Fusion Protein

Abstract

The present invention relates to a fusion protein for use in the treatment and/or prevention of a hepatitis B virus infection comprising at least one hepatitis B PreS polypeptide or fragment thereof fused to at least one peptide consisting of an amino acid sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.

Claims

1-7. (canceled)

8. A method for treating and/or preventing a hepatitis B virus infection in a subject which comprises administering a fusion protein to said subject, wherein the fusion protein comprises i) at least one hepatitis B PreS polypeptide; ii) a peptide sequence consisting of SEQ ID No. 1; iii) a peptide sequence consisting of SEQ ID No. 2; iv) a peptide sequence consisting of SEQ ID No. 3; and v) a peptide sequence consisting of SEQ ID No. 4.

9. The method of claim 8, wherein the amino acid sequence of the PreS polypeptide consists of SEQ ID No. 5.

10. The method of claim 8, wherein the peptides consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4 are fused to the N- and/or C-terminus of the PreS polypeptide.

11. The method of claim 8, wherein the fusion protein consists of amino acid sequence SEQ ID No. 6.

12. The method of claim 8, wherein the hepatitis B virus infection is caused by a hepatitis B virus genotype A, B, C, D, E, F, G, H or a subtype thereof.

13. The method of claim 8, wherein the fusion protein is administered to an individual at least once in an amount of 0.01 mg/kg body weight to 5 mg/kg body weight.

14. The method of claim 8, wherein the fusion protein is administered together with at least one adjuvant and/or pharmaceutical acceptable excipient.

15. The method of claim 8, wherein the fusion protein is administered to an individual at least once in an amount of 0.1 mg/kg body weight to 2 mg/kg body weight.

Description

[0031] The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

[0032] FIG. 1 shows the allocation of PreS peptides to aligned PreS sequences from different genotypes. Identical amino acids are indicated by points, the PreS1 domain includes amino acid residues 1 to 118 and the PreS2 domain amino acid residues 119 to 173 (see also SEQ ID No. 5) and amino acid residues 19 to 28 (grey box) play a crucial role in liver cell attachment of HBV and infection.

[0033] FIG. 2 shows IgG responses of rabbits immunized with PreS (n=1; FIG. 2A), or 20 μg of a PreS-fusion vaccine Mix (n=2; FIG. 2B), before (left bars in grey) and after (right bars in black) immunization. Optical density values (y-axes: OD values at 405 nm) correspond to IgG levels towards PreS and PreS-derived synthetic overlapping peptides P1-P8 (x-axes). Results represent mean values with SD from triplicate determinations.

[0034] FIGS. 3A to 3C show IgG responses towards PreS (FIG. 3A) and synthetic PreS-derived overlapping peptides P1-P8 (FIGS. 3B, and 3C) of subjects vaccinated with PreS-fusion vaccine Mix or placebo. Shown are optical density values (y-axes: OD values, means of triplicate determinations) corresponding to IgG levels towards PreS and peptides P1-P8 measured in subjects with or without prior hepatitis B vaccination who had been immunized with PreS-fusion vaccine Mix (n=22) or placebo (n=8) before (V5) and at different time points after immunization (V8 and V15) (x-axes). Results are represented as mean values with SD and significant differences (in all PreS-fusion vaccine Mix-vaccinated individuals at V5, V8, and V15) are indicated: *p<0.05, **p<0.01, ***p<0.001.

[0035] FIG. 4 shows PreS-specific antibody responses of subjects vaccinated with PreS-fusion vaccine Mix (PreS-FVM) or placebo and antibodies present in hepatitis B-infected individuals. Shown are optical density values (y-axes: OD values) corresponding to IgA, IgE, IgM, IgG and IgG subclass (IgG1-IgG4) levels specific for PreS of subjects immunized with placebo (n=8), 20 μg (n=10) or 40 μg of PreS-fusion vaccine Mix (n=12) as well as of hepatitis B-infected individuals (n=19) (x-axes). Graphs show mean values with SD. Significant differences are indicated: ***p<0.001.

[0036] FIG. 5 shows IgG responses specific for PreS peptides P1-P8 of subjects vaccinated with PreS-fusion vaccine Mix (PreS-FVM) or placebo and IgG present in hepatitis B-infected individuals. Shown are optical density values (y-axes: OD values) corresponding to IgG levels specific for PreS-derived peptides (P1-P8) of subjects immunized with placebo (n=8), 20 μg (n=10) or 40 μg of PreS vaccine mixture (n=12) at V15 as well as of hepatitis B-infected individuals (n=19) (x-axes). Results are represented as mean values with SD.

[0037] FIG. 6 shows PreS- and peptide-specific T cell responses. FIG. 6A: PreS-specific PBMC proliferations (y-axis: stimulation indices SIs) assessed by [3H] thymidine incorporation in subjects immunized with PreS-fusion vaccine Mix (n=19) at different time points (x-axis). Mean values with SD and significant differences are indicated: *p<0.05, **p<0.01, ***p<0.001. FIG. 6B, and FIG. 6C: Percentages of proliferated CD4 (B) and CD8 (C) T cells (y-axes) after stimulation with PreS peptides (P1-P8), PreS or an equimolar peptide mix (x-axes) in blood samples of subjects immunized with PreS-fusion vaccine Mix (n=11) at time point M2. Results are represented as mean values with SD.

[0038] FIG. 7 shows the antibody-induced inhibition of hepatitis B virus infection in an in-vitro virus neutralization assay which is based on in-vitro cultured liver cells. Percentages of the inhibition of hepatitis B infection of cultured HepG2-hNTCP (x-axis) achieved by pre-incubation of virus with anti-sera containing virus neutralizing antibodies. FIG. 7A: Inhibition of virus infection by Ma 18/7 (Positive control), serum from a placebo-treated human subject, sera from human subjects after immunization with PreS-fusion vaccine Mix (n=7), all subjects without prior hepatitis B vaccination. FIG. 7B: Inhibition of virus infection by sera from rabbits immunized with the commercial hepatitis B vaccine Engerix or the PreS-fusion vaccine Mix.

[0039] FIG. 8 shows a comparison of total serum IgG towards PreS in sera of New Zealand White (NZW) rabbits which have undergone immunization, either with recombinant PreS or PreS-fusion-proteins (PreS-F1-PreS-F4), as emulsion in Complete Freund's Adjuvant. The x-axis indicates the dilution of sera and on the y-axis, the OD values, measured at 405 nm are depicted. The experiment was assayed in duplicates.

EXAMPLES

Example 1: Expression and Purification of Recombinant PreS, Synthesis of PreS Overlapping Peptides, Sequence Alignments

[0040] Expression and purification of a hexahistidine-tagged recombinant PreS protein (PreS1+PreS2 (SEQ ID No. 5; genotype A; subtype adw2, derived from GenBank: AAT28735.1) in Escherichia coli BL21 (DE3, Stratagene, USA) has been performed as described in Niespodziana K et al. (J Allergy Clin Immunol 127(2011):1562-70).

[0041] Eight peptides of a length of approximately 30 amino acids and an overlap of 10 amino acids spanning the complete sequence of PreS (genotype A, subtype adw2; Table A; FIG. 1) were synthesized by a Fmoc (9-fluorenylmethoxycarbonyl)-strategy with HBTU [2-(1H-Benzotriazol-1-yl)1,1,3,3 tetramethyluronium hexafluorophosphat] activation (CEM-Liberty, Matthews, N.C.; Applied Biosystems, Life technologies, USA).

TABLE-US-00002 TABLE A  SEQ ID  Peptide Sequence No. P1 GGWSSKPRKGMGTNLSVPNPLGFFPDHQLD 14 P2 [00001] 15 P3 [00002] 16 P4 [00003] 17 P5 [00004] 18 P6 [00005] 19 P7 [00006] 20 P8 PAGGSSSGTVNPAPNIASHISSISARTGDPVTN 21 [0042] (overlapping regions of the peptides are underlined)

[0043] Peptides were purified by preparative HPLC and their identity was confirmed by mass spectrometry (Microflex MALDI-TOF, Bruker, USA).

[0044] An alignment of the PreS genotype A, serotype adw2 sequence and peptide sequences thereof with HBV genotypes B-H was performed with CLUSTAL W using reference sequences from the HBV data base (HBVdb: https://hbvdb.ibcp.fr/HBVdb/HBVdbIndex) (Hayer J et al. Nucleic Acids Res 2012; gks1022) (see FIG. 1).

Example 2: Immunization of Rabbits

[0045] Specific rabbit antibodies against recombinant PreS were raised by immunization of a New Zealand white rabbit with purified PreS (200 μg per injection) using Freund's complete adjuvant (CFA) for the first and incomplete Freund's adjuvant (IFA) for the second and third injection (Charles River, Germany). In addition, New Zealand white rabbits were immunized three times with a mix containing 20 μg (n=2) or 40 μg (n=2) of each of the four PreS vaccine mixture components (PreS vaccine mixture-20/PreS vaccine mixture-40) using Al(OH).sub.3 as adjuvant. The four PreS vaccine mixture components include PreS fusion proteins PreSF1, PreSF2, PreSF3 and PreSF4 having the following amino acid sequences:

TABLE-US-00003 PreSF1 (SEQ ID No. 22): MVRYTTEGGTKTEAEDVIPEGWKADTSYESKVRYTTEGGTKTEAEDVIPE GWKADTSYESKGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNN PDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVST IPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGLY FPAGGSSSGTVNPAPNIASHISSISARTGDPVTNVRYTTEGGTKTEAEDV IPEGWKADTSYESKVRYTTEGGTKTEAEDVIPEGWKADTSYESK PreSF2 (SEQ ID No. 23): MFRFLTEKGMKNVFDDVVPEKYTIGATYAPEEFRFLTEKGMKNVFDDVVP EKYTIGATYAPEEGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANS NNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTV STIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRG LYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTNFRFLTEKGMKNVFD DVVPEKYTIGATYAPEEFRFLTEKGMKNVFDDVVPEKYTIGATYAPEE PreSF3 (SEQ ID No. 6): MEAAFNDAIKASTGGAYESYKFIPALEAAVKAEEVKVIPAGELQVIEKVD AAFKVAATAANAAPANDKGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPA FGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQG ILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQD PRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTNADLGYGPAT PAAPAAGYTPATPAAPAEAAPAGKATTEEQKLIEKINAGFKAALAAAAGV QPADKYR PreSF4 (SEQ ID No. 24): MGKATTEEQKLIEDVNASFRAAMATTANVPPADKGKATTEEQKLIEDVNA SFRAAMATTANVPPADKGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAF GANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGI LTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDP RVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTNGKATTEEQKL IEDVNASFRAAMATTANVPPADKGKATTEEQKLIEDVNASFRAAMATTAN VPPADK

[0046] Furthermore, rabbit antibodies specific for the registered hepatitis B vaccine ENGERIX-B were obtained by immunizing New Zealand white rabbits (n=2) three-times with commercially available ready-to-use pre-filled syringes at an interval of one month.

[0047] Serum samples were obtained before immunization and approximately four weeks after the third immunization and stored at −20° C. until analysis.

[0048] Immunization with PreS vaccine mixture showed induction of IgG antibodies with specificity for sequential PreS epitopes. FIG. 2 shows a comparison of the IgG antibody responses towards PreS and synthetic PreS-derived peptides induced in rabbits with CFA-formulated PreS or aluminium hydroxide-adsorbed PreS vaccine mixture (FIG. 2B). Rabbit antibodies induced with CFA-formulated PreS recognized PreS and each of the PreS-derived peptides except of P7 (FIG. 2A). Aluminium-hydroxide adsorbed PreS vaccine mixture in a 20 μg dose induced PreS-specific IgG antibodies and IgG antibodies directed mainly to the N-terminal peptides P1, P2, peptide P6 and towards the C-terminal peptide P8 (FIG. 2B). No PreS or peptide-specific IgG responses were found in rabbits before immunization (FIG. 2, left bars).

Example 3: Assessment of PreS- and PreS Peptide-Specific Humoral Immune Responses

[0049] Serum samples were obtained from patients who have received three injections of Al(OH).sub.3-adsorbed PreS vaccine mixture (i.e., mixes of 10, 20 or 40 μg of each PreS vaccine mixture component or placebo, i.e., Al(OH).sub.3). Sera were collected before and four weeks after the third immunization and stored at −20° C. until use. A second set of serum samples was obtained from patients who were treated over a period of two years with seven subcutaneous injections of Al(OH).sub.3-adsorbed PreS vaccine mixture (i.e., mixes of 20 or 40 μg of each PreS vaccine mixture component or Al(OH).sub.3 as placebo). In addition, serum samples were obtained from patients suffering from hepatitis B infection which was diagnosed based on clinical data, liver function testing and HBV serum markers.

[0050] All sera analyzed, were screened for serological markers for HBV (i.e., hepatitis B surface antigen [HBsAg]; antibodies to the hepatitis B surface antigen [anti-HBs] as well as antibodies to the hepatitis B core antigen [anti-HBc].

[0051] ELISA plates (NUNC MaxiSorp®, Denmark) were coated with the antigens (recombinant PreS, synthetic PreS-overlapping peptides: P1-P8) or human serum albumin (negative control) (Behring, USA). Incubation was performed with rabbit sera in a dilution of 1:10,000 (CFA) or 1:500 (PreS vaccine mixture-20/PreS vaccine mixture-40), with mouse sera in a dilution of 1:1,000 and with human sera diluted differently for the isotypes and IgG subclasses. For the detection of human total IgG, sera were diluted 1:100, for IgA, IgG1, IgG2, IgG3, IgG4 as well as IgM, sera were diluted 1:20 and for detection of IgE antibodies sera were diluted 1:10.

[0052] Rabbit IgG was detected with donkey anti-rabbit horse radish peroxidase-conjugated IgG antibodies, diluted 1:2,500 (GE Healthcare, Buckinghamshire, Great Britain). Bound mouse IgG1 was detected with monoclonal rat anti-mouse IgG1 (BD Pharmingen, USA) diluted 1:1,000, followed by horse radish peroxidase-conjugated goat anti-rat IgG antibodies (Amersham Bioscience, Sweden) diluted 1:2,500.

[0053] Human IgG was detected with rabbit anti-human IgG Fc-specific antibody (Jackson-Dianova, Germany) diluted 1:10,000, followed by peroxidase-linked donkey anti-rabbit IgG (GE Healthcare) at a dilution of 1:2,500. Human IgA, IgG subclasses IgG1, IgG2 and IgG4 as well as human IgM were detected with purified mouse anti-human IgA1/IgA2, IgG1, IgG2, IgG4 and IgM (BD Pharmingen) antibodies, diluted 1:1,000 respectively, followed by peroxidase-linked sheep anti mouse IgG (GE Healthcare) at a dilution of 1:2,500. Monoclonal anti-human IgG3 (Sigma Aldrich, USA) was diluted 1:5,000. Human IgE was detected with goat anti-human horse radish peroxidase-conjugated IgE antibodies (KPL, USA).

Example 4: PreS-Specific Antibody Responses of PreS Vaccine Mixture Immunized Subjects are not Influenced by Prior Hepatitis B Immunity

[0054] Serum samples from human subjects who received immunotherapy with PreS vaccine mixture or with placebo were tested for IgG reactivity to PreS and synthetic PreS peptides (FIGS. 3a to 3c). These patients (n=30) had been screened for hepatitis B-specific serum markers (HBsAg, anti-HBs and anti-HBc antibodies) before treatment and found to be negative for HBsAg and anti-HBc antibodies. Due to previous vaccination with a hepatitis B vaccine, twenty-two of the subjects contained anti-HBs antibodies (FIGS. 3a to 3c). It was found that each of the patients who received immunotherapy with PreS vaccine mixture, regardless if they had been HB-vaccinated before or not, but not placebo-treated patients developed robust PreS-specific IgG responses when sera were tested after the third (V8; three months after first injection) as well as after the seventh injection (V15; 15 months after first injection) (FIGS. 3a to 3c). The PreS-specific IgG responses increased significantly from baseline before immunotherapy (i.e., V5 versus V8) and further increased significantly between V8 and V15 (i.e., after the seventh injection) (FIGS. 3a to 3c). The Pres-specific IgG responses in these patients were directed mainly towards the N-terminal peptides P1, P2 and P3 and again P1- and P2-specific IgG responses showed significant increases from baseline V5 to V8 and from V8 to V15 (FIGS. 3a to 3c). Also increases of IgG responses against the other PreS-derived peptides P4, P5, P6, P7 and P8 were found in sera from patients who received immunotherapy with PreS vaccine mixture but not in placebo-treated patients (FIGS. 3a to 3c).

Example 5: PreS-Specific Antibody Responses of PreS Vaccine Mixture Immunized Subjects are Directed Against Neutralizing Epitopes and Differ from Those of Hepatitis B-Infected Individuals

[0055] FIG. 4 shows a comparison of the PreS-specific isotype and IgG subclass responses of patients after immunotherapy with PreS vaccine mixture or placebo with that of hepatitis B-infected individuals. Immunotherapy with both doses of PreS vaccine mixture induced a robust Pres-specific IgG response in each of the treated patients which was significantly higher than the IgG response in hepatitis B-infected individuals (FIG. 4). No relevant PreS-specific IgA, IgE or IgM responses were detected in sera from patients who were treated with PreS vaccine mixture or placebo as well as in hepatitis B-infected individuals (FIG. 4). The PreS-specific IgG subclass response was different between PreS vaccine mixture-treated subjects and hepatitis B-infected individuals. PreS vaccine mixture-treated subjects showed a preferential IgG1 and IgG4 subclass response to PreS whereas hepatitis B-infected individuals mounted some IgG1 and IgG2 responses towards PreS (FIG. 4).

[0056] Also striking differences regarding the epitope specificity of PreS-specific antibodies in PreS vaccine mixture-treated patients versus hepatitis B-infected individuals were found (FIG. 5). PreS vaccine mixture-immunized patients but not hepatitis B-infected individuals showed strong IgG responses towards P1 and P3 (FIG. 5). This finding is surprising because the region defined by P1 corresponds to a motif within PreS1 (see also FIG. 1) that has been reported to contain the essential residues for inhibition of hepatitis B-infections. Furthermore, P7 was recognized only by PreS vaccine mixture-treated subjects but not by hepatitis B-infected individuals whereas IgG responses towards P2 and P6 were also found in hepatitis B-infected individuals (FIG. 5).

Example 6: Assessment of T Cell Responses

[0057] Peripheral Blood Mononuclear Cells (PBMC) were obtained from heparinized blood samples through density gradient centrifugation using Ficoll (Amersham Biosciences, Sweden). When blood samples could be obtained, PreS-specific PBMC proliferation was determined in PreS vaccine mixture-vaccinated subjects (n=19) at V5, V8, M1 (5 months after first vaccination) and M2 (17 months after first vaccination) by [3H]-thymidine incorporation.

[0058] For certain PreS vaccine mixture-immunized patients (n=11) CD4 and CD8 T cell responses could be assessed at M2 by carboxyfluorescein succinimidyl ester (CFSE) labelling.

[0059] Fluorescent dye-labelled cells were seeded at 200,000 cells/well in Ultra Culture™ serum-free medium (Lonza, Belgium) supplemented with 2 mmol/L L-glutamine (Sigma Aldrich, USA), 50 mmol/L β-mercaptoethanol (Sigma Aldrich), and 0.02 mg of gentamicin per milliliter (Sigma Aldrich), in a total volume of 200 μl in 96 well microplates with U shaped bottom (Thermo Fisher, USA). Cells were either left unstimulated (negative control) or were stimulated with Dynabeads® Human T-Activator CD3/CD28 (3 μg/well (Invitrogen, USA)) as positive control or with PreS (0.15 μg/well), equimolar quantities of PreS-overlapping peptides (0.03 μg/well) or with a mixture of the PreS-derived overlapping peptides containing 0.03 μg/well of each peptide and cultured at 37° C. in 5% CO.sub.2 for 7 days before antibody staining and FACS analysis was conducted.

[0060] For flow cytometry the following reagents were used: PerCP/Cy5.5 anti-human CD3 antibody (Clone HIT3a), Brilliant Violet 421™ anti-human CD4 antibody (Clone RPA-T4), APC anti-human CD8a antibody (Clone HIT8a), as well as isotype controls, i.e., PerCP/Cy5.5 mouse IgG2a, Brilliant Violet 421™ mouse IgG1, APC mouse IgG1 (BioLegend, USA) and Fixable Viability Dye eFluor® 780 (eBioscience, USA).

[0061] Flow Cytometry was performed on a BD FACS Canto II (Becton, Dickinson and Company, USA). Twenty thousand events were acquired per sample and analysis was performed via FlowJo Software, Version 10. Lymphocytes were gated according to morphological criteria on a forward and sideward scatter dot blot, dead cells were excluded by staining of viability dye and gating was focused on CD3CD4 and CD3CD8-positive T cells. Those cells that proliferated in response to antigen stimulation were identified by their reduction in CFSE fluorescence intensity. Results represent means of triplicate cultures and 235 median percentages stimulation of CD3+CD4+ and CD3+CD8+ above background are shown for the different antigens and the analysed patients.

[0062] FIG. 6 shows the development of PreS-specific T cell responses in patients who received immunotherapy with PreS vaccine mixture. A gradually increasing PreS-specific T cell response was found which was significantly higher at V8, M1 and M2 as compared to baseline at V5 (FIG. 6A). When the epitope specificity of the PreS-specific CD4 cell responses was analyzed by CFSE staining we found that P1, P2, P5 and P6 induced the strongest CD4 cell proliferation but CD4 responses towards P3, P4 and P7 were also found (FIG. 6B). Interestingly, the peptides and the peptide mix induced stronger CD4 cell proliferation than the PreS protein (FIG. 6B). Albeit at low frequency, some PreS and PreS peptide-specific CD8 cell response was detected which was mainly directed towards P2, P3, P6 and P8 and complete PreS (FIG. 6B).

Example 7: Hepatitis B Virus Neutralization Assays

[0063] The HBV inoculum for infection was prepared from supernatants of HepAd38 cells using a heparin column (GE Healthcare, Great Britain) to isolate viral particles. HepG2-hNTCP cells20 were seeded at a density of 3×10.sup.5 cells/well in a 24 well plate. At day two after seeding, the infection medium (DMEM, Invitrogen, USA) was supplemented with 2.5% DMSO (Merck, Germany) and at day three cells were infected with HBV. For the neutralization of HBV particles, patients' sera (10 μl) were pre-incubated with the HBV inoculum (6.9×10.sup.7 genome equivalents (GE)/well) for 30 minutes at 37° C., followed by co-incubation of cells with the patients' sera and virus in presence of 4% polyethylene glycol 800 (Sigma Aldrich, USA) for 16 hours at 37° C. The neutralizing monoclonal antibody Ma18/721 was used as positive control.

[0064] After 16 hours of inoculation, cells were washed extensively with PBS and fresh differentiation medium, supplemented with 2.5% DMSO (Invitrogen) was added. Additional medium changes were performed at day three and day five post infection.

[0065] Quantification of HBV infection was conducted by the measurement of secreted hepatitis B e antigen (HBeAg) in the supernatant from cells at day five to seven after infection. HBeAg was determined by ADVIA Centaur XPT automated chemiluminescence system (Siemens, Germany). Samples were considered as positive at a signal above 1 Index.

[0066] The expression of HBV core protein was detected by specific immunofluorescence. The supernatant was removed and the cells were washed with PBS prior to the fixation with 4% paraformaldehyde (Sigma Aldrich) for 30 minutes at room temperature (RT). Next, cells were washed with PBS followed by the permeabilization with 0.25% Triton X 100 (AppliChem GmbH, Germany) in PBS for 30 minutes at RT. Then, cells were incubated overnight at 4° C. with the primary antibody (anti-HBV core, rabbit polyclonal AK, DAKO Deutschland GmbH, Hamburg, Germany) diluted in 2% w/v BSA, PBS. On the next day, cells were washed with PBS and finally incubated with the secondary antibody (goat a rabbit Alexa 488; Invitrogen, Carlsbad, Calif.) and 4′, 6-Diamidin-2-phenylindo/Hoechst 33342 (Roche Applied Science, Germany) in the dark. For the detection of HBV core protein, the secondary antibody was incubated for 2 hours at RT, protected from light. Cells were examined under fluorescence microscope using 480 nm for Alexa-488-labeled secondary antibodies (Invitrogen, Carlsbad, Calif.) and 360 nm for the nuclear staining.

[0067] In the first type of assay the expression of hepatitis B core antigen (HBcAg) after infection of cells is detected by specific immunofluorescence. No HBcAg has been detected in uninfected cells but in infected and untreated cells and that expression can be prevented by pre-incubation of virus with the neutralizing monoclonal antibody Ma18/721 which is directed against the PreS1 domain of the large hepatitis B surface protein. Likewise it was found that pre-incubation of hepatitis B virus with rabbit antibodies induced by the commercial vaccine Engerix-B or with rabbit anti-PreS vaccine mixture (20 μg dose) antibodies inhibited infection of HepG2-hNTCP cells. A similar set of experiments was performed with sera from PreS vaccine mixture- or placebo-treated patients. Sera obtained from a patient before and after immunization with placebo did not inhibit infection of HepG2-hNTCP cells whereas sera obtained from a patient after immunization with 20 μg or from a patient after immunization with 40 μg inhibited infection of HepG2-hNTCP cells.

[0068] In addition to the staining of the HBcAg an assay based on the measurement of secreted hepatitis B e antigen (HBeAg) by HepG2-hNTCP cells was used seven days post infection with HBV as another surrogate marker to quantify the inhibition of HBV infection. It was found that sera from PreS vaccine mixture-treated inhibited HBV infection between 50-99% (FIG. 7A). No relevant difference was found depending on the dose and number of PreS vaccine mixture injections because a similar inhibition was observed for sera from patients who had received three injections (FIG. 7A) as well as for sera from patients who had received seven injections (FIG. 7A, black). Also, there was no obvious difference regarding the degree of inhibition between patients who either received the 20 μg or 40 μg dose of PreS vaccine mixture (FIG. 7A). No inhibition was observed for serum from a placebo treated patient and a more than 90% inhibition was observed for the monoclonal antibody Ma 18/7 (FIG. 7A). Rabbit anti-Engerix-B and rabbit anti-PreS vaccine mixture antibodies caused a more than 99% inhibition of HBV infection (FIG. 7B).

Example 8

[0069] Recombinant PreS and human serum albumin (Behring, USA) as negative control were coated onto Nunc Maxisorb microplates (Thermo-Fisher Scientific, USA) at a concentration of 2 μg/ml in 100 mM sodium phosphate buffer, pH 9.6 overnight at 4° C. Wash buffer was comprised of PBS, 0.05% v/v Tween20 (PBS/T) and the blocking procedures were performed with 2% w/v BSA, PBS/T for 2 hours at 37° C. All subsequent serum and reagent dilutions were done in 0.5% w/v BSA, PBS/T.

[0070] To determine humoral immune responses of rabbits, which have undergone complete immunization, either with recombinant PreS or PreS-fusion-proteins, as emulsion in Complete Freund's Adjuvant (CFA), sera were used in different dilutions (4° C., overnight) and bound total rabbit IgG was detected using donkey anti-rabbit horse radish peroxidase-conjugated IgG antibodies diluted 1:2.000 (GE Healthcare, Great Britain). The color reaction was induced by ABTS [2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid] and absorbance detection, corresponding to the levels of antigen-specific antibodies was performed at 405 nm and 490 nm using a microplate reader (Molecular Devices, USA). All determinations were performed in triplicates.

[0071] It surprisingly turned out that only a fusion protein comprising one or more peptides having the amino acid sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and/or SEQ ID No. 4 and PreS (PreS-F3) were able to induce the formation of PreS specific IgG to a much higher extend compared to PreS alone or other fusion proteins comprising also PreS fused to different peptides (PreS-F1, PreS-F2, PreS-F4) as depicted in FIG. 8.