

2. The combination product according to claim 1, wherein at least one nucleic acid sequence encoding HDAg includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or any combination thereof.

3. The combination product according to claim 1 or 2, wherein at least one nucleic acid sequence encoding PreS1 includes the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10, or both.

4. The combination product according to any one of claims 1 to 3, wherein in the nucleic acid, each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and in each group, the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence.

5. The combination product according to claim 4, further comprising at least one nucleic acid sequence encoding a self-catalytic peptide cleavage site, wherein the HDAg nucleic acid sequence and the PreS1 nucleic acid sequence are separated by at least one nucleic acid sequence encoding the self-catalytic peptide cleavage site.

6. The combination product according to claim 5, wherein at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site includes a nucleic acid sequence selected from the group consisting of the nucleic acid sequence of 2A (P2A) derived from porcine rhinitis virus type 1, the nucleic acid sequence of 2A (F2A) derived from foot-and-mouth disease virus, the nucleic acid sequence of 2A (E2A) derived from equine rhinitis A virus (ERAV), and the nucleic acid sequence of 2A (T2A) derived from Thosea asigna virus, and the encoded autocatalytic peptide cleavage site may include a GSG (glycine-serine-glycine) motif at its N-terminus.

7. The combination product according to claim 5 or 6, wherein at least one nucleic acid sequence encoding the autocatalytic peptide cleavage site includes the sequence shown in Sequence ID No.

13.

8. The combination product according to any one of claims 1 to 7, wherein the nucleic acid is codon-optimized for human expression.

9. The combination product according to any one of claims 1 to 8, wherein the nucleic acid comprises a sequence having at least 95% sequence identity with any one of the sequences indicated by sequence numbers 15 to 24, 35, or 36.

10. The combination product according to claim 9, wherein the nucleic acid includes a sequence having at least 95% sequence identity with any one of the sequences indicated by SEQ ID NO: 18, SEQ ID NO: 35, or SEQ ID NO:

36.

11. The combination product according to any one of claims 1 to 10, wherein the at least one HDAg polypeptide sequence includes one of the sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or any combination thereof.

12. The combination product according to any one of claims 1 to 11, wherein the at least one PreS1 polypeptide sequence includes the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, or both.

13. The combination product according to any one of claims 1 to 12, wherein the at least one PreS1 polypeptide sequence is located downstream of the at least one HDAg polypeptide sequence.

14. The combination product according to any one of claims 1 to 13, wherein the polypeptide comprises a sequence having at least 95% sequence identity with any one of the sequences indicated by sequence numbers 25 to 34 or 37.

15. The combination product according to any one of claims 1 to 14, wherein the polypeptide comprises a sequence having at least 95% sequence identity with any one of the sequences indicated by sequence numbers 29, 31, 32, or 37.

16. The combination product according to any one of claims 1 to 15, wherein the polypeptide is recombinantly expressed.

17. The combination product according to claim 16, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

18. The combination product according to any one of claims 1 to 17, further comprising an adjuvant.

19. The combination product according to any one of claims 1 to 18, wherein the nucleic acid includes DNA.

20. The combination product according to any one of claims 1 to 19, wherein the nucleic acid is provided in the form of a recombinant vector.

21. A combination product according to any one of claims 1 to 20, for use in the treatment of hepatitis B or hepatitis D, or in the reduction or prevention of hepatitis B virus infection or hepatitis D virus infection.

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