Patent Application
20180244726
The present invention relates to a fusion protein for use in the treatment and/or prevention of a hepatitis B virus (HBV) infection.
Hepatitis B is a liver disease caused by hepatitis B viruses. The disease affects millions of people per year throughout the world. The HBV is present in the blood and body fluids of infected people and can therefore be spread by contacting these fluids with fluids of healthy people.
HBV primarily interferes with the functions of the liver by replicating in liver cells. During HBV infection, the host immune response causes both hepatocellular damage and viral clearance.
Acute HBV infections are usually not treated because most people are able to clear the infection spontaneously. However, chronic HBV infections have to be treated in order to reduce the risk of cirrhosis and liver cancer. Antiviral drugs currently used in the treatment of HBV infections include lamivudine, adefovir, tenofovir, telbivudine and entecavir. Furthermore, interferon alpha-2a acting as immune system modulator can also be used in the treatment. However, none of these drugs can clear HBV infections. These drugs can only stop the HBV from replicating, thus minimizing liver damage.
It is an object of the present invention to provide new means in the treatment and/or prevention of HBV infections which overcome the drawbacks of the present HBV treatments. A particularly important objective is viral clearance of HBV in chronically infected patients by restoring an efficient humoral and cellular immune response.
These objectives are achieved by 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.
It surprisingly turned out that a fusion protein comprising a hepatitis B PreS polypeptide or fragment thereof and 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 induces the formation of PreS specific antibodies in an individual to a much higher extent compared to PreS alone or other fusion proteins comprising peptides different from SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. Furthermore the antibodies produced in response to the administration of the fusion protein of the present invention show superior hepatitis B neutralizing effects and are able to inhibit hepatitis B virus infections. This is the first time that the administration of a fusion protein comprising PreS can be successfully be used in the treatment and/or prevention of a hepatitis B virus infection in a human subject.
As shown in
The fusion protein of the present invention may comprise one or more hepatitis B PreS polypeptides or one or more fragments thereof. The presence of more than one hepatitis B PreS polypeptides or fragments thereof in the fusion protein has the advantage that more antigens are presented to the immune system allowing the formation of even more antibodies directed to PreS. In a particularly preferred embodiment of the present invention the fusion protein comprises one, two, three, four, five, six, seven, eight, nine or ten hepatitis B PreS polypeptides or fragments thereof. The HBV PreS polypeptides as well as their fragments as defined herein being part of the fusion protein of the present invention may be derived from the same HBV genotype or from different genotypes. For instance, the fusion protein of the present invention may comprise the PreS polypeptide or a fragment thereof of HBV genotype A only or may be combined with a further PreS polypeptide or fragment thereof derived from HBV genotype B, C, D, E, F, G or H.
In a particularly preferred embodiment of the present invention the fusion protein comprises at least one peptide consisting of an amino acid sequence having at least 80% identity to SEQ ID No. 1, at least one peptide consisting of an amino acid sequence having at least 80% identity to SEQ ID No. 2, at least one peptide consisting of an amino acid sequence having at least 80% identity to SEQ ID No. 3 and at least one peptide consisting of an amino acid sequence having at least 80% identity to SEQ ID No. 4. Alternatively the fusion protein of the present invention may comprise one, two, three, four, five six, seven, eight, nine or ten of these peptides in any possible combination or even only one specific peptide in the same amount.
The terms “fused to” or “fusion protein”, as used herein, refer to a protein comprising a hepatitis B PreS polypeptide or fragment thereof that are expressed and prepared as one single recombinant polypeptide chain.
Methods for the production of fusion proteins are well known in the art and can be found in standard molecular biology references such as Sambrook et al. (Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al. (Short Protocols in Molecular Biology, 3rd ed; Wiley and Sons, 1995). In general, a fusion protein is produced by first constructing a fusion gene which is inserted into a suitable expression vector, which is, in turn, used to transfect a suitable host cell. In general, recombinant fusion constructs are produced by a series of restriction enzyme digestions and ligation reactions which result in the desired sequences being incorporated into a plasmid. If suitable restriction sites are not available, synthetic oligonucleotide adapters or linkers can be used as is known by those skilled in the art and described in the references cited above. The polynucleotide sequences encoding allergens and native proteins can be assembled prior to insertion into a suitable vector or the sequence encoding the allergen can be inserted adjacent to a sequence encoding a native sequence already present in a vector. Insertion of the sequence within the vector should be in frame so that the sequence can be transcribed into a protein. It will be apparent to those of ordinary skill in the art that the precise restriction enzymes, linkers and/or adaptors required as well as the precise reaction conditions will vary with the sequences and cloning vectors used. The assembly of DNA constructs, however, is routine in the art and can be readily accomplished by a person skilled in the art.
A fragment of a hepatitis B PreS polypeptide consists preferably of at least 30, preferably at least 40, more preferably at least 50, consecutive amino acid residues and may comprise PreS1 and/or PreS2 of the hepatitis B PreS polypeptide. In a particularly preferred embodiment of the present invention a fragment of a hepatitis B PreS polypeptide may comprise amino acid residues 1 to 70, preferably amino acid residues 1 to 65, more preferably amino acid residues 1 to 60, more preferably amino acid residues 1 to 55, more preferably amino acid residues 1 to 50, more preferably 1 to 45, more preferably amino acid residues 1 to 40, more preferably amino acid residues 1 to 35, more preferably amino acid residues 5 to 70, more preferably amino acid residues 5 to 65, more preferably amino acid residues 5 to 60, more preferably amino acid residues 5 to 55, more preferably amino acid residues 5 to 50, more preferably 5 to 45, more preferably amino acid residues 5 to 40, more preferably amino acid residues 5 to 35, more preferably amino acid residues 10 to 70, more preferably amino acid residues 10 to 65, more preferably amino acid residues 10 to 60, more preferably amino acid residues 10 to 55, more preferably amino acid residues 10 to 50, more preferably 10 to 45, more preferably amino acid residues 10 to 40, more preferably amino acid residues 10 to 35, more preferably amino acid residues 15 to 70, more preferably amino acid residues 15 to 65, more preferably amino acid residues 15 to 60, more preferably amino acid residues 15 to 55, more preferably amino acid residues 15 to 50, more preferably 15 to 45, more preferably amino acid residues 15 to 40, more preferably amino acid residues 15 to 35, of the hepatitis B PreS polypeptide, preferably of the HBV PreS polypeptides consisting of SEQ ID Nos. 5, 7, 8, 9, 10, 11, 12, 13 or 14, whereby SEQ ID Nos. 8 to 14 belong to HBV genotypes B to H, respectively.
The at least one peptide to be fused to at least one hepatitis B PreS polypeptide or fragment thereof has an identity of at least 80%, preferably of at least 85%, more preferably of at least 90%, more preferably of at least 92%, more preferably of at least 94%, more preferably of at least 96%, more preferably of at least 98%, more preferably of at least 99%, in particular of 100%, to SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. The degree of identity of a first amino acid sequence to a second amino acid can be determined by a direct comparison between both amino acid sequences using certain algorithms. Sequence identity is preferably determined by BLAST alignment (http://blast.ncbi.nlm.nih.gov/; Altschul S F et al J. Mol. Biol. 215 (1990): 403-410) using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.
According to a preferred embodiment of the present invention the amino acid sequence of the PreS polypeptide is at least 80% identical to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13, most preferably to SEQ ID No. 5.
The hepatitis B PreS polypeptide to be fused to at least one of the peptides described above has an identity of at least 80%, preferably of at least 85%, more preferably of at least 90%, more preferably of at least 92%, more preferably of at least 94%, more preferably of at least 96%, more preferably of at least 98%, more preferably of at least 99%, in particular of 100%, to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13.
According to a further preferred embodiment of the present invention the at least one peptide is fused to the N- and/or C-terminus of the PreS polypeptide.
“Fused to the N- and/or C-terminus”, as used herein, means that at least one peptide is fused to the N- and/or C-terminus of the PreS polypeptide or fragment thereof. The fusion protein of the present invention may comprise one or more peptides fused to the N-terminus of the PreS polypeptide or fragment thereof or to its C-terminus.
According to a preferred embodiment of the present invention the fusion protein comprises an amino acid sequence which is at least 80% identical to SEQ ID No. 6.
The fusion protein of the present invention has an identity of at least 80%, preferably of at least 85%, more preferably of at least 90%, more preferably of at least 92%, more preferably of at least 94%, more preferably of at least 96%, more preferably of at least 98%, more preferably of at least 99%, in particular of 100%, to SEQ ID No. 6.
According to a further preferred embodiment of the present invention the hepatitis B virus infection is caused by a hepatitis B virus genotype A, B, C, D, E, F, G, H or a subtype therof. It is preferred to use a HBV PreS polypeptide or fragment thereof of the same genotype to treat and/or prevent a HBV caused by this HBV genotype (e.g. PreS of HBV genotype A is used to treat/prevent an infection of HBV genotype A or one of its subtypes). Due to the conserved amino acid sequences in those parts of the PreS polypeptide which is known to be involved in the HBV infection, it is of course also possible to use a HBV PreS polypeptide or fragment thereof of one genotype to treat/prevent an infection of another HBV genotype (e.g. PreS of HBV genotype A is used to treat/prevent an infection of HBV genotype B, C, D, E, F, G and/or H or a subtype thereof).
The fusion protein of the present invention may be used in the treatment and/or prevention of HBV infections of various genotypes and subtypes thereof. Subtypes of hepatitis B viruses include A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, C1, C2, C3, C4, C5, D1, D2, D3, D4, D5, F1, F2, F3 and F4 as discussed in Schaefer et al. (World J Gastroenterol 13(2007):14-21).
According to a particularly preferred embodiment of the present invention the fusion protein is administered to an individual at least once in an amount of 0.01 μg/kg body weight to 5 mg/kg body weight, preferably 0.1 μg/kg body weight to 2 mg/kg body weight. According to further preferred embodiment of the present invention the fusion protein is administered to a patient in an amount of 5 to 50 μg, preferably 10 to 40 μg, more preferably 15 to 30 μg, either independent from the body weight (i.e. a dose may comprise 15, 20, 25 or 30 μg) or per kg body weight.
The amount of fusion protein that may be combined with excipients to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The dose of the fusion protein may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability to elicit the desired antibody response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances. For instance, the polypeptides and vaccine of the present invention may be administered to an individual at intervals of several days, one or two weeks or even months depending always on the level of hepatitis B PreS specific IgG induction.
In a preferred embodiment of the present invention the fusion protein of the present invention is applied between 2 and 10, preferably between 2 and 7, even more preferably up to 5 and most preferably up to 3 times. In a particularly preferred embodiment the time interval between the subsequent vaccinations is chosen to be between 2 weeks and 5 years, preferably between 1 month and up to 3 years, more preferably between 2 months and 1.5 years. The repeated administration of the fusion protein of the present invention may maximize the final effect of the treatment.
According to a further preferred embodiment of the present invention the fusion protein is administered together with at least one adjuvant and/or pharmaceutical acceptable excipient.
The fusion protein of the present invention can be administrated subcutaneously, intramuscularly, intravenously, mucosally etc. Depending on the dosage form and administration route the fusion protein of the present invention may be combined with excipients, diluents, adjuvants and/or carriers. A preferred adjuvant is alum. Suitable protocols for the production of vaccine formulations are known to the person skilled in the art and can be found e.g. in “Vaccine Protocols” (A. Robinson, M. P. Cranage, M. Hudson; Humana Press Inc., U. S.; 2nd edition 2003).
The fusion protein of the present invention may be formulated also with other adjuvants regularly used in vaccines. For instance, suitable adjuvants may be MF59, aluminum phosphate, calcium phosphate, cytokines (e.g. IL-2, IL-12, GM-CSF), saponins (e.g. QS21), MDP derivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes, emulsions (e.g. Freund's, SAF), liposomes, virosomes, iscoms, cochleates, PLG microparticles, poloxamer particles, virus-like particles, heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g. LTK63 and LTR72), microparticles and/or polymerized liposomes. Suitable adjuvants are commercially available as, for example, AS01B (MPL and QS21 in a liposome formulation), AS02A, AS15, AS-2, AS-03 and derivatives thereof (GlaxoSmithKline, USA); CWS (cell-wall skeleton), TDM (trehalose-6,6′-dimycolate), LeIF (Leishmania elongation initiation factor), aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7 or -12 may also be used as adjuvants. Preferred adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-O-deacylated monophosphoryl lipid A (3D-MPL), optionally with an aluminum salt. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO 98/43670.
Another preferred adjuvant is a saponin or saponin mimetics or derivatives, preferably QS21 (Aquila Biopharmaceuticals Inc.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation is QS21, 3D-MPL and tocopherol in an oil-in-water emulsion. Additional saponin adjuvants for use in the present invention include QS7 (described in WO 96/33739 and WO 96/11711) and QS17 (described in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1).
The SEQ ID Nos. disclosed herein have the following amino acid sequence (Genbank Acc. No.:
The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.
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).
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;
PAGGSSSGTVNPAPNIASHISSISARTGDPVTN
Peptides were purified by preparative HPLC and their identity was confirmed by mass spectrometry (Microflex MALDI-TOF, Bruker, USA).
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
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)3 as adjuvant. The four PreS vaccine mixture components include PreS fusion proteins PreSF1, PreSF2, PreSF3 and PreSF4 having the following amino acid sequences:
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.
Serum samples were obtained before immunization and approximately four weeks after the third immunization and stored at −20° C. until analysis.
Immunization with PreS vaccine mixture showed induction of IgG antibodies with specificity for sequential PreS epitopes.
Serum samples were obtained from patients who have received three injections of Al(OH)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)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)3-adsorbed PreS vaccine mixture (i.e., mixes of 20 or 40 μg of each PreS vaccine mixture component or Al(OH)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.
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].
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.
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.
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).
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 (
Also striking differences regarding the epitope specificity of PreS-specific antibodies in PreS vaccine mixture-treated patients versus hepatitis B-infected individuals were found (
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.
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.
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% CO2 for 7 days before antibody staining and FACS analysis was conducted.
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).
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.
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 3×105 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 (6.9×107 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.
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.
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.
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.
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.
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% (
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.
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.
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
| Number | Date | Country | Kind |
|---|---|---|---|
| 15183983.4 | Sep 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/070824 | 9/5/2016 | WO | 00 |
