RU2812764C1 - Compositions and methods of treating and preventing hepatitis b and d - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a combination of products containing (a) a nucleic acid with 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, preferably components (a) and (b) of the said product combination being provided as separate compositions within the said product combination.

EFFECT: invention is effective for inducing an immune response to hepatitis B virus and hepatitis D virus in a subject.

49 cl, 12 dwg, 3 tbl, 10 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/966970, filed January 28, 2020, the contents of which are hereby expressly incorporated by reference herein in their entirety.

LINK TO LIST OF SEQUENCES

[0002] This application is filed together with a Sequence Listing in electronic format. The sequence listing is provided as a file called SVF005SeqListing.TXT, which was created and last modified on January 26, 2021, and is 141377 bytes in size. The information provided in the electronic Sequence Listing is hereby incorporated by reference into this application in its entirety.

TECHNICAL FIELD

[0003] Aspects of the present invention generally provide immunogenic compositions or combinations of engineered hepatitis B (HBV) and hepatitis D (HDV) nucleic acid products, genes, peptides or proteins that can be used to induce an immune response against HBV infection and/or HDV. This immune response includes, consists essentially of, or consists of activated immune cells that produce neutralizing antibodies, and activated immune cells, such as T cells and B cells, against HBV and/or HDV. The present invention also generally provides methods for using said immunogenic compositions or combinations of products in subjects to induce immune responses against HBV and/or HDV by administering said compositions or combinations using an approach involving priming with a homologous or heterologous nucleic acid and/or polypeptide and stimulation (boost) with nucleic acid and/or polypeptide.

BACKGROUND OF THE ART

[0004] Hepatitis is a disease that causes swelling and inflammation of the liver. This disorder is usually caused by viruses, of which five types are currently known (hepatitis A, B, C, D and E viruses). Hepatitis B virus infection can be either acute or chronic, with severe chronic infections causing chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatitis B virus has a partially double-stranded circular DNA genome that enters the host nucleus and is transcribed by the host RNA polymerase to produce four molecules of viral mRNA. They are used to translate viral proteins such as capsid proteins and surface antigens, and to produce more DNA genomes using reverse transcriptase. Hepatitis D is a virusoid that requires coinfection or superinfection with hepatitis B for replication. Hepatitis D circular single-stranded RNA is amplified using host RNA polymerases but also contains a separate hepatitis D antigen (HDAg) gene. During coinfection or superinfection with hepatitis B and D viruses, intact hepatitis D viruses are packaged into an envelope containing hepatitis B surface antigens surrounding an RNA genome coated with the HDAg protein. Incorporation of hepatitis B surface antigens is necessary for hepatitis D virus infectivity, since hepatitis D virus does not encode its own receptor binding proteins. Coinfection or superinfection with hepatitis D virus causes more severe complications, with an increased risk of liver failure, cirrhosis, and liver cancer. There is a need for effective immunogenic compositions and vaccines to provide prophylactic immunity against both hepatitis B and D virus infections.

BRIEF DESCRIPTION OF THE INVENTION

[0005] The present invention generally provides the use of recombinant nucleic acids, DNA, RNA, proteins, polypeptides or peptides containing HBV and/or HDV antigens to induce immune responses, antibody production, immune protection or immunity against HBV or HDV infections. In some embodiments, recombinant nucleic acids, DNA, RNA, proteins, polypeptides, or peptides containing HBV and/or HDV antigens are used in an approach comprising a DNA priming/protein stimulation composition. In some embodiments, this DNA priming/protein stimulation composition approach results in greater immune response, antibody production, immune protection, or immunity against HBV or HDV infections compared to DNA-only, protein-only, or organism-based immunogenic compositions.

[0006] Chronic infections with hepatitis B and D viruses (HBV/HDV) can cause cancer. Current nucleoside analogue (NA) therapy for HBV is lifelong and reduces, but does not eliminate, the risk of cancer. A hallmark of chronic hepatitis B is an impaired response of HBV-specific T cells. Some embodiments provide immunotherapy driven by unprimed healthy HDV antigen-specific (HDAg) T cells to bypass the need for HBV-specific T cells to prime PreS1-specific T cells and produce anti-PreS1 antibodies that block HBV entry. In some embodiments, the induction of anti-PreS1 antibodies and HBV- and HDV-specific T cells by combinations of PreS1 and/or HDAg sequences is assessed in vitro and in vivo. In some embodiments, HBV neutralization by mouse and rabbit PreS1-specific antibodies was assessed in cell culture, and HBV neutralization by rabbit anti-PreS1 antibodies was examined in mice repopulated with human hepatocytes. In some embodiments, adoptive transfer of anti-PreS1 antibodies prevented or modulated HBV infection following subsequent challenge in humanized mice.

[0007] In some embodiments, the nucleic acid or polypeptide compositions comprise HBV, HDV, PreS1, or HDAg sequences, genes, or polypeptides. In some embodiments, PreS1 is PreS1 A or PreS1 B. In some embodiments, the HDAg is a genotype 1 strain A HDAg (1A), a genotype 1 strain B HDAg (1B), a genotype 2 strain A HDAg (2A), or a genotype 2 HDAg strain B (2B). In some embodiments, the compositions also contain an autocatalytic peptide cleavage site. In some embodiments, the peptide autocatalytic cleavage site is a P2A peptide autocatalytic cleavage site. In some embodiments, the PreS1 and HDAg components are grouped together in compositions. In some embodiments, PreS1 is located downstream or immediately downstream of the HDAg sequence. In some embodiments, the PreS1 and HDAg moieties are separated by an autocatalytic peptide cleavage site. In some embodiments, the PreS1 and HDAg moieties are separated by an autocatalytic cleavage site of the P2A peptide.

[0008] In some embodiments, the nucleic acid compositions are 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 compositions are circular or linear. In some embodiments, the nucleic acid compositions are produced in a biological system, including, but not limited to, mammalian cells, human cells, bacterial cells, E. coli, yeast, S. cerevisiae, or other suitable biological systems. In some embodiments, the HBV and/or HDV nucleic acids or genes are contained in a cassette that contains elements necessary for the transcription and translation of the nucleic acids or genes in a biological system.

[0009] In some embodiments, the polypeptide compositions are properly folded or denatured. In some embodiments, the polypeptide compositions are produced in a biological system, including, but not limited to, recombinant expression systems in mammals, bacteria, yeast, insects, or a cell-free system. In some embodiments, the polypeptide compositions are produced 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, bacterial cells, Escherichia coli cells yeast, Saccharomyces cerevisiae, Pichia pastoris, insect cells, Spodoptera frugiperda Sf9 or S. frugiperda Sf21, or in a cell-free system. In some embodiments, the polypeptide compositions are purified using techniques known in the art, including, but not limited to, extraction, freezing/thawing, homogenization, permeabilization, centrifugation, density gradient centrifugation, ultracentrifugation, precipitation, polyacrylamide gel electrophoresis in denaturing conditions (PAGE/SDS), native EP in 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 affinity chromatography, nickel column chromatography , epitope tag purification or lyophilization.

[0010] In some embodiments, the nucleic acid or polypeptide compositions are administered to an animal, including, but not limited to, humans, mice, rats, rabbits, cats, dogs, horses, cows, pigs, sheep, monkeys, primates, or chickens. In some embodiments, the nucleic acid or polypeptide compositions are administered at intervals of 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or any time within the range specified by any two of the above time points between each dose. In some embodiments, the nucleic acid compositions are administered before the polypeptide compositions are administered. In some embodiments, the polypeptide compositions are administered before the nucleic acid compositions are administered.

[0011] In some embodiments, the nucleic acid or polypeptide composition is administered in an amount of 1, 10, 100, 1000 ng, or 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg, or 1, 10, 100 or 1000 mg or any amount within the range specified by any two of the above amounts. In some embodiments, the nucleic acid or polypeptide compositions are administered with excipients. In some embodiments, the nucleic acid or polypeptide compositions are administered with adjuvants. In some embodiments, the nucleic acid compositions are administered by in vivo electroporation.

[0012] In some embodiments, the immunogenicity of nucleic acid or polypeptide compositions is assessed by measuring interferon gamma (IFNγ)-producing immune cells using techniques known in the art, including enzyme-linked immunospot spot (ELISpot) testing, HBV-specific IgG antibody titer measurement, HDV, HBV proteins, HBV nucleic acids, HDV proteins, HDV nucleic acids, PreS1 or HDAg, or measurement of neutralizing activity of sera or purified antibodies from immunized animals in an in vitro or in vivo assay.

[0013] In some embodiments, administration of nucleic acid or polypeptide compositions provides temporary, long-term, or permanent protection against HBV or HDV infection. In some embodiments, temporary, long-term, or persistent protection against HBV or HDV infection is superior to that of other immunogenic compositions. In some embodiments, the administration of nucleic acid or polypeptide compositions is carried out in combination with antiviral therapy. In some embodiments, administration of nucleic acid or polypeptide compositions to provide temporary, long-term, or permanent protection against HBV or HDV infection is effective in humans. In some embodiments, nucleic acid or polypeptide compositions are used as vaccines against HBV or HDV.

[0014] Preferred aspects of the present invention relate to the following numbered alternatives:

[0015] 1. An immunogenic composition or combination of products containing:

(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.

[0016] 2. The immunogenic composition or combination of products according to Alternative 1, characterized in that at least one nucleic acid sequence encoding an HDAg contains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or any combination thereof.

[0017] 3. An immunogenic composition or combination of products according to Alternative 1 or 2, characterized in that at least one nucleic acid sequence encoding PreS1 contains SEQ ID NO: 9 or SEQ ID NO: 10, or both.

[0018] 4. An immunogenic composition or combination of products according to any of alternatives 1-3, characterized in that the nucleic acid is designed such that each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and in that the PreS1 nucleic acid sequence is located directly downstream of the HDAg nucleic acid sequence.

[0019] 5. The immunogenic composition or combination of products according to Alternative 4, further comprising at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site, wherein the grouped HDAg and PreS1 nucleic acid sequences are separated by at least one nucleic acid sequence encoding an autocatalytic cleavage site peptide cleavage.

[0020] 6. The immunogenic composition or combination of products according to Alternative 5, characterized in that at least one nucleic acid sequence encoding the autocatalytic cleavage site of the peptide contains a nucleic acid sequence selected from the group consisting of porcine teschovirus-1 nucleic acids 2A (P2A), foot and mouth disease virus 2A (F2A), equine rhinitis virus A (ERAV) 2A (E2A) and Thosea asigna virus 2A (T2A), and the fact that each encoded peptide autocatalytic cleavage site may optionally contain a GSG (glycine-serine) motif -glycine) at its N-terminus.

[0021] 7. The immunogenic composition or combination of products according to alternative 5 or 6, characterized in that at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site contains SEQ ID NO: 13.

[0022] 8. An immunogenic composition or combination of products according to any of alternatives 1-7, characterized in that the nucleic acid is codon-optimized for expression in humans.

[0023] 9. An immunogenic composition or combination of products according to any of alternatives 1-8, characterized in that the nucleic acid contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to the sequence SEQ ID NO: 15-24 or 35-36.

[0024] 10. An immunogenic composition or combination of products according to any of alternatives 1-9, characterized in that the nucleic acid contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to the sequence SEQ ID NO: 18 or SEQ ID NO: 35-36.

[0025] 11. An immunogenic composition or combination of products according to any of alternatives 1-10, characterized in that at least one HDAg polypeptide contains SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO : 8, or any combination thereof.

[0026] 12. An immunogenic composition or combination of products according to any of alternatives 1-11, wherein at least one PreS1 polypeptide sequence comprises SEQ ID NO: 11 or SEQ ID NO: 12, or both.

[0027] 13. An immunogenic composition or combination of products according to any of alternatives 1-12, characterized in that at least one PreS1 polypeptide sequence is located downstream of at least one HDAg polypeptide sequence.

[0028] 14. An immunogenic composition or combination of products according to any of alternatives 1-13, characterized in that the polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to the sequence of SEQ ID NO: 25-34 or 37.

[0029] 15. An immunogenic composition or combination of products according to any of alternatives 1-14, characterized in that the polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to the sequence of SEQ ID NO: 29, 31, 32 or 37.

[0030] 16. An immunogenic composition or combination of products according to any of alternatives 1-15, characterized in that the polypeptide is expressed in a recombinant manner.

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

[0032] 18. An immunogenic composition or combination of products according to any of alternatives 1-17, further containing an adjuvant.

[0033] 19. The immunogenic composition or combination of products of Alternative 18, wherein the adjuvant is alum, QS-21 or MF59, or any combination thereof.

[0034] 20. An immunogenic composition or combination of products according to any of alternatives 1-19, wherein the nucleic acid includes DNA.

[0035] 21. An immunogenic composition or combination of products according to any of alternatives 1-20, characterized in that the nucleic acid is provided in a recombinant vector.

[0036] 22. A method of inducing an immune response in a subject using an immunogenic composition or combination of products presented in any of Alternatives 1-21, comprising:

administering to said subject at least one priming dose containing the nucleic acid; And

administering to said subject at least one stimulant dose comprising the polypeptide.

[0037] 23. The method according to alternative 22, characterized in that at least one stimulating dose further contains an adjuvant.

[0038] 24. The method of Alternative 23, wherein the adjuvant is alum, QS-21 or MF59, or any combination thereof.

[0039] 25. The method according to any of alternatives 22-24, characterized in that at least one stimulating dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 24, 36 or 48 days or weeks after administration of at least one priming dose or within a time range specified by any two of the above time points, for example, within 1-48 days or 1-48 weeks.

[0040] 26. The method according to any of alternatives 22-25, characterized in that the administration is enteral, oral, intranasal, parenteral, subcutaneous, intramuscular, intradermal or intravenous, or any combination of these routes.

[0041] 27. The method according to any of alternatives 22-26, characterized in that the administration is carried out in combination with antiviral therapy.

100421 28. The method according to alternative 27, wherein the antiviral therapy includes the administration of entecavir, tenofovir, lamivudine, adefovir, telbivudine, emtricitabine, interferon-a, pegylated interferon-a or interferon alpha-2b, or any combination thereof.

10043] 29. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D, containing:

[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) a polypeptide comprising at least one HDAg polypeptide sequence and at least one PreS1 polypeptide sequence.

[0046] 30. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to alternative 29, characterized in that at least one nucleic acid sequence encoding an 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 combination of products for use in the treatment of hepatitis B or hepatitis D according to alternative 29 or 30, characterized in that at least one nucleic acid sequence encoding PreS1 contains SEQ ID NO: 9 or SEQ ID NO : 10, or both.

[0048] 32. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-31, wherein the nucleic acid is designed such that each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and the fact that the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence.

10049] 33. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to Alternative 32, further comprising at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site, wherein the grouped HDAg and PreS1 nucleic acid sequences are separated by at least one nucleic acid sequence encoding the site of autocatalytic cleavage of the peptide.

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

[0051] 35. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to alternative 33 or 34, characterized in that at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site contains SEQ IDNO: 13.

[0052] 36. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-35, characterized in that the nucleic acid is codon-optimized for expression in humans.

[0053] 37. An immunogenic composition or combination of products for use in treating or inhibiting hepatitis B or hepatitis D according to any of alternatives 29-36, characterized in that the nucleic acid contains at least 80%, 85%, 90% sequence , 95%, 99% or 100% homologous to SEQ ID NO: 15-24 or 35-36.

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

[0055] 39. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-38, characterized in that at least one HDAg polypeptide contains 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 combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-39, characterized in that at least one PreS1 polypeptide sequence contains SEQ ID NO: 11 or SEQ ID NO: 12 , or both.

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

[0058] 42. An immunogenic composition or combination of products for use in treating or inhibiting hepatitis B or hepatitis D according to any of alternatives 29-41, characterized in that the polypeptide contains the sequence of at least 80%, 85%, 90%, 95%, 99% or 100% homologous to SEQ ID NO: 25-34 or 37.

[0059] 43. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-42, characterized in that the polypeptide contains at least 80%, 85%, 90%, 95% of the sequence , 99% or 100% homologous to SEQ ID NO: 29, 31, 32 or 37.

[0060] 44. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-43, characterized in that the polypeptide is expressed in a recombinant manner.

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

[0062] 46. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-45, further containing an adjuvant.

[0063] 47. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to Alternative 46, wherein the adjuvant is alum, QS-21 or MF59, or any combination thereof.

[0064] 48. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-47, wherein the nucleic acid includes DNA.

[0065] 49. An immunogenic composition or combination of products for use in the treatment of hepatitis B or hepatitis D according to any of alternatives 29-48, characterized in that the nucleic acid is provided in a recombinant vector.

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In addition to the features described above, additional features and variations will be apparent from the following description of the drawings and exemplary embodiments. It should be understood that these drawings depict typical embodiments and are not intended to limit the scope of the invention.

[0067] FIGURES 1A-B depict nucleic acid or polypeptide constructs containing HBV and/or HDV antigens used in this application. Ten designs 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 the ten polypeptide constructs were correctly expressed (FIGURE 1B). GFP was used as a control for Western blotting.

[0068] FIGURE 2 depicts constructs used for the DNA priming/protein stimulation composition approach. The DNA composition contains a Δ-4 nucleic acid sequence, and the protein composition contains a polypeptide sequence of either Δ-7 or Δ-8, or a fusion of Δ-7 and Δ-8.

[0069] FIGURES 3A-3E depict quantitative analysis of interferon gamma (IFNγ) spots generated per 10 ⁶ cells in the ELISpot assay, which correspond to the activation of T lymphocytes, a purified population of leukocytes from sera derived from mice immunized with HBV/HDV DNA compositions, in response to contact with various HBV or HDV antigens. Antigens include purified polypeptides including PreS1 A (SEQ ID NO: 11), PreS1 A (SEQ ID NO: 12), HDAg genotype 1 A (SEQ ID NO: 5, "HDAg gtr 1A-pool 1" and "HDAg gtr 1A -pool 2"), HDAg genotype 1 B (SEQ ID NO: 6, "HDAg gtr 1B-pool 3" and "HDAg gtr 1B-pool 4"), HDAg genotype 2 A (SEQ ID NO: 7, "HDAg gtr 2C-pool 5" and "HDAg gtr 2C-pool 6") and HDAg genotype 2B (SEQ ID NO: 8, "HDAg gtr 20-pool 7" and "HDAg gtr 20-pool 8"). Mice were sacrificed 6 weeks after the first immunization and pooled splenocytes from each group were stimulated for 48 hours with pools of HDV 18 peptides corresponding to genotype 1 (pools 1-4) and genotype 2 (pools 5-8). Pools 1 and 2 of genotype 1 belong to sequence/isolate A, while pools 3 and 4 correspond to sequence/isolate B. Similarly, pools 5 and 6 of genotype 2 belong to sequence/isolate C and pools 7 and 8 of genotype 2 belong to sequence/ isolate D. Each pool contained 20 or 221 (for pools 1 and 5) 15-mer peptides with an overlap of 10 AA. Concanavalin A (“ConA”) was used as a positive control, and two ovalbumin peptides (“OVA Th” and “OVA CTL”) and growth medium (“medium”) were used as negative controls. For each peptide-stimulated group, runs were run in triplicate, and the bars indicate the average number of IFNγ spot-forming cells (SFC) per 10 ⁶ cells with standard error. The cutoff was set at 100 SFC/10 ⁶ splenocytes. Antigen concentrations are presented.

[0070] FIGURES 4A-4C depict quantitative analysis of anti-PreS1 IgG antibody titer in sera derived from mice immunized with HBV/HDV DNA compositions. The Δ-1-Δ-10 constructs were tested for the production of IgG antibodies against the consensus sequences PreS1 A and PreS1 B in mice (5 mice per group). FIGURES 4A-4B show reactivity against amino acids 2-48 of PreS1. FIGURE 4C examines cross-reactivity against HBV (sub-)genotypes A1, A2, B, B2, C, D1, E1 hF.

[0071] FIGURES 5A-5C depict quantification of interferon gamma (IFNγ) spot formation per 10 ⁶ cells in an ELISpot assay of a purified population of leukocytes from sera derived from C57BL/6 mice or HLA-A2 transgenic HHD mice immunized with the Δ DNA composition -4, or unprimed C57BL/6 mice, in response to exposure to various HBV or HDV antigens or peptides. Antigens include purified polypeptides including PreS1 A (SEQ ID NO: 11), PreS1 A (SEQ ID NO: 12), a pool containing HDAg genotypes 1 A and 1 B (SEQ ID NO: 5 and 6, "gtr 1-pool 1", "gtr 1-pool 2", "gtr 1-pool 3", "gtr 1-pool 4"), a pool containing HDAg genotypes 2 A and 2 B (SEQ ID NO: 7 and 8, "gtr 2 -pool B1", "gtr 2-pool B2", "gtr 2-pool B3", "gtr 2-pool B4"), pools of HDAg peptide fragments containing peptides KLEDDNPWL, KLEEENPWL and FPWDILFPA ("pep-3-pool" ), and individual HDAg peptides KLEDDNPWL, KLEEENPWL and FPWDILFPA. Concanavalin A (“ConA”) was used as a positive control, and two ovalbumin peptides (“OVA Th” and “OVA CTL”) and growth medium (“medium”) were used as negative controls. Antigen concentrations are presented.

[0072] FIGURES 6A-6C depict quantitative analysis of anti-PreS1 IgG titer in New Zealand White rabbits immunized with Δ-3 or Δ-4 DNA compositions. Sera were collected from rabbits and tested by ELISA against the consensus peptides PreS1A and PreS1B (FIGURE 6B). Antiserum from vaccinated rabbits was also tested for cross-reactivity with HBV (sub-)genotypes A1, A2, B, B2, C, D1, E1 and F (FIGURE 6C). The graph bars show the mean final anti-PreS1 titers for each group, defined as the final final dilution of serum yielding an OD at 405 nm three times higher than the OD of nonimmunized sera at the same dilution. Sera were serially titrated in sixfold dilutions starting at 1:60.

[0073] FIGURE 6D shows the percentage reactivity of antisera from D-4 vaccinated rabbits against PreS1 of various HBV (sub-)genotypes. Antisera from vaccinated D-4 rabbits at six weeks of age were tested for reactivity (OD at 405 nm) against HBV (sub-)genotypes D1, F, A1, C, A2, B, B2 and E1 using ELISA. Using individual 20-mer PreS1 peptides overlapping ten AAs corresponding to each HBV type from AAs 2-21, 12-31, 22-41 and 32-48, neutralizing epitopes were predominantly localized to the region of AAs 22-41 and 32-48 of genotype D1 , as evidenced by the highest percentage of reactivity, followed by (sub-)types C, E1 and A1 at the same AK site.

[0074] FIGURES 7A-7C depict quantitative analysis of interferon gamma (IFNy) spot formation in a 10 ⁶ cell ELISpot assay of a purified leukocyte population from sera obtained from C57BL/6 mice immunized with D-4 DNA, Δ-7 protein only or a priming/stimulating DNA Δ-4/protein Δ-8 composition. Antigens include purified polypeptides including PreS1 A (SEQ ID NO: 11), PreS1 A (SEQ ID NO: 12), a pool containing HDAg genotypes 1 A and 1 B (SEQ ID NO: 5 and 6, "gtr 1-pool 1", "gtr 1-pool 2", "gtr 1-pool 3", "gtr 1-pool 4"), and a pool containing HDAg genotypes 2 A and 2 B (SEQ ID NO: 7 and 8, "gtr 2-pool 5", "gtr 2-pool 6", "gtr 2-pool 7" _z "gtr 2-pool 8"). Concanavalin A (“ConA”) was used as a positive control, and two ovalbumin peptides (“OVA Th” and “OVA CTL”), DMSO, and growth medium (“medium”) were used as negative controls. Antigen concentrations are presented.

[0075] FIGURES 8A-8C depict quantitative analysis of anti-PreS1 IgG titer in C57BL/6 mice immunized with DNA alone, protein alone, or DNA priming/protein boosting compositions of HBV/HDV.

[0076] FIGURE 9 depicts quantitative analysis of anti-PreS1 IgG titer in rabbits immunized with DNA alone, protein alone, or HBV/HDV DNA priming/protein boosting compositions.

[00771 FIGURES 10A-10B depict protective effects against HBV infection at 1, 2, 3, 4, 6 and 8 weeks after the first inoculation, as determined by HBV titers at each time point. Each line shows one individual mouse (FIGURE 10A). Two negative control mice (gray lines) received IgG from nonimmunized rabbits and three mice (red lines) received D4 PreS1 IgG. One mouse in the PreS1-IgG group died at week 4, therefore only measurements at weeks 1, 2, and 3 are available for this mouse. No significant differences were observed between groups with respect to serum levels of alanine transferase, asparagine transferase, alkaline phosphatase, or bilirubin (FIGURE 10B).

[0078] FIGURES 11A-D depict the evaluation of mixtures of peptides D-7 and D-8 (10 μg each for administration to mice) with various adjuvants. The adjuvants QS-21, MF59 and alum were studied. D-4 DNA compositions that were administered intramuscularly by electroporation were used as controls. FIGURE 11A shows the dosing schedule and end titer examples with the adjuvants studied. FIGURE 11B shows the % reactivity of individual mice for each condition as assessed by ELISA. The x-axis (“1, 3, 10, 30, 0”) corresponds to the identification numbers of individual mice. FIGURE PS shows IFNy activation of splenocytes by HBV PreS1 consensus peptides and HDV antigens, as assessed by ELISpot. FIGURE 11D shows the final titers of the anti-PreS1 antibody against the PreS1A and PreS1B peptides.

[0079] FIGURES 12A-D depict a comparison of priming/stimulation with a mixture of peptides D-7 and D-8, only with a fusion peptide D-7+D-8, and DNA priming with D-4 and stimulation with a mixture of peptides D-7 and D- 8 with D-4 DNA primed/stimulated only and unprimed controls. FIGURE 12A shows IFNγ activation of splenocytes by HBV PreS1 consensus peptides and HDV antigens as assessed by ELISpot. FIGURE 12B shows anti-PreS1A antibody levels assessed 2 weeks after the first round of administration. FIGURE 12C shows anti-PreS1A antibody levels assessed 2 weeks after the second round of administration. FIGURE 12D shows anti-PreS1B antibody levels assessed 2 weeks after the second round of administration. The legends to FIGURES 12B-D correspond to the identification numbers of individual mice.

DETAILED DESCRIPTION OF THE INVENTION

[0080] Despite preventive vaccines and antiviral treatments, chronic hepatitis B virus (HBV) infection currently affects more than 250 million people worldwide. One million chronic carriers die each year due to liver-related complications caused by HBV, such as cirrhosis and eventually hepatocellular carcinoma (HCC). Hepatitis D virus (HDV), a satellite RNA virus for HBV that “steals” the surface antigen from HBV (HBsAg), coinfects 15–25 million HBV carriers worldwide and exacerbates disease progression. There is still no effective functional treatment for chronic HBV or HDV infection. Current standard treatment for HBV consists of nucleoside analogues (NAs), which inhibit the reverse transcriptase (RT) function of HBV polymerase. This prevents virus maturation by blocking the synthesis of partial dsDNA within the capsid. Thus, NAs only inhibit viral replication during therapy. This is because blocking RT does not affect either protein production and release (including HBsAg) or the synthesis of covalently closed circular DNA, the main cause of HBV persistence. Lifelong use of NA reduces but does not eliminate the risk of HCC. A regimen including at least one year of pegylated interferon (1G>1)-alpha is currently the recommended treatment for chronic HDV; however, lasting responses are rare. Combination treatment with pegylated IFN-alpha and NA has been shown to have limited efficacy against HDV and HBV.

[0081] HBV uses several strategies to evade the host immune response. The chronic presence of HBV proteins causes T cell dysfunction. HBV-infected cells produce increased amounts of subviral HBsAg particles, predominantly containing small HBsAg (SHBsAg), to block the population of neutralizing antibodies directed against SHBsAg. This guarantees the survival of viral particles whose surface contains more medium HBsAg proteins (MHBsAg; containing S and PreS2) and large HBsAg proteins (LHBsAg; containing S, PreS2 and PreS1). It is important to note that the PreS1 domain is responsible for binding to the Ia ⁺ -taurocholate cotransporting polypeptide (NTCP) receptor for HBV on hepatocytes. Thus, an obvious way to target infectious HBV particles and prevent infection of new hepatocytes would be to increase antibody levels against the PreS1 domain of the virus.

[0082] This application discloses that, to create an immunotherapy targeting both HBV and HDV infection, chimeric genes containing PreS1 and HDV large antigen were generated to induce the production of antibodies against PreS1 and T cells specific for HBV and HDV in various combinations (Figure 1A). The advantage of coupling PreS1 to HDAg is that the HDAg will act as a heterologous carrier of the T cell epitope in patients who are monoinfected with HBV. Thus, these HDAg-specific T cells support the long-term endogenous production of anti-PreS1 antibodies that block viral entry and bypass the requirement for HBV-specific T cells. In fact, >90% of HBV carriers are monoinfected with HBV, and in these patients heterologous HDAg will prime healthy unprimed T cells that support the priming of HBV-specific responses. In addition, it is likely that HDAg-specific T cells and anti-PreS1 antibodies prevent HDV superinfection in these patients. Genetic immunization has been used to induce both neutralizing antibodies and T cells, as this strategy has been shown to activate the immune response to HBV. Overall, this strategy to block viral entry complements the maturation inhibitors and capsid assembly inhibitors currently in development to achieve durable responses beyond therapy against HBD and/or HDV infection.

[0083] Embodiments provided herein relate to immunogenic compositions or combinations of engineered hepatitis B (HBV) and hepatitis D (HDV) nucleic acid products, genes, peptides or proteins that can be used to induce an immune response against HBV infection or HDV. The use of chimeric genes and chimeric proteins containing HBV and HDV nucleic acids, genes, peptides or proteins has been described, for example, in WO 2017/132332, which is hereby expressly incorporated by reference herein in its entirety.

[0084] The following detailed description makes reference to the accompanying drawings which form a part thereof. In the drawings, like symbols typically represent like components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings and claims are not intended to be limiting. Other embodiments may be used and other changes may be made without departing from the spirit and scope of the invention as provided herein. It will be immediately apparent that the aspects of the present invention described generally in this application and illustrated in the Figures can be arranged, substituted, combined, divided and developed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0085] Unless otherwise specified, all technical and scientific terms used in this application have the same meanings as commonly understood by one of ordinary skill in the art. All patents, patent applications, published patent applications and other publications referenced in this application are expressly incorporated herein by reference in their entirety unless otherwise noted. In the event that there are multiple definitions of a term in this application, the definitions given in this section shall prevail unless otherwise stated.

[0086] Singular forms are used herein to refer to one or more than one (eg, at least one) of the grammatical entities. By way of example, "element" means one element or more than one element.

[0087] The terms "about" or "about" in this application refer to an amount, level, value, number, frequency, percentage, measurement, size, magnitude, mass or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% relative to the specified amount, level, value, number, frequency, percentage, measurement, size, magnitude, mass or length.

[0088] Throughout this specification, unless the context otherwise requires, the terms “comprise,” “comprises,” and “comprising” are to be understood to imply the inclusion of a specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of stages or elements.

[0089] The phrase “consisting of” is intended to be inclusive and is limited to what follows the phrase “consisting of.” Thus, the wording "consisting of" indicates that the listed elements are necessary or required and that other elements cannot be present. By “consisting essentially of” is meant to include any of the elements listed after said language and to be limited to such other elements as do not interfere with or facilitate the activity or effect specified herein for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or required, but that other elements are optional and may or may not be present depending on whether they do or do not have a significant effect on the activity or effect of the listed elements.

[0090] The practice of the present invention will employ, unless specifically stated to the contrary, conventional molecular biology techniques and recombinant DNA technologies within the scope of the art, many of which are described below for purposes of illustration. Such techniques are fully explained in the literature. See, for example, Sambrook, el al., Molecular Cloning: A Laboratory Manual (Ze edition, 2000); DNA Cloning: A Practical Approach, volumes 1 and 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, R.I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5th ed. Hoboken NJ, John Wiley &Sons; B. Perbal, A Practical Guide to Molecular Cloning (Ze edition, 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3rd edition, 2005).

10091] The term “purity” of any given substance, compound or material in this application refers to the actual presence of the substance, compound or material compared to the expected presence. For example, the substance, compound or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% pure, including all decimals between those values. Purity may be affected by undesirable impurities, including, but not limited to, by-products, isomers, enantiomers, degradation products, solvent, carrier, media or impurities, or any combination thereof. Purity can be measured using technologies including, but not limited to, chromatography, liquid chromatography, gas chromatography, spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry or titration, or any combination thereof.

[0092] The terms “function” and “functional” as used herein refer to biological, enzymatic or therapeutic function.

[0093] The terms "effective amount" or "effective dose" as used herein refer to an amount sufficient to produce a desired result and will accordingly depend on the ingredient and the desired result. However, once the desired effect is known, determination of the effective amount is within the skill of the person skilled in the art.

[0094] Typically, the “error bars” shown in the figures represent the standard error of the mean.

[0095] The terms "formulation" and "composition", used interchangeably in this application, are equivalent terms referring to a mixture of chemically related substances for administration to a subject.

[0096] The term “isolated” as used herein refers to a material that is substantially or substantially free of the components that normally accompany it in its natural state. For example, an “isolated cell” as used herein includes a cell that has been purified from its environment or organisms in its naturally occurring state, a cell that has been removed from a subject or from a culture, e.g., it is not substantially associated with substances in vivo or in vitro.

[0097] The term “subject” as used herein has its usual meaning as understood in light of the present disclosure, and refers to an animal that is the subject of treatment, inhibition or enhancement, observation or experiment. The term "animal" has its usual meaning, as understood in light of the present description, and includes cold-blooded and warm-blooded vertebrates and/or invertebrates, such as fish, molluscs or reptiles and, in particular, mammals. The term "mammal" has its usual meaning as understood in light of the present description, and includes, but is not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates such as humans, monkeys, chimpanzees or great apes. In some embodiments, the subject is a human.

[0098] In some embodiments described herein, selection of the subject or patient in need is provided. In some embodiments, a patient is selected who is in need of treatment for a viral infection. In some embodiments, a patient who has previously received treatment for a viral infection is selected. In some embodiments, a patient is selected who has previously received treatment for a risk of viral infection. In some embodiments, a patient who has developed a relapse of the viral infection is selected. In some embodiments, a patient who has developed resistance to treatments for the viral infection is selected. In some embodiments, a patient is selected who may have any combination of the above selection criteria.

[0099] The terms “treat,” “cure,” “therapeutic,” or “therapy” as used herein have their usual meaning as understood in light of the present disclosure and do not necessarily mean complete cure or elimination of a disease or condition. The term “treat” or “treatment” (and as is well known in the art) also means an approach to obtaining useful or desirable results with respect to a subject's condition, including clinical results. Beneficial or desirable clinical outcomes may include, but are not limited to: alleviation or reduction in the severity of one or more symptoms or conditions, reduction in the extent of the disease, stabilization (ie, non-worsening) of the disease state, prevention of transmission or spread of the disease, delay or slowing progression of the disease, reduction in severity or temporary relief of the disease state, reduction in the recurrence of the disease and remission, either partially or completely and either detectable or undetectable. The terms “treat” and “treatment” as used herein also include prophylactic treatment. Methods of treatment include administering to a subject a therapeutically effective amount of an active agent. The administration step may consist of a single administration or may include a series of administrations. The compositions are administered to a subject in an amount and for a period of time sufficient to treat the patient. The length of the treatment period depends on various factors, such as the severity of the condition, the age and genetic profile of the patient, the concentration of the active agent, the potency of the compositions used for treatment, or a combination thereof. It will also be understood that the effective dosage of an agent used for treatment or prophylaxis may be increased or decreased during a particular treatment or prophylactic regimen. Changes in dosage may result from and become clear from standard diagnostic tests known in the art. In some cases, continuous administration may be required.

[0100] The term “inhibit” as used herein has its usual meaning as understood in light of the present disclosure, and may refer to reducing or preventing viral infection. The reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or an amount that is within the range specified by any two of the above values. As used herein, the term “delay” has its usual meaning, as understood in light of the present description, and refers to slowing down, postponing or postponing an event, such as a viral infection, to a later time than would otherwise be expected. The deferral may be a deferral of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range specified by any two of the above values. The terms inhibit and delay do not necessarily indicate 100% inhibition or delay. Partial inhibition or delay may occur.

[0101] The term "immunogenic composition" as used herein refers to a substance or mixture of substances, 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, designed to elicit an immune response when administered to a host. The immune response includes both innate and acquired immune responses, the latter of which creates long-lasting immunological memory through cells such as memory T cells and memory B cells. Antibodies generated during a primary immune response to an immunogenic composition may be raised by subsequent challenges with the same antigens, epitopes, nucleic acids, peptides, polypeptides, proteins, polysaccharides, lipids, haptens, toxoids, inactivated organisms or attenuated organisms, or a living organism or a pathogen exhibiting said antigens, epitopes, nucleic acids, peptides, polypeptides, proteins, polysaccharides, lipids, haptens or toxoids, or any combination thereof. Thus, the immunogenic composition can serve as a vaccine against a particular pathogen. Immunogenic compositions may also contain one or more adjuvants to stimulate an immune response and enhance the effectiveness of protective immunity.

[0102] The term “product combination” as used herein refers to a collection of two or more separate compounds, substances, materials or compositions that can be used together for a single function. In some embodiments, the combination of products comprises at least one nucleic acid composition and at least one polypeptide composition that are used together to elicit an immune response when administered to a host, not necessarily to a greater extent than would be elicited if only one type of composition was to be introduced.

[0103] The terms “nucleic acid” or “nucleic acid molecule” as used herein refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, polymerase chain reaction (PCR) fragments, and fragments obtained by any of the methods of ligation, cleavage, endonuclease action and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogues of naturally occurring nucleotides (such as enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides may have changes in sugar groups and/or pyrimidine or purine base groups. Sugar modifications include, for example, the replacement of one or more hydroxyl groups with halogens, alkyl groups, amines and azido groups, or sugars can be functionalized to form ethers or esters. Moreover, the entire sugar group can be replaced by sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogues. Examples of modifications on the base group include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well known heterocyclic substituents. Nucleic acid monomers may be linked by phosphodiester bonds or analogues of such bonds. Phosphodiester linkage analogues include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphodiselenoate, phosphoroanilotioate, phosphoranilidate, or phosphoramidate linkages. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids", which contain naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. The term "oligonucleotide" can be used interchangeably with the term "nucleic acid" and can refer to either double- or single-stranded DNA or RNA. The nucleic acid or nucleic acids may be contained in a nucleic acid vector or nucleic acid construct (e.g., plasmid, virus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC) ), which can be used to amplify and/or express a nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including, but not limited to: promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting 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 various peptides, polypeptides or proteins. Such one or more sequences may be joined in the same nucleic acid or nucleic acid molecule adjacent to each other or with additional nucleic acids in between, such as linkers, repeats or restriction enzyme sites, or any other sequence that is 1 in length , 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 bases, or any length within the range specified by any two of the above lengths. The term "downstream" on a nucleic acid as used herein refers to the sequence that is located after the 3' end of the preceding sequence, on the strand containing the coding sequence (sense strand), if the nucleic acid is double-stranded. The term "upstream of a nucleic acid" as used herein refers to the sequence that is located upstream of the 5' end of the next sequence on the strand containing the coding sequence (sense strand) if the nucleic acid is double-stranded. The term "clustered" on a nucleic acid as used herein refers to two or more sequences that are in proximity either directly or with additional nucleic acids between them, such as linkers, repeats or restriction enzyme sites, or any other sequence that is 1 in length , 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 bases, or any length in the range given by any two of the above lengths, but generally not with a sequence between them, which encodes a functional or catalytic polypeptide, protein or protein domain.

[0105] The term "codon-optimized" as used in this application refers to the replacement of nucleic acid codons to enhance or maximize translation in a host species without changing the polypeptide sequence, based on species-specific codon usage preferences and the relative availability of each aminoacyl-tRNA in the cytoplasm of the target cell. Codon optimization and techniques for performing such optimization are known in the art. Programs containing algorithms for codon optimization are known to those skilled in the art. Programs may include, for example, OptimumGene, GeneGPS®, etc. algorithms. In addition, synthetic codon-optimized sequences can be obtained from commercial sources, such as Integrated DNA Technologies and other commercially available DNA sequencing services. One skilled in the art will appreciate that gene expression levels depend on many factors, such as promoter sequences and regulatory elements. In most bacteria, tRNA species have been found to recognize small subsets of codons, leading to translational selection that may serve as an important constraint on protein expression. In this aspect, a variety of synthetic genes can be designed to increase the protein expression level thereof.

[0106] Nucleic acids described in this application contain nucleobases. Primary, canonical, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil. Other nucleobases include, but are not limited to: purines, pyrimidines, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases or biotin-labeled bases.

[0107] The terms "peptide", "polypeptide" and "protein" as used herein refer to macromolecules consisting of amino acids linked by peptide bonds. A variety of functions of peptides, polypeptides and proteins are known in the art, and these include, but are not limited to, enzyme functions, structural, transport, defense, hormone, or signaling functions. Peptides, polypeptides and proteins are often, but not always, produced biologically using a ribosomal complex using a nucleic acid template, although chemical synthesis methods are also available. By manipulating the nucleic acid template, peptide, polypeptide and protein, mutations such as substitutions, deletions, truncations, additions, duplications or fusions of more than one peptide, polypeptide or protein can be made. Such fusions of more than one peptide, polypeptide or protein can be carried out in the same molecule next to each other or with additional amino acids between them, for example, linkers, repeats, epitopes or tags, or any other sequence that is 1, 2 in length , 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 bases, or any length in the range specified by any two of the above lengths.

[0108] In some embodiments, the nucleic acid or peptide sequences provided herein and used in the examples are functional in a variety of biological systems, including, but not limited to, human, mouse, rabbit, E. coli, yeast, and mammalian cells. In other embodiments, nucleic acid or peptide sequences similar to 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage within the range specified by any two of the above percentages of similarity to the nucleic acid or peptide sequences provided in this application and used in the examples can also be used without affecting the function of the sequences in biological systems. As used herein, the term “similarity” refers to a nucleic acid or peptide sequence having the same general nucleotide or amino acid order, respectively, as the reference nucleic acid or peptide sequence, with certain changes such as substitutions, deletions, repeats, or insertions within sequences. In some embodiments, two nucleic acid sequences that are as little as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92% similar. 93%, 94%, 95%, 96%, 97%, 98% or 99% can code for the same polypeptide due to the content of different codons that code for the same amino acid during translation.

[0109] The term “recombinantly expressed” as used herein refers to the production of proteins in optimized or tailored biological systems. These systems have advantages over natural host protein expression, including, but not limited to, high expression (overexpression), ease of purification, ease of transformation, inducibility, low cost, or protein stability. In some embodiments, the proteins are expressed in mammalian, bacterial, yeast, insect, or cell-free recombinant protein expression systems. Each system has its own advantages or disadvantages. For example, bacterial expression systems are highly optimized for overexpression but can cause misfolding or aggregation of the resulting protein, yeast systems are suitable when post-translational modifications are required, and insect and mammalian expression systems are suitable for the correct RNA splicing that occurs in higher level organisms. In some embodiments, Δ-7, Δ-8, and other recombinant polypeptides are obtained and purified from mammalian, human, primary, immortal cells, cancer cells, stem cells, fibroblasts, human embryonic kidney (HEK) 293 cells, ovarian cells Chinese hamster (CHO), bacterial cells, Escherichia coli cells, yeast, Saccharomyces cerevisiae, Pichia pastoris, insect, Spodoptera frugiperda SJ9 or S. frugiperda Sf21l, or from a cell-free system. In some embodiments, genes, vectors, or expression constructs are delivered into recombinant expression systems as plasmids, bacteriophages, viruses, adeno-associated viruses (AAV), baculovirus, cosmids, fosmids, phagemids, BAC, YAC, or HAC. For a more detailed discussion of recombinant expression systems, see Gomes et al. "An Overview of Heterologous Expression Host Systems for the Production of Recombinant Proteins" ((2016) Adv. Anim. Vet. Sci. 4(7):346-356), which is hereby expressly incorporated by reference herein in its entirety.

[0110] The term “HDAg” as used herein refers to the hepatitis D antigen gene or protein. There are small (24 kDa) and large (27 kDa, 213 amino acids, excluding the methionine start codon) isoforms of HDAg, and they are translated from a single open frame reads in the HDV genome. Deamination of the adenosine at the UAG stop codon at codon 196 of the coding sequence allows translation to continue, resulting in the formation of the larger isoform. Unless explicitly stated otherwise, embodiments described herein include the large HDAg isoform. In some embodiments, the HDAg sequences comprise at least one of four different HDAg strain sequences: “Genotype 1 A HDAg,” “Genotype 1 B HDAg,” “Genotype 2 A HDAg,” or “Genotype 2 B HDAg.” In some embodiments, the nucleic acid sequence encoding at least one HDAg polypeptide comprises an HDAg genotype 1 A (SEQ ID NO: 1), HDAg genotype 1 B (SEQ ID NO: 2), HDAg genotype 2 A (SEQ ID NO: 2) nucleic acid sequence ID NO: 3) or HDAg genotype 2B (SEQ ID NO: 4). In some embodiments, the polypeptide comprising at least one HDAg polypeptide comprises a polypeptide sequence of a genotype 1 HDAg A (SEQ ID NO: 5), a genotype 1 HDAg B (SEQ ID NO: 6), a genotype 2 HDAg A (SEQ ID NO: 7) or HDAg genotype 2B (SEQ ID NO: 8).

[0111] The term “PreS1” as used herein refers to a fragment of the N-terminal domain on the HBV large surface antigen (HBsAg). The 47 amino acid PreS1 fragment from the 108-119 amino acid N-terminal domain from large HBsAg effectively elicits an immune response and induces high titers of anti-PreS1/anti-HBV antibodies in mammalian models. In some embodiments, the PreS1 sequences comprise at least one of two different PreS1 consensus sequences: “PreS1 A” and/or “PreS1 B.” In some embodiments, the nucleic acid sequence encoding the at least one PreS1 polypeptide comprises a PreS1A (SEQ ID NO: 9) or PreS1 B (SEQ ID NO: 10) nucleic acid sequence. In some embodiments, the polypeptide comprising at least one PreS1 polypeptide comprises a PreS1 A (SEQ ID NO: 11) or PreS1 B (SEQ ID NO: 12) polypeptide sequence.

[0112] In some embodiments, the HBV PreS1A and PreS1B consensus sequences are derived from or derived from the similarity of PreS1 sequences in known HBV genotypes. There are ten known or common genotypes of HBV (genotypes A, B, C, D, E, F, G, H, I, and J), having up to or approximately 8% nucleotide differences in the genomic sequence. Among them, there are additional subgenotypes that exhibit up to or approximately 4%-8% nucleotide differences in the genomic sequence. HBV subgenotypes include, but are not limited to: Al, A2, A3, A4, A5, A6, A7, B2, V3, B4, B5, B6, B7, B9, C1, C2, C3, C4, C5, C6, C7 , C8, C9, C10, D1, D2, D3, D4, D5, D6, D7, Fl, F2, F2a, F3 or F4. For a more detailed discussion of HBV genotypes, see Sunbul, “Hepatitis B virus genotypes: Global distribution and clinical importance”) ((2014) World. J. Gastroenterology. 20(18): 5427-5434, which is hereby expressly incorporated herein in its entirety via link.

[0113] The terms “autocatalytic peptide cleavage site” or “peptide 2A” as used herein refer to a peptide sequence in which cleavage of the peptide bond between its two constituent amino acids occurs, resulting in separation of the two proteins that flank the sequence. It is believed that this cleavage is the result of ribosomal slippage through the formed peptide bond between the C-terminal proline and glycine in the 2A peptide sequence. Four autocatalytic peptide cleavage site sequences identified so far have actual use in biomedical research: foot-and-mouth disease virus 2A (F2A); Equine rhinitis virus A (ERAV) 2A (E2A); 2A porcine teschovirus-1 (P2A) and 2A Thosea asigna virus (T2A). In some embodiments, the nucleic acid (SEQ ID NO: 13) and polypeptide (SEQ ID NO: 14) sequences of the autocatalytic cleavage site of the P2A peptide are used.

[0114] The term “HBeAg” as used herein refers to the HBV antigen protein located between the nucleocapsid core and the lipid envelope of the virus. HBeAg produced in the host is secreted into the serum and is a reliable marker of active HBV infection. Quantitative in vitro HBeAg secretion assays in a cell culture model can be used to evaluate the effect of biological or pharmaceutical compounds or compositions on HBV infectivity.

[0115] The term "excipient" has its usual meaning as understood in light of the present description, and refers to other substances, compounds or materials found in an immunogenic composition or vaccine. Excipients with desirable properties include, but are not limited to: preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, 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, whey, 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. Some excipients may be in residual quantities or be impurities left over from the manufacturing process of an immunogenic composition or vaccine, including, but not limited to: serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, p-propiolactone, gelatin, debris cells, nucleic acids, peptides, amino acids or growth medium components, or any combination thereof. The amount of excipient may be present in the immunogenic composition or vaccine at the percentage level: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7 %, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40% , 50%, 60%, 70%, 80%, 90%, 95%, 100% by weight, or any percentage by weight within the range specified by any two of the above values.

[0116] The term “adjuvant” as used herein refers to a substance, compound or material that stimulates an immune response and enhances the effectiveness of protective immunity and which is administered in combination with an immunogenic antigen, epitope or composition. Adjuvants serve to improve immune responses by allowing continuous release of antigen, up-regulation of cytokines and chemokines, recruitment of cells to the site of administration, increased antigen uptake and presentation to 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 hydroxyphosphate, potassium aluminum sulfate, oils, mineral oil, paraffin oil, oil-in-water emulsions, detergents, MF59®, squalene , AS03, α-tocopherol, polysorbate 80, AS04, monophosphoryl lipid A, virosomes, nucleic acids, polyinosinic:polycytidylic acid, saponins, QS-21, proteins, flagellin, cytokines, chemokines, IL-1, IL-2, IL -12, IL-15, IL-21, imidazoquinolines, CpG oligonucleotides, lipids, phospholipids, dioleoyl-phosphatidylcholine (DOPC), trehalose dimycolate, peptide o glycans, bacterial extracts, lipopolysaccharides or Freund's adjuvant, or any combination thereof.

[0117] The terms “priming” and “boosting” as used herein refer to distinct immunogenic compositions used in a heterologous priming-boosting immunization approach. Immunizations or vaccines typically require more than one administration of an immunogenic composition to induce effective immunity to the target pathogen in the host. Compared to this homologous approach, in which the same composition is provided for all administrations, heterologous priming-boosting may more effectively generate strong immunity with greater antibody levels and improved clearance or resistance to certain pathogens such as HBV or HDV. When performing heterologous priming-boosting, at least one priming dose containing one type of immunogenic composition is first provided. After providing said at least one priming dose, at least one stimulating dose is then provided containing another type of immunogenic composition. Administration of at least one stimulating dose is carried out at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36 or 48 days or weeks after administration of at least one priming dose or within a time range specified by any two of the above time points, for example, within 1-48 days or 1-48 weeks. In some embodiments, the priming dose contains a nucleic acid (eg, DNA or RNA) that encodes one or more antigens or epitopes, and the stimulating dose contains a polypeptide that contains one or more antigens or epitopes. In the host, the priming nucleic acid is translated in vivo to induce an immune response and produces a greater response against subsequent stimulation by the polypeptide. In some embodiments, the priming nucleic acid comprises sequences that encode at least one HDAg polypeptide, at least one PreS1 peptide, and at least one peptide autocatalytic cleavage site. In some embodiments, the stimulatory polypeptide comprises at least one HDAg polypeptide and at least one PreS1 polypeptide.

[0118] In some embodiments, administration of a priming nucleic acid and a stimulatory polypeptide containing HBV and HDV components to an experimental organism results in a greater titer of anti-HDAg, anti-PreS1, anti-HBV, or anti-HDV antibodies at a ratio of 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 100000 or 1000000, or any ratio within range defined by any two of the above ratios to that of a nucleic acid-only or polypeptide-only or non-immunized control organism, which was analyzed quantitatively using techniques known in the art, such as ELISA. In some embodiments, administration of a priming nucleic acid and stimulatory polypeptide containing HBV and HDV components to an experimental organism results in the serum neutralizing HBV or HDV infectivity in vitro more effectively and reducing the incidence of infection to a ratio of 0.00001 to 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 ratio within the range specified by any two of the above ratios, compared with sera from a nucleic acid-only or polypeptide-only or non-immunized control organism. In some embodiments, administration of a priming nucleic acid and a stimulatory polypeptide containing HBV and HDV components to an experimental organism results in a greater number of interferon gamma (IFNγ) positive cells (e.g., T cells) at a ratio of 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400 . acid or polypeptide alone or a non-immunized control organism.

[0119] In some embodiments, immunogenic compositions or combinations of products are administered with an adjuvant. In some embodiments, the immunogenic compositions or combinations of products are administered enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally or intravenously, or any combination of these routes. In some embodiments, immunogenic compositions or combinations of products are administered in combination with an antiviral therapy compound that has known activity against HBV or HDV, including, but not limited to, entecavir, tenofovir, lamivudine, adefovir, telbivudine, emtricitabine, interferon- α, pegylated interferon-α or interferon alpha-2b, or any combination thereof.

[0120] The terms "in vivo electroporation", "electroporation" and "EP" as used herein refer to the delivery of genes, nucleic acids, DNA, RNA, proteins or vectors into cells of living tissues or organisms using electrical currents using techniques known in the art. in this field of technology. Electroporation can be used as an alternative to other gene transfer methods such as viruses (transduction), lipofection, gene gun (biolistics), microinjection, vesicle fusion or chemical transformation. Electroporation limits the risk of immunogenicity and unfavorable integration or mutagenesis of the cell genome. DNA vectors, such as plasmids, are able to enter the cell nucleus, allowing the transcription and translation of their constituent genes. In some embodiments, genes, nucleic acids, DNA, RNA, proteins, or vectors are introduced into a target tissue or target organism by subcutaneous, intramuscular, or intradermal injection. The electroporator then delivers short electrical pulses through electrodes placed in or near the injected sample. As used herein, the term “IM/EP” refers to in vivo electroporation of a sample delivered intramuscularly (“IM”).

[0121] The term "uPA ^+/+ -SCID" as used herein refers to a mouse model of immunodeficiency used to study liver diseases, including hepatitis virus infections. These mice are homozygous for Prkdc^, which causes a deficiency of functional T and B lymphocytes. Overexpression of urokinase-type plasminogen activator (uPA) also causes severe liver cytotoxicity and liver failure during development. Subsequent transplantation and grafting of human liver tissue into these mice results in a model ideal for studying liver disease in humans. For more information on uPA ^+/+ -SCID mice, 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), which is hereby expressly incorporated by reference herein in its entirety.

[0122] The term "% by weight" or "% w/w" as used herein has its usual meaning as understood in light of the present specification, and refers to the percentage, expressed as the weight of the ingredient or agent, of the total weight of the composition multiplied by 100 The term "% by volume" or "% v/v" as used herein has its usual meaning as understood in light of the present specification, and refers to the percentage, expressed as liquid volume of a compound, substance, ingredient or agent, of the total liquid volume of the composition , multiplied by 100.

[0123] The present invention is described in this application in general terms, using affirmative terminology to describe a variety of embodiments. Also included within the scope of the present invention are embodiments in which the subject matter is excluded, in whole or in part, such as substances or materials, method steps and conditions, protocols or procedures.

Immunogenic compositions and product combinations.

[0124] This application provides immunogenic compositions or combinations of products. In some embodiments, the immunogenic compositions or combinations of products are designed to induce an immunogenic response to a specific antigen. In some embodiments, the immunogenic compositions or product combinations comprise (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 HDAg-encoding nucleic acid sequence comprises 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 comprises SEQ ID NO: 9 or SEQ ID NO: 10, or both. In some embodiments, the nucleic acid is designed such that each HDAg nucleic acid sequence is clustered with a PreS1 nucleic acid sequence, and that the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence. In some embodiments, the immunogenic compositions or product combinations further comprise at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site, wherein the grouped HDAg and PreS1 nucleic acid sequences are separated by at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site. In some embodiments, the at least one nucleic acid sequence encoding an autocatalytic cleavage site of the peptide comprises a nucleic acid sequence selected from the group consisting of porcine techovirus-1 (P2A) nucleic acids, foot-and-mouth disease virus 2A (F2A), virus 2A equine rhinitis A (ERAV) (E2A) and Thosea asigna virus 2A (T2A), and each encoded peptide autocatalytic cleavage site may optionally contain a GSG (glycine-serine-glycine) motif at its N-terminus. In some embodiments, the at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site comprises SEQ ID NO: 13. In some embodiments, the nucleic acid is codon-optimized for expression in humans. In some embodiments, the nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 15-24 or 35-36. In some embodiments, the nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 18 or SEQ ID NO: 35-36. In some embodiments, 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. In some embodiments, at least one PreS1 polypeptide sequence comprises SEQ ID NO: 11 or SEQ ID NO: 12, or both. In some embodiments, at least one PreS1 polypeptide sequence is located downstream of at least one HDAg polypeptide sequence. In some embodiments, the polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 25-34 or 37. In some embodiments, the polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 29, 31, 32, or 37. In some embodiments, the polypeptide is recombinantly expressed. In some embodiments, the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system. In some embodiments, the immunogenic compositions or product combinations further comprise an adjuvant. In some embodiments, the adjuvant is alum, 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 a recombinant vector.

[0125] Also provided herein are methods of inducing an immune response in a subject using an immunogenic composition or combination of products. In some embodiments, the immunogenic composition or product combination is any of the immunogenic compositions or product combinations described herein. In some embodiments, the methods comprise administering to the subject at least one priming dose comprising a nucleic acid; and administering to said subject at least one stimulant dose comprising the polypeptide. In some embodiments, the at least one stimulating dose further comprises an adjuvant. In some embodiments, the adjuvant is alum, QS-21 or MF59, or any combination thereof. In some embodiments, at least one stimulant dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeks after administration of the at least one priming dose or within a time range specified by any two of the above time points, for example, within 1-48 days or 1-48 weeks. In some embodiments, administration is via enteral, oral, intranasal, parenteral, subcutaneous, intramuscular, intradermal, or intravenous routes, or any combination of these routes. In some embodiments, administration is carried out in combination with antiviral therapy. In some embodiments, the antiviral therapy includes administration of entecavir, tenofovir, lamivudine, adefovir, telbivudine, emtricitabine, interferon-a, pegylated interferon-a, or interferon alfa-2b, or any combination thereof.

[0126] Also provided herein are immunogenic compositions or product combinations for use in treating or inhibiting hepatitis B or hepatitis D. In some embodiments, the immunogenic compositions or product combinations are any of the immunogenic compositions or product combinations described herein. In some embodiments, the immunogenic compositions or product combinations comprise (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 HDAg-encoding nucleic acid sequence 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 the nucleic acid sequence encoding PreS1 contains SEQ ID NO: 9 or SEQ ID NO: 10, or both. In some embodiments, the nucleic acid is designed such that each HDAg nucleic acid sequence is clustered with a PreS1 nucleic acid sequence, and that the PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence. In some embodiments, the immunogenic compositions or product combinations further comprise at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site, wherein the grouped HDAg and PreS1 nucleic acid sequences are separated by at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site. In some embodiments, the at least one nucleic acid sequence encoding an autocatalytic cleavage site of the peptide comprises a nucleic acid sequence selected from the group consisting of porcine techovirus-1 (P2A) nucleic acids, foot-and-mouth disease virus 2A (F2A), virus 2A equine rhinitis A (ERAV) (E2A) and Thosea asigna virus 2A (T2A), and each encoded peptide autocatalytic cleavage site may optionally contain a GSG (glycine-serine-glycine) motif at its N-terminus. In some embodiments, the at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site comprises SEQ ID NO: 13. In some embodiments, the nucleic acid is codon-optimized for expression in humans. In some embodiments, the nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 15-24 or 35-36. In some embodiments, the nucleic acid comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 18, or SEQ ID NO: 35-36. In some embodiments, 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. In some embodiments, at least one PreS1 polypeptide sequence comprises SEQ ID NO: 11 or SEQ ID NO: 12, or both. In some embodiments, at least one PreS1 polypeptide sequence is located downstream of at least one HDAg polypeptide sequence. In some embodiments, the polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 25-34 or 37. In some embodiments, the polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% homologous to SEQ ID NO: 29, 31, 32, or 37. In some embodiments, the polypeptide is recombinantly expressed. In some embodiments, the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system. In some embodiments, the immunogenic compositions or product combinations further comprise an adjuvant. In some embodiments, the adjuvant is alum, 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 a recombinant vector.

EXAMPLES

[0127] Certain aspects of the embodiments discussed above are described in more detail in the following examples, which are not intended to limit the scope of the present invention in any way. Those skilled in the art will appreciate that many other embodiments are also within the scope of the present invention as described hereinbefore and in the claims.

Example 1. Methodology.

Animals.

[0128] Female C57BL/6 (H-2b) mice were obtained from Charles River Laboratories.

Human leukocyte antigen A2 (HLA-A2) transgenic HHD mice were self-bred. All mice were 8–10 weeks old at the start of the experiments and were housed under standard conditions. uPA ^+/+ -SCID mice with humanized liver were obtained and maintained. New Zealand White rabbits were purchased from commercial suppliers.

DNA plasmids.

[0129] Plasmids encoding L-HDAg genotypes 1 and 2 and the PreS1 domain (AK 2 -48) of HBsAg were used in this study as optionally P2A-cleavable fusion constructs consisting of various combinations of HDAg/PreS1 sequences. HDAg genotypes 1 and 2 sequences were obtained from four different clinical isolates; US-2 and NE, and 7/18/83 and TW2476, respectively. All genes were cloned into the pVAXl backbone (Invitrogen, Carlsbad, CA) using EcoR I and HindIII restriction sites. Plasmids were propagated in E. coli TOP10 cells (Life Technologies, Carlsbad, CA) and purified for in vivo injection using the Qiagen Endofree DNA purification kit (Qiagen GmbH) following the manufacturer's instructions. Correct gene size was confirmed by restriction enzyme digestion using EcoR I and HindIII (Fast Digest, Thermo Fisher Scientific).

Western blotting.

[0130] Western blotting was performed essentially as is generally known in the art. Hela cells were transfected with each of the plasmid DNA pVAXl Dl-D10 and pVAX1 with the GFP reporter gene as a control using Lipofectamine® 3000 transfection reagent (Thermo Fisher Scientific). For protein detection, serum from D4 vaccinated rabbits diluted 1:1000 (primary antibody) and goat anti-rabbit immunoglobulin-HRP antibody, 0.25 g/L (DAKO), diluted 1:4000 (secondary antibody) were used. Pierce™ ECL Plus Western Blotting Substrate was used for chemiluminescence detection and images were acquired using a Gel Doc XR+ system (Biorad).

Peptides.

[0131] A total of 168 15-mer HDAg peptides with an overlap of 10 AA were purchased from Sigma-Aldrich (St. Louis, MO). These 168 peptides were divided into 8 pools, each of which contained 20 or 21 peptides. Four pools corresponded to genotype 1 (pool 1 _1-21 , pool 2 _22-42 , pool 3 _43-63 and pool 4 _64-84 ) and four pools corresponded to genotype 2 (pool 1 _1-21 , pool 2 _22-42 , pool 3 _43-63 and pool 4 _64-84 ) For sequences A, B, C and D, each sequence belonging to each clinical isolate.

[0132] Two PreS1 HBsAg consensus sequences (PreS1A and PreS1B), consisting of 47 AA, and 20-mer PreS1 peptides with 10 AA overlap for HBV (sub-)genotypes A1, A2, B, B2, C, Dl, E1 and F, purchased from Sigma-Aldrich (St. Louis, MO). All peptides passed quality control (QC) (Sigma-Aldrich PEPscreen® Directory) and had a purity of >70%. Ovalbumin peptides OVA 257-264 CTL (SIINFEKL (SEQ ID NO: 38)) and OVA 323-339 Th (ISQAVHAAHAEINEAGR (SEQ ID NO: 39)) were used as negative peptide controls, while concanavalin A (ConA), purchased from Sigma Aldrich (St. Louis, MO) was used as a positive control at a final concentration of 0.5 μg/μl.

Immunization protocols for assessing the immunogenicity of HBV/HDV plasmids in mice and rabbits.

[0133] To evaluate the immunogenicity of the constructs in vivo, mice and rabbits were immunized essentially as described, stimulated at one-month intervals, and sacrificed two weeks later for collection of spleens and blood. Briefly, female C57BL/6 mice (five per group) were immunized intramuscularly (IM) into the tibialis anterior (TA) muscle with 50 μg of plasmid DNA in a volume of 50 μl in sterile PBS by conventional needle injection (27G), followed by electroporation ( EP) in vivo using the Cliniporator2 device (IGEA, Carpi, Italy). During in vivo electroporation, a pulse pattern of 1 ms 600 V/cm followed by a 400 ms pulse 60 V/cm was used to promote better DNA uptake. Mice were given an analgesic before vaccine injection and were kept under isoflurane anesthesia during vaccinations. For the rabbit studies, two New Zealand White rabbits per group were immunized with 300 μg of D3 and D4 DNA vaccines. Vaccines were administered by intramuscular injection in 300 μl of sterile PBS into the right TA muscle, followed by in vivo EP.

Detection of IFNy-producing T cells using enzyme-linked immunospot spot (ELISpot) assay.

[0134] Two weeks after the last vaccination, splenocytes from each immunized group of mice were pooled (five mice/group) and their ability to induce HBV/HDV-specific T cells based on IFN-y secretion after stimulation with the peptide for 48 hours was examined. , as is known in the art, using a commercially available ELISpot assay kit (Mabtech, Naka Strand, Sweden).

Antibody detection using ELISA .

[0135] Detection of mouse and rabbit IgG against PreS1 consensus and overlapping 20-mer peptides (10 μg/ml) was performed using protocols known in the art. Antibody titers were defined as the end point of sera titration at which the OD value at 405 nm was at least twice the OD of the negative control (nonimmunized or control animal sera) at the same dilution.

HBV Neutralization Assay in the uPA-SCID Human Liver Mouse Model .

[0136] HepG2-NTCP-A3 is a selected cell clone derived from a HepG2 cell expressing human NTCP, as previously described. It was cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin. During and after inoculation, 2.5% DMSO was added to the medium to improve HBV infection and replication. The HBV stock solution used for infection was prepared from HepAD38 cells by PEG precipitation as described. Culture medium was collected between days 3 and 6 postinfection and diluted 1:5 with PBS for quantitative analysis of HBeAg by ELISA using commercially available antibodies.

Statistical analysis.

[0137] The results were analyzed using GraphPad Prism V.5 and V.8 software and Microsoft Excel V. 16.13.1.

Example 2: HBV and HDV immunogenic constructs .

[0138] The use of recombinant HBV and HDV polypeptide constructs has been shown to effectively induce antibody production and immune protection against these two hepatitis viruses, for example in WO 2017/132332, which is hereby expressly incorporated by reference herein in its entirety. These recombinant polypeptide constructs were assembled by combining an HDAg selected from four different HDV genotypes (HDAg genotype 1 A, HDAg genotype 1 B, HDAg genotype 2 A and HDAg genotype 2 B), PreS1 selected from two genotypes of consensus sequences (PreS1A and PreS1B) , and one or more autocatalytic cleavage sites of the P2A peptide. Schematic representations of the eleven recombinant constructs are shown in Figures 1A and 2, and the corresponding SEQ ID NOs of DNA sequences and polypeptides, when available, are presented in Table 1. Polypeptides were confirmed to be correctly expressed from the Δ-1-Δ- recombinant constructs by Western blotting. 10 (Figure 1B).

Example 3 HBV/HDV DNA compositions induce an immunogenic response in mice.

[0139] Although immunogenic compositions and vaccines have traditionally been either whole organisms or antigenic proteins, it has recently been shown that introduction of DNA in vivo into living tissue and subsequent transcription and translation of antigenic proteins is also highly effective in triggering an immune response. Such immunogenic DNA compositions have been investigated as potential vaccine candidates against various diseases.

[0140] 2 weeks after the second administration of the DNA construct compositions, the immunity of mice against HBV and HDV antigens was assessed. Leukocytes were purified from mouse whole blood samples and incubated with purified polypeptide antigens, including PreS1 A, PreS1 B, HDAg genotypes 1 A, 1 B, 2 A and 2 B. Cells were also incubated with concanavalin A (“ConA”) as a positive control and two ovalbumin peptides (“OVA Th” and “OVA CTL”) as negative controls. The frequency of interferon gamma (IFNγ)-producing cells in a population in response to antigen was assessed using enzyme-linked immunospot spot (ELISpot) assays. Briefly, leukocytes were incubated with antigen in wells coated with anti-IFNγ antibodies. Cells were then removed and biotinylated anti-IFNγ antibodies, alkaline phosphatase cross-linked streptavidin, and alkaline phosphatase substrates-colorimetric reagents were added sequentially to the wells, with thorough washing between the indicated reagents. The plate was then allowed to dry and the remaining stained spots, which corresponded to IFNγ-secreting cells, were counted by microscopy. The calculated total numbers of IFNγ spot-forming cells per 10 ⁶ total cells in response to various peptide antigens for each of the mice are shown in Figures 3A (Δ-1 and Δ-2), 3B (Δ-3 and Δ-4), 3 (Δ -5 and Δ-6), 3D (Δ-7 and Δ-8) and 3E (Δ-9 and Δ-10).

[0141] The reactivity of antisera against the consensus peptides PreS1A and PreS1B (AK 2-48) and their cross-reactivity with HBV (sub-)types A1, A2, B, B2, C, D1, E1 and F were studied using pools of 20-mer PreS1 peptides. Immunogenic compositions containing Δ-1, Δ-2, Δ-3, Δ-4, Δ-7 and Δ-8 resulted in strong immunogenicity against both HBV PreS1 antigens (Figures 4A-B). Δ-3 and Δ-4 induced antibody titers >10 ⁴ in mice, followed by Δ-1, Δ-2, Δ-7 and Δ-8 constructs. Importantly, antisera from Δ-4 and Δ-7 immunized mice cross-reacted effectively with all HBV types tested (Figure 4C). The immune response to HDAg peptides was more variable, likely due to genotype sequence differences, but was generally greater than for ovalbumin controls. Notably, the Δ-3 and Δ-4 treated groups showed a slight reduction in T cell responses to HDV, for example, compared to constructs that contained HDAg alone (Δ-5, Δ-6, Δ-9, Δ-10), which can be explained by competition for epitope recognition while simultaneously priming PreS1-specific T cells. Overall, this demonstrates that active immunization is able to induce functional T cells against PreS1 and HDAg antigens and leads to the suggestion that broadly functional immunotherapies should contain both HDV genotypes 1 and 2 to ensure the induction of specific T cells.

[0142] Similar experiments were performed with HLA-A2-restricted T cells purified from HLA-A2 transgenic HHD mice. ELISpot IFNγ for normal C57BL/6 (Figure 5A) and HLA-A2 HHD (Figure 5B) mice electroporated with a formulation containing Δ-4, along with unprimed HLA-A2 HHD mice as a control (Figure 5C), confirmed immunogenicity in transgenic mice, suggesting the effectiveness of DNA compositions for the treatment of humans.

Example 4 HBV/HDV DNA compositions induce an immunogenic response in rabbits.

[0143] Experiments similar to those described in Example 3 were also performed with rabbits (Oryctolagus cuniculus). New Zealand White rabbits were administered by intramuscular injection a saline solution containing 900 μg of DNA compositions containing either Δ-3 or Δ-4 and electroporated. Doses were administered at weeks 0 and 4. After immunization, antibody titers against PreS1 in rabbit sera were observed for both DNA compositions containing Δ-3 or Δ-4, with Δ-4 being more effective (>10 ³ ) (Figures 6A-B). The cross-reactivity of rabbit antisera against HBV (sub-)types A1, A2, B, B2, C, D1, E1, and F was also examined using pools of 20-mer PreS1 peptides (Figure 6C). The precise specificity of the rabbit anti-D4 antisera was determined using individual 20-mer HBV type A1, A2, B, B2, C, Dl, E1, and F PreS1 peptides (Figure 6D). This allowed mapping of epitopes on PreS1 located at AA 22-48 in genotype D1, as evidenced by higher reactivity followed by lower reactivity to genotypes C, E1 and A1. This overlaps with the NTCP binding site and partially with previously identified epitopes recognized by neutralizing antibodies.

[0144] Table 2 summarizes the immune effects of ten immunogenic DNA compositions. DNA compositions containing D-4 resulted in the highest titers of anti-PreS1/anti-HBV antibodies in both mice and rabbits and were used for priming/challenge immunizations in the following Examples. For D-4, the widest reactivity towards various HBV genotypes was also revealed. “n/a” means low or undetectable levels of antibody activity, “n/a” means the experiment was not performed.

Example 5 DNA priming/protein stimulation approach using HBV/HDV constructs improves immunogenic response in mice.

[0145] DNA compositions containing Δ-4 (SEQ ID NO: 18) and polypeptide compositions containing Δ-7 (SEQ ID NO: 31) or Δ-8 (SEQ ID NO: 32) were used in the immunization approach by DNA priming/protein stimulation to create acquired immunity and induce production of antibodies against HBV and/or HDV in vivo (Figure 2).

[0146] C57BL/6 mice were immunized with (1) a DNA composition containing Δ-4 (3 consecutive doses of 50 μg DNA), (2) a polypeptide composition containing Δ-7 (3 consecutive doses of 20 μg protein with alum as adjuvant), or (3) a DNA composition containing Δ-4, followed by a polypeptide composition containing Δ-8 (2 doses of 50 μg DNA, then 2 doses of 20 μg protein with alum). Following administration of the compounds, IFNγ production by purified leukocytes in response to HBV and HDV antigens was examined using ELISpot (as described in Examples 1 and 2). Mice treated with (1) showed responses to hepatitis antigens (Figure 7A) comparable to those observed in Example 3 and Figure 3B, but mice treated with DNA priming/protein stimulation compositions (3) showed comparatively stronger overall immune cell response (Figure 7C). Since Δ-8 contains genotype 2 HDAg polypeptide sequences, the immune response analyzed was particularly enhanced against these antigens (Figure 7C, gtr 2-pool 5, 6, 7 and 8). In contrast, the protein (2)-only approach using Δ-7 polypeptides was not able to elicit as effective an immune response to both HBV and HDV antigens (Figure 7B). This demonstrates that this DNA priming/protein stimulation approach can effectively induce a potent immunogenic response, greater than conventional protein or organism based compositions, against several pathogens, including HBV and HDV.

[0147] Other DNA priming/protein stimulation combinations have also been evaluated in mice. Anti-PreS1 IgG titers in mice were measured after immunization with (1) DNA-only compositions containing Δ-4 (“D4”), (2) protein-only compositions containing Δ-7 (“D7-D7”), Δ-8 (“ D8-D8"), Δ-9 ("D9-D9") or Δ-10 ("D10-D10"), or (3) DNA-protein compositions containing Δ-4 DNA with Δ-7 protein ("D4 -D7"), Δ-8 ("D4-D8"), Δ-9 ("D4-D9") or Δ-10 ("D4-D10"). The formulations were administered three times at weeks 0, 4, and 8, with either 50 μg DNA IM/EP or 20 μg protein with alum administered at each dose. For DNA-protein formulations (3), 50 μg DNA IM/EP was administered as the first dose at week 0 and 20 μg protein with alum was administered as the second and third doses at weeks 4 and 8. Anti-PreS1 IgG titers in sera were assessed 2 weeks (Figure 8A), 6 weeks (Figure 8B) and 10 weeks (Figure 8C) after the first dose (ie, 2 weeks after each dose). The DNA priming/D4-D7 protein stimulation formulation resulted in higher anti-PreS1 titers after completion of the dosing schedule.

Example 6: A DNA priming/protein stimulation approach using HBV/HDV constructs improved the immunogenic response in rabbits.

[0148] 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-priming/protein-boosting composition containing Δ-4 DNA and protein Δ-4. The formulations were administered four times at weeks 0, 4, 8, and 12, with either 900 μg DNA IM/EP or 300 μg protein with alum administered at each dose. For DNA-protein formulations (3), 900 μg DNA IM/EP was administered as the first dose at week 0, and 300 μg protein with alum was administered as the second, third, and fourth doses at weeks 4, 8, and 12. Anti-PreS1 IgG titers in sera were assessed at weeks 0, 2, 10, and 14 (ie, 2 weeks after each dose) (Figure 9). The DNA priming/protein stimulation composition (3) not only resulted in greater overall titers compared to the DNA-only (1) and protein-only (2) compositions, but also induced robust antibody production more rapidly, by week 2, compared to the DNA-only (2) composition. squirrel.

Example 7 Adoptive transfer of sera or purified IgG from immunized animals protected humanized mice from HBV and HDV challenges.

[0149] The ability of D4-induced antibodies to neutralize HBV infection in vivo was determined using a human liver chimeric uPA ^+/+ -SCID mouse model as described. Total IgG was purified from D4-immunized and nonimmunized rabbits and injected into uPA ^+/+ -SCID mice that had been repopulated with human hepatocytes three days before HBV challenge. D4-induced IgG antibodies against PreS1 protected or significantly delayed the peak of viremia in all challenged mice (Figure 10A). Of the three challenged mice, one was protected (weeks 1–3), while the other two had serum HBV levels < ¹⁰⁴ IU/mL up to monthly screening and remained lower than controls until 8 weeks later. observations. All control mice treated with unprimed rabbit IgG achieved serum HBV DNA levels greater than 10 ⁸ IU/ml. No significant differences were observed between groups in serum levels of alanine transferase, asparagine transferase, alkaline phosphatase, or bilirubin (Figure 10B). In conclusion, passive immunization with D4-specific IgG antibodies against PreS1, given as a single dose, was able to prevent or significantly delay HBV infection in vivo in mice repopulated with human hepatocytes (Table 3). Importantly, the inoculum contained high levels of SHBsAg subviral particles, indicating that these antibodies actually avoided blocking by SHBsAg. Anti-PreS1 antibodies present at inoculation and during the first weeks clearly blocked infection, or the first rounds of infection, and limited the number of infected hepatocytes. This limited the spread of the virus and delayed the development of peak viremia.

Example 8. Challenge with HBV/HDV peptide constructs with various adjuvants.

[0150] Mixtures of peptides D-7 and D-8 were evaluated using various adjuvants. C57BL/6J mice were treated with 2 rounds of 20 μg of a mixture of D-7 and D-8 peptides (10 μg each of D-7 and D-8) at week 0 and week 3 (Figure 11A). Peripheral blood samples were collected at week 2 (between the two rounds) to determine final titers of reactivity against HBV and HDV by ELISA (Figures 11A and 11B), and splenocytes were isolated to determine reactivity against HBV and HDV by ELISpot (Figures 11C-D ) at 5 weeks. Peptide compositions were administered subcutaneously with adjuvants QS-21, MF59 and alum. Unprimed mice and mice treated with D-4 DNA plasmid by intramuscular electroporation were used as controls. IFNγ ELISpot was performed as described above using pools of HDAg peptides, PreS1A and PreS1B peptides, with OVA peptides and concanavalin A as controls (Figures 11C-D). Formulations administered with the adjuvant QS-21 showed increased reactivity against HDAg compared to other adjuvants. 5 mice per group were studied.

Example 9 Comparison of Typical HBV/HDV DNA and/or Peptide Constructs.

[0151] Immunogenicity was compared for 1) a mixture of D-7 and D-8 peptides only, 2) a D-7+D-8 fusion peptide only, 3) D-4 DNA priming and stimulation with a mixture of D-7 and D- peptides 8 using D-4 DNA only and unprimed conditions as controls. Mice were injected with either 20 μg of the D-7+D-8 fusion protein or 10 μg of each of the D-7 and D-8 peptides in mixed conditions with QS-21 adjuvant, subcutaneously at the base of the tail in a volume of 100 μl. Two rounds of administration were performed at 0 and 4 weeks. Control DNA D-4 was administered at a dose of 50 μg intramuscularly in 50 μl of PBS with electroporation. At week 6 (after two rounds of administration), T cell responses to PreS1 and HDV genotypes 1 and 2 antigen were determined using IFNγ ELISpot (Figure 12A). In addition, antibody levels against the consensus peptides PreS1A (Figure 12B-C) and PreS1B (Figure 12D) were assessed at week 2 (after one round of administration) and week 6 (after two rounds of administration). The greatest reactivity towards HBV and HDV was observed under conditions of DNA priming and peptide stimulation.

Example 10 Immunization by DNA or protein priming with DNA or protein stimulation against HBV and/or HDV in human clinical trials.

[0152] The following example describes embodiments using an immunogenic composition or combination of products, optionally consisting of a nucleic acid component and a polypeptide component, to treat or prevent viral infections caused by viruses such as HBV and HDV.

[0153] The DNA priming/protein stimulation compositions described in Example 5 are administered enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally, or intravenously to human patients. These human patients may be currently infected with HBV and/or HDV, previously infected with HBV and/or HDV, at risk for HBV and/or HDV infection, or not infected with HBV and/or HDV.

[0154] First, DNA priming doses are administered in amounts of 1, 10, 100, 1000 ng, or 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg, or 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg, or any amount within the range specified by any two of the above amounts, or any other amount suitable for optimal effectiveness in people. After the first priming dose of DNA, 1, 2, 3, 4 or 5 additional priming doses of DNA can be administered after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36 or 48 days or weeks, or any time within the range specified by any two of the above times, after administration of the previous priming dose of DNA, for example, within 1-48 days or 1-48 weeks. Doses of protein stimulation are administered after DNA priming doses in amounts of 1, 10, 100, 1000 ng, or 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg, or 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg, or any amount within the range specified by any two of the above amounts, or any other amount suitable for optimal effectiveness in people. The first stimulating dose of protein is administered after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36 or 48 days or weeks or any time in the range specified by any two of these higher than the time after the last priming dose of DNA was administered. After the first stimulating dose of protein, 1, 2, 3, 4 or 5 additional stimulating doses of protein can be administered after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36 or 48 days or weeks, or any time within the range specified by any two of the above times, after administration of the previous stimulating dose of protein.

[0155] Patients will be monitored for successful anti-HBV and/or HDV responses, such as production of anti-HBV, anti-HDV, anti-PreS1, or anti-HDAg antibodies in sera, rapid activation of T cells and other immune cells following exposure to antigens. HBV and/or HDV, and protection against future HBV and/or HDV infections.

[0156] In patients currently infected, previously infected, or at risk of infection with HBV and/or HDV, administration of DNA priming/protein stimulation compositions can be done in combination with antiviral therapy. Potential antiviral therapy drugs that have been shown to be effective against HBV or HDV include, but are not limited to: entecavir, tenofovir, lamivudine, adefovir, telbivudine, emtricitabine, interferon-a, pegylated interferon-a or interferon alfa-2b, or any their combination. Patients will be monitored for side effects such as dizziness, nausea, diarrhea, depression, sleep disturbance, headaches, itching, rash, fever or other known side effects of the proposed antiviral drugs.

[0157] In at least some of the previously described embodiments, one or more elements used in one implementation may be used interchangeably in another implementation, unless such replacement is technically impossible. Those skilled in the art will appreciate that various other omissions, additions and modifications to the methods and structures described above can be made without deviating from the scope of the claimed subject matter. All such modifications and changes are intended to be within the scope of the invention as defined by the appended claims.

[0158] With respect to the use of substantially any plural and/or singular terms in this application, those skilled in the art may shift from the plural to the singular and/or from the singular to the plural as appropriate for the context and /or application. Various conversions from singular to plural and vice versa may be expressly indicated in this application for the sake of clarity.

[0159] Those skilled in the art will recognize that, in general, terms used in this application and particularly in the appended claims (e.g., in the restrictive portion of the appended claims) are generally considered to be “open” terms (e.g., the term “ including" should be understood as "including but not limited to", the term "having" should be understood as "having at least", the term "includes" should be understood as "including but not limited to", etc.) . Those skilled in the art will further understand that if a certain number of claims are intended to be listed, then that number of claims will be substantially listed in the claims, and the absence of such listing indicates that this intent was not present. For example, to facilitate understanding, the following appended claims may use the introductory phrases “at least one” and “one or more” to introduce the enumerated claims. However, the use of such phrases should not be construed as implying that the singular representation of an enumerated claim limits any particular claim containing such enumerated claim to alternative embodiments containing only one such enumeration, even if the same claim contains the introductory phrases "one or more" or "at least one" and singular forms (for example, the singular form should be interpreted as "at least one" or "one or more") to list the claims claims. Moreover, even when explicitly stating a specific number of an enumerated claim, those skilled in the art will understand that such enumeration should be understood to mean at least the enumeration number (e.g., simply listing "two enumerations", without other modifiers, generally means at least two listings or two or more than two listings). Moreover, when a conventional expression similar to “at least one of A, B, and C, etc.” is used, such language is usually intended in a sense that would be understood by one skilled in the art (e.g., “ a system containing at least one of "A, B, and C" will include, but will not be limited to, systems that contain A alone, B alone, C alone, A and B together, A and C together, B and C together, and/ or A, B and C together, etc.). Where a conventional expression similar to “at least one of A, B or C, etc.” is used, such language is generally intended in a sense that would be understood by one skilled in the art (for example, the phrase “a system comprising at least one of "A, B or C" will include, but will not be limited to, systems that contain A alone, B alone, C alone, A and B together, A and C together, B and C together and/or A, B and C together, etc. Those skilled in the art will further understand that virtually any disjunctive word and/or language presenting two or more alternative terms, either in the description, claims, or figures, is to be understood as the possibility of including one of the terms, either of these terms, or both terms. For example, it should be understood that the statement "A or B" includes the possible options "A" or "B" or "A and B".

[0160] In addition, if properties or aspects of the present description are presented in terms of Markush groups, then those skilled in the art will understand that the present description is thereby also described in terms of any individual representative or subset of representatives of a Markush group.

[0161] One skilled in the art will understand that, for any and all purposes, such as providing a written description, all ranges described in this application also include any and all possible sub-bands and combinations of sub-bands. Any range listed can easily be regarded as sufficiently descriptive to allow the range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be easily broken down into a lower third, a middle third, and an upper third, etc. One skilled in the art will also understand that all statements such as “up to,” “at least,” “more than,” “less than,” and the like include the number indicated and refer to ranges that can then be break down into sub-bands as discussed above. Finally, one skilled in the art will understand that the range contains each individual representative. Thus, for example, a group containing 1-3 elements refers to groups containing 1, 2 or 3 elements. Likewise, a group containing 1-5 elements refers to groups containing 1, 2, 3, 4 or 5 elements, and so on.

[0162] Although various aspects and embodiments have been described in this application, other aspects and embodiments will be apparent to those skilled in the art. These various aspects and embodiments disclosed herein are offered for illustrative purposes and are not intended to be limiting, with the actual scope and spirit of the present invention being defined by the following claims.

[0163] All references cited in this application, including, but not limited to, published and unpublished patent applications, patents and literature, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent that publications and patents or patent applications incorporated herein by reference conflict with the disclosure contained herein, this specification is intended to supersede and/or take precedence over any such conflicting material.

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LIST OF SEQUENCES

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<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of HDAg genotype 2 A

<400> 3

agccagagcg aaacccgccg cggccgccgc ggcacccgcg aagaaaccct ggaaaaatgg

60

attaccgcgc gcaaaaaagc ggaagaactg gaaaaagatc tgcgcaaaac ccgcaaaacc

120

attaaaaaac tggaagaaga aaacccgtgg ctgggcaaca ttgtgggcat tattcgcaaa

180

ggcaaagatg gcgaaggcgc gccgccggcg aaacgcccgc gcaccgatca gatggaagtg

240

gatagcggcc cgggcaaacg cccgcataaa agcggcttta ccgataaaga acgcgaagat

300

catcgccgcc gcaaagcgct ggaaaacaaa aaaaaacagc tgagcgcggg cggcaaaatt

360

ctgagcaaag aagaagaaga agaactgcgc cgcctgaccg atgaagatga agaacgcaaa

420

cgccgcgtgg cgggcccgcg cgtgggcgat gtgaacccga gccgcggcgg cccgcgcggc

480

gcgccgggcg gcggctttgt gccgcagatg gcgggcgtgc cggaaagccc gtttagccgc

540

accggcgaag gcctggatat tcgcggcacc cagggctttc cgtgggtgag cccgagcccg

600

ccgcagcagc gcctgccgct gctggaatgc accccgcag

639

<210> 4

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of HDAg genotype 2 B

<400> 4

agccagagcg aaagcaaaaa aaaccgccgc ggcggccgcg aagatattct ggaaaaatgg

60

attaccaccc gccgcaaagc ggaagaactg gaaaaagatc tgcgcaaagc gcgcaaaacc

120

attaaaaaac tggaagatga aaacccgtgg ctgggcaaca ttattggcat tattcgcaaa

180

ggcaaagatg gcgaaggcgc gccgccggcg aaacgcccgc gcaccgatca gatggaaatt

240

gatagcggca ccggcaaacg cccgcataaa agcggcttta ccgataaaga acgcgaagat

300

catcgccgcc gcaaagcgct ggaaaacaaa aaaaaacagc tgagcagcgg cggcaaaaac

360

ctgagccgcg aagaagaaga agaactgggc cgcctgaccg tggaagatga agaacgccgc

420

cgccgcgtgg cgggcccgcg caccggcgat gtgaacctga gcggcggcgg cccgcgcggc

480

gcgccgggcg gcggctttgt gccgcgcatg gaaggcgtgc cggaaagccc gtttacccgc

540

accggcgaag gcctggatat tcgcggcaac cagggctttc cgtgggtgcg cccgagcccg

600

ccgcagcagc gcctgccgct gctggaatgc accccgcag

639

<210> 5

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> Polypeptide sequence of HDAg genotype 1 A

<400> 5

Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu Ile Leu

1 5 10 15

Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu Arg Asp

20 25 30

Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu Asn Pro

35 40 45

Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg Glu Gly

50 55 60

Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met Glu Val

65 70 75 80

Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr Asp Lys

85 90 95

Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg Lys

100 105 110

Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu Glu Glu

115 120 125

Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg Val Ala

130 135 140

Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr Arg Gly

145 150 155 160

Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu Ser

165 170 175

Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn Gln Gly

180 185 190

Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro Gln

195 200 205

Ser Cys Arg Pro Gln

210

<210> 6

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> Polypeptide sequence of HDAg genotype 1 B

<400> 6

Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu Val Leu

1 5 10 15

Glu Gln Trp Val Asn Gly Arg Lys Lys Leu Glu Glu Leu Glu Arg Glu

20 25 30

Leu Arg Arg Ala Arg Lys Lys Ile Lys Lys Leu Glu Asp Asp Asn Pro

35 40 45

Trp Leu Gly Asn Val Lys Gly Ile Leu Gly Lys Lys Asp Lys Asp Gly

50 55 60

Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Thr Asp Gln Met Glu Ile

65 70 75 80

Asp Ser Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp Arg

85 90 95

Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu Lys Asn Lys Lys Lys

100 105 110

Gln Leu Ser Ala Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu Glu Glu

115 120 125

Leu Lys Arg Leu Thr Arg Glu Asp Glu Glu Arg Lys Lys Glu Glu His

130 135 140

Gly Pro Ser Arg Leu Gly Val Asn Pro Ser Glu Gly Gly Pro Arg Gly

145 150 155 160

Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Ile Pro Glu Ser

165 170 175

Arg Phe Thr Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Ser Arg Gly

180 185 190

Phe Pro Gln Asp Ile Leu Phe Pro Ser Asp Pro Pro Phe Ser Pro Gln

195 200 205

Ser Cys Arg Pro Gln

210

<210> 7

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> Polypeptide sequence of HDAg genotype 2 A

<400> 7

Ser Gln Ser Glu Thr Arg Arg Gly Arg Arg Gly Thr Arg Glu Glu Thr

1 5 10 15

Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys Ala Glu Glu Leu Glu Lys

20 25 30

Asp Leu Arg Lys Thr Arg Lys Thr Ile Lys Lys Leu Glu Glu Glu Asn

35 40 45

Pro Trp Leu Gly Asn Ile Val Gly Ile Ile Arg Lys Gly Lys Asp Gly

50 55 60

Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met Glu Val

65 70 75 80

Asp Ser Gly Pro Gly Lys Arg Pro His Lys Ser Gly Phe Thr Asp Lys

85 90 95

Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Lys Lys

100 105 110

Gln Leu Ser Ala Gly Gly Lys Ile Leu Ser Lys Glu Glu Glu Glu Glu

115 120 125

Leu Arg Arg Leu Thr Asp Glu Asp Glu Glu Arg Lys Arg Arg Val Ala

130 135 140

Gly Pro Arg Val Gly Asp Val Asn Pro Ser Arg Gly Gly Pro Arg Gly

145 150 155 160

Ala Pro Gly Gly Gly Phe Val Pro Gln Met Ala Gly Val Pro Glu Ser

165 170 175

Pro Phe Ser Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Thr Gln Gly

180 185 190

Phe Pro Trp Val Ser Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu

195 200 205

Glu Cys Thr Pro Gln

210

<210> 8

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> Polypeptide sequence of HDAg genotype 2 B

<400> 8

Ser Gln Ser Glu Ser Lys Lys Asn Arg Arg Gly Gly Arg Glu Asp Ile

1 5 10 15

Leu Glu Lys Trp Ile Thr Thr Arg Arg Lys Ala Glu Glu Leu Glu Lys

20 25 30

Asp Leu Arg Lys Ala Arg Lys Thr Ile Lys Lys Leu Glu Asp Glu Asn

35 40 45

Pro Trp Leu Gly Asn Ile Ile Gly Ile Ile Arg Lys Gly Lys Asp Gly

50 55 60

Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met Glu Ile

65 70 75 80

Asp Ser Gly Thr Gly Lys Arg Pro His Lys Ser Gly Phe Thr Asp Lys

85 90 95

Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Lys Lys

100 105 110

Gln Leu Ser Ser Gly Gly Lys Asn Leu Ser Arg Glu Glu Glu Glu Glu

115 120 125

Leu Gly Arg Leu Thr Val Glu Asp Glu Glu Arg Arg Arg Arg Val Ala

130 135 140

Gly Pro Arg Thr Gly Asp Val Asn Leu Ser Gly Gly Gly Pro Arg Gly

145 150 155 160

Ala Pro Gly Gly Gly Phe Val Pro Arg Met Glu Gly Val Pro Glu Ser

165 170 175

Pro Phe Thr Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Asn Gln Gly

180 185 190

Phe Pro Trp Val Arg Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu

195 200 205

Glu Cys Thr Pro Gln

210

<210> 9

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> PreS1 A DNA sequence

<400> 9

ggcaccaacc tgagcaccag caacccgctg ggcttttttc cggatcatca gctggatccg

60

gcgtttcgcg cgaacagcgc gaacccggat tgggatttta acccgaacaa agatacctgg

120

ccggatgcga acaaagtggg c

141

<210> 10

<211> 141

<212> DNA

<213> Artificial sequence

<220>

<223> PreS1 B DNA sequence

<400> 10

ggccagaacc tgagcaccag caacccgctg ggcttttttc cggatcatca gctggatccg

60

gcgtttcgcg cgaacaccgc gaacccggat tgggatttta acccgaacaa agatacctgg

120

ccggatgcga acaaagtggg c

141

<210> 11

<211> 47

<212> PRT

<213> Artificial sequence

<220>

<223> PreS1 A polypeptide sequence

<400> 11

Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

35 40 45

<210> 12

<211> 47

<212> PRT

<213> Artificial sequence

<220>

<223> PreS1 B polypeptide sequence

<400> 12

Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His

1 5 10 15

Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp

20 25 30

Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

35 40 45

<210> 13

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence of the autocatalytic cleavage site

P2A peptide

<400> 13

ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct

60

ggacct

66

<210> 14

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Polypeptide sequence of the autocatalytic site

P2A peptide cleavage

<400> 14

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 15

<211> 3198

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-1

<400> 15

atggccagca gaagtgaatc aaaaaagaat cggggagggc gggaagaaat cctggaacag

60

tgggtcggag cacggaagaa actggaagaa ctggagaggg acctgcgcaa gatcaagaag

120

aagatcaaga agctggagga ggagaacccc tggctgggca atatcaaggg catcctgggc

180

aagaaggatc gggagggaga gggagcacca cctgcaaaga gggccagagc cgaccagatg

240

gaggtggata gcggaccaag gaagcgccct ttcagaggag agtttaccga caagggagcgg

300

agagatcaca ggcgccggaa ggccctggag aacaagagga agcagctgag ctccggcggc

360

aagtccctgt ctaagggagga ggaggaggag ctgcgcaagc tgacagagga ggacgagaga

420

agggagga gggtggcagg acctagggtg ggaggcgtga acccactgga gggaggaacc

480

agaggagcac ctggaggagg attcgtgcca tccatgcagg gagtgcccga gtctcctttt

540

gcccggacag gcgagggcct ggatgtgaga ggcaatcagg gcttcccctg ggacatcctg

600

tttcctgccg atccaccctt ctctcctcag agctgccggc cacagagcag atccgagtct

660

aagaagaaca ggggaggaag agaggaggtg ctggagcagt gggtgaatgg ccggaagaag

720

ctggagggagc tggagcggga gctgagaagg gccagaaaga agatcaagaa gctggaagac

780

gataatcctt ggctgggcaa tgtgaaaggc atcctgggca agaaggacaa ggatggagag

840

ggagcacctc cagcaaagag ggcaagaacc gaccagatgg agatcgattc tggaccaagg

900

aagcgccccc tgagaggagg cttcacagac cgggagagac aggatcaccg ccggagaaag

960

gccctgaaga acaagaagaa gcagctgtcc gccggaggca agagcctgtc caaagaagag

1020

gaagaggagc tgaagaggct gacccgcgag gacgaggaga ggaagaagga ggagcacgga

1080

ccaagcaggc tgggagtgaa tccttccgag ggaggaccta ggggagcacc aggaggaggc

1140

ttcgtgccat ctatgcaggg catccccgag agccggttta ccagaacagg agagggcctg

1200

gacgtgaggg gctcccgcgg ctttcctcag gacatcctgt tcccatctga tccccctttt

1260

tccccccagt cttgtaggcc tcagggcacc aacctgtcta caagcaatcc actgggcttc

1320

tttcccgacc accagctgga tcctgccttc cgcgccaaca gcgccaatcc cgactgggac

1380

ttcaacccaa ataaggacac ctggccagat gccaacaagg tcggcggcca gaacctgtcc

1440

acatctaatc ctctgggctt ctttccagac caccagctgg atccagcctt ccgggccaac

1500

acagctaacc ctgactggga cttcaacccc aataaggata cttggcccga cgccaacaag

1560

gtcggcggaa gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag

1620

aaccctggac ctatgagcca gtccgagaca aggaggggcc ggagaggaac cagggaggag

1680

acactggaga agtggatcac agcccgcaag aaggccgagg agctggagaa ggacctgcgg

1740

aagaccagaa agacaatcaa gaagctggaa gaagagaacc catggctggg caatatcgtg

1800

ggcatcatca gaaagggcaa ggacggcgag ggagcaccac cagcaaagag gcccaggact

1860

gatcagatgg aagtcgatag cggaccaggc aagcggcctc acaagtccgg cttcacagac

1920

aaggagagag aggaccatag gcgccggaag gccctggaaa acaagaagaa gcaattatcc

1980

gccggcggca agatcctgtc caaagaggaa gaagaggagc tgagaaggct gaccgacgag

2040

gatgaggaga ggaaaagaag ggtggcagga ccaagggtgg gcgacgtgaa tcccagcagg

2100

ggaggaccaa gaggcgcccc tggcggcggc ttcgtgccac agatggcagg agtgccagag

2160

agcccctttt cccaggacagg agagggcctg gatatcagag gcacccaggg ctttccttgg

2220

gtgtctccaa gccctccaca gcagcggctg ccactgctgg agtgcacccc tcagtcccag

2280

tctgagagca agaagaacag aaggggcggc agagaggaca tcctggagaa gtggatcacc

2340

acacgcagaa aagctgaaga actggaaaag gacctgagga aggcccgcaa aacaatcaag

2400

aagctggagg atgaaaatcc atggctggga aacatcatcg gcatcatcag gaagggcaag

2460

gacggggaag gcgcaccacc tgcaaagcgg cctagaacag atcagatgga aatcgattct

2520

ggcaccggca agaggccaca caagagcggc ttcaccgaca aggagcgcga ggatcacaga

2580

aggcgcaagg ccctggagaa caagaagaag caattaagca gcggcggcaa gaatctgtcc

2640

agagaagaag aggaggagct gggccgcctg accgtggagg acgaggagcg gagaaggcgc

2700

gtggcaggac cacgcacagg cgatgtgaac ctgtccggag gaggaccaag gggagcacct

2760

ggaggcggct tcgtgcctag aatggaggga gtgcctgagt cccccttcac ccgcaccgga

2820

gagggcctgg acatcagagg caatcaggga ttcccatggg tgaggcccag cccaccacag

2880

cagcgcctgc cactgctgga gtgtaccccc cagggcacaa acctgtccac ctctaatccc

2940

ctgggcttct ttcctgatca tcagctggac ccagccttca gggccaactc cgccaatcca

3000

gattgggact tcaacccgaa taaggatact tggccagatg caaacaaggt cggaggacag

3060

aacctgagca catccaaccc tctgggcttc tttcctgacc atcagctgga tcccgccttt

3120

cgcgccaata ccgccaaccc tgattgggac ttcaacccta ataaggatac ttggcctgat

3180

gctaataagg tcggggtga

3198

<210> 16

<211> 3198

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-2

<400> 16

atggccggca ctaacctgtc tacatcaaac cctctgggat ttttccccga tcatcagctg

60

gaccccgcat ttcgcgctaa ctctgctaac cctgactggg atttcaaccc taataaggac

120

acatggccag atgccaacaa ggtcggcggc cagaacctgt ccacctctaa tcccctgggc

180

ttctttcctg accaccagct ggatcctgcc ttcagggcca acaccgccaa tcccgactgg

240

gacttcaacc caaataagga tacctggcct gacgctaaca aggtcggcag ccggtccgag

300

tctaagaaga ataggggagg aagggaggag atcctggagc agtgggtggg cgccagaaag

360

aagctggagg agctggagcg ggacctgaga aagatcaaga agaagatcaa gaagctggag

420

gaggagaacc cctggctggg caatatcaag ggcatcctgg gcaagaagga tcgggaggga

480

gagggagcac cacctgcaaa gagggccaga gccgaccaga tggaggtgga ttccggccct

540

aggaagcgcc cattcagagg cgagtttaca gacaaggagc ggagagatca caggcgccgg

600

aaggccctgg agaacaagag gaagcagctg agctccggcg gcaagagcct gtccaaggag

660

gaggaggagg agctgcgcaa gctgaccgag gaggacgaga gaagggagag gagggtggca

720

ggacctaggg tgggaggcgt gaacccactg gagggaggaa caagaggagc acccggagga

780

ggcttcgtgc cttctatgca gggcgtgcct gagagcccat ttgccaggac cggagagggc

840

ctggacgtga gaggcaatca gggcttccca tgggacatcc tgtttcccgc cgatccaccc

900

ttcagcccac agtcctgcag gccccagtct cgcagcgagt ccaagaagaa cagaggcgga

960

agggaggagg tgctggagca gtgggtgaat ggcaggaaga agctggaaga actggagagg

1020

gagctgagaa gggcccgcaa gaagatcaag aagctggaag acgataatcc ttggctgggc

1080

aatgtgaaag gcatcctggg caagaaggac aaggatggag agggagcacc tccagcaaag

1140

agggcaagaa cagaccagat ggagatcgat tccggaccaa ggaagcgccc tctgagggga

1200

ggcttcaccg accgggagag acaggatcac cgccggagaa aggccctgaa gaacaagaag

1260

aagcagctga gcgccggcgg caagtctctg agtaaagaag aagaggagga gctgaagcgg

1320

ctgacaagag aggacgagga gaggaagaag gaggagcacg gaccatccag gctgggagtg

1380

aatccttctg agggaggacc aaggggcgcc cctggcggag gcttcgtgcc tagcatgcag

1440

ggcatcccag agtccaggtt taccaggaca ggcgaaggcc tggacgtgcg gggctctaga

1500

ggctttcccc aggacatcct gttccctagc gatccccctt tttctcctca gagctgtaga

1560

ccacagggaa gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag

1620

aaccctggac ctatgggcac caacctgtcc acatctaacc ctctgggctt ctttccagat

1680

catcagctgg acccagcctt cagggccaac agcgccaatc cagactggga cttcaacccc

1740

aataaggaca catggcctga cgcaaacaag gtcggagaggac agaacctgag cacctccaat

1800

ccactgggct tctttcccga ccaccagctg gatccagcct tccgcgccaa cactgctaac

1860

cctgattggg acttcaaccc taataaggat acatggcctg atgccaataa ggtcggctct

1920

cagagcgaga caaggagggg ccggagagga accagggagg agacactgga gaagtggatc

1980

accgcccgca agaaggccga ggagctggag aaggacctga ggaagacccg caagacaatc

2040

aagaagctgg aagaagagaa cccatggctg ggcaatatcg tgggcatcat cagaaagggc

2100

aaggacggcg agggagcacc accagcaaag aggccccgca cagatcagat ggaagtggat

2160

tccggacctg gcaagcggcc acacaagtct ggcttcaccg acaagggagag agaggaccat

2220

aggcgccgga aggccctgga aaacaagaag aagcaattat ctgccggcgg caagatcctg

2280

agtaaagaag aggaagagga gctgagaagg ctgaccgacg aggatgagga gaggaagcgc

2340

cgggtggccg gcccacgcgt gggcgacgtg aatccctcca ggggaggacc aagaggagca

2400

cctggaggcg gcttcgtgcc ccagatggcc ggcgtgcccg agtccccttt ttctcggacc

2460

ggcgagggcc tggatatcag aggcacacag ggctttccat gggtgtcccc ctctcctcca

2520

cagcagaggc tgccactgct ggagtgcaca ccccagagcc agagcgaatc taagaagaac

2580

agaaggggag gccgcgagga catcctggaa aaatggatca ccacacgcag aaaagctgaa

2640

gaactggaaa aggacctgcg gaaggccaga aagaccatca agaagctgga ggatgaaaat

2700

ccatggctgg gaaacatcat cggcatcatc cggaagggca aggacgggga aggcgcacca

2760

cctgcaaagc ggcctagaac cgatcagatg gaaatcgata gcggcacagg caagaggcca

2820

cacaagtccg gcttcaccga taaagagcgc gaggatcaca gaaggcgcaa ggccctggag

2880

aacaagaaga agcaattaag cagcggcggc aagaatctgt ccagagaaga ggaggaagag

2940

ctgggccgcc tgacagtgga ggacgaggag cggagaaggc gcgtggcagg acccagaacc

3000

ggcgatgtga acctgtccgg aggaggacct aggggagcac caggaggcgg cttcgtgcct

3060

agaatggagg gcgtgccaga gtctcccttt acccggacag gcgagggcct ggacatcaga

3120

ggcaatcagg gctttccctg ggtccgcccc tccccccctc agcagagact gccactgctg

3180

gaatgcacac cacagtga

3198

<210> 17

<211> 3762

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-3

<400> 17

atggccggca ccaatctgtc tacctcaaat cccctgggct tcttccccga tcatcagctg

60

gaccctgcct tccgagcaaa ttccgctaat cctgattggg atttcaaccc aaataaggac

120

acatggccag atgccaacaa ggtcggcggc cagaacctgt ccacctctaa tcctctgggc

180

ttctttccag accaccagct ggatcccgcc ttcaggcca acacagccaa tcccgactgg

240

gacttcaacc ctaataagga cacctggcct gacgccaaca aggtcggcag caggtccgag

300

tctaagaaga ataggggagg aagggaggag atcctggagc agtgggtggg agcacgcaag

360

aagctggagg agctggagcg ggacctgaga aagatcaaga agaagatcaa gaagctggag

420

gaggagaacc cctggctggg caatatcaag ggcatcctgg gcaagaagga tcgggaggga

480

gagggagcac cacctgcaaa gagggccaga gccgaccaga tggaggtgga ttccggacca

540

aggaagcgcc ctttcagagg agagtttaca gacaaggagc ggagagatca caggcgccgg

600

aaggccctgg agaacaagcg gaagcagctg agctccggcg gcaagagcct gtccaaggag

660

gaggaggagg agctgagaaa gctgaccgag gaggacgaga gaagggagag gagggtggcc

720

ggccccaggg tgggcggcgt gaaccctctg gagggaggaa caaggggagc accaggagga

780

ggcttcgtgc cttccatgca gggcgtgccc gagtctcctt ttgccaggac cggagagggc

840

ctggacgtgc gcggcaatca gggcttccca tgggacatcc tgtttcccgc cgatccaccc

900

ttctctcccc agagctgcag gcctcagtct cgcagcgagt ccaagaagaa cagaggcgga

960

agggaggagg tgctggagca gtgggtgaat ggcaggaaga agctggaaga actggagagg

1020

gagctgagaa gggcccgcaa gaagatcaag aagctggaag acgataatcc ttggctgggc

1080

aatgtgaaag gcatcctggg caagaaggac aaggatggag agggagcacc tccagcaaag

1140

agggcaagaa cagaccagat ggagatcgat tctggaccaa ggaagcgccc cctgagggga

1200

ggcttcaccg accgggagag acaggatcac cgccggagaa aggccctgaa gaacaagaag

1260

aagcagctga gcgccggcgg caagtctctg agtaaagaag aagaggagga gctgaagcgg

1320

ctgaccagag aggacgagga gcggaagaag gaggagcacg gcccaagcag actgggagtg

1380

aatccatccg agggaggacc tagaggcgcc cctggcggcg gcttcgtgcc ttctatgcag

1440

ggcatcccag agagcaggtt taccaggaca ggcgaaggcc tggacgtgcg gggctccaga

1500

ggctttcccc aggacatcct gttcccttct gatccccctt tttccccaca gtcttgtagg

1560

ccccagggca ccaacctgtc cacatctaac ccactgggct tctttcctga tcaccagctg

1620

gatccagcct tccgcgccaa ctccgccaat ccagactggg acttcaaccc caataaggac

1680

acatggcctg atgctaacaa ggtcggaggc cagaacctga gcacctccaa tcccctgggc

1740

ttctttcctg accaccagct ggatcctgcc ttccgcgcca acacagctaa ccctgattgg

1800

gacttcaacc caaataagga tacctggcct gatgcaaaca aggtcggagg aagcggagct

1860

actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctatgggc

1920

accaacctgt ctacaagcaa tccactgggc ttctttcccg accatcagct ggacccagcc

1980

ttcagggcca acagcgccaa ccctgactgg gacttcaacc caaataagga cacgtggcct

2040

gatgccaaca aggtcggagg acaaaacctg tccacctcta accccctggg cttctttccc

2100

gatcatcaat tagacccagc cttccgcgct aacactgcta accctgactg ggacttcaac

2160

ccgaataagg atacttggcc tgatgccaat aaggtcggca gccagtccga gacaaggagg

2220

ggccggagag gaaccagga ggagacactg gagaagtgga tcaccgccag aaagaaggcc

2280

gaggagctgg agaaggacct gaggaagacc cgcaagacaa tcaagaagct ggaagaagag

2340

aacccttggc tgggcaatat cgtgggcatc atcagaaagg gcaaggacgg cgagggagca

2400

ccaccagcca agaggccacg cacagatcag atggaagtgg atagcggacc aggcaagagg

2460

cctcacaagt ccggcttcac cgacaaggag agggaggacc ataggcgccg gaaggccctg

2520

gaaaacaaga agaagcaatt atccgccggc ggcaagatcc tgtctaaaga agaggaagaa

2580

gagctgagaa ggctgaccga cgaggatgag gagaggaaga ggagggtggc aggacctaga

2640

gtgggcgacg tgaatccatc caggggagga ccaagaggag caccaggagg cggcttcgtg

2700

ccacagatgg caggagtgcc agagagcccc ttttccagga caggagaggg cctggatatc

2760

aggggaaccc agggctttcc ttgggtgtct ccaagccctc cacagcagcg gctgccactg

2820

ctggagtgca caccccagtc ccagtctgag agcaagaaga acagaagggg cggcagagag

2880

gacatcctgg aaaaatggat caccacacgc agaaaagctg aagaactgga aaaggacctg

2940

cggaaggcca gaaagaccat caagaagctg gaggatgaaa atccatggct gggaaatatc

3000

atcggcatca tccggaaggg caaggacggg gaaggcgcac cacctgcaaa gcggcccagg

3060

accgatcaga tggaaatcga ttctggaacc ggcaagcggc ctcacaagag tggcttcacc

3120

gataaggaga gagaggatca cagaaggcgc aaggccctgg agaacaagaa gaagcaatta

3180

agcagcggcg gcaagaatct gtccagagaa gaggaagagg agctgggcag actgacagtg

3240

gaggacgagg agcggagaag gcgcgtggca ggaccaagaa ccggcgatgt gaacctgtcc

3300

ggaggaggac caaggggagc acctggggga ggcttcgtgc caaggatgga gggagtgcct

3360

gagtccccct tcaccagaac cggcgaaggc ctggacatca ggggcaatca gggattccca

3420

tgggtgcggc cctccccacc ccagcagaga ctgcctctgc tggagtgtac cccacagggc

3480

actaacctgt ccacctctaa cccgttaggc ttctttcctg accatcaatt agatcccgcc

3540

ttccgggcca acagcgccaa tcctgattgg gacttcaacc cgaataagga cacctggccc

3600

gacgcaaaca aggtcggagg gcaaaacctg agcacctcca accctttagg cttctttcca

3660

gatcatcagc tggatccagc ctttagagcc aataccgcca accctgactg ggatttcaac

3720

cctaacaaag atacctggcc cgacgctaac aaagtgggat ga

3762

<210> 18

<211> 3900

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-4

<400> 18

atggccagtc ggagcgaatc aaagaaaaat aggggagggc gggaagaaat cctggagcag

60

tgggtcggag cacgaaagaa actggaagaa ctggagaggg acctgcgcaa gatcaagaag

120

aagatcaaga agctggagga ggagaacccc tggctgggca atatcaaggg catcctgggc

180

aagaaggatc gggagggaga gggagcacca cctgcaaaga gggccagagc cgaccagatg

240

gaggtggata gcggccctag gaagcgccca ttcagaggcg agtttacaga caaggagcgg

300

agagatcaca ggcgccggaa ggccctggag aacaagcgga agcagctgag ctccggcggc

360

aagtccctgt ctaagggagga ggaggaggag ctgagaaagc tgaccgagga ggacgagaga

420

agggagga gggtggcagg acctagggtg ggaggcgtga acccactgga gggaggaaca

480

aggggagcac ctggaggagg attcgtgcca tccatgcagg gagtgcctga gtctccatt

540

gccaggaccg gagagggcct ggatgtgcgc ggaaatcagg gcttcccctg ggacatcctg

600

tttcctgccg atccaccctt ctccccacag tcttgcaggc cacagggaac caacctgagc

660

acatccaatc ctctgggctt ctttccagac caccagctgg atcctgcctt cagagccaac

720

tccgccaatc cagactggga cttcaacccc aataaggaca catggcctga tgccaacaag

780

gtcggcggcc agaacctgtc taccagcaat cccctgggct tctttcctga ccaccagctg

840

gatccagcct tccggggccaa cactgctaac cctgattggg acttcaaccc taataaggat

900

acctggccag acgccaacaa ggtcggcgga agcggagcta ctaacttcag cctgctgaag

960

caggctggag acgtggagga gaaccctgga cctatgtcca ggtctgagag caagaagaat

1020

aggggaggaa gagaggaggt gctggagcag tgggtgaacg gccgcaagaa gctggaggag

1080

ctggagaggg agctgagaag ggcccgcaag aagatcaaga agctggaaga cgataatcct

1140

tggctgggca atgtgaaagg catcctgggc aagaaggaca aggatggaga gggagcacct

1200

ccagcaaaga gggcaagaac agaccagatg gagatcgatt ctggaccaag gaagcgccct

1260

ctgaggggag gcttcaccga ccgggagaga caggatcacc gccggagaaa ggccctgaag

1320

aacaagaaga agcagctgtc cgccggcggc aagtccctga gcaaagaaga ggaagaggag

1380

ctgaagaggc tgacccgcga ggacgaggag cggaagaagg aggagcacgg accaagcaga

1440

ctggggagtga atccttccga ggggaggacca agaggagcac ccggaggagg cttcgtgcca

1500

tctatgcagg gcatccccga gagccggttt accagaacag gagagggcct ggacgtgagg

1560

ggctcccgcg gctttcctca ggacatcctg ttcccatctg atcccccttt tagcccacag

1620

tcctgtaggc cccagggcac taacctgagc acatccaacc cactgggctt ctttcctgat

1680

catcagctgg acccagcctt ccgcgccaac agcgccaacc ctgactggga cttcaaccca

1740

aataaggaca catggccaga tgctaacaag gtcggaggac aaaacctgtc taccagcaac

1800

cctctgggct tctttcccga tcatcagctg gaccccgcct tcagggccaa cacagccaat

1860

cccgactggg acttcaaccc gaataaggac acctggccag atgcaaacaa ggtcggagga

1920

agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga

1980

cctatgagcc agtctgagac aaggaggggc cggagaggaa ccagggagga gacactggag

2040

aagtggatca ccgccagaaa gaaggccgag gagctggaga aggacctgcg gaagaccaga

2100

aagacaatca agaagctgga agaagagaac ccatggctgg gcaatatcgt gggcatcatc

2160

cgcaagggca aggacggcga gggagcacca ccagcaaaga ggccccgcac agatcagatg

2220

gaagtggata gcggccctgg caagaggcca cacaagtccg gcttcaccga caaggagagg

2280

gaggaccata ggcgccggaa ggccctggaa aacaagaaga agcaattatc cgccggcggc

2340

aagatcctgt ccaaagagga agaagaggag ctgagaaggc tgaccgacga ggatgaggag

2400

aggaaaagaa gggtggcagg accaagagtg ggcgacgtga atcccagcag aggcggacca

2460

agaggagcac ctggaggcgg cttcgtgccc cagatggccg gcgtgcccga gtctcctttt

2520

agcagaactg gagagggcct ggatatcagg ggaacacagg gctttccatg ggtgagccca

2580

tcccctccac agcagaggct gccactgctg gagtgcaccc ctcagggaac caacctgtct

2640

accagcaacc cgctgggctt ctttcccgac catcagctgg accctgcctt ccgcgccaac

2700

tccgccaacc ctgattggga cttcaacccg aataaggata cctggcccga cgctaacaag

2760

gtcggaggcc agaacctgtc cacctctaac cccttaggct tctttcccga tcaccagctg

2820

gatcccgcct tcagagccaa cactgctaac cccgattggg acttcaaccc gaataaggac

2880

acgtggccag acgctaacaa ggtcggggga agcggagcta ctaacttcag cctgctgaag

2940

caggctggag acgtggagga gaaccctgga cctatgtcgc agtccgagtc taagaagaat

3000

agaaggggcg gccgggagga tatcctggaa aaatggatca ccacacgcag aaaagctgaa

3060

gaactggaaa aggacctgag gaaggcccgc aagaccatca agaagctgga ggatgaaaat

3120

ccatggctgg gaaacatcat cggcatcatc agaaagggca aggacgggga aggcgcccca

3180

cctgcaaagc ggcctagaac cgatcagatg gaaatcgatt ctggcacagg caagcggcca

3240

cacaagagtg gcttcaccga taaggagaga gaggatcaca gaaggcgcaa ggccctggag

3300

aacaagaaga agcaattaag cagcggcggc aagaatctgt ccagagaaga agaggaggag

3360

ctgggcagac tgacagtgga ggacgaggag cggagaaggc gcgtggcagg accaaggacc

3420

ggcgatgtga acctgagcgg aggaggacct aggggagcac caggaggcgg cttcgtgcct

3480

aggatggagg gagtgccaga gtcccccttt accaggactg gcgagggcct ggacatcagg

3540

ggaaatcagg gattcccatg ggtgcggcct agcccaccac agcagagact gccactgctg

3600

gagtgtacac cccagggcac aaacctgagc acatccaatc cgctgggctt ctttccagat

3660

catcaattag atccagcctt cagggccaac tccgccaatc cggattgga cttcaacccg

3720

aataaggaca cttggcccga cgcaaacaag gtcggagggc aaaacctgtc taccagcaat

3780

ccacttggct tctttcctga ccatcagctg gatcccgcct ttcgcgccaa taccgccaat

3840

cctgactggg acttcaatcc taacaaagac acctggcccg acgcaaacaa agtgggatga

3900

<210> 19

<211> 2634

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-5

<400> 19

atggcctcac ggtcagagtc aaagaaaaat aggggggggc gggaagaaat cctggaacag

60

tgggtcggag cacggaaaaa actggaagag ctggagaggg acctgcgcaa gatcaagaag

120

aagatcaaga agctggagga ggagaacccc tggctgggca atatcaaggg catcctgggc

180

aagaaggatc gggagggaga gggagcacca cctgcaaaga gggccagagc cgaccagatg

240

gaggtggata gcggccctag gaagcgccca ttcagaggcg agtttaccga caagggagcgg

300

agagatcaca ggcgccggaa ggccctggag aacaagcgga agcagctgag ctccggcggc

360

aagtccctgt ctaagggagga ggaggaggag ctgagaaagc tgacagagga ggacgagaga

420

agggagcgcc gggtggccgg cccaagggtg ggcggcgtga accccctgga gggaggaacc

480

aggggagcac ctggaggagg cttcgtgcca tctatgcagg gcgtgcctga gagcccattt

540

gccaggacag gagagggcct ggatgtgcgc ggcaatcagg gcttcccctg ggacatcctg

600

tttcctgccg atccaccctt cagcccacag tcctgcaggc ctcagagcag atccgagtct

660

aagaagaaca ggggaggaag agaggaggtg ctggagcagt gggtgaatgg ccggaagaag

720

ctggagggagc tggagcggga gctgagaagg gccagaaaga agatcaagaa gctggaagac

780

gataatcctt ggctgggcaa tgtgaaaggc atcctgggca agaaggacaa ggatggagag

840

ggagcacctc cagcaaagag ggcaagaacc gaccagatgg agatcgatag cggaccaagg

900

aagcgccctc tgagaggagg cttcacagac cgggagagac aggatcaccg ccggagaaag

960

gccctgaaga acaagaagaa gcagctgtcc gccggaggca agagcctgtc caaagaagag

1020

gaagaggagc tgaagaggct gacccgcgag gacgaggagc ggaagaagga ggagcacggc

1080

ccttccagac tgggcgtgaa tccatctgag gggaggaccaa ggggagcacc aggcggcggc

1140

ttcgtgccaa gcatgcaggg catccccgag tcccggttta ccagaacagg agagggcctg

1200

gacgtgaggg gctctcgcgg ctttcctcag gacatcctgt tcccaagcga tccccctttt

1260

tctccacaga gctgtcgccc ccagggaagc ggagctacta acttcagcct gctgaagcag

1320

gctggagacg tggaggagaa ccctggacct atgtctcaga gcgagacaag gaggggccgg

1380

agaggaacca gggaggagac actggagaag tggatcacag ccagaaagaa ggccgaggag

1440

ctggagaagg acctgcggaa gaccagaaag acaatcaaga agctggaaga agaaaatcca

1500

tggctgggaa atatcgtggg catcatcagg aagggcaagg acggcgaggg agcaccacca

1560

gccaagaggc ctcgcactga tcagatggag gtggattccg gccctggcaa gaggccacac

1620

aagtctggct tcacagacaa ggagagggag gaccataggc gccggaaggc cctggaaaac

1680

aagaagaagc aattatctgc cggcggcaag atcctgagca aagaggaaga ggaggagctg

1740

agaaggctga ccgacgagga tgaggagagg aagaggaggg tggcaggacc aagagtgggc

1800

gacgtgaatc ctagcagagg cggaccaaga ggcgccccag gcgggggctt cgtgccacag

1860

atggcaggag tgccagagtc ccctttttct aggaccggag agggcctgga tatcagggga

1920

acacagggct ttccatgggt gtccccatct cctccacagc agaggctgcc actgctggag

1980

tgcacccctc agagccagtc cgagtctaag aagaatagaa ggggcggccg cgaggacatc

2040

ctggagaagt ggatcaccac acgcagaaaa gctgaagaac tggaaaagga cctgaggaag

2100

gcccgcaaaa caatcaagaa gctggaggat gagaaccctt ggctgggcaa tatcatcgga

2160

attatcagga agggcaagga tggcgaaggc gccccacctg caaagcggcc aaggactgat

2220

cagatggaaa tcgatagcgg aacaggcaag cggccccaca agtccggctt caccgacaag

2280

gagagagagg atcacagaag gcgcaaggcc ctggagaaca agaagaagca attaagcagc

2340

ggcggcaaga atctgtccag agaagaagag gaggagctgg gcagactgac cgtggaggac

2400

gaggagcgga gaaggcgcgt ggcaggacct cgcacaggcg atgtgaacct gtccggagga

2460

ggacctaggg gagcaccagg aggcggcttc gtgccacgca tggagggcgt gccagagtct

2520

ccctttaccc gcaccggaga gggcctggac atcaggggca atcagggctt tccctgggtc

2580

cgcccctccc cccctcagca gagactgccc ctgctggaat gcacaccaca gtga

2634

<210> 20

<211> 2772

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-6

<400> 20

atggcctcac ggtcagagtc aaagaagaac agaggcggaa gagaagaaat cctggagcag

60

tgggtcggag cacggaaaaa gctggaagaa ctggagaggg acctgcgcaa gatcaagaag

120

aagatcaaga agctggagga ggagaacccc tggctgggca atatcaaggg catcctgggc

180

aagaaggata gggagggaga gggagcacca cctgcaaaga gggccagagc cgaccagatg

240

gaggtggata gcggaccaag gaagcgcccc ttccgcggag agtttaccga caagggagcgg

300

agagatcaca ggcgccggaa ggccctggag aacaagagga agcagctgag ctccggcggc

360

aagtccctgt ctaagggagga ggaggaggag ctgcgcaagc tgacagagga ggacgagaga

420

agggagga gggtggcagg accaagggtg ggaggagtga atcctctgga gggaggaacc

480

agaggagcac caggaggagg cttcgtgcca agcatgcagg gagtgccaga gtcccccttt

540

gccaggacag gagagggcct ggacgtgaga ggcaaccagg gcttcccttg ggacatcctg

600

tttccagccg atccaccctt cagccctcag tcctgcaggc cacagggaag cggagctact

660

aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tatgagccgg

720

tccgagtcta agaagaatag gggaggaaga gaggaggtgc tggagcagtg ggtgaacggc

780

agaaagaagc tggaggagct ggagagggag ctgagaaggg cccgcaagaa gatcaagaag

840

ctggaagacg ataatccttg gctgggcaat gtgaaaggca tcctgggcaa gaaggacaag

900

gatggagagg gagcacctcc agcaaagagg gcaagaaccg accagatgga gatcgatagc

960

ggacctagga agcgcccact gaggggaggc tttacagacc gggagagaca ggatcaccgc

1020

cggagaaagg ccctgaagaa caagaagaag cagctgtccg ccggaggcaa gagcctgtcc

1080

aaagaagagg aagaggagct gaagaggctg acccgcgagg acgaggagag gaagaaggag

1140

gagcacggac catctaggct gggagtgaat cccagcgagg gaggaccaag gggagcacct

1200

ggaggaggct tcgtgccctc catgcagggc atccctgagt ctcggtttac cagaaccggc

1260

gagggcctgg acgtgagggg cagccgcggc tttccacagg acatcctgtt cccctccgat

1320

cccccttttt ctccccagag ctgtcgccct caaggaagcg gagctactaa cttcagcctg

1380

ctgaagcagg ctggagacgt ggaggagaac cctggaccta tgtctcagag cgagacaagg

1440

aggggccgga gaggaaccag ggaggagaca ctggagaagt ggatcacagc ccgcaagaag

1500

gccgaggagc tggagaagga cctgcggaag accagaaaga caatcaagaa gctggaagaa

1560

gagaaccctt ggctggggcaa tatcgtgggc atcatcagga agggcaagga cggcgaggga

1620

gcaccaccag ccaagaggcc acgcactgat cagatggagg tggattctgg accaggcaag

1680

cggccccaca agagcggctt cacagacaag gagagagagg accataggcg ccggaaggcc

1740

ctggaaaaca agaagaagca attaagcgcc ggcggcaaga tcctgtccaa agaggaagag

1800

gaggagctga gaaggctgac cgacgaggat gaggagagga aaagaagggt ggcaggacct

1860

agggtgggcg acgtgaatcc aagcagggga ggacctagag gagcaccagg aggcggcttc

1920

gtgccacaga tggcaggagt gcctgagtcc ccattttctc ggaccggcga gggcctggat

1980

atcagaggca cacagggctt cccctgggtg tccccttctc ctccacagca gcggctgcct

2040

ctgctggagt gcacccctca gggaagcgga gctactaact tcagcctgct gaagcaggct

2100

ggagacgtgg aggagaaccc tggacctatg tcgcagagcg aatctaagaa gaatagaagg

2160

ggcggcagag aggatatcct ggagaagtgg atcaccacac gcagaaaagc tgaagaactg

2220

gaaaaggacc tgaggaaggc ccgcaagacc atcaagaagc tggaggatga aaatccatgg

2280

ctgggaaata tcatcggcat catccggaag ggcaaggacg gggaaggcgc cccacctgca

2340

aagcggccca ggactgatca gatggaaatc gattccggca caggcaagag gcctcacaag

2400

tctggcttca cagataaaga gcgcgaggat cacagaaggc gcaaggccct ggagaacaag

2460

aagaagcaat tatctagcgg cggcaagaat ctgtccagag aagaagagga ggagctgggc

2520

cgcctgaccg tggaggacga ggagcggaga aggcgcgtgg caggaccaag aacaggcgat

2580

gtgaacctgt ctggaggcgg cccaaggggc gcccccggcg gaggcttcgt gccaagaatg

2640

gaaggcgtgc cagagtcccc ttttacccgg acaggggaag gcctggacat tagaggcaat

2700

cagggctttc cctgggtgcg accaagcccc cctcagcagc gactgcctct gctggagtgt

2760

acccctcagtga

2772

<210> 21

<211> 1569

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-7

<400> 21

atggcctcac ggtctgagtc aaagaagaat cgggggggaa gagaagaaat cctggaacag

60

tgggtcggcg cacggaaaaa actggaagaa ctggagcggg acctgagaaa gatcaagaag

120

aagatcaaga agctggagga agagaacccc tggctgggca atatcaaggg catcctgggc

180

aagaaggatc gggagggcga gggagcacca cctgcaaaga gggcaagggc agaccagatg

240

gaggtggatt ccggacctag gaagcggccc ttccggggag agtttaccga caaggagcgg

300

agagatcaca ggcgccggaa ggccctggag aacaagcgga agcagctgag ctccggcggc

360

aagtctctga gcaaggagga ggaggaggag ctgagaaagc tgacagagga ggacgagaga

420

agggagcgcc gggtggccgg cccaagggtg ggcggcgtga accccctgga gggaggaacc

480

aggggagcac caggaggagg cttcgtgcct tctatgcagg gcgtgccaga gagccccttt

540

gccaggacag gagagggcct ggatgtgcgc ggcaatcagg gcttcccatg ggacatcctg

600

tttcccgccg atccaccctt ctcccctcag tcttgcaggc cacagtcccg ctctgagagc

660

aagaagaaca ggggaggaag ggaggaggtg ctggagcagt gggtgaatgg caggaagaag

720

ctggagggagc tggagcggga gctgagaagg gccagaaaga agatcaagaa gctggaagac

780

gataatcctt ggctgggcaa tgtgaaaggc atcctgggca agaaggacaa ggatggagag

840

ggagcacctc cagcaaagag ggcacgcacc gaccagatgg agatcgattc cggaccaagg

900

aagcggcccc tgaggggagg cttcacagac agggagcgcc aggatcaccg ccggagaaag

960

gccctgaaga acaagaagaa gcagctgtct gccggcggca agtccctgtc taaagaagag

1020

gaggaggagc tgaagcggct gaccagagag gacgaggagc ggaagaagga ggagcacggc

1080

ccttccagac tgggcgtgaa tccatctgag gggaggaccaa gaggcgcccc tggcggaggc

1140

ttcgtgccta gcatgcaggg catcccagag tccaggttta ccagaaccgg agagggcctg

1200

gacgtgcggg gctctagagg ctttccccag gacatcctgt tccctagcga tccccctttt

1260

agcccccagt cctgtaggcc tcagggcacc aacctgagca catccaatcc actgggcttc

1320

tttccagacc accagctgga tccagccttc cgcgccaaca gcgccaatcc agactgggac

1380

ttcaacccca ataaggacac ctggcctgat gccaacaagg tcggcggcca gaacctgtct

1440

acaagcaatc ctctgggctt ctttcctgat caccagctgg atcctgcctt tcgggccaat

1500

acagccaacc ctgactggga cttcaatcct aacaaagaca cttggcccga tgctaataag

1560

gtcggctga

1569

<210> 22

<211> 1569

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-8

<400> 22

atggccagtc agagcgagac ccgcagagga cggagaggaa cacgagaaga gacactggag

60

aaatggatta cagcacggaa gaaggcagaa gagctggaga aggacctgag gaagacccgc

120

aagacaatca agaagctgga ggaggagaac ccctggctgg gcaatatcgt gggcatcatc

180

aggaagggca aggatggaga gggagcacca cctgccaaga ggcctcgcac agaccagatg

240

gaggtggata gcggaccagg caagcggcct cacaagtccg gcttcaccga caaggagaga

300

gaggatcacc ggagaaggaa ggccctggag aacaagaaga agcagctgtc cgccggcggc

360

aagatcctgt ctaaggagga ggaggaggag ctgcgccggc tgacagacga ggatgaggag

420

aggaagagaa gggtggcagg accaagggtg ggcgacgtga atccttctag gggaggacca

480

aggggagcac caggaggagg cttcgtgcct cagatggccg gcgtgccaga gtctcccttt

540

agccggacag gcgaggggcct ggatatcaga ggcacccagg gctttccttg ggtgtctcca

600

agcccaccac agcagcggct gccactgctg gagtgcacac cccagtccca gtctgagagc

660

aagaagaaca ggaggggagg aagagaggac atcctggaga agtggatcac cacaagaagg

720

aaggccgagg agctggagaa ggacctgcgg aaggccagaa agaccatcaa gaagctggag

780

gatgaaaatc cttggctggg aaatatcatc ggaattatta gaaaaggcaa ggacggagag

840

ggagcacctc cagcaaagcg gccaagaaca gaccagatgg agatcgattc tggaaccggc

900

aagaggcccc acaagagtgg cttcaccgat aaggagcgcg aggatcaccg ccggagaaag

960

gccctggaaa acaagaagaa gcaattaagc tccggcggca agaatctgag cagagaagaa

1020

gaggaggagc tgggccgcct gacagtggag gacgaggaga ggcgccggag agtggcagga

1080

cctagaaccg gcgatgtgaa cctgtccgga ggcggcccaa ggggagcacc tggaggcggc

1140

ttcgtgccac gcatggaggg cgtgcctgag tctcccttca cccaggacagg agaggcctg

1200

gacatcagag gcaatcaggg attcccatgg gtgcggccca gcccacctca gcagagactg

1260

cctctgctgg agtgtacccc acagggcaca aacctgtcca cctctaatcc tctgggcttc

1320

tttccagacc accagctgga tccagccttc agggccaact ccgccaaccc tgactgggac

1380

ttcaacccta ataaggacac atggccagat gccaacaagg tcggcggcca gaacctgagc

1440

acctccaatc ccctgggctt ctttcctgac caccagctgg atcccgcctt tcgcgccaat

1500

accgccaatc ccgactggga cttcaatcca aataaggaca cctggcccga tgctaacaaa

1560

gtgggatga

1569

<210> 23

<211> 1287

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-9

<400> 23

atggccagtc ggagcgaatc aaagaaaaat agagggggaa gagaagaaat cctggagcag

60

tgggtcgggg cacggaaaaa actggaagaa ctggagcggg acctgagaaa gatcaagaag

120

aagatcaaga agctggagga agagaacccc tggctgggca atatcaaggg catcctgggc

180

aagaaggata gggagggcga gggagcacca cctgcaaaga gggcaagggc agaccagatg

240

gaggtggatt ccggaccaag gaagcggccc ttccggggag agtttaccga caaggagcgg

300

agagatcaca ggcgccggaa ggccctggag aacaagcgga agcagctgag ctccggcggc

360

aagtctctga gcaaggagga ggaggaggag ctgagaaagc tgacagagga ggacgagaga

420

agggagga gggtggcagg acctagggtg ggaggcgtga acccactgga gggaggaacc

480

aggggagcac ctggaggagg ctttgtgcca tctatgcagg gagtgccaga gagccctttc

540

gccaggacag gagagggcct ggatgtgcgc ggcaatcagg gcttcccctg ggacatcctg

600

tttcctgccg atccaccctt cagcccacag tcctgcaggc cacagtcccg ctctgagagc

660

aagaagaaca ggggaggaag ggaggaggtg ctggagcagt gggtgaatgg ccggaagaag

720

ctggagggagc tggagcggga gctgagaagg gccagaaaga agatcaagaa gctggaagac

780

gataatcctt ggctgggcaa tgtgaaaggc atcctgggca agaaggacaa ggatggagag

840

ggagcacctc cagcaaagag ggcacgcacc gaccagatgg agatcgattc tggacctagg

900

aagcggcccc tgagaggagg ctttacagac agggagcgcc aggatcaccg ccggagaaag

960

gccctgaaga acaagaagaa gcagctgagc gccggcggca agtccctgtc taaagaagag

1020

gaggaggagc tgaagcggct gaccagagag gacgaggagc ggaagaagga ggagcacgga

1080

ccatccagac tgggagtgaa tccttctgag gggaggaccaa gaggcgcccc aggcggcggc

1140

tttgtgccaa gcatgcaggg catccccgag tccaggttca ccagaaccgg cgaaggcctg

1200

gatgtgcggg gcagcagagg cttcccccag gatattctgt ttccctccga cccccccttc

1260

agtccccagt cttgccgacc tcagtga

1287

<210> 24

<211> 1287

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized DNA sequence Delta-10

<400> 24

atggcctcac agagcgaaac acggcggggg cggaggggaa ctagagagga aacactggaa

60

aaatggatta cagcacggaa aaaggcagag gaactggaga aggacctgag gaagacccgc

120

aagacaatca agaagctgga ggaggagaac ccatggctgg gcaatatcgt gggcatcatc

180

cggaagggca aggatggaga gggagcacca cctgcaaaga ggccccgcac cgaccagatg

240

gaggtggatt ctggccctgg caagaggcca cacaagagcg gcttcacaga caagggagcgc

300

gaggatcacc ggagaaggaa ggccctggag aacaagaaga agcagctgag cgccggcggc

360

aagatcctgt ccaaggagga ggaggaggag ctgcgccggc tgaccgacga ggatgaggag

420

cggaagagaa gggtggcagg accaagagtg ggcgacgtga atccctctag gggaggacca

480

aggggagcac ctggaggagg cttcgtgcct cagatggcag gagtgccaga gtcccctttt

540

tctaggaccg gagagggcct ggatatcagg ggaacacagg gctttccatg ggtgtctcca

600

agcccaccac agcagaggct gccactgctg gagtgcaccc ctcagtccca gtctgagagc

660

aagaagaaca ggaggggagg aagggaggac atcctggaga agtggatcac cacaagaagg

720

aaggccgagg agctggagaa ggacctgcgg aaggccagaa aaacaatcaa gaagctggaa

780

gatgagaacc cctggctggg caatatcatc ggcatcatca gaaaaggcaa ggacggcgag

840

ggagcacctc cagcaaagcg gcctagaacc gaccagatgg agatcgattc cggcacaggc

900

aagcggccac acaagtctgg cttcaccgac aaggagagag aggatcaccg ccggagaaag

960

gccctggaaa acaagaagaa gcaattaagc tccggcggca agaatctgag cagagaagaa

1020

gaggaggagc tgggcagact gaccgtggag gacgaggaga ggcgccggag agtggcagga

1080

cccagaacag gcgatgtgaa cctgagcgga ggaggaccta ggggagcacc aggaggcggc

1140

ttcgtgccta gaatggaggg cgtgccagag tcccccttta cccaggacagg agagggcctg

1200

gacatcaggg gcaatcaggg ctttccctgg gtccgccctt caccaccaca gcagagactg

1260

cccctgctgg aatgcacacc acagtga

1287

<210> 25

<211> 1065

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-1 polypeptide sequence

<400> 25

Met Ala Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu

1 5 10 15

Ile Leu Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu

20 25 30

Arg Asp Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu

35 40 45

Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg

50 55 60

Glu Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr

85 90 95

Asp Lys Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Arg Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro

165 170 175

Glu Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn

180 185 190

Gln Gly Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser

195 200 205

Pro Gln Ser Cys Arg Pro Gln Ser Arg Ser Glu Ser Lys Lys Asn Arg

210 215 220

Gly Gly Arg Glu Glu Val Leu Glu Gln Trp Val Asn Gly Arg Lys Lys

225 230 235 240

Leu Glu Glu Leu Glu Arg Glu Leu Arg Arg Ala Arg Lys Lys Ile Lys

245 250 255

Lys Leu Glu Asp Asp Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu

260 265 270

Gly Lys Lys Asp Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala

275 280 285

Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Pro Arg Lys Arg Pro Leu

290 295 300

Arg Gly Gly Phe Thr Asp Arg Glu Arg Gln Asp His Arg Arg Arg Lys

305 310 315 320

Ala Leu Lys Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ser Leu

325 330 335

Ser Lys Glu Glu Glu Glu Glu Leu Lys Arg Leu Thr Arg Glu Asp Glu

340 345 350

Glu Arg Lys Lys Glu Glu His Gly Pro Ser Arg Leu Gly Val Asn Pro

355 360 365

Ser Glu Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser

370 375 380

Met Gln Gly Ile Pro Glu Ser Arg Phe Thr Arg Thr Gly Glu Gly Leu

385 390 395 400

Asp Val Arg Gly Ser Arg Gly Phe Pro Gln Asp Ile Leu Phe Pro Ser

405 410 415

Asp Pro Pro Phe Ser Pro Gln Ser Cys Arg Pro Gln Gly Thr Asn Leu

420 425 430

Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro

435 440 445

Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn

450 455 460

Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser

465 470 475 480

Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala

485 490 495

Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys

500 505 510

Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Ser Gly Ala Thr Asn

515 520 525

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

530 535 540

Met Ser Gln Ser Glu Thr Arg Arg Gly Arg Arg Gly Thr Arg Glu Glu

545 550 555 560

Thr Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys Ala Glu Glu Leu Glu

565 570 575

Lys Asp Leu Arg Lys Thr Arg Lys Thr Ile Lys Lys Leu Glu Glu Glu

580 585 590

Asn Pro Trp Leu Gly Asn Ile Val Gly Ile Ile Arg Lys Gly Lys Asp

595 600 605

Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met Glu

610 615 620

Val Asp Ser Gly Pro Gly Lys Arg Pro His Lys Ser Gly Phe Thr Asp

625 630 635 640

Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Lys

645 650 655

Lys Gln Leu Ser Ala Gly Gly Lys Ile Leu Ser Lys Glu Glu Glu Glu

660 665 670

Glu Leu Arg Arg Leu Thr Asp Glu Asp Glu Glu Arg Lys Arg Arg Val

675 680 685

Ala Gly Pro Arg Val Gly Asp Val Asn Pro Ser Arg Gly Gly Pro Arg

690 695 700

Gly Ala Pro Gly Gly Gly Phe Val Pro Gln Met Ala Gly Val Pro Glu

705 710 715 720

Ser Pro Phe Ser Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Thr Gln

725 730 735

Gly Phe Pro Trp Val Ser Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu

740 745 750

Leu Glu Cys Thr Pro Gln Ser Gln Ser Glu Ser Lys Lys Asn Arg Arg

755 760 765

Gly Gly Arg Glu Asp Ile Leu Glu Lys Trp Ile Thr Thr Arg Arg Lys

770 775 780

Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile Lys

785 790 795 800

Lys Leu Glu Asp Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly Ile Ile

805 810 815

Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg

820 825 830

Thr Asp Gln Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro His Lys

835 840 845

Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala

850 855 860

Leu Glu Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn Leu Ser

865 870 875 880

Arg Glu Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp Glu Glu

885 890 895

Arg Arg Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val Asn Leu Ser

900 905 910

Gly Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Arg Met

915 920 925

Glu Gly Val Pro Glu Ser Pro Phe Thr Arg Thr Gly Glu Gly Leu Asp

930 935 940

Ile Arg Gly Asn Gln Gly Phe Pro Trp Val Arg Pro Ser Pro Pro Gln

945 950 955 960

Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln Gly Thr Asn Leu Ser

965 970 975

Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala

980 985 990

Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys

995 1000 1005

Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser

1010 1015 1020

Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro

1025 1030 1035

Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro

1040 1045 1050

Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

1055 1060 1065

<210> 26

<211> 1065

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-2 polypeptide sequence

<400> 26

Met Ala Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro

1 5 10 15

Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp

20 25 30

Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val

35 40 45

Gly Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp

50 55 60

His Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp

65 70 75 80

Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

85 90 95

Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu Ile Leu

100 105 110

Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu Arg Asp

115 120 125

Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu Asn Pro

130 135 140

Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg Glu Gly

145 150 155 160

Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met Glu Val

165 170 175

Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr Asp Lys

180 185 190

Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg Lys

195 200 205

Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu Glu Glu

210 215 220

Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg Val Ala

225 230 235 240

Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr Arg Gly

245 250 255

Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu Ser

260 265 270

Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn Gln Gly

275 280 285

Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro Gln

290 295 300

Ser Cys Arg Pro Gln Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly

305 310 315 320

Arg Glu Glu Val Leu Glu Gln Trp Val Asn Gly Arg Lys Lys Leu Glu

325 330 335

Glu Leu Glu Arg Glu Leu Arg Arg Ala Arg Lys Lys Ile Lys Lys Leu

340 345 350

Glu Asp Asp Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu Gly Lys

355 360 365

Lys Asp Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Thr

370 375 380

Asp Gln Met Glu Ile Asp Ser Gly Pro Arg Lys Arg Pro Leu Arg Gly

385 390 395 400

Gly Phe Thr Asp Arg Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu

405 410 415

Lys Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ser Leu Ser Lys

420 425 430

Glu Glu Glu Glu Glu Leu Lys Arg Leu Thr Arg Glu Asp Glu Glu Arg

435 440 445

Lys Lys Glu Glu His Gly Pro Ser Arg Leu Gly Val Asn Pro Ser Glu

450 455 460

Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln

465 470 475 480

Gly Ile Pro Glu Ser Arg Phe Thr Arg Thr Gly Glu Gly Leu Asp Val

485 490 495

Arg Gly Ser Arg Gly Phe Pro Gln Asp Ile Leu Phe Pro Ser Asp Pro

500 505 510

Pro Phe Ser Pro Gln Ser Cys Arg Pro Gln Gly Ser Gly Ala Thr Asn

515 520 525

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

530 535 540

Met Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp

545 550 555 560

His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp

565 570 575

Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

580 585 590

Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His

595 600 605

Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp

610 615 620

Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Ser

625 630 635 640

Gln Ser Glu Thr Arg Arg Gly Arg Arg Gly Thr Arg Glu Glu Thr Leu

645 650 655

Glu Lys Trp Ile Thr Ala Arg Lys Lys Ala Glu Glu Leu Glu Lys Asp

660 665 670

Leu Arg Lys Thr Arg Lys Thr Ile Lys Lys Leu Glu Glu Glu Asn Pro

675 680 685

Trp Leu Gly Asn Ile Val Gly Ile Ile Arg Lys Gly Lys Asp Gly Glu

690 695 700

Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met Glu Val Asp

705 710 715 720

Ser Gly Pro Gly Lys Arg Pro His Lys Ser Gly Phe Thr Asp Lys Glu

725 730 735

Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Lys Lys Gln

740 745 750

Leu Ser Ala Gly Gly Lys Ile Leu Ser Lys Glu Glu Glu Glu Glu Leu

755 760 765

Arg Arg Leu Thr Asp Glu Asp Glu Glu Arg Lys Arg Arg Val Ala Gly

770 775 780

Pro Arg Val Gly Asp Val Asn Pro Ser Arg Gly Gly Pro Arg Gly Ala

785 790 795 800

Pro Gly Gly Gly Phe Val Pro Gln Met Ala Gly Val Pro Glu Ser Pro

805 810 815

Phe Ser Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Thr Gln Gly Phe

820 825 830

Pro Trp Val Ser Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu Glu

835 840 845

Cys Thr Pro Gln Ser Gln Ser Glu Ser Lys Lys Asn Arg Arg Gly Gly

850 855 860

Arg Glu Asp Ile Leu Glu Lys Trp Ile Thr Thr Arg Arg Lys Ala Glu

865 870 875 880

Glu Leu Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile Lys Lys Leu

885 890 895

Glu Asp Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly Ile Ile Arg Lys

900 905 910

Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp

915 920 925

Gln Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro His Lys Ser Gly

930 935 940

Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu

945 950 955 960

Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn Leu Ser Arg Glu

965 970 975

Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp Glu Glu Arg Arg

980 985 990

Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val Asn Leu Ser Gly Gly

995 1000 1005

Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Arg Met Glu

1010 1015 1020

Gly Val Pro Glu Ser Pro Phe Thr Arg Thr Gly Glu Gly Leu Asp

1025 1030 1035

Ile Arg Gly Asn Gln Gly Phe Pro Trp Val Arg Pro Ser Pro Pro

1040 1045 1050

Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln

1055 1060 1065

<210> 27

<211> 1253

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-3 polypeptide sequence

<400> 27

Met Ala Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro

1 5 10 15

Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp

20 25 30

Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val

35 40 45

Gly Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp

50 55 60

His Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp

65 70 75 80

Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

85 90 95

Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu Ile Leu

100 105 110

Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu Arg Asp

115 120 125

Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu Asn Pro

130 135 140

Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg Glu Gly

145 150 155 160

Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met Glu Val

165 170 175

Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr Asp Lys

180 185 190

Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg Lys

195 200 205

Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu Glu Glu

210 215 220

Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg Val Ala

225 230 235 240

Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr Arg Gly

245 250 255

Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu Ser

260 265 270

Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn Gln Gly

275 280 285

Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro Gln

290 295 300

Ser Cys Arg Pro Gln Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly

305 310 315 320

Arg Glu Glu Val Leu Glu Gln Trp Val Asn Gly Arg Lys Lys Leu Glu

325 330 335

Glu Leu Glu Arg Glu Leu Arg Arg Ala Arg Lys Lys Ile Lys Lys Leu

340 345 350

Glu Asp Asp Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu Gly Lys

355 360 365

Lys Asp Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Thr

370 375 380

Asp Gln Met Glu Ile Asp Ser Gly Pro Arg Lys Arg Pro Leu Arg Gly

385 390 395 400

Gly Phe Thr Asp Arg Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu

405 410 415

Lys Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ser Leu Ser Lys

420 425 430

Glu Glu Glu Glu Glu Leu Lys Arg Leu Thr Arg Glu Asp Glu Glu Arg

435 440 445

Lys Lys Glu Glu His Gly Pro Ser Arg Leu Gly Val Asn Pro Ser Glu

450 455 460

Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln

465 470 475 480

Gly Ile Pro Glu Ser Arg Phe Thr Arg Thr Gly Glu Gly Leu Asp Val

485 490 495

Arg Gly Ser Arg Gly Phe Pro Gln Asp Ile Leu Phe Pro Ser Asp Pro

500 505 510

Pro Phe Ser Pro Gln Ser Cys Arg Pro Gln Gly Thr Asn Leu Ser Thr

515 520 525

Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe

530 535 540

Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp

545 550 555 560

Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser Thr Ser

565 570 575

Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg

580 585 590

Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr

595 600 605

Trp Pro Asp Ala Asn Lys Val Gly Gly Ser Gly Ala Thr Asn Phe Ser

610 615 620

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly

625 630 635 640

Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln

645 650 655

Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe

660 665 670

Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln

675 680 685

Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu

690 695 700

Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn

705 710 715 720

Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Ser Gln Ser

725 730 735

Glu Thr Arg Arg Gly Arg Arg Gly Thr Arg Glu Glu Thr Leu Glu Lys

740 745 750

Trp Ile Thr Ala Arg Lys Lys Ala Glu Glu Leu Glu Lys Asp Leu Arg

755 760 765

Lys Thr Arg Lys Thr Ile Lys Lys Leu Glu Glu Glu Asn Pro Trp Leu

770 775 780

Gly Asn Ile Val Gly Ile Ile Arg Lys Gly Lys Asp Gly Glu Gly Ala

785 790 795 800

Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met Glu Val Asp Ser Gly

805 810 815

Pro Gly Lys Arg Pro His Lys Ser Gly Phe Thr Asp Lys Glu Arg Glu

820 825 830

Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Lys Lys Gln Leu Ser

835 840 845

Ala Gly Gly Lys Ile Leu Ser Lys Glu Glu Glu Glu Glu Leu Arg Arg

850 855 860

Leu Thr Asp Glu Asp Glu Glu Arg Lys Arg Arg Val Ala Gly Pro Arg

865 870 875 880

Val Gly Asp Val Asn Pro Ser Arg Gly Gly Pro Arg Gly Ala Pro Gly

885 890 895

Gly Gly Phe Val Pro Gln Met Ala Gly Val Pro Glu Ser Pro Phe Ser

900 905 910

Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Thr Gln Gly Phe Pro Trp

915 920 925

Val Ser Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr

930 935 940

Pro Gln Ser Gln Ser Glu Ser Lys Lys Asn Arg Arg Gly Gly Arg Glu

945 950 955 960

Asp Ile Leu Glu Lys Trp Ile Thr Thr Arg Arg Lys Ala Glu Glu Leu

965 970 975

Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile Lys Lys Leu Glu Asp

980 985 990

Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly Ile Ile Arg Lys Gly Lys

995 1000 1005

Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln

1010 1015 1020

Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro His Lys Ser Gly

1025 1030 1035

Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu

1040 1045 1050

Glu Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn Leu Ser

1055 1060 1065

Arg Glu Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp Glu

1070 1075 1080

Glu Arg Arg Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val Asn

1085 1090 1095

Leu Ser Gly Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val

1100 1105 1110

Pro Arg Met Glu Gly Val Pro Glu Ser Pro Phe Thr Arg Thr Gly

1115 1120 1125

Glu Gly Leu Asp Ile Arg Gly Asn Gln Gly Phe Pro Trp Val Arg

1130 1135 1140

Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro

1145 1150 1155

Gln Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro

1160 1165 1170

Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro

1175 1180 1185

Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn

1190 1195 1200

Lys Val Gly Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe

1205 1210 1215

Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala

1220 1225 1230

Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp

1235 1240 1245

Ala Asn Lys Val Gly

1250

<210> 28

<211> 1299

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-4 polypeptide sequence

<400> 28

Met Ala Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu

1 5 10 15

Ile Leu Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu

20 25 30

Arg Asp Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu

35 40 45

Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg

50 55 60

Glu Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr

85 90 95

Asp Lys Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Arg Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro

165 170 175

Glu Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn

180 185 190

Gln Gly Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser

195 200 205

Pro Gln Ser Cys Arg Pro Gln Gly Thr Asn Leu Ser Thr Ser Asn Pro

210 215 220

Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn

225 230 235 240

Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro

245 250 255

Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu

260 265 270

Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr

275 280 285

Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp

290 295 300

Ala Asn Lys Val Gly Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys

305 310 315 320

Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Arg Ser Glu

325 330 335

Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu Val Leu Glu Gln Trp Val

340 345 350

Asn Gly Arg Lys Lys Leu Glu Glu Leu Glu Arg Glu Leu Arg Arg Ala

355 360 365

Arg Lys Lys Ile Lys Lys Leu Glu Asp Asp Asn Pro Trp Leu Gly Asn

370 375 380

Val Lys Gly Ile Leu Gly Lys Lys Asp Lys Asp Gly Glu Gly Ala Pro

385 390 395 400

Pro Ala Lys Arg Ala Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Pro

405 410 415

Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp Arg Glu Arg Gln Asp

420 425 430

His Arg Arg Arg Lys Ala Leu Lys Asn Lys Lys Lys Gln Leu Ser Ala

435 440 445

Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu Glu Glu Leu Lys Arg Leu

450 455 460

Thr Arg Glu Asp Glu Glu Arg Lys Lys Glu Glu His Gly Pro Ser Arg

465 470 475 480

Leu Gly Val Asn Pro Ser Glu Gly Gly Pro Arg Gly Ala Pro Gly Gly

485 490 495

Gly Phe Val Pro Ser Met Gln Gly Ile Pro Glu Ser Arg Phe Thr Arg

500 505 510

Thr Gly Glu Gly Leu Asp Val Arg Gly Ser Arg Gly Phe Pro Gln Asp

515 520 525

Ile Leu Phe Pro Ser Asp Pro Pro Phe Ser Pro Gln Ser Cys Arg Pro

530 535 540

Gln Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp

545 550 555 560

His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp

565 570 575

Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

580 585 590

Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His

595 600 605

Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp

610 615 620

Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly

625 630 635 640

Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu

645 650 655

Glu Asn Pro Gly Pro Met Ser Gln Ser Glu Thr Arg Arg Gly Arg Arg

660 665 670

Gly Thr Arg Glu Glu Thr Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys

675 680 685

Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Thr Arg Lys Thr Ile Lys

690 695 700

Lys Leu Glu Glu Glu Asn Pro Trp Leu Gly Asn Ile Val Gly Ile Ile

705 710 715 720

Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg

725 730 735

Thr Asp Gln Met Glu Val Asp Ser Gly Pro Gly Lys Arg Pro His Lys

740 745 750

Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala

755 760 765

Leu Glu Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ile Leu Ser

770 775 780

Lys Glu Glu Glu Glu Glu Leu Arg Arg Leu Thr Asp Glu Asp Glu Glu

785 790 795 800

Arg Lys Arg Arg Val Ala Gly Pro Arg Val Gly Asp Val Asn Pro Ser

805 810 815

Arg Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Gln Met

820 825 830

Ala Gly Val Pro Glu Ser Pro Phe Ser Arg Thr Gly Glu Gly Leu Asp

835 840 845

Ile Arg Gly Thr Gln Gly Phe Pro Trp Val Ser Pro Ser Pro Pro Gln

850 855 860

Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln Gly Thr Asn Leu Ser

865 870 875 880

Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala

885 890 895

Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys

900 905 910

Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser Thr

915 920 925

Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe

930 935 940

Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp

945 950 955 960

Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Ser Gly Ala Thr Asn Phe

965 970 975

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met

980 985 990

Ser Gln Ser Glu Ser Lys Lys Asn Arg Arg Gly Gly Arg Glu Asp Ile

995 1000 1005

Leu Glu Lys Trp Ile Thr Thr Arg Arg Lys Ala Glu Glu Leu Glu

1010 1015 1020

Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile Lys Lys Leu Glu Asp

1025 1030 1035

Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly Ile Ile Arg Lys Gly

1040 1045 1050

Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp

1055 1060 1065

Gln Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro His Lys Ser

1070 1075 1080

Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala

1085 1090 1095

Leu Glu Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn Leu

1100 1105 1110

Ser Arg Glu Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp

1115 1120 1125

Glu Glu Arg Arg Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val

1130 1135 1140

Asn Leu Ser Gly Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe

1145 1150 1155

Val Pro Arg Met Glu Gly Val Pro Glu Ser Pro Phe Thr Arg Thr

1160 1165 1170

Gly Glu Gly Leu Asp Ile Arg Gly Asn Gln Gly Phe Pro Trp Val

1175 1180 1185

Arg Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr

1190 1195 1200

Pro Gln Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe

1205 1210 1215

Pro Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn

1220 1225 1230

Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala

1235 1240 1245

Asn Lys Val Gly Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly

1250 1255 1260

Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr

1265 1270 1275

Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro

1280 1285 1290

Asp Ala Asn Lys Val Gly

1295

<210> 29

<211> 877

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-5 polypeptide sequence

<400> 29

Met Ala Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu

1 5 10 15

Ile Leu Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu

20 25 30

Arg Asp Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu

35 40 45

Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg

50 55 60

Glu Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr

85 90 95

Asp Lys Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Arg Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro

165 170 175

Glu Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn

180 185 190

Gln Gly Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser

195 200 205

Pro Gln Ser Cys Arg Pro Gln Ser Arg Ser Glu Ser Lys Lys Asn Arg

210 215 220

Gly Gly Arg Glu Glu Val Leu Glu Gln Trp Val Asn Gly Arg Lys Lys

225 230 235 240

Leu Glu Glu Leu Glu Arg Glu Leu Arg Arg Ala Arg Lys Lys Ile Lys

245 250 255

Lys Leu Glu Asp Asp Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu

260 265 270

Gly Lys Lys Asp Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala

275 280 285

Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Pro Arg Lys Arg Pro Leu

290 295 300

Arg Gly Gly Phe Thr Asp Arg Glu Arg Gln Asp His Arg Arg Arg Lys

305 310 315 320

Ala Leu Lys Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ser Leu

325 330 335

Ser Lys Glu Glu Glu Glu Glu Leu Lys Arg Leu Thr Arg Glu Asp Glu

340 345 350

Glu Arg Lys Lys Glu Glu His Gly Pro Ser Arg Leu Gly Val Asn Pro

355 360 365

Ser Glu Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser

370 375 380

Met Gln Gly Ile Pro Glu Ser Arg Phe Thr Arg Thr Gly Glu Gly Leu

385 390 395 400

Asp Val Arg Gly Ser Arg Gly Phe Pro Gln Asp Ile Leu Phe Pro Ser

405 410 415

Asp Pro Pro Phe Ser Pro Gln Ser Cys Arg Pro Gln Gly Ser Gly Ala

420 425 430

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

435 440 445

Gly Pro Met Ser Gln Ser Glu Thr Arg Arg Gly Arg Arg Gly Thr Arg

450 455 460

Glu Glu Thr Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys Ala Glu Glu

465 470 475 480

Leu Glu Lys Asp Leu Arg Lys Thr Arg Lys Thr Ile Lys Lys Leu Glu

485 490 495

Glu Glu Asn Pro Trp Leu Gly Asn Ile Val Gly Ile Ile Arg Lys Gly

500 505 510

Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln

515 520 525

Met Glu Val Asp Ser Gly Pro Gly Lys Arg Pro His Lys Ser Gly Phe

530 535 540

Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu Asn

545 550 555 560

Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ile Leu Ser Lys Glu Glu

565 570 575

Glu Glu Glu Leu Arg Arg Leu Thr Asp Glu Asp Glu Glu Arg Lys Arg

580 585 590

Arg Val Ala Gly Pro Arg Val Gly Asp Val Asn Pro Ser Arg Gly Gly

595 600 605

Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Gln Met Ala Gly Val

610 615 620

Pro Glu Ser Pro Phe Ser Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly

625 630 635 640

Thr Gln Gly Phe Pro Trp Val Ser Pro Ser Pro Pro Gln Gln Arg Leu

645 650 655

Pro Leu Leu Glu Cys Thr Pro Gln Ser Gln Ser Glu Ser Lys Lys Asn

660 665 670

Arg Arg Gly Gly Arg Glu Asp Ile Leu Glu Lys Trp Ile Thr Thr Arg

675 680 685

Arg Lys Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr

690 695 700

Ile Lys Lys Leu Glu Asp Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly

705 710 715 720

Ile Ile Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg

725 730 735

Pro Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro

740 745 750

His Lys Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg

755 760 765

Lys Ala Leu Glu Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn

770 775 780

Leu Ser Arg Glu Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp

785 790 795 800

Glu Glu Arg Arg Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val Asn

805 810 815

Leu Ser Gly Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro

820 825 830

Arg Met Glu Gly Val Pro Glu Ser Pro Phe Thr Arg Thr Gly Glu Gly

835 840 845

Leu Asp Ile Arg Gly Asn Gln Gly Phe Pro Trp Val Arg Pro Ser Pro

850 855 860

Pro Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln

865 870 875

<210> 30

<211> 923

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-6 polypeptide sequence

<400> 30

Met Ala Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu

1 5 10 15

Ile Leu Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu

20 25 30

Arg Asp Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu

35 40 45

Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg

50 55 60

Glu Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr

85 90 95

Asp Lys Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Arg Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro

165 170 175

Glu Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn

180 185 190

Gln Gly Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser

195 200 205

Pro Gln Ser Cys Arg Pro Gln Gly Ser Gly Ala Thr Asn Phe Ser Leu

210 215 220

Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Arg

225 230 235 240

Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu Val Leu Glu Gln

245 250 255

Trp Val Asn Gly Arg Lys Lys Leu Glu Glu Leu Glu Arg Glu Leu Arg

260 265 270

Arg Ala Arg Lys Lys Ile Lys Lys Leu Glu Asp Asp Asn Pro Trp Leu

275 280 285

Gly Asn Val Lys Gly Ile Leu Gly Lys Lys Asp Lys Asp Gly Glu Gly

290 295 300

Ala Pro Pro Ala Lys Arg Ala Arg Thr Asp Gln Met Glu Ile Asp Ser

305 310 315 320

Gly Pro Arg Lys Arg Pro Leu Arg Gly Gly Phe Thr Asp Arg Glu Arg

325 330 335

Gln Asp His Arg Arg Arg Lys Ala Leu Lys Asn Lys Lys Lys Gln Leu

340 345 350

Ser Ala Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu Glu Glu Leu Lys

355 360 365

Arg Leu Thr Arg Glu Asp Glu Glu Arg Lys Lys Glu Glu His Gly Pro

370 375 380

Ser Arg Leu Gly Val Asn Pro Ser Glu Gly Gly Pro Arg Gly Ala Pro

385 390 395 400

Gly Gly Gly Phe Val Pro Ser Met Gln Gly Ile Pro Glu Ser Arg Phe

405 410 415

Thr Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Ser Arg Gly Phe Pro

420 425 430

Gln Asp Ile Leu Phe Pro Ser Asp Pro Pro Phe Ser Pro Gln Ser Cys

435 440 445

Arg Pro Gln Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala

450 455 460

Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Gln Ser Glu Thr Arg

465 470 475 480

Arg Gly Arg Arg Gly Thr Arg Glu Glu Thr Leu Glu Lys Trp Ile Thr

485 490 495

Ala Arg Lys Lys Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Thr Arg

500 505 510

Lys Thr Ile Lys Lys Leu Glu Glu Glu Asn Pro Trp Leu Gly Asn Ile

515 520 525

Val Gly Ile Ile Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala

530 535 540

Lys Arg Pro Arg Thr Asp Gln Met Glu Val Asp Ser Gly Pro Gly Lys

545 550 555 560

Arg Pro His Lys Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg

565 570 575

Arg Arg Lys Ala Leu Glu Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly

580 585 590

Lys Ile Leu Ser Lys Glu Glu Glu Glu Glu Leu Arg Arg Leu Thr Asp

595 600 605

Glu Asp Glu Glu Arg Lys Arg Arg Val Ala Gly Pro Arg Val Gly Asp

610 615 620

Val Asn Pro Ser Arg Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe

625 630 635 640

Val Pro Gln Met Ala Gly Val Pro Glu Ser Pro Phe Ser Arg Thr Gly

645 650 655

Glu Gly Leu Asp Ile Arg Gly Thr Gln Gly Phe Pro Trp Val Ser Pro

660 665 670

Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln Gly

675 680 685

Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu

690 695 700

Glu Asn Pro Gly Pro Met Ser Gln Ser Glu Ser Lys Lys Asn Arg Arg

705 710 715 720

Gly Gly Arg Glu Asp Ile Leu Glu Lys Trp Ile Thr Thr Arg Arg Lys

725 730 735

Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile Lys

740 745 750

Lys Leu Glu Asp Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly Ile Ile

755 760 765

Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg

770 775 780

Thr Asp Gln Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro His Lys

785 790 795 800

Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala

805 810 815

Leu Glu Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn Leu Ser

820 825 830

Arg Glu Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp Glu Glu

835 840 845

Arg Arg Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val Asn Leu Ser

850 855 860

Gly Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Arg Met

865 870 875 880

Glu Gly Val Pro Glu Ser Pro Phe Thr Arg Thr Gly Glu Gly Leu Asp

885 890 895

Ile Arg Gly Asn Gln Gly Phe Pro Trp Val Arg Pro Ser Pro Pro Gln

900 905 910

Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln

915 920

<210> 31

<211> 522

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-7 polypeptide sequence

<400> 31

Met Ala Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu

1 5 10 15

Ile Leu Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu

20 25 30

Arg Asp Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu

35 40 45

Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg

50 55 60

Glu Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr

85 90 95

Asp Lys Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Arg Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro

165 170 175

Glu Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn

180 185 190

Gln Gly Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser

195 200 205

Pro Gln Ser Cys Arg Pro Gln Ser Arg Ser Glu Ser Lys Lys Asn Arg

210 215 220

Gly Gly Arg Glu Glu Val Leu Glu Gln Trp Val Asn Gly Arg Lys Lys

225 230 235 240

Leu Glu Glu Leu Glu Arg Glu Leu Arg Arg Ala Arg Lys Lys Ile Lys

245 250 255

Lys Leu Glu Asp Asp Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu

260 265 270

Gly Lys Lys Asp Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala

275 280 285

Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Pro Arg Lys Arg Pro Leu

290 295 300

Arg Gly Gly Phe Thr Asp Arg Glu Arg Gln Asp His Arg Arg Arg Lys

305 310 315 320

Ala Leu Lys Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ser Leu

325 330 335

Ser Lys Glu Glu Glu Glu Glu Leu Lys Arg Leu Thr Arg Glu Asp Glu

340 345 350

Glu Arg Lys Lys Glu Glu His Gly Pro Ser Arg Leu Gly Val Asn Pro

355 360 365

Ser Glu Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser

370 375 380

Met Gln Gly Ile Pro Glu Ser Arg Phe Thr Arg Thr Gly Glu Gly Leu

385 390 395 400

Asp Val Arg Gly Ser Arg Gly Phe Pro Gln Asp Ile Leu Phe Pro Ser

405 410 415

Asp Pro Pro Phe Ser Pro Gln Ser Cys Arg Pro Gln Gly Thr Asn Leu

420 425 430

Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro

435 440 445

Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn

450 455 460

Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser

465 470 475 480

Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala

485 490 495

Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys

500 505 510

Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

515 520

<210> 32

<211> 522

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-8 polypeptide sequence

<400> 32

Met Ala Ser Gln Ser Glu Thr Arg Arg Gly Arg Arg Gly Thr Arg Glu

1 5 10 15

Glu Thr Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys Ala Glu Glu Leu

20 25 30

Glu Lys Asp Leu Arg Lys Thr Arg Lys Thr Ile Lys Lys Leu Glu Glu

35 40 45

Glu Asn Pro Trp Leu Gly Asn Ile Val Gly Ile Ile Arg Lys Gly Lys

50 55 60

Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Gly Lys Arg Pro His Lys Ser Gly Phe Thr

85 90 95

Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Lys Lys Gln Leu Ser Ala Gly Gly Lys Ile Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Arg Leu Thr Asp Glu Asp Glu Glu Arg Lys Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Asp Val Asn Pro Ser Arg Gly Gly Pro

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Gln Met Ala Gly Val Pro

165 170 175

Glu Ser Pro Phe Ser Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Thr

180 185 190

Gln Gly Phe Pro Trp Val Ser Pro Ser Pro Pro Gln Gln Arg Leu Pro

195 200 205

Leu Leu Glu Cys Thr Pro Gln Ser Gln Ser Glu Ser Lys Lys Asn Arg

210 215 220

Arg Gly Gly Arg Glu Asp Ile Leu Glu Lys Trp Ile Thr Thr Arg Arg

225 230 235 240

Lys Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile

245 250 255

Lys Lys Leu Glu Asp Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly Ile

260 265 270

Ile Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro

275 280 285

Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro His

290 295 300

Lys Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys

305 310 315 320

Ala Leu Glu Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn Leu

325 330 335

Ser Arg Glu Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp Glu

340 345 350

Glu Arg Arg Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val Asn Leu

355 360 365

Ser Gly Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Arg

370 375 380

Met Glu Gly Val Pro Glu Ser Pro Phe Thr Arg Thr Gly Glu Gly Leu

385 390 395 400

Asp Ile Arg Gly Asn Gln Gly Phe Pro Trp Val Arg Pro Ser Pro Pro

405 410 415

Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln Gly Thr Asn Leu

420 425 430

Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro

435 440 445

Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn

450 455 460

Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser

465 470 475 480

Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala

485 490 495

Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys

500 505 510

Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

515 520

<210> 33

<211> 428

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-9 polypeptide sequence

<400> 33

Met Ala Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu

1 5 10 15

Ile Leu Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu

20 25 30

Arg Asp Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu

35 40 45

Asn Pro Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg

50 55 60

Glu Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr

85 90 95

Asp Lys Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Arg Lys Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro

165 170 175

Glu Ser Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn

180 185 190

Gln Gly Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser

195 200 205

Pro Gln Ser Cys Arg Pro Gln Ser Arg Ser Glu Ser Lys Lys Asn Arg

210 215 220

Gly Gly Arg Glu Glu Val Leu Glu Gln Trp Val Asn Gly Arg Lys Lys

225 230 235 240

Leu Glu Glu Leu Glu Arg Glu Leu Arg Arg Ala Arg Lys Lys Ile Lys

245 250 255

Lys Leu Glu Asp Asp Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu

260 265 270

Gly Lys Lys Asp Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala

275 280 285

Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Pro Arg Lys Arg Pro Leu

290 295 300

Arg Gly Gly Phe Thr Asp Arg Glu Arg Gln Asp His Arg Arg Arg Lys

305 310 315 320

Ala Leu Lys Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ser Leu

325 330 335

Ser Lys Glu Glu Glu Glu Glu Leu Lys Arg Leu Thr Arg Glu Asp Glu

340 345 350

Glu Arg Lys Lys Glu Glu His Gly Pro Ser Arg Leu Gly Val Asn Pro

355 360 365

Ser Glu Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser

370 375 380

Met Gln Gly Ile Pro Glu Ser Arg Phe Thr Arg Thr Gly Glu Gly Leu

385 390 395 400

Asp Val Arg Gly Ser Arg Gly Phe Pro Gln Asp Ile Leu Phe Pro Ser

405 410 415

Asp Pro Pro Phe Ser Pro Gln Ser Cys Arg Pro Gln

420 425

<210> 34

<211> 428

<212> PRT

<213> Artificial sequence

<220>

<223> Delta-10 polypeptide sequence

<400> 34

Met Ala Ser Gln Ser Glu Thr Arg Arg Gly Arg Arg Gly Thr Arg Glu

1 5 10 15

Glu Thr Leu Glu Lys Trp Ile Thr Ala Arg Lys Lys Ala Glu Glu Leu

20 25 30

Glu Lys Asp Leu Arg Lys Thr Arg Lys Thr Ile Lys Lys Leu Glu Glu

35 40 45

Glu Asn Pro Trp Leu Gly Asn Ile Val Gly Ile Ile Arg Lys Gly Lys

50 55 60

Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met

65 70 75 80

Glu Val Asp Ser Gly Pro Gly Lys Arg Pro His Lys Ser Gly Phe Thr

85 90 95

Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys

100 105 110

Lys Lys Gln Leu Ser Ala Gly Gly Lys Ile Leu Ser Lys Glu Glu Glu

115 120 125

Glu Glu Leu Arg Arg Leu Thr Asp Glu Asp Glu Glu Arg Lys Arg Arg

130 135 140

Val Ala Gly Pro Arg Val Gly Asp Val Asn Pro Ser Arg Gly Gly Pro

145 150 155 160

Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Gln Met Ala Gly Val Pro

165 170 175

Glu Ser Pro Phe Ser Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Thr

180 185 190

Gln Gly Phe Pro Trp Val Ser Pro Ser Pro Pro Gln Gln Arg Leu Pro

195 200 205

Leu Leu Glu Cys Thr Pro Gln Ser Gln Ser Glu Ser Lys Lys Asn Arg

210 215 220

Arg Gly Gly Arg Glu Asp Ile Leu Glu Lys Trp Ile Thr Thr Arg Arg

225 230 235 240

Lys Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Ala Arg Lys Thr Ile

245 250 255

Lys Lys Leu Glu Asp Glu Asn Pro Trp Leu Gly Asn Ile Ile Gly Ile

260 265 270

Ile Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Pro

275 280 285

Arg Thr Asp Gln Met Glu Ile Asp Ser Gly Thr Gly Lys Arg Pro His

290 295 300

Lys Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg Lys

305 310 315 320

Ala Leu Glu Asn Lys Lys Lys Gln Leu Ser Ser Gly Gly Lys Asn Leu

325 330 335

Ser Arg Glu Glu Glu Glu Glu Leu Gly Arg Leu Thr Val Glu Asp Glu

340 345 350

Glu Arg Arg Arg Arg Val Ala Gly Pro Arg Thr Gly Asp Val Asn Leu

355 360 365

Ser Gly Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Arg

370 375 380

Met Glu Gly Val Pro Glu Ser Pro Phe Thr Arg Thr Gly Glu Gly Leu

385 390 395 400

Asp Ile Arg Gly Asn Gln Gly Phe Pro Trp Val Arg Pro Ser Pro Pro

405 410 415

Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln

420 425

<210> 35

<211> 3434

<212> DNA

<213> Artificial sequence

<220>

<223> Fusion nucleic acid sequence

Delta-7/Delta-8 wild type

<400> 35

aagcttgcac catggccggc accaacctga gcaccagcaa cccgctgggc ttttttccgg

60

atcatcagct ggatccggcg tttcgcgcga acagcgcgaa cccggattgg gattttaacc

120

cgaacaaaga tacctggccg gatgcgaaca aagtgggcgg ccagaacctg agcaccagca

180

acccgctggg cttttttccg gatcatcagc tggatccggc gtttcgcgcg aacaccgcga

240

acccggattg ggattttaac ccgaacaaag atacctggcc ggatgcgaac aaagtgggca

300

gccgcagcga aagcaaaaaa aaccgcggcg gccgcgaaga aattctggaa cagtgggtgg

360

gcgcgcgcaa aaaactggaa gaactggaac gcgatctgcg caaaattaaa aaaaaaatta

420

aaaaactgga agaagaaaac ccgtggctgg gcaacattaa aggcattctg ggcaaaaaag

480

atcgcgaagg cgaaggcgcg ccgccggcga aacgcgcgcg cgcggatcag atggaagtgg

540

atagcggccc gcgcaaacgc ccgtttcgcg gcgaatttac cgataaagaa cgccgcgatc

600

atcgccgccg caaagcgctg gaaaacaaac gcaaacagct gagcagcggc ggcaaaagcc

660

tgagcaaaga agaagaagaa gaactgcgca aactgaccga agaagatgaa cgccgcgaac

720

gccgcgtggc gggcccgcgc gtgggcggcg tgaacccgct ggaaggcggc acccgcggcg

780

cgccgggcgg cggctttgtg ccgagcatgc agggcgtgcc ggaaagcccg tttgcgcgca

840

ccggcgaagg cctggatgtg cgcggcaacc agggctttcc gtgggatatt ctgtttccgg

900

cggatccgcc gtttagcccg cagagctgcc gcccgcagag ccgcagcgaa agcaaaaaaa

960

accgcggcgg ccgcgaagaa gtgctggaac agtgggtgaa cggccgcaaa aaactggaag

1020

aactggaacg cgaactgcgc cgcgcgcgca aaaaaattaa aaaactggaa gatgataacc

1080

cgtggctggg caacgtgaaa ggcattctgg gcaaaaaaga taaagatggc gaaggcgcgc

1140

cgccggcgaa acgcgcgcgc accgatcaga tggaaattga tagcggcccg cgcaaacgcc

1200

cgctgcgcgg cggctttacc gatcgcgaac gccaggatca tcgccgccgc aaagcgctga

1260

aaaacaaaaa aaaacagctg agcgcgggcg gcaaaagcct gagcaaagaa gaagaagaag

1320

aactgaaacg cctgacccgc gaagatgaag aacgcaaaaa agaagaacat ggcccgagcc

1380

gcctgggcgt gaacccgagc gaaggcggcc cgcgcggcgc gccgggcggc ggctttgtgc

1440

cgagcatgca gggcattccg gaaagccgct ttacccgcac cggcgaaggc ctggatgtgc

1500

gcggcagccg cggctttccg caggatattc tgtttccgag cgatccgccg tttagcccgc

1560

agagctgccg cccgcagggc accaacctga gcaccagcaa cccgctgggc ttttttccgg

1620

atcatcagct ggatccggcg tttcgcgcga acagcgcgaa cccggattgg gattttaacc

1680

cgaacaaaga tacctggccg gatgcgaaca aagtgggcgg ccagaacctg agcaccagca

1740

acccgctggg cttttttccg gatcatcagc tggatccggc gtttcgcgcg aacaccgcga

1800

acccggattg ggattttaac ccgaacaaag atacctggcc ggatgcgaac aaagtgggca

1860

gccagagcga aacccgccgc ggccgccgcg gcacccgcga agaaaccctg gaaaaatgga

1920

ttaccgcgcg caaaaaagcg gaagaactgg aaaaagatct gcgcaaaacc cgcaaaacca

1980

ttaaaaaact ggaagaagaa aacccgtggc tgggcaacat tgtgggcatt attcgcaaag

2040

gcaaagatgg cgaaggcgcg ccgccggcga aacgcccgcg caccgatcag atggaagtgg

2100

atagcggccc gggcaaacgc ccgcataaaa gcggctttac cgataaagaa cgcgaagatc

2160

atcgccgccg caaagcgctg gaaaacaaaa aaaaacagct gagcgcgggc ggcaaaattc

2220

tgagcaaaga agaagaagaa gaactgcgcc gcctgaccga tgaagatgaa gaacgcaaac

2280

gccgcgtggc gggcccgcgc gtgggcgatg tgaacccgag ccgcggcggc ccgcgcggcg

2340

cgccgggcgg cggctttgtg ccgcagatgg cgggcgtgcc ggaaagcccg tttagccgca

2400

ccggcgaagg cctggatatt cgcggcaccc agggctttcc gtgggtgagc ccgagcccgc

2460

cgcagcagcg cctgccgctg ctggaatgca ccccgcagag ccagagcgaa agcaaaaaaa

2520

accgccgcgg cggccgcgaa gatattctgg aaaaatggat taccacccgc cgcaaagcgg

2580

aagaactgga aaaagatctg cgcaaagcgc gcaaaaccat taaaaaactg gaagatgaaa

2640

acccgtggct gggcaacatt attggcatta ttcgcaaagg caaagatggc gaaggcgcgc

2700

cgccggcgaa acgcccgcgc accgatcaga tggaaattga tagcggcacc ggcaaacgcc

2760

cgcataaaag cggctttacc gataaagaac gcgaagatca tcgccgccgc aaagcgctgg

2820

aaaacaaaaa aaaacagctg agcagcggcg gcaaaaacct gagccgcgaa gaagaagaag

2880

aactgggccg cctgaccgtg gaagatgaag aacgccgccg ccgcgtggcg ggcccgcgca

2940

ccggcgatgt gaacctgagc ggcggcggcc cgcgcggcgc gccgggcggc ggctttgtgc

3000

cgcgcatgga aggcgtgccg gaaagcccgt ttacccgcac cggcgaaggc ctggatattc

3060

gcggcaacca gggctttccg tgggtgcgcc cgagcccgcc gcagcagcgc ctgccgctgc

3120

tggaatgcac cccgcagggc accaacctga gcaccagcaa cccgctgggc ttttttccgg

3180

atcatcagct ggatccggcg tttcgcgcga acagcgcgaa cccggattgg gattttaacc

3240

cgaacaaaga tacctggccg gatgcgaaca aagtgggcgg ccagaacctg agcaccagca

3300

acccgctggg cttttttccg gatcatcagc tggatccggc gtttcgcgcg aacaccgcga

3360

acccggattg ggattttaac ccgaacaaag atacctggcc ggatgcgaac aaagtgggct

3420

gatgagaatt ccgt

3434

<210> 36

<211> 3431

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized nucleic acid sequence

merged Delta-7/Delta-8

<400> 36

aagcttgcac catggcaggc accaatctgt caacctctaa ccctctgggc tttttccccg

60

accatcagct ggaccccgca ttccgagcaa actccgctaa ccctgactgg gatttcaacc

120

caaataagga cacctggccc gatgccaata aggtcggcgg ccagaacctg tccacatcta

180

atccactggg cttctttccc gaccaccagc tggatcctgc cttccggggcc aacacagcca

240

atcctgactg ggacttcaac cctaataagg atacctggcc cgatgccaac aaggtcggca

300

gcaggtccga gtctaagaag aataggggag gaagggagga gatcctggag cagtgggtgg

360

gagcacgcaa gaagctggag gagctggagc gggacctgag aaagatcaag aagaagatca

420

agaagctgga ggaggagaac ccctggctgg gcaatatcaa gggcatcctg ggcaagaagg

480

atcgggaggg agagggagca ccacctgcaa agaggggccag agccgaccag atggaggtgg

540

attccggccc taggaagcgc ccattcagag gcgagtttac cgacaaggag cggagagatc

600

acaggcgccg gaaggccctg gagaacaagc ggaagcagct gagctccggc ggcaagagcc

660

tgtccaagga ggaggaggag gagctgagaa agctgacaga ggaggacgag agaagggaga

720

ggagggtggc aggacctagg gtgggaggcg tgaacccact ggagggagga accaggggag

780

cacctggagg aggattcgtg ccatccatgc agggagtgcc tgagtctcca tttgccagga

840

caggagaggg cctggacgtg cgcggaaatc agggcttccc ctgggacatc ctgtttcctg

900

ccgatccacc cttctcccct cagtcttgca ggccacagtc tcgcagcgag tccaagaaga

960

acagaggcgg aagggaggag gtgctggagc agtgggtgaa tggccggaag aagctggaag

1020

aactggagag ggagctgaga agggcccgca agaagatcaa gaagctggaa gacgataatc

1080

cttggctggg caatgtgaaa ggcatcctgg gcaagaagga caaggatgga gagggagcac

1140

ctccagcaaa gagggcaaga accgaccaga tggagatcga ttctggacca aggaagcgcc

1200

ctctgagagg aggcttcaca gaccgggaga gacaggatca ccgccggaga aaggccctga

1260

agaacaagaa gaagcagctg agcgccggcg gcaagtctct gagtaaagaa gaagaggagg

1320

agctgaagcg gctgaccaga gaggacgagg agcggaagaa ggaggagcac ggaccaagca

1380

gactgggagt gaatccttcc gagggaggac caagaggagc acccggagga ggcttcgtgc

1440

catctatgca gggcatcccc gagagcaggt ttaccagaac cggcgaaggc ctggacgtgc

1500

ggggctccag aggctttcct caggacatcc tgttcccatc tgatccccct tttagccccc

1560

agtcctgtag gcctcagggc accaacctgt ccacatctaa ccctctgggc ttctttcctg

1620

atcatcagct ggacccagcc ttccgcgcca acagcgccaa tcccgattgg gacttcaacc

1680

caaataagga tacctggcca gacgctaaca aggtcggagg acagaacctg agcacatcca

1740

atcctctggg cttctttcca gaccaccagc tggatccagc cttcagggct aatacagcca

1800

atcccgactg ggacttcaac ccaaataagg acacgtggcc agacgcaaac aaggtcggct

1860

ctcagagcga gacaaggagg ggccggagag gaaccaggga ggagacactg gagaagtgga

1920

tcaccgccag aaagaaggcc gaggagctgg agaaggacct gaggaagacc cgcaagacaa

1980

tcaagaagct ggaagaagag aacccatggc tgggcaatat cgtgggcatc atccgcaagg

2040

gcaaggacgg cgagggagca ccaccagcaa agaggcccag gactgatcag atggaggtgg

2100

atagcggccc tggcaagagg ccacacaagt ccggcttcac agacaaggag agggaggacc

2160

ataggcgccg gaaggccctg gaaaacaaga agaagcaatt atccgccggc ggcaagatcc

2220

tgtctaaaga agaggaagaa gagctgagaa ggctgaccga cgaggatgag gagaggaaaa

2280

gaagggtggc aggaccaaga gtgggcgacg tgaatccctc cagaggcgga ccaagaggag

2340

cacctggagg cggcttcgtg ccccagatgg ccggcgtgcc cgagtctcct tttagcagaa

2400

ccggagaggg cctggatatc aggggaaccc agggctttcc atgggtgtcc ccatctcctc

2460

cacagcagcg gctgccactg ctggagtgca cccctcagag cccagcgaa tctaagaaga

2520

acagaagggg cggcagagag gacatcctgg aaaaatggat caccacacgc agaaaagctg

2580

aagaactgga aaaggacctg cggaaggcca gaaaaacaat caagaagctg gaggatgaaa

2640

atccatggct gggaaacatc atcggcatca tcagaaaggg caaggacggg gaaggcgcac

2700

cacctgcaaa gcggcctaga acagatcaga tggaaatcga ttctggaacc ggcaagcggc

2760

cacacaagag cggcttcacc gacaaggaga gagaggatca cagaaggcgc aaggccctgg

2820

agaacaagaa gaagcaatta agcagcggcg gcaagaatct gtccagagaa gaggaagagg

2880

agctgggcag actgaccgtg gaggacgagg agcggagaag gcgcgtggca ggacccagga

2940

caggcgatgt gaacctgagc ggaggaggac ctaggggagc accaggaggc ggcttcgtgc

3000

ctaggatgga gggagtgcca gagtccccct ttaccagaac cggcgagggc ctggacatca

3060

ggggaaatca gggattccca tgggtgcggc cttccccacc acagcagaga ctgccactgc

3120

tggagtgtac cccccagggc acaaacctga gcacctccaa tcccctgggc ttctttcctg

3180

accatcagct ggaccctgcc ttcagggcca acagcgccaa cccagattgg gacttcaacc

3240

ctaataagga cacctggcca gatgcaaaca aggtcggcgg ccaaaacctg tctacaagca

3300

accccctggg cttctttcca gatcaccagc tggatcccgc ctttcgcgcc aataccgcca

3360

accctgattg ggatttcaac cctaacaaag acacttggcc tgacgctaat aaggtcggct

3420

gatgagaatt c

3431

<210> 37

<211> 1136

<212> PRT

<213> Artificial sequence

<220>

<223> Polypeptide sequence of Delta-7/Delta-8 fusion

<400> 37

Met Ala Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro

1 5 10 15

Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser Ala Asn Pro Asp

20 25 30

Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val

35 40 45

Gly Gly Gln Asn Leu Ser Thr Ser Asn Pro Leu Gly Phe Phe Pro Asp

50 55 60

His Gln Leu Asp Pro Ala Phe Arg Ala Asn Thr Ala Asn Pro Asp Trp

65 70 75 80

Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro Asp Ala Asn Lys Val Gly

85 90 95

Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly Arg Glu Glu Ile Leu

100 105 110

Glu Gln Trp Val Gly Ala Arg Lys Lys Leu Glu Glu Leu Glu Arg Asp

115 120 125

Leu Arg Lys Ile Lys Lys Lys Ile Lys Lys Leu Glu Glu Glu Asn Pro

130 135 140

Trp Leu Gly Asn Ile Lys Gly Ile Leu Gly Lys Lys Asp Arg Glu Gly

145 150 155 160

Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Ala Asp Gln Met Glu Val

165 170 175

Asp Ser Gly Pro Arg Lys Arg Pro Phe Arg Gly Glu Phe Thr Asp Lys

180 185 190

Glu Arg Arg Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Arg Lys

195 200 205

Gln Leu Ser Ser Gly Gly Lys Ser Leu Ser Lys Glu Glu Glu Glu Glu

210 215 220

Leu Arg Lys Leu Thr Glu Glu Asp Glu Arg Arg Glu Arg Arg Val Ala

225 230 235 240

Gly Pro Arg Val Gly Gly Val Asn Pro Leu Glu Gly Gly Thr Arg Gly

245 250 255

Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln Gly Val Pro Glu Ser

260 265 270

Pro Phe Ala Arg Thr Gly Glu Gly Leu Asp Val Arg Gly Asn Gln Gly

275 280 285

Phe Pro Trp Asp Ile Leu Phe Pro Ala Asp Pro Pro Phe Ser Pro Gln

290 295 300

Ser Cys Arg Pro Gln Ser Arg Ser Glu Ser Lys Lys Asn Arg Gly Gly

305 310 315 320

Arg Glu Glu Val Leu Glu Gln Trp Val Asn Gly Arg Lys Lys Leu Glu

325 330 335

Glu Leu Glu Arg Glu Leu Arg Arg Ala Arg Lys Lys Ile Lys Lys Leu

340 345 350

Glu Asp Asp Asn Pro Trp Leu Gly Asn Val Lys Gly Ile Leu Gly Lys

355 360 365

Lys Asp Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg Ala Arg Thr

370 375 380

Asp Gln Met Glu Ile Asp Ser Gly Pro Arg Lys Arg Pro Leu Arg Gly

385 390 395 400

Gly Phe Thr Asp Arg Glu Arg Gln Asp His Arg Arg Arg Lys Ala Leu

405 410 415

Lys Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ser Leu Ser Lys

420 425 430

Glu Glu Glu Glu Glu Leu Lys Arg Leu Thr Arg Glu Asp Glu Glu Arg

435 440 445

Lys Lys Glu Glu His Gly Pro Ser Arg Leu Gly Val Asn Pro Ser Glu

450 455 460

Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro Ser Met Gln

465 470 475 480

Gly Ile Pro Glu Ser Arg Phe Thr Arg Thr Gly Glu Gly Leu Asp Val

485 490 495

Arg Gly Ser Arg Gly Phe Pro Gln Asp Ile Leu Phe Pro Ser Asp Pro

500 505 510

Pro Phe Ser Pro Gln Ser Cys Arg Pro Gln Gly Thr Asn Leu Ser Thr

515 520 525

Ser Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe

530 535 540

Arg Ala Asn Ser Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp

545 550 555 560

Thr Trp Pro Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser Thr Ser

565 570 575

Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg

580 585 590

Ala Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr

595 600 605

Trp Pro Asp Ala Asn Lys Val Gly Ser Gln Ser Glu Thr Arg Arg Gly

610 615 620

Arg Arg Gly Thr Arg Glu Glu Thr Leu Glu Lys Trp Ile Thr Ala Arg

625 630 635 640

Lys Lys Ala Glu Glu Leu Glu Lys Asp Leu Arg Lys Thr Arg Lys Thr

645 650 655

Ile Lys Lys Leu Glu Glu Glu Asn Pro Trp Leu Gly Asn Ile Val Gly

660 665 670

Ile Ile Arg Lys Gly Lys Asp Gly Glu Gly Ala Pro Pro Ala Lys Arg

675 680 685

Pro Arg Thr Asp Gln Met Glu Val Asp Ser Gly Pro Gly Lys Arg Pro

690 695 700

His Lys Ser Gly Phe Thr Asp Lys Glu Arg Glu Asp His Arg Arg Arg

705 710 715 720

Lys Ala Leu Glu Asn Lys Lys Lys Gln Leu Ser Ala Gly Gly Lys Ile

725 730 735

Leu Ser Lys Glu Glu Glu Glu Glu Leu Arg Arg Leu Thr Asp Glu Asp

740 745 750

Glu Glu Arg Lys Arg Arg Val Ala Gly Pro Arg Val Gly Asp Val Asn

755 760 765

Pro Ser Arg Gly Gly Pro Arg Gly Ala Pro Gly Gly Gly Phe Val Pro

770 775 780

Gln Met Ala Gly Val Pro Glu Ser Pro Phe Ser Arg Thr Gly Glu Gly

785 790 795 800

Leu Asp Ile Arg Gly Thr Gln Gly Phe Pro Trp Val Ser Pro Ser Pro

805 810 815

Pro Gln Gln Arg Leu Pro Leu Leu Glu Cys Thr Pro Gln Ser Gln Ser

820 825 830

Glu Ser Lys Lys Asn Arg Arg Gly Gly Arg Glu Asp Ile Leu Glu Lys

835 840 845

Trp Ile Thr Thr Arg Arg Lys Ala Glu Glu Leu Glu Lys Asp Leu Arg

850 855 860

Lys Ala Arg Lys Thr Ile Lys Lys Leu Glu Asp Glu Asn Pro Trp Leu

865 870 875 880

Gly Asn Ile Ile Gly Ile Ile Arg Lys Gly Lys Asp Gly Glu Gly Ala

885 890 895

Pro Pro Ala Lys Arg Pro Arg Thr Asp Gln Met Glu Ile Asp Ser Gly

900 905 910

Thr Gly Lys Arg Pro His Lys Ser Gly Phe Thr Asp Lys Glu Arg Glu

915 920 925

Asp His Arg Arg Arg Lys Ala Leu Glu Asn Lys Lys Lys Gln Leu Ser

930 935 940

Ser Gly Gly Lys Asn Leu Ser Arg Glu Glu Glu Glu Glu Leu Gly Arg

945 950 955 960

Leu Thr Val Glu Asp Glu Glu Arg Arg Arg Arg Val Ala Gly Pro Arg

965 970 975

Thr Gly Asp Val Asn Leu Ser Gly Gly Gly Pro Arg Gly Ala Pro Gly

980 985 990

Gly Gly Phe Val Pro Arg Met Glu Gly Val Pro Glu Ser Pro Phe Thr

995 1000 1005

Arg Thr Gly Glu Gly Leu Asp Ile Arg Gly Asn Gln Gly Phe Pro

1010 1015 1020

Trp Val Arg Pro Ser Pro Pro Gln Gln Arg Leu Pro Leu Leu Glu

1025 1030 1035

Cys Thr Pro Gln Gly Thr Asn Leu Ser Thr Ser Asn Pro Leu Gly

1040 1045 1050

Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg Ala Asn Ser

1055 1060 1065

Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr Trp Pro

1070 1075 1080

Asp Ala Asn Lys Val Gly Gly Gln Asn Leu Ser Thr Ser Asn Pro

1085 1090 1095

Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala Phe Arg Ala

1100 1105 1110

Asn Thr Ala Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys Asp Thr

1115 1120 1125

Trp Pro Asp Ala Asn Lys Val Gly

1130 1135

<210> 38

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Ovalbumin 257-264 CTL

<400> 38

Ser Ile Ile Asn Phe Glu Lys Leu

15

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Ovalbumin 323-339 Th

<400> 39

Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly

1 5 10 15

Arg

<---

Claims (55)

1. A combination of products suitable for inducing an immune response to hepatitis B virus and hepatitis D virus, which contains: (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, preferably components (a) and (b) of said product combination being provided as separate compositions within said product combination. 2. The combination of products according to claim 1, characterized in that said at least one nucleic acid sequence encoding HDAg contains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or any combination thereof. 3. The combination of products according to claim 1 or 2, characterized in that said at least one nucleic acid sequence encoding PreS1 contains SEQ ID NO: 9 or SEQ ID NO: 10, or both. 4. Combination of products according to any one of paragraphs. 1-3, characterized in that said nucleic acid is designed such that each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and wherein said PreS1 nucleic acid sequence is located immediately downstream of the HDAg nucleic acid sequence. 5. The product combination of claim 4, further comprising at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site, wherein said grouped HDAg and PreS1 nucleic acid sequences are separated by said at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site. 6. The combination of products according to claim 5, characterized in that said at least one nucleic acid sequence encoding the site of autocatalytic cleavage of the peptide contains a nucleic acid sequence selected from the group consisting of porcine teschovirus 2A nucleic acids-1 (P2A), Foot and mouth disease virus 2A (F2A), equine rhinitis virus A (ERAV) 2A (E2A) and Thosea asigna virus 2A (T2A), and each encoded peptide autocatalytic cleavage site may optionally contain a GSG (glycine-serine-glycine) motif on its N-terminal 7. The combination of products according to claim 5 or 6, characterized in that said at least one nucleic acid sequence encoding the autocatalytic cleavage site of the peptide contains SEQ ID NO: 13. 8. Combination of products according to any one of paragraphs. 1-7, characterized in that said nucleic acid is codon-optimized for expression in humans. 9. Combination of products according to any one of paragraphs. 1-8, characterized in that said nucleic acid contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 15-24 or 35-36. 10. Combination of products according to any one of paragraphs. 1-9, characterized in that the specified nucleic acid contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 18 or SEQ ID NO: 35-36 . 11. Combination of products according to any one of paragraphs. 1-10, wherein said at least one HDAg polypeptide contains SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or any combination thereof. 12. Combination of products according to any one of paragraphs. 1-11, characterized in that said at least one PreS1 polypeptide sequence contains SEQ ID NO: 11 or SEQ ID NO: 12, or both. 13. Combination of products according to any one of paragraphs. 1-12, characterized in that said at least one PreS1 polypeptide sequence is located downstream of said at least one HDAg polypeptide sequence. 14. Combination of products according to any one of paragraphs. 1-13, characterized in that said polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 25-34 or 37. 15. Combination of products according to any one of paragraphs. 1-14, characterized in that said polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 29, 31, 32 or 37. 16. Combination of products according to any one of paragraphs. 1-15, characterized in that said polypeptide is expressed in a recombinant manner. 17. The product combination of claim 16, wherein said polypeptide is expressed recombinantly in a mammalian, bacterial, yeast, insect or cell-free system. 18. Combination of products according to any one of paragraphs. 1-17, additionally containing an adjuvant. 19. The product combination of claim 18, wherein said adjuvant is alum, QS-21 or MF59, or any combination thereof. 20. Combination of products according to any one of paragraphs. 1-19, characterized in that said nucleic acid includes DNA. 21. Combination of products according to any one of paragraphs. 1-20, characterized in that said nucleic acid is provided in a recombinant vector. 22. A method of inducing an immune response to hepatitis B virus and hepatitis D virus in a subject using a combination of products according to any one of paragraphs. 1-21, including: administering to said subject at least one priming dose containing said nucleic acid; And administering to said subject at least one stimulant dose containing said polypeptide. 23. The method according to claim 22, characterized in that said at least one stimulating dose additionally contains an adjuvant. 24. The method of claim 23, wherein said adjuvant is alum, QS-21 or MF59, or any combination thereof. 25. Method according to any one of paragraphs. 22-24, characterized in that said at least one stimulating dose is administered at least after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36 or 48 days or weeks after administration of said at least one priming dose or within a time range specified by any two of the above time points, for example, within 1-48 days or 1-48 weeks. 26. Method according to any one of paragraphs. 22-25, characterized in that said administration is provided enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally or intravenously, or any combination of these routes. 27. Method according to any one of paragraphs. 22-26, characterized in that said administration is carried out in combination with antiviral therapy. 28. The method of claim 27, wherein said antiviral therapy comprises administration of entecavir, tenofovir, lamivudine, adefovir, telbivudine, emtricitabine, interferon-α, pegylated interferon-α or interferon alfa-2b, or any combination thereof. 29. A combination of products for use in the treatment or prevention of hepatitis B virus (HBV) and hepatitis D virus (HDV) infection, containing: (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. 30. The combination of products for use in the treatment or prevention of HBV and HDV infection according to claim 29, characterized in that said at least one nucleic acid sequence encoding HDAg contains SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or any combination thereof. 31. The combination of products for use in the treatment or prevention of HBV and HDV infection according to claim 29 or 30, characterized in that said at least one nucleic acid sequence encoding PreS1 contains SEQ ID NO: 9 or SEQ ID NO: 10, or both. 32. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-31, wherein said nucleic acid is designed such that each HDAg nucleic acid sequence is grouped with a PreS1 nucleic acid sequence, and wherein said PreS1 nucleic acid sequence is located immediately downstream of said HDAg nucleic acid sequence. 33. The combination of products for use in the treatment or prevention of HBV and HDV infection according to claim 32, further comprising at least one nucleic acid sequence encoding an autocatalytic peptide cleavage site, wherein said grouped HDAg and PreS1 nucleic acid sequences are separated by said at least one a nucleic acid sequence encoding the site of autocatalytic cleavage of the peptide. 34. The combination of products for use in the treatment or prevention of HBV and HDV infection according to claim 33, characterized in that said at least one nucleic acid sequence encoding the autocatalytic cleavage site of the peptide contains a nucleic acid sequence selected from the group consisting of nucleic acid porcine teschovirus 1 acid 2A (P2A), foot and mouth disease virus 2A (F2A), equine rhinitis virus A (ERAV) 2A (E2A), and Thosea asigna virus 2A (T2A), and each encoded peptide autocatalytic cleavage site may optionally contain a motif GSG (glycine-serine-glycine) at its N-terminus. 35. The combination of products for use in the treatment or prevention of HBV and HDV infection according to claim 33 or 34, characterized in that said at least one nucleic acid sequence encoding a peptide autocatalytic cleavage site contains SEQ ID NO: 13. 36. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-35, characterized in that said nucleic acid is codon-optimized for expression in humans. 37. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-36, characterized in that said nucleic acid contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 15-24 or 35, 36. 38. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-37, characterized in that said nucleic acid contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 18 or SEQ ID NO: 35, 36 . 39. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-38, characterized in that said at least one HDAg polypeptide contains SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or any combination thereof. 40. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-39, characterized in that said at least one PreS1 polypeptide sequence contains SEQ ID NO: 11 or SEQ ID NO: 12, or both. 41. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-40, characterized in that said at least one PreS1 polypeptide sequence is located downstream of said at least one HDAg polypeptide sequence. 42. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-41, characterized in that said polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 25-34 or 37. 43. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-42, characterized in that said polypeptide contains a sequence that is at least 80%, 85%, 90%, 95%, 99% or 100% homologous to the sequence of SEQ ID NO: 29, 31, 32 or 37. 44. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-43, characterized in that said polypeptide is expressed in a recombinant manner. 45. The combination of products for use in the treatment or prevention of HBV and HDV infection according to claim 44, wherein said polypeptide is expressed recombinantly in a mammalian, bacterial, yeast, insect or cell-free system. 46. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-45, additionally containing an adjuvant. 47. The combination of products for use in the treatment or prevention of HBV and HDV infection according to claim 46, wherein said adjuvant is alum, QS-21 or MF59, or any combination thereof. 48. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-47, characterized in that said nucleic acid includes DNA. 49. A combination of products for use in the treatment or prevention of HBV and HDV infection according to any one of paragraphs. 29-48, characterized in that said nucleic acid is provided in a recombinant vector.