MEANS AND METHODS FOR TREATING HBV

Abstract

The present invention relates to an improved recombinant vaccination vector for the treatment or vaccination against hepatitis B virus (HBV) as well as pharmaceutical compositions or vaccines comprising said recombinant vaccination vector. The present invention also relates to a recombinant vaccination vector for use in a method of vaccination against HBV, as well as kits comprising a vaccine comprising the recombinant vaccination vector.

Claims

1. A recombinant vaccination vector expressing (a) an envelope protein (HBs-antigen) from hepatitis B virus serotype adw, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and (b) a core protein (HBc-antigen) from hepatitis B virus serotype ayw, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw; and at least one of the following: (c) an immunogenic envelope protein (HBs-antigen) from hepatitis B virus having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and/or (d) an immunogenic core protein (HBc-antigen) from hepatitis B virus having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; and/or (e) an immunogenic RT domain of a polymerase from hepatitis B virus having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

2. The recombinant vaccination vector of claim 1, wherein the HBs-antigen in (c) and/or the HBc-antigen in (d) is/are from hepatitis B virus genotype C.

3. The recombinant vaccination vector of claim 1, wherein the immunogenic HBs-antigen in (c) has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and/or the immunogenic HBc-antigen in (d) has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and/or the immunogenic RT domain of a polymerase in (e) has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

4. The recombinant vaccination vector of claim 1, wherein the core protein from hepatitis B virus serotype ayw in (b) is a C-terminally truncated core protein comprising or consisting of amino acids 1-149 of the HBc-antigen from hepatitis B virus genotype D serotype ayw.

5. The recombinant vaccination vector of claim 1, further expressing (f) a CD70.

6. The recombinant vaccination vector of claim 5, wherein the CD70 is a human CD70.

7. The recombinant vaccination vector of claim 5, wherein the CD70 has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26.

8. The recombinant vaccination vector of claim 1, wherein the recombinant vaccination vector is a virus, a virus like particle or a bacterium.

9. The recombinant vaccination vector of claim 1, wherein the recombinant vaccination vector is a MVA virus.

10. The recombinant vaccination vector of claim 1, wherein the recombinant vector is an attenuated Salmonella strain, a CMV-, a VSV-based vector, an Adenoviral vector or a Measles vector.

11. The MVA virus of claim 9, wherein at least one, preferably at least two, preferably at least three, preferably at least four, preferably five nucleic acid sequence(s) encoding for (a), (b), (c), (d), and/or (e) is/are comprised in one expression cassette.

12. The MVA virus of claim 9, wherein at least one of the nucleic acids encoding for (a), (b), (c), (d), and/or (e) is/are under control of a poxviral promoter, wherein the poxviral promoter is preferably P7.5 or PH5.

13. The MVA virus of claim 11, wherein die expression cassette encodes for an amino acid sequence set forth in SEQ ID NO: 7.

14. The MVA virus of claim 9, wherein a nucleic acid sequence encoding at least one of (a), (b), (c), (d), and/or (e) is inserted into deletion I (del I), deletion II (del II), deletion III (del III), deletion IV (del IV), deletion V (del V), or deletion VI (del VI), preferably deletion III (del III) of the MVA genome.

15. A MVA virus expressing (a) an envelope protein (HBs-antigen) from hepatitis B virus serotype adw, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and a MVA virus expressing (b) a core protein (HBc-antigen) from hepatitis B virus serotype ayw, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw; for use in a vaccination method against hepatitis B, wherein the method comprises: (i) administering to a subject (a) an envelope protein from hepatitis B virus serotype adw, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and/or (b) a core protein (HBc-antigen) from hepatitis B virus serotype ayw, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw; and (ii) administering the MVA virus expressing (a) and the MVA virus expressing (b) to the subject.

16. The MVA virus for use of claim 15, wherein the MVA virus expressing (a) and/or the MVA virus expressing (b) further expresses a CD70.

17. A MVA virus expressing (a) an envelope protein (HBs-antigen) from hepatitis B virus serotype adw, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and (b) a core protein (HBc-antigen) from hepatitis B virus serotype ayw, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw; for use in a vaccination method against hepatitis B, wherein the method comprises: (i) administering a subject (a) an envelope protein from hepatitis B virus serotype adw, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and/or (b) a core protein (HBc-antigen) from hepatitis B virus serotype ayw, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw; and (ii) administering the MVA virus to the subject.

18. The MVA virus for use of claim 17, wherein the MVA virus further expresses a CD70.

19. A MVA virus according to claim for use in therapy or vaccination.

20. The MVA virus for use according to claim 19 wherein the use is a vaccination method against hepatitis B, wherein the use comprises (i) administering to a subject (a) an envelope protein from hepatitis B virus serotype adw, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and/or (b) a core protein (HBc-antigen) from hepatitis B virus serotype ayw, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw; and (ii) administering the MVA virus to the subject.

21. The MVA virus for use according to claim 15, wherein the vaccination method is preferably a method for therapeutic vaccination.

22. The MVA virus for use according to claim 15, wherein (i) of the vaccination method is a priming step and (ii) of the vaccination method is a boosting step.

23. The MVA virus for use according to claim 15, wherein the envelope protein and/or the core protein in (i) is co-administered with at least one adjuvant, wherein the adjuvant is preferably selected from the group consisting of poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP), an immune stimulatory oligonucleotide, a toll like receptor (TLR) agonist, a saponin or combinations thereof, wherein the TLR agonist is preferably a TLR 3 agonist, a TLR 4 agonist, a TLR 7 agonist, a TLR 8 agonist, or a TLR 9 agonist, and wherein the immune stimulatory oligonucleotide is preferably poly I/C, CpG, a RIG-I ligand, a STING ligand, cyclic di-AMP, cyclic di-CMP, cyclic di-GMP, a TLR 7 agonist, a TLR 8 agonist, CTA1DD, or dmLT.

24. The MVA virus for use according to claim 23, wherein the adjuvant is PCEP and/or a CpG adjuvant.

25. The MVA virus for use according to claim 23, wherein the adjuvant is cyclic di-AMP.

26. The MVA virus for use according to claim 15, wherein (i) is conducted at least about 1 day before conducting (ii), preferably at least about 5 days, preferably at least about 1 week, preferably about 1 week to about 8 weeks, preferably about 2 weeks to about 5 weeks, preferably about 3 weeks to about 4 weeks.

27. The MVA virus for use according to claim 15, wherein the vaccination method further comprises after (i) and prior to (ii): (i) administering to a subject (a) an envelope protein from hepatitis B virus genotype A, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and/or (b) a core protein (HBc-antigen) from hepatitis B virus genotype D, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw, wherein (i) is preferably a boosting step.

28. The MVA virus for use of claim 27, wherein (i) is conducted at least about 1 day before conducting (i), preferably at least about 5 days, preferably at least about 1 week, preferably about 1 week to about 8 weeks, preferably about 2 weeks to about 5 weeks, preferably about 3 weeks to about 4 weeks, and wherein (i) is conducted at least about 1 day before conducting (ii), preferably at least about 5 days, preferably at least about 1 week, preferably about 1 week to about 8 weeks, preferably about 2 weeks to about 5 weeks, preferably about 3 weeks to about 4 weeks.

29. The MVA virus for use of claim 15, wherein administration is by a parenteral or mucosal route.

30. The MVA virus for use of claim 29, wherein administration is intramuscular, and wherein step (i) and/or (i) comprises administration of an adjuvant, wherein the adjuvant comprises cyclic di-AMP.

31. The MVA virus for use of claim 29, wherein administration is subcutaneous or intramuscular, and wherein step (i) and/or (i) comprises administration of an adjuvant, wherein the adjuvant comprises poly I/C or RIG-I-ligand.

32. A vaccine or a pharmaceutical composition comprising the recombinant vaccination vector or the MVA virus of claim 1.

33. The vaccine of claim 32, wherein the recombinant vaccination vector is a MVA virus or a Salmonella strain.

34. The vaccine of claim 32, wherein the vaccine is a parenteral or mucosal vaccine.

35. A kit comprising: (i) a protein composition comprising: (a) an envelope protein from hepatitis B virus genotype A, wherein the envelope protein is preferably a small or large envelope protein from hepatitis B virus genotype A serotype adw, wherein the small or large envelope protein is preferably a small envelope protein; and/or (b) a core protein (HBc-antigen) from hepatitis B virus genotype D, wherein the core protein is preferably from hepatitis B virus genotype D serotype ayw; (ii) a vaccine of claim 32.

36. The kit of claim 35, wherein the protein composition is suitable for parenteral administration, wherein the composition preferably comprises at least one adjuvant that is PCEP and/or a CpG adjuvant.

37. The kit of claim 35, wherein the protein composition is suitable for mucosal administration, wherein the composition preferably comprises an adjuvant selected from the group consisting of CTA1DD, dmLT, PCEP, poly I/C, RIG-I-ligand, c-di-AMP, c-di-CMP and c-diGMP or combinations thereof.

38. The kit of claim 35, wherein the protein composition is suitable for intramuscular administration, wherein the composition preferably comprises at least one adjuvant that is cyclic di-AMP.

39. The kit of claim 35, wherein the protein composition is suitable for subcutaneous or intramuscular administration, wherein the composition preferably comprises at least one adjuvant that is poly I/C.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0096] FIG. 1: Protein-prime/MVA-boost vaccination is highly immunogenic. (A) S and core open reading frames of HBV subtype ayw were inserted into deletion III (del III) of the MVA genome under control of poxviral promoters P7.5 and PH5, respectively. (B) Wildtype mice (n=3) were vaccinated once (day 0; filled bars) or twice (day 0 and 21; striped bars) with MVA-S (110.sup.8 i. u.), MVA-core (110.sup.8 i. u.) or MVAwt (110.sup.8 i. u.). On day 8 (post prime) or 27(post boost), splenocytes were stimulated with HBsAg (S.sub.190 and S.sub.208)- or HBcAg (C.sub.93)-derived peptides and analyzed for IFN expression by intracellular cytokine staining. (C) C57BL/6 mice were vaccinated with 12 g recombinant HBsAg or HBcAg. CpG was used as adjuvant. On day 8, splenocytes were stimulated with HBsAg- or HBcAg-derived peptides and analyzed for IFN expression by ICS. (D) C57BL/6 mice were prime vaccinated with 12 g recombinant, CpG adjuvanted HBcAg or HBsAg, and on day 21, boosted with MVA-core (110.sup.8 i. u.) or MVA-S (110.sup.8 i. u.). On day 27, splenocytes were stimulated with HBsAg- or HBcAg-derived peptides and analyzed for IFN expression by ICS. Sera were analyzed for anti-HBs by immunoassay (middle panel) or anti-HBc by competitive ELISA (right panel). Frequencies of IFN-producing T-cells shown are background subtracted. S/CO signal to cutoff; i. u. infectious units.

[0097] FIG. 2: Vaccination-induced HBV-specific antibody- and CD4+ T-cell responses inversely correlate with antigenemia. (A-D) HBVtg mice of low, medium and high antigenemia (n=4) were immunized with CpG adjuvanted HBsAg or HBcAg. On day 21, mice were boosted with MVA-S (110.sup.8 i. u.) or MVA-core (110.sup.8 i. u.), respectively. On day 27 (day 6 post boost), sera were analyzed for (A) anti-HBs and (B) anti-HBc antibodies. (C) Splenocytes and (D) liver-associated lymphocytes of low-antigenemic HBVtg mice were stimulated with HBsAg and analyzed for IFN-expressing CD4+ T-cells by intracellular cytokine staining. Frequencies of IFN-producing T-cells shown are background subtracted. S/CO signal to cutoff; neg.=negative; i. u. infectious units.

[0098] FIG. 3: Vaccination-induced HBV-specific CD8+ T-cell frequencies inversely correlate with antigenemia. HBVtg mice of low, medium and high antigenemia (n=4) were immunized with 12 g CpG adjuvanted HBsAg or HBcAg. On day 21, mice were boosted with MVA-S (110.sup.8 i. u.) or MVA-core (110.sup.8 i. u.). On day 27 post boost, splenocytes (A) and liver-associated lymphocytes (B) were stimulated with HBsAg (S.sub.109 and S.sub.208)- or HBcAg (C.sub.93)-specific peptides and analyzed for IFN expression by intracellular cytokine staining. Upper and middle panels show exemplary animals, the lowest panel gives frequencies of IFN-producing T-cells after background subtraction. i. u. infectious units.

[0099] FIG. 4: Functionality of vaccination-induced HBV-specific CD8+ T-cells. Wildtype mice as well as low and medium antigenemic HBVtg mice were immunized with CpG adjuvanted HBsAg. On day 21, mice were boosted with MVA-S (110.sup.8 i. u.). On day 6 post boost, S-specific spleen-derived CD8+ T-cells were analyzed (A) for CD107a and IFN expression. (B) S-specific spleen or liver derived CD8+ T-cells were analyzed for IFN-, IL-2- and TNFa-expression after stimulation with peptide S.sub.190. (C) Multimer-staining of S.sub.190-specific CD8+ T cells and co-staining for CD127 and KLRG-1 surface expression. i. u. infectious units.

[0100] FIG. 5: Heterologous vaccination breaks T-cell tolerance in high-antigenemic HBVtg mice. (A) S subtype ayw and adw open reading frames were placed into deletion III (del III) of the MVA genome under control of the strong poxvirus promoter PH5. (B) to (D) High-antigenemic HBVtg mice (n>4) were vaccinated with 16 g HBsAg (subtype ayw or adw) and 16 g HBcAg (subtype ayw) together with the indicated adjuvants on days 0 and 14. On day 28, mice were boosted with MVA-PH5-S (510.sup.7 i. u.; subtype ayw or adw) and MVA-core (510.sup.7 i. u.). On day 6 post boost (B) splenocytes (left panel) and liver-associated lymphocytes (LAL, right panel) were isolated and stimulated with peptides S.sub.109 and S.sub.208 (subtype adw if indicated) or C.sub.93 and analyzed for IFN expression by intracellular cytokine staining. (C) Splenocytes and LAL were stimulated with a C-terminal pool of HBsAg-specific 15-mer peptides (covering amino acids 145 to 226, subtype ayw) and analyzed for IFN-expressing CD4+ T-cells by ICS. (D) Representative FACS plot of S.sub.208-specific CD8+ T-cells expressing IFN were analyzed for TNFa and IL-2 expression. MeanSEM of IFN-producing T cell frequencies are shown background subtraction. ns: not significant. * p<0.05, ** p<0.01 students t-test; i. u. infectious units

[0101] FIG. 6: Heterologous vaccination induces seroconversion in high-antigenemic HBVtg mice. (A) to (D) High-antigenemic HBVtg mice (n>4) were vaccinated with 16 g HBcAg (subtype ayw) and 16 g HBsAg (subtype ayw or adw as indicated) adjuvanted with CpG and PCEP on days 0 and 14. On day 28, mice were boosted with MVA-PH5-S (510.sup.7 i.u.; subtype ayw or adw) and MVA-core (510.sup.7 i.u.). As control, 5 high-antigenemic HBVtg mice were vaccinated with adjuvants CpG and PCEP on days 0 and 14 followed by a boost with 110.sup.8 MVA-wt. On day 6 post boost, sera were analyzed for presence of (A) anti-HBs or (B) anti-HBc antibodies. (C) Anti-HBs positive sera from HBVtg mice were analyzed for neutralization capacity of subtype ayw HBsAg. (D) shows serum HBsAg levels before and after vaccination. MeanSEM is shown. ns: not significant S/CO signal to cutoff. ** p<0.01 students t-test.

[0102] FIG. 7: Expression of HBV antigens by MVA vectors. (A) and (B) Murine NIH-3T3 cells were infected with MVA-S, MVA-core or MVAwt (MOI of 10). 16 h post infection (A) total cellular lysates were analyzed for HBsAg and HBcAg expression by Western blot. (B) secreted HBsAg in the supernatant was determined by HBsAg-specific ELISA. S/CO: signal to cutoff.

[0103] FIG. 8: Vaccination with CpG adjuvanted HBsAg. High-antigenemic HBVtg mice were immunized with 12 g HBsAg containing CpG as adjuvant. On day 21, mice were boosted with MVA-S. On days 0, 27, 35, 42, 49 and 56 post prime immunization, sera were analyzed for levels of HBsAg and anti-HBs. S/CO: signal to cutoff.

[0104] FIG. 9: Comparison of adjuvants for protein-prime vaccination. (A) to (C) Wildtype mice were vaccinated with 16 g HBsAg (subtype ayw) and 16 g HBcAg (subtype ayw) together with the indicated adjuvant(s) on days 0. On day 28, mice were boosted with MVA-PH5-S (510.sup.7 i.u.; subtype ayw) and MVA-core (510.sup.7 i.u.). On day 6 post boost sera were analyzed for presence of (A) anti-HBs and (B) anti-HBc. (C) On day 6 post boost splenocytes were analyzed by ICS after stimulation with HBsAg (S.sub.190 and S.sub.208adw)- or HBcAg (C.sub.93)-specific peptides. Bars show percentage (meanSEM) of CD8+cells staining positive for IFN after background subtraction. i. u. infectious units.

[0105] FIG. 10: Grouping of HBVtg mice.

[0106] Mice were grouped according to their antigen levels which correlates in the case of HBVtg mice closely to the virus titers determined in serum.

[0107] FIG. 11: Peptides used for stimulating T-cells.

[0108] The table gives sequences of peptides used to determine CD8+ T cell responses restricted by the murine K.sup.b/K.sup.d MHS-I alleles. The S pool was used to broadly determine CD4+ and CD8+ T cell responses of mice and humans.

[0109] FIG. 12: Graphical overview of the vaccination scheme of Example 4.

[0110] High-antigenemic HBVtg mice (were vaccinated with 16 g HBsAg (subtype ayw or adw) and 16 g HBcAg (subtype ayw) together with the indicated adjuvants on days 0 and 14. On day 28, mice were boosted with MVA-PH5-S (510e7 i.u.; subtype ayw or adw) and MVA-core (510e7 i.u.). On day 6 post boost, (B) splenocytes (left panel) and liver-associated lymphocytes (LAL, right panel) were isolated and stimulated with peptides S109 and S208 (subtype adw if indicated) or C93 and analyzed for IFN expression by intracellular cytokine staining (ICS).

[0111] FIG. 13: Correlation of multimer and intracellular cytokine stainings of HBV specific CD8+ T cells. HBVtg mice were immunized with 12 g HBsAg containing CpG as adjuvant. On day 21, mice were boosted with MVA-S. HBV-specific T cell responses were detected at day 28 by either S190 multimer staining or ICS after ex vivo restimulation with peptide S190.

[0112] FIG. 14: Detection of serum immune complexes. HBVtg mice were immunized with CpG adjuvanted HBsAg. On day 21, mice were boosted with MVA-S (110.sup.8 i. u.). On day 6 post boost, sera were analyzed for anti-HBs, HBsAg and HBsAg in precipitated immune complexes. i. u.; infectious units; nd; not detectable

[0113] FIG. 15: Comparison of different vaccine adjuvants. Wild type CH57131/6 mice were immunized with HBcAg and HBsAg complexed in different adjuvant formulations. 1:CpG plus alumn hydroxide; 2: polyphosphazene PCEP plus alum; 3: PCEP plus CpG plus alumn; 4: CpG only; 5: PCEP only; 6: CpG plus PCEP. At day 28, all animals were boosted with MVA expressing core and S subtype adw. FIG. 15A-B shows anti-HBs (FIG. 15A) and anti-HBc (FIG. 15B) antibody responses after 28 (protein prime only) and 34 days (protein prime plus MVA boost). FIG. 15C-D shows CD8+ T cell responses against S and core epitopes after prime (FIG. 15C) and after the MVA-boost (FIG. 15D) FIG. 15E shows a neutralization assay in which HBV subtype ayw was incubated with indicated serum dilutions before infection and HBsAg secretion by infected cells was measured after 4, 7 and 10 days.

[0114] FIG. 16: Multiantigenic open reading frame. FIG. 16A depicts the structure of the multiantigenic polypeptide chain represented by SEQ ID NO: 07. FIG. 16B schematically depicts the formation of subviral particles comprising HBs A/adw and C/ayw antigens. FIG. 16C schematically depicts the formation of empty capsids comprising HBc D/ayw and C/ayw antigens.

[0115] FIG. 17: Schematic illustration of completely processed proteins derived from the multiantigenic polypeptide chain represented by SEQ ID NO: 07 and their fate.

[0116] FIG. 18: Schematic illustration of partially unprocessed proteins derived from the multiantigenic polypeptide chain represented by SEQ ID NO: 07 and their expected fate. Most of the partially unprocessed are assumed to increase the immune response (especially enhance and broaden the adaptive immune response) due to incorporation into secreted virus-like particles/filaments:

[0117] FIG. 19: Amino acid sequence of small envelope protein of HBV A2/adw2 including C-terminal overhang (SEQ ID NO: 10). The underlined sequence corresponds to the amino acid sequence of small envelope protein of HBV A2/adw2 without C-terminal overhang (SEQ ID NO: 08). The C-terminal overhang is a P2A cleavage fragment that corresponds to SEQ ID NO: 09.

[0118] FIG. 20: Amino acid sequence of core protein fragment 1-149 of HBV D/ayw including N- and C-terminal overhangs (SEQ ID NO: 12). The underlined sequence corresponds to the amino acid sequence of core protein fragment 1-149 of HBV D/ayw without N- and C-terminal overhangs (SEQ ID NO: 11). The C-terminal overhang is a P2A cleavage fragment that corresponds to SEQ ID NO: 09.

[0119] FIG. 21: Amino acid sequence of RT domain of HBV polymerase including N- and C-terminal overhangs (SEQ ID NO: 16). The underlined sequence corresponds to the amino acid sequence of RT domain of HBV polymerase without N- and C-terminal overhangs (SEQ ID NO: 06). The C-terminal overhang is a T2A cleavage fragment that corresponds to SEQ ID NO: 13.

[0120] FIG. 22: Amino acid sequence of large envelope protein of HBV C/ayw including N- and C-terminal overhangs (SEQ ID NO: 14). The underlined sequence corresponds to the amino acid sequence of large envelope protein of HBV C/ayw without N- and C-terminal overhangs (SEQ ID NO: 04). The C-terminal overhang is a T2A cleavage fragment that corresponds to SEQ ID NO: 13.

[0121] FIG. 23: Amino acid sequence of core protein of HBV C/ayw including N-terminal overhang (SEQ ID NO: 15). The underlined sequence corresponds to the amino acid sequence of core protein of HBV C/ayw without N-terminal overhangs (SEQ ID NO: 05).

[0122] FIG. 24: Amino acid sequence of consensus sequence of RT-domain of HBV polymerase (SEQ ID NO: 03)

[0123] FIG. 25: Amino acid sequence of Consensus sequence of large envelope proteins of genotype C HBV strains (SEQ ID NO: 01).

[0124] FIG. 26: Amino acid sequence of consensus sequence of core protein of genotype C HBV strains (SEQ ID NO: 02).

[0125] FIG. 27: Combination of RNAi and therapeutic vaccination. (A) Experimental set-up. HBVxfs transgenic mice received HBV-specific siRNA (siHBV), an irrelevant siRNA (siNEG) or were left untreated. Eight weeks later, all mice received protein primeMVA boost therapeutic immunization with HBV core and surface antigens (HBcAg and HBsAg). (B) Schematic illustration of the HBV-specific siRNA/shRNA design.

[0126] FIG. 28: HBV antigen levels and antibody response and CD8+ T cell responses. (A) Kinetics of serum HBeAg and HBeAg levels. (B) Anti-HBs antibodies in the serum of mice at the time point of sacrifice (day 91, week (W)13). (C) CD8+ T cell responses measured in liver and spleen after prime-boost vaccination.

[0127] FIG. 29: Estimation of optimal MVA dosage.

[0128] HBVtg mice of low and medium antigenemia were grouped according to serum HBeAg levels. (A) Groups of HBVtg mice (n=3-4) were immunized twice in two weeks' intervals with 15 g of particulate HBcAg adjuvanted with c-di-AMP. On day 28, mice were boosted with 4 different dosages of MVA-core (310.sup.6, 110.sup.7, 310.sup.7, 110.sup.8 PFU, respectively). Sera of mice from day 0 and 35 (day 7 post boost) were analysed for HBsAg, HBeAg, anti-HBs and anti-HBc antibodies (B), and ALT levels (C). (D) On day 35 splenocytes and liver-associated lymphocytes of HBVtg mice were isolated, stimulated with MVA-derived peptide B8R or HBcAg-derived peptide c93 and analyzed for IFN-expressing CD8+ T-cells by intracellular cytokine staining. Frequencies of IFN-producing T-cells shown are background subtracted. i.m.intramuscular immunization; S/COsignal to cutoff; PFUplague forming units; U-units; IU-international units.

[0129] FIG. 30: Evaluation of c-di-AMP as an adjuvant for protein priming.

[0130] HBVtg mice of medium and high antigenemia were grouped according to serum HBeAg levels. (A) Groups of HBVtg mice (n=7) were immunized in two weeks' intervals with mixture of 15 g of particulate HBsAg and 15 g of HBcAg adjuvanted with c-di-AMP, or combination of CpG/PCEP. On day 28, mice were boosted with 10.sup.8 MVA-S/core. HBVtg mice (n=4) injected twice with c-di-AMP and boosted with empty MVA (MVAwt) were used as controls. Sera of mice from day 0 and 34 (day 6 post boost) were analysed for ALT levels (B), HBsAg, HBeAg, anti-HBs and anti-HBc antibodies (C-D). (E) On day 34 splenocytes and liver-associated lymphocytes of HBVtg mice (n=4) were isolated, stimulated with HBcAg-derived peptide c93 or HBsAg-derived peptide s208 and analyzed for IFN-expressing CD8+ T-cells by intracellular cytokine staining. Frequencies of IFN-producing T-cells shown are background subtracted. i.m.intramuscular immunization; S/COsignal to cutoff; PFUplague forming units; IU-international units.

[0131] FIG. 31: Estimation of optimal delivery route for various adjuvants: c-di-AMP, poly-IC and RIG-I ligand.

[0132] HBVtg mice of low and medium antigenemia were grouped according to serum HBeAg levels. (A) Groups of HBVtg mice (n=5) were immunized in two weeks' intervals with mixture of 15 g of particulate HBsAg and 15 g of HBcAg adjuvanted with c-di-AMP, poly-IC, or RIG-I ligand. On day 28, mice were boosted with 610.sup.7 MVA-S/core. Immunizations were performed either exclusively by intramuscular route, or protein priming was administered subcutaneously followed by intraperitoneal boost. Sera of mice from day 0 and 34 (day 6 post boost) were analysed for HBsAg, HBeAg, anti-HBs and anti-HBc antibodies (A). The weight of HBVtg mice was monitored weekly over the experiment (B). (C) On day 34 splenocytes and liver-associated lymphocytes of HBVtg mice were isolated and stimulated with MVA-derived peptide B8R, HBsAg-derived peptide s208, or HBcAg-derived peptide c93. Cells were then analyzed for IFN-expressing CD8+ T-cells by intracellular cytokine staining. Frequencies of IFN-producing T-cells shown are background subtracted. i.m., s.c., i.p.intramuscular, subcutaneous, intraperitoneal immunization, respectively; S/COsignal to cutoff; PFUplague forming units; IU-international units.

[0133] FIG. 32: Evaluation of the new MVA construct (MVA HBVvac) in C57BL/6 mice.

[0134] (A) Schematic depiction of MVA-S/core and MVA-HBVVac. B) Western blot analysis of lysates from cells producting indicated MVA-clones. Staining for non-glycosylated and glycosylated S using polyclonal antibodies. (C) Groups of C57BL/6 mice (n=5) were primed once with mixture of 20 g of particulate HBsAg and 20 g of HBcAg adjuvanted with c-di-AMP. Two weeks later, mice were boosted with either 610.sup.7 MVA-S/core, or with 610.sup.7 MVA-HBVvac, expressing HBsAg, HBcAg and RT domain of HBV polymerase. (D) Sera of mice from and 21 (day 7 post boost) were analysed for anti-HBs and anti-HBc antibodies. (E) On day 21 splenocytes isolated and stimulated with MVA-derived peptide B8R, HBsAg-, HBcAg- and HBV RT-specific peptides and peptide pools. Ovalbumine-derived peptide SIINFEKL served as negative control. Cells were then analyzed for IFN-expressing CD8+ T-cells by intracellular cytokine staining. Red arrows indicate positive RT-specific CD8+ T cell responses. i.m.intramuscular immunization, respectively; S/COsignal to cutoff; PFUplague forming units; IU-international units.

[0135] FIG. 33: Nucleotide sequence of the construct of the recombinant vaccination vector (rMVA) further expressing CD70 (SEQ ID NO: 27).

[0136] The different domains of said construct are depicted as follows: Del III-flanking sequence 1, mH5 promoter, HBcore protein, P2A, human CD70 molecule, IRES (EMCV), eGFP, Del III-flanking sequence 2.

[0137] FIG. 34: Evaluation of MVA expressing CD70 in C57BL/6 mice. (A) Schematic depiction of MVAcore and MVAcore-CD70. Both vectors express the HBV core genotype D sequence and GFP to allow easier purification. MVAcore-CD70 expresses in addition a CD70 gene. (B) Groups of C57BL/6 mice (n=6-7) were primed once intramuscularly with 20 g particulate HBcAg adjuvanted with PCEP and CpG. Three weeks later, mice were boosted with either 10.sup.8 i.u. MVAcore or 10.sup.8 MVAcore-CD70 or 10.sup.8 MVA-wildtype (MVAwt) injected intraperitoneally. (C) On day 35 splenocytes were isolated and stimulated with MVA-derived peptide B8R, or core-peptide C93. Ovalbumine-derived peptide SIINFEKL served as negative control. Cells were then analyzed for IFN-expressing CD8+ T-cells by intracellular cytokine staining. Data are given as meanSD per group. Dots indicate values determined in individual mice. i.u.-infectious units.

[0138] FIG. 35: Evaluation of MVA expressing CD70 in HBV transgenic mice.

[0139] (A) Schematic depiction of MVAcore and MVAcore-CD70. Both vectors express the HBV core genotype D sequence and GFP to allow easier purification. MVAcore-CD70 expresses in addition a CD70 gene. (B) Groups of transgenic mice carrying a 1.3-fold overlength genome (n=5-6) were primed once intramuscularly with 20 g particulate HBcAg adjuvanted with PCEP and CpG and three weeks later, boosted with either 10.sup.8 i.u. MVAcore or 10.sup.8 MVAcore-CD70 injected intraperitoneally. 2 mice treated accordingly with 10.sup.8 MVA-wildtype (MVAwt) served as control. (C) On day 35 liver-associated lymphocytes (LAL) were isolated and stimulated with MVA-derived peptide B8R, or core-peptide C93. Unstimulated cells served as negative control. Cells were analyzed by flow cytometry after intracellular cytokine staining for IFN (red) and IL-2 (blue). FACS plots for three representative animals are shown.

EXAMPLES

[0140] Mice and Vaccinations

[0141] C57BL/6 wildtype (wt) and HBV-transgenic mice (Strain HBV1.3.32 (Guidotti et al., J Virol 1995; 69:6158-69) (HBV genotype D, subtype ayw), kindly provided by F. Chisari, The Scripps Institute, La Jolla, Calif., USA) were derived from in-house breeding under specific pathogen-free conditions following institutional guidelines. For protein vaccinations, mice were immunized subcutaneously with recombinant yeast HBsAg or E. coli HBcAg (APP Latvijas BiomedicT nas, Riga, Latvia) mixed with 31.91 g of synthetic phosphorothioated CpG ODN 1668 and/or 25 or 50 g poly[di(sodiumcarboxylatoethyl-phenoxy)phosphazene] (PCEP) in 50 l PBS. For MVA vaccination, mice were vaccinated intraperitoneally with 110.sup.8 infectious units of respective recombinant MVA in 500 l PBS.

[0142] Intracellular Cytokine Staining, Multimer Staining and Degranulation Assay

[0143] Splenocytes and liver-associated lymphocytes (LAL) were isolated as described previously (Stross et al., Hepatology 2012; 56:873-83) and stimulated with H2-k.sup.b- or H-2D.sup.b-restricted peptides (FIG. 10) (jpt Peptide Technologies, Berlin, Germany) or recombinant HBsAg (kindly provided by Rheinbiotech-Dynavax, Dusseldorf, Germany) for 5 h in presence of 1 mg/ml Brefeldin A (Sigma-Aldrich, Taufkirchen, Germany). Cells were live/dead-stained with ethidium monoazide bromide (Invitrogen, Karlsruhe, Germany) and blocked with anti-CD16/CD32-Fc-Block (BD Biosciences, Heidelberg, Germany). Surface markers were stained with PB-conjugated anti-CD8a and PE-conjugated anti-CD4 (eBiosciences, Eching, Germany). Intracellular cytokine staining (ICS) was performed with FITC anti-IFN (XMG1.2), PE-Cy7 anti-TNFa and APC anti-IL-2 (eBiosciences, Eching, Germany) using the Cytofix/Cytoperm kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer's recommendations.

[0144] For degranulation assay, splenocytes were stimulated with peptide in the presence of Monensin, Brefeldin A, FITC-conjugated anti-CD107a antibody and APC-conjugated anti-CD107b antibody for 5 h followed by surface Pacific Blue CD8a staining and ICS with PerCP-Cy5.5 IFN (eBiosciences, Eching, Germany) using the Cytofix/Cytoperm kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer's recommendations.

[0145] For multimer staining, splenocytes and LAL were stained with PE-conjugated S.sub.190 (VWLSVIWM, SEQ ID NO: 19) or MVA B8R (TSYKFESV, SEQ ID NO: 20) multimers for 20 minutes followed by staining with Pacific Blue CD8a, FITC KLRG1 and APC CD127 (eBiosciences, Eching, Germany) in the presence of anti-Fc receptor antibody (clone 2.4G2) for 20 minutes. Data were acquired by FACS analysis on aFACSCanto II (BD Biosciences, Heidelberg, Germany) and analyzed using FlowJo software (Treestar, Ashland, USA).

[0146] Serological Analysis

[0147] Serum levels of HBsAg, HBeAg, anti-HBs and anti-HBc were determined in 1:20 dilutions using AXSYM assays (Abbott Laboratories, Abbott Park, Ill., USA). Quantification of serum HBV titers by real-time polymerase chain reaction was performed as described previously (Untergasser et al., Hepatology 2006; 43:539-47).

[0148] Neutralization Assay

[0149] HepaRG cells differentiated and cultured as described (Lucifora et al., J Hepatol 2011; 55:996-1003) were infected with 200 DNA-containing, enveloped HBV particles/cell (subtype ayw) in duplicate in the presence of a serial dilution of sera from vaccinated mice (1:33, 1:100, 1:333, and 1:1000). As positive control 0.8 international units of the Hepatect CP antibody (Biotest Pharma GmbH, Dreieich, Germany) were used. 24 hours post infection, cells were washed three times with PBS and 1 ml of differentiation medium was added. Supernatants were collected on day 4, 7 and 10 post infection and HBsAg was detected by immunoassay in 1:20 dilutions.

[0150] Statistical Analysis

[0151] Statistical analyses were performed using Prism5 software (GraphPad, San Diego, USA). Results are expressed as meanstandard error of the mean. Differences between groups were analyzed for statistical significance using two-tailed Student's t-tests.

[0152] Generation of MVA Vaccines

[0153] Recombinant MVA were generated by homologous recombination and host range selection as described previously (Staib et al., Biotechniques 2003; 34:694-6, 698, 700). The entire HBcAg (genotype D, subtype ayw) and HBsAg open reading frames (genotype D, subtype ayw or adw) were cloned into MVA transfer plasmids pIIIHR-PH5 or pIIIHR-P7.5, thereby placing the HBV proteins under the control of the early/late Vaccinia virus-specific promoters PH5 (HBcAg ayw/HBsAg ayw/HBsAg adw) or P7.5 (HBsAg ayw). After construction of each virus, gene expression, sequence of inserted DNA, and viral purity were verified. For generation of vaccine preparations, MVA were routinely propagated in CEF, purified by ultracentrifugation through sucrose, reconstituted in 1 mM Tris-HCL pH 9.0 and titrated following standard methodology (Staib et al., Methods Mol Biol 2004; 269:77-100).

[0154] Immunoblot

[0155] NIH-3T3 mouse fibroblasts (CRL-1658) were cultured in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin and 100 g/ml streptomycin. Cells were harvested in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 0.02% NaN.sub.3, and 100 g/ml phenylmethylsulfonyl fluoride) 16 h post infection, dissolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto a nitrocellulose membrane (0.45 M; Bio-Rad, Munich, Germany). Membranes were incubated at 4 C. with anti-HBc (antiserum H800; kindly provided by H. Schaller), anti-HBs (Murex HBsAg version 3; Abbott, Abbott Park, Ill., USA) or anti-actin (Sigma, Munich, Germany) antibodies at 1:10000, 1:50 and 1:10000 dilutions, respectively. Horseradish peroxidase-labeled secondary mouse and rabbit antibodies (Dianova, Hamburg, Germany) were used at a 1:5000 dilution for 1 h at 21 C. Antibodies were diluted in phosphate-buffered saline containing 5% skim milk. Enhanced chemiluminescence was used as directed (Roche, Mannheim, Germany).

[0156] Secreted HBsAg in supernatant of cultured cells was determined using Abbott AxSYM HBsAg assay (Abbott Laboratories, Abbott Park, Ill., USA).

Example 1: Protein-Prime/MVA-Boost Vaccination Induces Strong Anti-HBV Immunity

[0157] Two MVA vaccines expressing either HBs (MVA-S) or HBc (MVA-core) were generated from HBV genotype D, subtype ayw (FIG. 1A). Western blotting and HBsAg ELISA confirmed correct protein expression from either MVA (FIGS. 7A, B).

[0158] To examine immunogenicity, C57BL/6 (wt) mice were immunized with MVA-S, MVA-core or MVAwt (10.sup.8 infectious units) i.p. and frequencies of IFN-producing CD8+ T-cells were determined by intracellular cytokine staining (ICS) on day 8 post immunization. The i.p. route was used because systemic MVA distribution was intended. MVA-S, MVA-core and MVAwt immunization induced comparable CD8+ T-cell responses to the MVA-derived immunodominant B8R peptide, which was boosted after a second immunization (FIG. 1B). In contrast, no HBV-specific CD8+ T cell responses were detected (FIG. 1B).

[0159] In order to induce HBV-specific immunity, we performed heterologous prime-boost vaccinations. Mice received 12 g of recombinant, particulate HBsAg or HBcAg (subtype ayw) with CpG as adjuvant. 8 days after HBsAg vaccination, frequencies of splenic CD8+ T-cells secreting IFN in response to stimulation with S.sub.190 and S.sub.208 peptides were around 0.6% whereas hardly any CD8+ T-cell responses against C.sub.93 were detectable following HBcAg immunization (FIG. 1C). A boost vaccination on day 21 with either MVA-S or MVA-core was able to induce high frequencies of HBsAg- or HBcAg-specific CD8+ T-cells as well as high anti-HBs or anti-HBc titers, respectively (FIG. 1D). Taken together, those data indicate that heterologous prime-boost vaccination was needed to induce HBV-specific T-cells.

Example 2: High Antigenemia Prevents Induction of Anti-HBV Immunity

[0160] In order to investigate the impact of HBV antigen load on the induction of HBV-specific immune responses, HBV1.3.32 transgenic (HBVtg) mice were sorted into low, medium and high-antigenemic groups according to their serum HBeAg levels before vaccination (FIG. 10). 6 days post boost, anti-HBs titers in mice receiving HBsAg/MVA-S were higher in the low-antigenemic group as compared to mice from the medium-antigenemic group, and remained undetectable in high-antigenemic mice (FIG. 2A). Even when it was monitored for 35 days after MVA-S boost, high-antigenemic mice did not develop detectable anti-HBs titers, and HBsAg persisted at low levels (FIG. 11). To study whether anti-HBs may be complexed by the excess amounts of HBsAg and thus escape detection, we dissolved precipitated 131 protein complexes with urea and repeated the immunoassay. Hereby, we found HBsAg-anti-HBs immune complexes in vaccinated high and intermediate but not low antigenemic HBVtg mice (FIG. 14). HBcAg-specific antibodies, however, were detected in sera of HBcAg/MVA-core vaccinated mice from all groups, but titers again showed the tendency to inversely correlate with antigenemia (FIG. 2B). In order to analyze the vaccination-induced CD4+ T-cell response, we stimulated splenocytes and liver-associated lymphocytes (LAL) with HBsAg. Although we found high frequencies of HBsAg-specific CD4+ T-cells in wt mice, we did not detect HBV-specific CD4+ T-cells in HBVtg mice in any of the groups (FIG. 2C, D).

[0161] Similar to antibody titers, we observed an inverse correlation between antigenemia and HBV-specific CD8+ T-cell responses. In high-antigenemic mice, HBsAg/MVA-S as well as HBcAg/MVA-core immunization failed to induce detectable HBsAg- or HBcAg-specific CD8+ T cellsneither in the periphery (spleen) nor in the liver, the site of HBV-replication (FIG. 3A, B). Mice with a medium or low HBeAg burden developed S.sub.190.sup. and S.sub.208-specific CD8+ T-cell responses to HBsAg/MVA-S, and C.sub.93-specific T-cell responses to HBcAg/MVA-C vaccination. HBV-specific CD8+ T-cell frequencies detected in low-antigenemic mice were higher as those found in medium-antigenemic mice. Importantly, MVA B8R-specific CD8+ T-cell frequencies were independent of antigenemia and comparable between all groups indicating equal vaccination efficiency (FIG. 3A,B).

[0162] During chronic infection and antigen persistence, CD8+ T-cells can develop an exhausted, dysfunctional phenotype. In such conditions, the number of antigen-specific T-cells is largely underestimated through functional tests such as IFN production. Therefore it was performed S.sub.190-, C.sub.93- and B8R-specific multimer-staining, which did not detect HBV-specific CD8+ T-cells in spleens or livers of immunized high-antigenemic mice while B8R-multimer positive CD8+ T-cells were readily detectable. This suggested that the vaccine indeed failed to induce HBV-specific T-cells in this group. In low- and medium-antigenemic groups, HBV-S.sub.190specific and MVA-B8R-specific responses displayed a similar ratio of multimer-positive and IFN-positive CD8+ T-cells (FIG. 13). Taken together, HBV antigen levels influence how efficiently HBV-specific antibody as well as T-cell responses can be induced by heterologous prime-boost vaccination.

Example 3: Antigenemia Influences the Quality of Vaccination-Induced Responses

[0163] Important effector functions of CD8+ T-cells include the production of IL-2 and TNF in addition to IFN as well as the ability to degranulate in response to peptide stimulation, which can be analyzed by the surface expression of CD107a. Upon S190 peptide stimulation, IFN+S190-specific splenic CD8+ T-cells induced in wt mice and low-antigenemic HBVtg mice by HBsAg/MVA-S immunization degranulated to similar ratios (72.3% and 73.4%, respectively) (FIG. 4A). and showed comparable expression of IL-2 and TNFa in spleen and liver CD8+ T-cells derived from medium-antigenemic mice, which were also able to degranulate upon peptide stimulation although to a lesser extent (62%), but lacked IL-2 expression (FIG. 4B).

[0164] To determine the differentiation status of multimer-binding cells to proliferate, we stained CD127 and KLRG-1. Vaccination of wt and low-antigenemic HBVtg mice induced a high percentage of S.sub.190-specific CD127+ KLRG-1-multimer-binding cells in the livers and spleens, that are considered to be a transient precursors of long-lived cells with the potential to proliferate and to give rise to new effector cell progeny (FIG. 4C). In medium-antigenemic mice, these cells, however, were hardly detectable (FIG. 4C). These data indicate that HBV antigen expression diminishes polyfunctionality of CD8+ T-cells and in particular effector cell IL-2 secretion and proliferation capacity.

Example 4: Comparison of Adjuvants for Protein-Prime Vaccination

[0165] In order to investigate the polyphosphazene adjuvant PCEP will enhance the immune stimulatory effect of CpG, PCEP was used instead of CpG or was added to CpG for the protein vaccine formulation and combined HBsAg and HBcAg.

[0166] Wildtype mice were vaccinated with 16 g HBsAg (subtype ayw) and 16 g HBcAg (subtype ayw) together with the respective adjuvant(s) on days 0. On day 28, mice were boosted with MVA-PH5-S (510.sup.7 i.u.; subtype ayw) and MVA-core (510.sup.7 i.u.). On day 6 post boost sera were analyzed for presence of anti-HBs (FIG. 9A) and anti-HBc (FIG. 9B). On day 6 post boost splenocytes were analyzed by ICS after stimulation with HBsAg (S.sub.190 and S.sub.208adw)- or HBcAg (C.sub.93)-specific peptides (FIG. 9C). Bars show percentage (meanSEM) of CD8+cells staining positive for IFN after background subtraction. i. u. infectious units.

[0167] The use of PCEP (alone or in combination with CpG) was superior in inducing anti-HBs and anti-HBc antibody responses to CpG alone after protein-prime/MVA-boost vaccination in wt mice, while CD8+ T-cell responses were comparable (FIG. 9A-C).

Example 5: Comparison of Adjuvant Combinations

[0168] Next it was aimed at determining the effect of different adjuvants on humoral and cellular immune responses. Therefore, wild type CH57Bl/6 mice were primed with particulate HBcAg and HBsAg complexed in different adjuvant formulations. In groups 1 to 3 (n=3) HBsAg was complexed with alumn hydroxide and combined with HBcAg, CpG or polyphosphazene adjuvant or both. Groups 4 to 6 (n=3) were vaccinated using particulate HBcAg and HBsAg adjuvanted with either CpG or polyphosphazene or both but without any alumn. At day 28, all animals were booster with MVA expressing core and S subtype adw.

[0169] Antibody responses against HBs and HBc were determined after 28 days (protein prime only) and after 34 days (protein prime plus MVA boost). FIGS. 15A and 15B show that antibody responses in particular against HBs were unexpectedly much more pronounced when alum was avoided. FIG. 15C-D shows CD8+ T cell responses against S and core epitopes after prime (FIG. 15C) and after the MVA-boost (FIG. 15D).

[0170] T cell responses were detectable already after prime when vaccine formulations contained no alumn. Interestingly, after the MVA boost with equal efficiency in all groups (indicated by B8R-specific responses), all mice developed core-specific T cell responses, while again animals vaccinated without alumn developed much more pronounced S-specific CD8+ T cell responses.

Example 6: Vaccination with Heterologous HBsAg Subtype Breaks T-Cell Tolerance and Induces Strong Antibody Production in High-Antigenemic HBVtg Mice

[0171] Mouse sera were analyzed for their neutralization capacity after vaccination. Mice vaccinated with HBsAg subtype adw, were able to cross-neutralize HBV subtype ayw even in high dilutions of up to 1:1000 (FIG. 15E).

[0172] Next, it was aimed at enhancing the immunogenicity of heterologous protein-prime/MVA-boost vaccination to break tolerance in the presence of higher HBV antigen load.

[0173] To test whether a stronger antigen trigger would improve vaccination efficiency, new MVAs expressing HBsAg were engineered also under control of the stronger promoter PH5 (FIG. 5A) and a second protein vaccination on day 14 was performed (FIG. 12). In addition, HBsAg of subtype ayw (identical to HBVtg mice) and adw were compared. In addition, PCEP was added to CpG for the protein vaccine formulation and combined HBsAg and HBcAg during prime and boost in order to achieve immune responses to multiple HBV antigens. When this modified vaccination regimen (combined HBsAg/HBcAg prime adjuvanted with CpG and PCEP on days 0 and 14 followed by boost on day 28 using MVA-S/MVA-core which express the antigens under the stronger PH5 promoter) was applied, there was the ability to break tolerance in high-antigenemic HBVtg mice and induced HBsAg- and HBcAg-specific CD8+ and CD4+ T-cells (FIGS. 5B, C).

[0174] Next, it was investigated whether a partial mismatch between vaccine and target antigen would improve vaccine efficacy (Schirmbeck et al., Eur J Immunol 2003; 33:3342-52). Either vaccine antigen, S.sub.ayw or S.sub.adw, induced CD8+ T-cells against both subtypes (FIG. 5B) as determined with subtype-specific peptide S.sub.208 (FIG. 11). However, the heterologous S.sub.adw-containing vaccine induced stronger CD8+ and detectable CD4+ T-cell responses (FIGS. 5B, C). Similar to what we had observed with S-specific CD8+ T-cells derived from medium-antigenemic mice (FIG. 4B), splenic S.sub.208-specific CD8+ T-cells were found to secrete IFN and to certain extend TNFa, but showed only marginal expression of IL-2 (FIG. 5D).

[0175] Notably, only the S.sub.adw, but not the S.sub.ayw containing vaccine formulation was able to induce detectable anti-HBs antibody responses in high-antigenemic mice (FIG. 6A), while anti-HBc antibodies were induced by both formulations (FIG. 6B). Importantly, anti-HBs antibodies generated by S.sub.adw were able to neutralize HBV particles of subtype ayw (FIG. 6C). Concomitantly to the induction of neutralizing anti-HBs in the S.sub.adw vaccination group, levels of HBsAg significantly dropped to low levels (FIG. 6D). Taken together, this indicated that the modified vaccination scheme indeed allowed breaking B- and T-cell tolerance in HBVtg mice.

Example 7: Broad Immune Response Induced by Multi-Antigenic MVA

[0176] Next, it was aimed at comparing the induction of immune responses against one, two and several HBV antigens using a multi-antigenic MVA. Unexpectedly, humoral as well as cellular immune responses against either S or S and Pol were improved when core was co-expressed by the MVA vaccine vector.

Example 8: Combination of RNAi and Therapeutic Vaccination

[0177] HBV transgenic mice, strain HBVxfs, expressing high titer HBV antigens were treated with HBV-specific siRNAs targeting the 3 region of all HBV RNAs. siRNA treatment reduced HBeAg and HBsAg levels by 90%. After 8 weeks, animals were vaccinated with a protein primeMVA-HBV boost vaccine to induce anti-HBs antibodies and HBV-specific T cells (FIG. 27). As a protein vaccine, particulate HBsAg and HBcAg were adjuvanted with CpG and PCEP. The MVA vaccine vector expressed the complete open reading frame of HBV S and core proteins. Controls were no siRNA, no vaccination and a combination thereof.

[0178] 6 days after boost vaccination, mice were sacrificed and HBeAg and HBsAg levels as well as anti-HBs titers were determined (FIG. 28A, B). From livers and spleen, T cells were isolated, ex vivo stimulated with HBV-specific peptides, stained for interferon gamma expression by intracellular cytokine staining and analyzed by flow cytometry (FIG. 28C).

Example 9: Estimation of Optimal MVA Dosage

[0179] In the first sets of experiments we aimed to assess the lowest MVA dosage for heterologous protein-prime/MVA-boost vaccination that would show satisfactory immunogenicity and would be able to break immune tolerance in HBVtg mice. Therefore, groups of low and middle antigenemic HBVtg mice were immunized twice in two weeks' intervals with 15 g of particulate HBcAg adjuvanted with bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP). On day 28, mice were boosted with 4 different dosages of MVA-core (310.sup.6, 110.sup.7, 310.sup.7, 110.sup.8 PFU, respectively) (FIG. 29A). Humoral and cellular immune responses elicited by immunization regimens employing various MVA dosages were evaluated 7 days after the boost immunization (day 35).

[0180] Sera of mice from day 0 and 35 (day 7 post boost) were analyzed for HBsAg, HBeAg, anti-HBs and anti-HBc antibodies (FIG. 29B). All immunization regimens elicited similar levels of anti-HBc antibodies detected in the serum of HBVtg mice at day 35. In addition, all groups of mice showed significant reduction of HBsAg in the blood. This effect was not mediated by anti-HBs antibodies as the immunization regimen did not include HBsAg, crucial for HBsAg serocoversion in HBVtg model. Interestingly, HBVtg mice from the groups that received higher dosages of MVA-core (310.sup.7 and 110.sup.8 PFU) as a boost showed considerable decrease in serum HBeAg levels (FIG. 29B). Moreover, slight elevation of liver alanine transferase (ALT) was observed also in the groups of mice that received higher dosages of MVA-core (310.sup.7 and 110.sup.8 PFU) (FIG. 29C). These data suggest that immunization protocols in these two groups of mice resulted in suppressed HBV replication in the liver possibly due to the enhanced activity of HBcAg-specific T cells. Indeed, intracellular IFN staining of liver-associated lymphocytes (LALs) and splenocytes showed, that HBVtg mice that were immunized with higher dosages of MVA-core could mount more effective HBV-specific CD8+ T cell responses, particularly in the liver (FIG. 29D). Simultaneously, MVA-specific CD8+ T cell responses were not significantly increased with the higher MVA dosage used for immunization.

[0181] It can be concluded from these results that MVA-core dosage of 310.sup.7 PFU for heterologous protein-prime/MVA-boost vaccination is sufficient to break immune tolerance in low and middle antigenemic HBVtg mice.

Example 10: Evaluation of c-Di-AMP as an Adjuvant for Protein Priming

[0182] Lack of a safe and effective adjuvant inducing a balanced Th1/Th2 CD4+ T cell response may be an obstacle for initiating clinical trials. Moreover, triggering the newly identified cytoplasmic pattern recognition receptor STING is an interesting alternative for therapeutic vaccination. Therefore, we aimed to investigate the efficacy of c-di-AMP as a potential new adjuvant for a therapeutic hepatitis B vaccine. To this purpose, groups of middle and high antigenemic HBVtg mice (n=7) mice received two protein primes and a MVA boost immunization. Particulate HBsAg and HBcAg for protein priming were combined and adjuvanted with c-di-AMP or a previously established combination of CpG with polyphosphazenes (PCEP). On day 28, mice were boosted with mixture of MVA-S/core (FIG. 30A). HBVtg mice (n=4) that received c-di-AMP injection and empty MVA (MVAwt) were used as controls. The efficacy of the vaccine formulations to induce humoral and cellular immune responses was compared at day 34 (6 days after the boost).

[0183] Neither c-di-AMP nor CpG/PCEP immunization protocol had an impact on serum HBeAg levels in high antigenemic HBVtg mice (FIG. 30C). Both tested vaccine formulations induced significant anti-HBc responses. However, immunization with c-di-AMP induced significantly higher titers of anti-HBc antibodies, as compared to CpG/PCEP regimen (p<0.05) (FIG. 30C). Interestingly, both immunization protocols resulted in HBsAg to anti-HBs seroconversion in all examined HBVtg mice (FIG. 30D). High levels of anti-HBs antibodies elicited by the c-di-AMP- or CpG/PCEP-adjuvanted vaccines complexed circulating HBsAg and removed it from the serum of the mice. By contrast, HBVtg mice that received c-di-AMP only followed by MVAwt boost did not develop any anti-HBs, and the levels of HBsAg in the serum of these mice remained unchanged. Importantly, both vaccine formulations induced significant HBsAg-specific (s208) and HBcAg-specific (c93) CD8+ T cell responses detectable in spleen (p<0.05) and, in particular, liver-associated lymphocytes in the HBVtg mice (p<0.05) (FIG. 30E), accompanied by mild T-cell-induced liver damage due to an increase in ALT (FIG. 30B). There was no statistically significant difference in the magnitude of HBV-specific CD8+ T cell responses elicited by c-di-AMP or CpG/PCEP regimens.

[0184] In view of these data, c-di-AMP is considered being a potent adjuvant for therapeutic protein prime-MVA boost vaccination even in high antigenemic HBVtg mice.

Example 11: Estimation of Optimal Delivery Route for Various Adjuvants: c-Di-AMP, Poly-LCIC and RIG-I Ligand

[0185] For an appropriate adjuvant for the protein priming for the therapeutic heterologous protein-prime/MVA-boost vaccination the screening expanded. Our objective was to compare the efficacy of c-di-AMP to two potential new adjuvants for a therapeutic hepatitis B vaccine: poly-LCIC and RIG-I ligand. Moreover, we examined the various immunization protocols to find the most efficacious application route. To this purpose, groups of low and middle antigenemic HBVtg mice (n=5) received two protein primes and a MVA boost immunization (FIG. 30A). Particulate HBsAg and HBcAg for protein priming were combined and adjuvanted with c-di-AMP, poly-LCIC, or RIG-I ligand. On day 28, mice were boosted with mixture of MVA-sAg and MVA-core. Immunizations were performed either exclusively by intramuscular (i.m.) route, or protein priming was administered subcutaneously (s.c.) followed by intraperitoneal (i.p.) boost. The efficacy of the different vaccine formulations and application routes were compared with respect to inducing humoral and cellular immune responses at day 34 (6 days after the boost).

[0186] All vaccination protocols potently reduced HBsAg levels in the sera of HBVtg mice. This was due to the fact, that all examined adjuvants and delivery routes could elicit high titers of anti-HBs antibodies that complexed HBsAg in the blood of mice. Similarly, the levels of induced anti-HBc antibodies was comparable between the groups of mice, with a slight tendency that intramuscular immunization route was more potent in induction anti-HBc. Nevertheless, lower HBV replication, detected indirectly by HBeAg levels, was observed only in the groups of HBVtg mice that received c-di-AMP via i.m. or s.c./i.p. routes, or poly-LCIC via i.m. route (FIG. 31A). Unfortunately, immunization with poly-LCIC in intramuscular manner was the only examined protocol that resulted in considerable body weight loss in HBVtg mice (FIG. 31B). C-di-AMP can be considered as being also superior in inducing both HBcAg-specific (c93) and HBsAg-specific (s208) CD8+ T cell responses in the spleens and especially in the livers of immunized HBVtg mice, independently which administration route was used (FIG. 31C). Interestingly poly-LCIC resulted in vigorous intrahepatic HBcAg-specific CD8+ T cell response when delivered intramuscularly, whereas when delivered in s.c./i.p. route predominantly elicited HBsAg-specific (s208) CD8+ T cell responses. RIG-I ligand was able to induce HBV-specific humoral responses, but failed to induce prime HBV-specific CD8+ T cell responses. MVA-specific CD8+ T cell responses, used as controls, were comparable in all immunized groups in spleen and liver, indicating equal vaccination efficiency.

[0187] These data demonstrate that c-di-AMP is very potent adjuvant, poly-LCIC shows intermediate efficacy, and RIG-I ligand is not effective enough for therapeutic protein prime-MVA boost vaccination. C-di-AMP was equally effective in both i.m. and s.c./i.p. application routes.

Example 12: Evaluation of the New MVA Construct (MVA-HBVvac) in C57BL/6 Mice

[0188] Further, in vivo immunogenicity of the newly constructed polycistronic MVA expressing HBsAg, HBcAg (sequences covering the main HBV genotypes A, B, C, D) and RT domain of HBV polymerase (MVA-HBVvac) was evaluated. Schematic depiction of the two polycystronic vaccination constructs was generated: HBVVac covering HBV core and S of all major HBV genotypes as well as the RT domain of HBV polymerase, and C/S (C/S) expressing HBV core and S (FIG. 32A). Protein expression was confirmed by Western blotting. FIG. 32B shows S-expression by different recombinant MVA-clones expressing either HBVVAc or S/C.

[0189] Groups of C57BL/6 mice (n=5) were primed once with mixture of particulate HBsAg and HBcAg adjuvanted with c-di-AMP. Two weeks later, mice were boosted with either mixture of MVA-S and MVA-core, or with equal amount of the new MVA-HBVvac. Mice were sacrificed at day 21 to evaluate HBV-specific humoral and cellular immune responses (FIG. 32C).

[0190] The new polycistronic MVA did elicit significant anti-HBs and anti-HBc antibody responses, comparable to the mixture of a combination of MVA-S and MVA-core constructs (FIG. 32D). Moreover, immunization with MVA-HBVvac elicited vigorous HBsAg-specific (s190, s208 and Spool) and HBcAg-specific (c93, Cpool) CD8+ T cell responses (determined by analysing splenocytes) that were similar in magnitude to these induced by the mixture of MVA-S and MVA-core (FIG. 32E). In addition, immunization with MVA-HBVvac resulted in the detection of RT-specific CD8+ T cell responses for peptides RT61, RT333 and RT peptide pool 2 (marked with arrows) at low levels, even though no RT protein was used for priming.

[0191] These data showed that the polycistronic MVA (MVA-HBVvac) expressed all proteins expected and showed excellent in vivo immunogenicity in C57BL/6 mice.

Example 13: Increasing Immunogenicity of MVA Constructs by Co-Expression of CD70

[0192] To improve in vivo immunogenicity of MVA-based vaccine vectors, a MVA vector that expresses CD70 in a bicistronic fashion was constructed (FIG. 34A).

[0193] Groups of C57BL/6 mice (n=6-7) were primed once with particulate HBcAg adjuvanted with CpG and PCEP. Two weeks later, mice were boosted with either MVA-core or with an equal amount MVAcore-CD70 expressing CD70 in addition or a wild type MVA as control (FIG. 34B). Mice were sacrificed at day 35 to evaluate HBV-specific humoral and cellular immune responses. While humoral immune responses were identical after MVAcore and MVAcore-CD70 boost, CD8+ T cell responses against the MVA(B8R)-specific cytokine were slightly and against HBV(C93)-specific cytokine were significantly increased in mice boosted with MVAcore-CD70 (FIG. 34C). A repeat experiment gave identical results.

[0194] In a third experiment, groups of HBV-transgenic mice bread on a C57BL/6 background (n=5-6) were vaccinated. In these animals, immune tolerance can be broken upon therapeutic vaccination. After priming once with particulate HBcAg adjuvanted with CpG and PCEP, mice were boosted with either MVA-core or with an equal amount MVAcore-CD70 expressing CD70 (FIGS. 35A and B). Mice vaccinated with a wild type MVA served as control. Mice were sacrificed at day 35 to evaluate MVA- and HBV-specific T cell responses. CD8+ T cells gated onto in liver associated lymphocytes showed a more pronounced secretion of IFNg and IL2 upon re-stimulation with MVA- and HBVcore-specific peptides, respectively, when mice had been vaccinated MVAcore-CD70 compared to mice vaccinated with MVAcore. In mice boosted with MVAwt, MVA-specific, but no HBV-specific T cell responses were detected (FIG. 35C).

[0195] Concerning the results above it can be concluded that coexpression of CD70 with an HBV-specific antigen increased MVA-as HBV-specific T cell responses in vivo significantly.

[0196] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms comprising, including, containing, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0197] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0198] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0199] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.