WO2008031878A1 - Composition pour la thérapie et/ou pour la prophylaxie d'infections au vhb et de maladies dont la médiation est assurée par le vhb - Google Patents

Composition pour la thérapie et/ou pour la prophylaxie d'infections au vhb et de maladies dont la médiation est assurée par le vhb Download PDF

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Publication number
WO2008031878A1
WO2008031878A1 PCT/EP2007/059672 EP2007059672W WO2008031878A1 WO 2008031878 A1 WO2008031878 A1 WO 2008031878A1 EP 2007059672 W EP2007059672 W EP 2007059672W WO 2008031878 A1 WO2008031878 A1 WO 2008031878A1
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WIPO (PCT)
Prior art keywords
solution
range
hbv
antigen
adjuvant
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PCT/EP2007/059672
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English (en)
Inventor
Karl Melber
Pascale Buchmann
Zbigniew Janowicz
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Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH
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Priority claimed from PCT/EP2006/009014 external-priority patent/WO2007031334A2/fr
Priority claimed from EP06019910A external-priority patent/EP1902727A1/fr
Application filed by Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH filed Critical Rhein Biotech Gesellschaft für neue Biotechnologische Prozesse und Produkte mbH
Publication of WO2008031878A1 publication Critical patent/WO2008031878A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • composition for therapy and/or for prophylaxis of HBV-infections and HBV- mediated diseases A composition for therapy and/or for prophylaxis of HBV-infections and HBV- mediated diseases
  • the present invention relates to a composition, to a process for production of a composition, to the composition obtainable by this process, to a pharmaceutical formulation, to the use of the composition or of the pharmaceutical formulation for therapy and/or prophylaxis of hepatitis B virus (HBV)-infections and HBV- mediated diseases, to the use of the composition or of the pharmaceutical formula- tion for production of a pharmaceutical for therapy and/or for prophylaxis of HBV-infections and HBV-mediated diseases as well as to a process for therapy and/or for prophylaxis of HBV-infections and HBV-mediated diseases.
  • HBV hepatitis B virus
  • HBV hepatitis B virus
  • HBV represents a hepatotropic virus, which can cause both acute and chronic infections. While an infection at birth or in the first month of life leads, in 90 % of cases, to a chronic infection, this is only observed in about 10 % of cases with infection in adulthood. In about 2 % of infected adults, a fulminant hepatitis (acute liver failure) can occur, which is linked to a high death rate.
  • a chronic infection is without clinical symptoms, but can develop after a latency period of 10 to 30 years into an acute liver disease (active, chronic hepatitis B) because of the persisting HBV and the pathological interaction of the immune system with the infected liver cells.
  • the number of chronic HBV carriers who develop chronic hepatitis B is around 25 %.
  • Chronic hepatitis B can finally lead to liver cirrhosis and/or primary liver cell carcinoma. Both diseases are linked with a high death rate. It is estimated that there are currently around 350 million chronically infected HBV-carriers worldwide. Every year, around a million people die from the consequences of a chronic HBV-infection.
  • HBV infections can be prevented by prophylactic hepatitis B vaccines.
  • Such vaccines based on purified hepatitis B surface antigen (HBsAg) from the plasma of chronic carriers were first available at the start of the 1980's. Later, these were replaced by HBsAg produced recombinantly. Since, however, vaccina- tion is not currently routinely introduced in all countries, HBV will continue to represent a threat to human health. In this context, it is significant that chronic HBV carriers are considered as infectious and can transfer the virus.
  • Chronic hepatitis B is a very dynamic disease with a number of forms of appear- ance.
  • the immunotolerant phase which is characterised by the presence of viral markers in the serum (HBV- DNA, HBsAg, HBeAg) as well as the lack of liver problems.
  • HBV-DNA, HBsAg, HBeAg a virus-DNA
  • serum-transaminases increase batchwise.
  • HBsAg While HBsAg continues to be present in the serum, there is a small percentage (around 1 %) of patients, which spontaneously undergoes a serum conversion from HBeAg positive to HBeAg negative. This serum conversion is a sign for the entry into the inactive carrier stage, which is characterised by a low virus activity. In few cases, a seroconversion from HBsAg positive to anti- HBsAg positive is additionally observed, which is considered a sign of a healed infection.
  • the active chronic hepatitis B is characterised by necro-inflammation and fibrosis of the liver tissues, which leads in the course of time to cirrhosis and decompensated liver disease. As further complication, cirrhosis can lead to pri- mary liver cell carcinoma, whereby some chronic HBV carriers can also develop liver cancer without signs of cirrhosis.
  • Interferon- ⁇ for example Intron A ® or Roferon A ®
  • antiviral substances Livudine, Adefovir, Entecavir
  • Interferon- ⁇ is preferably used with HBeAg positive patients, whereby around 30 % react with a HBeAg-serum conversion, which correlates with an inactive carrier status.
  • a few patients around 3 %) also show an HBsAg seroconversion as sign of a full healing.
  • the treatment with Interferon- ⁇ is, however, linked with severe side effects, which often necessitate an early stop to the therapy.
  • Interferon- ⁇ shows low response rates compared with HBeAg positive patients.
  • antiviral substances which belong to the class of nucleoside/nucleotide reverse transcriptase inhibitors, can be used here. These antiviral substances inhibit the replication of the virus and, as a result, prevent advance of the liver disease.
  • the serum conversion rates for HBeAg positive patients are lower, compared with Interferon- ⁇ , and are not higher than the spontaneously occurring remission.
  • the target of the antiviral therapy is thus to delay the disease by reduction of the viral activity for an unlimited time.
  • the long-term use of the antiviral substances is, however, jeopardised by the appear- ance of resistant virus mutants.
  • Lamivudine In the case of the most frequently used substance, Lamivudine, a resistance rate of up to 68 % within four years has been observed. The most recently introduced, Adefovir, seems to cause resistance less frequently. The basic problem of virus resistance represents, however, an inherent threat for long-term treatment, even if the antiviral substances should be used in combina- tion. There exists, therefore, an urgent need for additional therapeutic means of treating chronic hepatitis B. Particularly desirable are immunomodulatory agents with few side effects and with the perspective for a short therapy for the patients.
  • Such an alternative is the specific immunotherapy in the form of a therapeutic vaccine.
  • the concept of a therapeutic vaccine is to stimulate specifically the immune system of a chronic hepatitis B patient in such a way that an adequate immune response is initiated against the virus, which finally leads to control of the virus and curbs the advance of the liver disease.
  • One of the obvious effects which a therapeutic vaccine must show is the breaking through of the present immu- no tolerance in the presence of large amounts of viral antigens.
  • the therapy should, in particular, generate virus-specific T-cell responses, in particular cytotoxic T-lymphocytes (CTL) against diverse antigens of HBV.
  • CTL cytotoxic T-lymphocytes
  • Recombinant subunit vaccines are generally not able to cause CTL responses.
  • DNA-vaccines in particular DNA-vaccines based on a hepatitis B core antigen (HBcAg)-coding plas- mid vector can be achieved, however, current vaccines show to date overall in humans only very weak immune response and do not stimulate any potent CTL response.
  • HBcAg hepatitis B core antigen
  • Prior vaccines show to date overall in humans only very weak immune response and do not stimulate any potent CTL response.
  • so-called "Prime-Boost- Vaccines” are currently being developed, whereby immunization takes place alternately with a DNA-vaccine (e.g. coding for HBsAg) and a recombinant virus vector (e.g. modified Vaccinia Ankara Stem), which, e.g.
  • WO-A-01/98333 describes the use of an HBc-antigen, in which one or more of the four arginine repeat units at the C-terminus was removed, but the C-terminal cysteine residue was retained, inter alia for therapeutic treatment of HBV.
  • the removed arginine repeat units can be replaced by sequences which represent epitopes from bacterial or viral pathogens, parasites, allergens or antigens associated with cancer, which are recognised by T-helper cells, B-cells or cytotoxic T- lymphocytes.
  • WO-A-2005/023297 describes a composition for treatment of HBV-infections and HBV-mediated diseases, which comprise at least two hepatitis B surface antigens (HBsAg's), fragments thereof and/or nucleic acids coding these, whereby the HBsAg's in the HBV genotype in the S-region and/or the pre-Sl-region are different and whereby the composition comprises no HBcAg or nucleic acids coding this.
  • HBsAg's hepatitis B surface antigens
  • WO-A-2005/056051 discloses inter alia a recombinant polypeptide composition for treatment or prophylaxis of a hepatitis B infection, whereby the composition comprises a plurality of components, preferably at least three or all four selected from a) a core-polypeptide element, which comprises a sequence of the immunogenic region of the HBV-core-protein, b) a surface antigen-polypeptide element, which comprises a sequence of the immunogenic region of the HBV-surface anti- gen-protein (S, M or L), c) an X-polypeptide element, which comprises a sequence of the immunogenic region of the HBV-X-protein and d) a polymerase- polypeptide-element, which comprises a sequence of the immunogenic region of the HBV-polymerase-proteins, whereby at least one of the elements comprises at least 100 amino acids of the respective HBV proteins.
  • EP-A-I 136 077 discloses a vaccine composition for nasal
  • the present invention has the object of overcoming the disadvantages known from the prior art for vaccines for treatment or prophylaxis of a hepatitis B infection in humans.
  • the present invention had the object of providing a composition suitable for treatment of a human, which, in comparison to the compositions known from the prior art, leads to strong immune responses, in particular strong CTL responses against the HBcAg, in patients with a chronic HBV infection.
  • the composition should lead to breaking through of the immunological tolerance in as many patients as possible with a chronic HBV infection, irrespective of their HLA type.
  • composition according to the invention should enable a breaking through of the immunological tolerance, irrespective of the HBV genotypes of the chronic patient and thus lead to generation of an immune response which keeps the virus under control.
  • the present invention further had the object of providing a process with which a composition with the above-described advantages can be obtained with as little preparative complexity as possible.
  • HBsAg a fragment of this antigen, a variant of this antigen or of the fragment of this antigen or a mixture of at least two thereof.
  • the adjuvant comprises as saponin 50 to 90 wt.%, particularly preferably 60 to 80 wt.%, yet more preferably 65 to 75 wt.% and most preferably about 70 wt.% of the fraction A obtained by the herein-described test method from Quillaja Saponaria Molina extract and 10 to 50 wt.%, particularly preferably 20 to 40 wt.%, yet more preferably 25 to 35 wt.% and most preferably about 30 wt.% of the fraction C obtained by the herein-described test method from Quillaja Saponaria Molina extract, whereby the total amount of saponins of the fraction A and saponins of the fraction C is 100 wt.%;
  • the adjuvant comprises, besides the saponin, cholesterol, phosphatidylcholine and decanoyl-N-methylglucamide.
  • polypeptide used in the following details relates to a polymer of amino acids and is not restricted to a particular minimum number of amino acids. It therefore comprises equally peptides, oligopeptides, dimers, trimers, oligomers, particles and the like. Furthermore, the term “polypeptides” comprises not only the pure polymers of amino acids, which are obtained directly after translation in the ribosomes, but also those polypeptides which are obtained post-translationally by glycosylation, acetylation, phosphorylation and the like.
  • the HBV genotype A comprises the reference nucleic acid sequence Genbank X02763 or respectively the HBV genotype Aa& comprises the reference nucleic acid sequence according to Genbank AF297621.
  • the reference nucleic acid sequence is Genbank D00330, for the genotype B,, the reference nucleic acid sequence AB073858.
  • the reference nucleic acid sequence is Genbank AY206389, for the genotype C aus , the reference nucleic acid sequence according to Genbank AB048704.
  • the reference nucleic acid sequence is Genbank X02496, for the genotype E, Genbank X75657, for the genotype F, Genbank X69798, for the genotype G, Genbank AF160501 and for the genotype H, Genbank AY090454.
  • fragment of an HBcAgi_ x is preferably understood a polypeptide in which a section of at least 25, preferably at least 50 and particularly preferably at least 100 amino acids is identical with the corresponding section of the HBcAgi_ x .
  • variant of the HBcAgi_ x or of the fragment of the HBcAgi_ x is preferably understood a polypeptide in which at most 10, preferably at most 5 amino acids, particularly preferably at most 2 amino acids and most preferably at most 1 amino acid of the HBcAgi_ x or of the fragment of the HBcAgi_ x has been deleted, inserted, substituted or appended at the C- and/or N-terminal ends, preferably sub- stituted.
  • variants also comprises fusion polypeptides, in particular fusions with HBsAg, which comprise HBcAgi_ x .
  • adjuvant a compound which enables the induction of an immune response, preferably the induction of a CTL response to the antigen.
  • the HBcAgi_ x 's which are comprised in the composition according to the invention and which are truncated compared to the natural HBcAg's, of which at least a part of the C-terminal nucleic acid binding region, preferably the whole C- terminal nucleic acid binding region is lacking, can be obtained by recombination processes known to the skilled person.
  • One possibility is to amplify the core or pre-core reading frame from a virus isolate by means of polymerase chain reaction (PCR). It is also conceivable to prepare the corresponding DNA-sequence synthetically and then to amplify by means of PCR. The obtained PCR fragment is then cloned in a plasmid vector.
  • the correspondingly shortened form can then be inserted into an expression vector, for example into an expression vector E. coli.
  • the insertion preferably occurs in such a way that the gene coding for HBcAgi_ x or for the fragment or the variant respectively is under the control of an active promoter.
  • the expression vector can simultaneously comprise induction elements for increasing the expression rate.
  • individual clones are obtained, which express the HBcAgi_ x or the fragment or the variants respectively.
  • an individual clone is injected into culture medium and cultivated overnight at 37°C in the shaking flask. The E.
  • HBcAg or HBcAg-variants respectively situated in the homogenisate are concentrated by precipitation with ammonium sulphate and then purified of bacterial host components by gel filtration. Particle-forming HBcAg-variants can be further purified by a speed zone centrifugation.
  • Particle-deficient HBcAgi_ x 's are ideally engineered with an N-terminal His-Tag-affmity marker and purified by means of a Ni-NTA-affmity chromatography and subsequent gel filtration. The thus-produced HBcAgi_ x 's are subjected to a sterile filtration and used as such for the formulation of the composition according to the invention.
  • HBcAgi_ x 's or their fragments or variants comprised in the composition according to the invention can also the obtained synthetically.
  • the HBcAgi_ x 's comprised in the composition according to the invention can be particle forming or non particle forming, whereby particle forming HBcAgi_ x 's are preferred.
  • Particle forming HBcAgi_ x 's can associate to form cap- sides with a diameter of approximately 27 nm.
  • 180 Or 240 dimers associate to form macromolecular structures.
  • the HBcAgi_ x 's are, therefore, present in the form of the above-described particles.
  • Preferred antigens according to the invention are in particular the HBcAgi_i24, HBcAgi_i27, the HBcAgM 44 , the HBcAgM 45 , the HBcAgM 46 , the HBcAgM 47 , the HBcAgi_i48, the HBcAgM 49 , (i.e. polypeptides which comprise, preferably which consist of the first 127, 144, 145, 146, 147, 148 or 149 amino acids of the natural HBcAg) and HBcAgsi-m (a fragment of HBcAg) which comprises, preferably which consists of the amino acids 81 to 133 of the naturally occurring HBcAg).
  • polypeptides which comprise, preferably which consist of the first 127, 144, 145, 146, 147, 148 or 149 amino acids of the natural HBcAg
  • HBcAgsi-m a fragment of HB
  • a further preferred antigen is HBcAgM 44+
  • a polypeptide which comprises the first 144 amino acids of the naturally occurring HBcAg 's and which as 145 th amino acid comprises an amino acid AS which is different to the glutamic acid appearing as 145 th amino acid in the naturally occurring HBcAg, whereby this amino acid AS which is different to glutamic acid is preferably iso leucine.
  • the HBcAgi_ x does not comprise any epitopes which are foreign to HBcAg, in particular no HBsAg- epitopes.
  • the HBcAgi_ x 's comprise no pre-Sl epitopes, in particular not the amino acids 3 to 55 of the pre-Sl antigen.
  • the HBcAgi_ x comprises no cysteine residue at the C- terminal position x, as long as x is not equal to 48, 61 or 107.
  • composition according to the invention comprises at least two HBcAgi_ x 's or fragments or variants thereof, whereby the HBcAgi_ x 's differ in the HBV-genotype.
  • the composition according to the invention comprises an adjuvant comprising a saponin, whereby the adjuvant preferably comprises as saponins exclusively the fractions A and C obtained from Quillaja Saponaria Molina extract and is substantially free of saponins of fraction B.
  • the adjuvant is present in the form of particles with an average particle diameter in a range from 20 to 200 nm, particularly preferably 30 to 100 nm and most preferably 35 to 45 nm.
  • the indivicual adjuvant particles comprise both saponins of fraction A and saponins of fraction C, the saponins of fraction A and of fraction C are therefore distributed substantially homogeneously over all adjuvant particles.
  • the particularly preferred adjuvants according to the invention are preferably saponin complexes obtainable by a process comprising the following process steps:
  • PBS solution Li with a pH value in a range from 7.2 to 7.6, particularly preferably 7.3 to 7.5 and most preferably with a pH value of about 7.4; lib) provision of a solution L 2 of 5 to 15 mg/1, particularly preferably 7.5 to 12.5 mg/ml and most preferably about 10 mg/ml cholesterol and 5 to 15 mg/ml, particularly preferably 7.5 to 12.5 mg/ml and most preferably about 10 mg/ml phosphatidylcholine in 10 to 30 wt.%, particularly preferably 15 to 25 wt.% and most preferably about 20 wt.% aqueous de- canoyl-N-methylglucamide solution; lie) provision of a PBS solution L 3 comprising 50 to 150 mg/ml, preferably 75 to 125 mg/ml and most preferably about 100 mg/ml of the mixture of the saponins from the fractions A and C with a pH value in a range from 6.0 to 6.4; Hd) addition of the solution
  • Hf dialysis of the solution L 4 against PBS solution with a pH value in a range from 6.0 to 6.4, particularly preferably 6.1 to 6.3 and most preferably with a pH value of about 6.2, whereby this dialysis preferably occurs at a tem- perature in a range from 4 to 30 0 C, particularly preferably 15 to 25 0 C and most preferably at ambient temperature and over a period from 0.5 to 24 hours, particularly preferably 1 to 12 hours and most preferably 2 to 6 hours;
  • Hg optionally repeated dialysis of the solution L 4 against PBS solution with a pH value in a range from 6.0 to 6.4, particularly preferably 6.1 to 6.3 and most preferably with a pH value of about 6.2, whereby this dialysis preferably occurs at a temperature in a range from 0 to 10 0 C, particularly preferably 2 to 6 0 C and most preferably at 4 0 C.
  • adjuvant particles which comprise respectively saponins of fraction A and saponins of fraction C.
  • ISCOMATRIX ® adjuvant for antigen delivery M.J. Pearse and D. Drane, Advanced Drug Delivery Reviews 57 (2005), pages 465-474, besides the saponins, cholesterol, dipalymitoyl phosphatidylcholine as phosphatidylcholine and MEGA-IO as decanoyl-N-methylglucamide.
  • this can also comprise as co-adjuvants, in addition to the saponin complex, further adjuvants which do not comprise saponin, whereby these co-adjuvants can be present separate from the complex or integrated into this complex.
  • co- adjuvant any adjuvant known to the skilled person can be used which is commonly used in vaccines.
  • Preferred adjuvants are selected from the group comprising particulate adjuvants, such as, for example, sub-micro oil-in-water-emulsion, to which belong, for example, MF59 (5 % Squalene, 0,5 % Tween ® 80 and 0,5 % Span ® 85), SAF (10 % Squalene, 0.4 % Tween ® 80, 5 % Pluron-blocked polymer) and thr-muramyl dipeptide (thr-MDP), mineral compounds such as, for example, aluminium hydroxide, aluminium sulphate or aluminium phosphate, incomplete Freund's adjuvant, lipopolysaccharides, N-acetylglucosaminyl-N-acetylmuramyl- L-alanyl-dipeptide (GMDP) or muramyldipeptide (MDP), GAL-ceramide, di- methyldioctadecylammonium bromide (
  • co-adjuvants are lipopolysaccharides, aluminium hydroxide, aluminium phosphate, GAL-ceramide, oligodesoxyribonucleotide with CpG-motif, as well as PEG-sorbitan fatty acid esters.
  • this further comprises, in addition to the antigen HBcAgi_ x and the adjuvant at least one HBsAg, a fragment from this antigen, a variant of this antigen or of the fragment of this antigen or a mixture of at least two thereof.
  • the antigen HBcAgi_ x and the adjuvant at least one HBsAg, a fragment from this antigen, a variant of this antigen or of the fragment of this antigen or a mixture of at least two thereof.
  • the HBsAg is a 226-amino acid protein (p24/gp27 or S-protein), which self as-flects in 20-30 nm lipoprotein particles, in which around 100-150 sub-units are cross-networked by means of multiple inter-and intra-molecular disulfide links.
  • the HBsAg's optionally comprised in the composition according to the invention can be derived from all 8 HBV-standard genotypes A, B, C, D, E, F, G and H known at this time, in the same way as the HBcAgi_ x 's.
  • HBsAg also further comprises, in addition to 226-amino acid protein, both M- and L-proteins, which, in addition to the HBsAg also comprise the 55-amino acid long pre-S2- peptide (M-protein) or the 55-amino acid long pre-S2-peptide and the 120-amino acid long pre-Sl -peptide (L-protein).
  • M-protein 55-amino acid long pre-S2- peptide
  • L-protein 120-amino acid long pre-Sl -peptide
  • fragment of an HBsAg is preferably understood a polypeptide in which a section of at least 50, preferably at least 150 and particularly preferably at least 200 amino acids is identical to a corresponding section of the HBsAg.
  • variants of the HBsAg or of the fragment of the HBsAg is preferably understood a polypeptide in which at most 5, preferably at most 2 and particularly preferably at most 1 amino acid of HBsAg or of the fragment of the HBsAg is deleted, inserted, appended at the ends or substituted, preferably substituted.
  • this comprises not only the HBsAg, but, as described in WO-A-2005/02397, at least two HBsAg's or fragments or variants thereof, whereby the HBsAg's differ in the HBV-genotype in the S-region and/or in the pre-Sl -region.
  • the two HBsAg's which differ in the HBV-genotype are present in the form of homologous particles, as described likewise in WO-A-2005/02397.
  • a "homologous" HBsAg particle is understood a particle which is composed solely of HBsAg of the same sub-type/genotype.
  • the HBsAg's can be obtained by processes known to the skilled person, either from the serum of patients with chronic hepatitis B, synthetically or as recombinant polypeptides. In this context, reference is made to the above-described methods in connection with the obtaining of HBcAgi_ x .
  • the composition according to the invention can also comprise further additives iv), such as pharmaceuticals carriers or additives, in addition to the components i), ii) and optionally iii), in particular if it is present as pharmaceutical formulation.
  • Preferred pharmaceutical carriers are in particular water, buffered water, preferably isotonic salt solutions such as PBS (Phosphate Buffered Saline), glucoses, dextroses, mannitol, lactoses, starches, magnesium stearate, celluloses, magnesium carbonate, 0.3 % glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides or the like.
  • the type of pharmaceutical carrier used is particularly dependent on whether the composition according to the invention is formulated for oral, intranasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • tonicity regulators, wetting agents, emulsifies or buffer substances can be comprised in the composition ac- cording to the invention.
  • the relative amount ratio between the antigen components i) or the antigen components i) and iii) to adjuvant ii) preferably lies within a range from 1 : 20 to 20 : 1, particularly preferably within a range from 1 : 10 to 10 : 1, yet more pref- erably within a range from 1 : 5 to 5 : 1 and most preferably within a range from 1 : 2 to 2 : 1.
  • the weight ratio between adjuvant and antigen which is optimal for the respective application case can be determined by the skilled person by means of simple trials, in which, for example, the strength of the CTL-response is determined dependent on this amount ratio.
  • the weight ratio between the HBcAgi_ x and the HBsAg preferably lies within a range from 1 : 20 to 20 : 1, particularly preferably within a range from 1 : 10 to 10 : 1, yet more preferably within a range from 1 : 5 to 5 : 1 and most preferably within a range from 1 : 2 to 2 : 1.
  • the skilled person can also determine the optimal weight ratio here by means of simple trials.
  • the total concentration of the components i), ii) and optionally iii) in the composi- tion according to the invention preferably lies within a range from 0.1 to 2,000 ⁇ g/ml, particularly preferably within a range from 0.5 to 1,500 ⁇ g/ml, yet more preferably within a range from 1 to
  • composition according to the invention is not present as lyophilisate.
  • composition according the invention comprises
  • composition according to the invention comprises
  • composition according to the invention comprises i) an HBcAgi_i45, ii) the above described saponin complex as adjuvant, and iii) an HBsAg.
  • composition according to the invention comprises
  • composition according to the invention is preferably further characterised in that is has an HBcAg-specif ⁇ c CD8 + T-cell frequency in the spleen of at least 30, particularly preferably at least 50, yet more preferably at least 75, even more pref- erably at least 100 and most preferably at least 300 CD8 + IFNy + T-cells per 10 5 CD8 + T-cells in C57BL/6-mice with an immunization according to the herein- described immunization process and subsequent re-stimulation described herein with MGLKFRQL (a synthetic fragment of HBcAg's).
  • composition according to the invention is further preferably characterised in that, if it comprises HBsAg, it induces in C57BL/6-mice potent antibody responses against HBsAg with an immunization according to the herein-described immunization process.
  • the total anti-HBs antibody count determined according to the herein-described AUSAB ® -assay at up to, on average, at least 100 mlU/mL, preferably at least 500 mlU/mL, particularly preferably at least 1,000 mlU/mL and most preferably at least 1,500 mlU/mL.
  • the determination of the anti-HBs- specific IgG-iso type-class shows that both IgGl- and IgG2b-counts are increased, which is an indication of the formation of both a ThI and a Th2 immune response.
  • HBcAgi_ x whereby x is a whole number from the range from 100 to 160, preferably the range from 120 to 150, particularly preferably the range from 140 to 149, yet more preferably the range from 144 to 146 and most preferably is equal to 145, of a fragment from this antigen, of a variant of this antigen or of the fragment of this antigen or of a mixture of at least two thereof,
  • the adjuvant comprises as saponin 50 to 90 wt.%, particularly preferably 60 to 80 wt.%, yet more preferably 65 to 75 wt.% and most pref- erably about 70 wt.% of the fraction A obtained by the herein- described test method from Quillaja Saponaria Molina extract and 10 to 50 wt.%, particularly preferably 20 to 40 wt.%, yet more preferably 25 to 35 wt.% and most preferably about 30 wt.% of the fraction C obtained by the herein-described test method from Quillaja Saponaria Molina extract, whereby the total amount of saponins of the fraction A and saponins of the fraction C is 100 wt.%;
  • the adjuvant comprises, besides the saponin, cholesterol, phosphatidylcholine and decanoyl-N-methylglucamide, III) provision of a HBsAg, of a fragment of this antigen, of a variant of this antigen or of the fragment of this antigen or of a mixture of at least two thereof, and
  • HBcAgi_ x or as fragment or variant thereof as adjuvant comprising a saponin, a saponin derivative or a mixture of at least two thereof as well as as HBsAg or as fragment or variant thereof, those compounds which were already mentioned as preferred components i), ii) and iii) in connection with the composition according to the invention are preferred.
  • the provision of the HBcAg and of the HBsAg occurs in such a way that the antigens or antigen fragments obtained from the plasma of patients with chronic hepatitis B (only in the case of HBsAg) by synthetic processes or by recombinant expression are presented in a suitable pharmaceutical carrier.
  • This pharmaceutical carrier is prefera- bly a liquid, particularly preferably an aqueous liquid, yet more preferably an aqueous salt solution, such as PBS.
  • the aqueous antigen solutions obtained in this way can be sterile filtered by processes known to the skilled person.
  • the provision of the HBcAg and optionally of HBsAg in process steps I) and III) occurs in such a way that these antigens are first presented in a pharmaceutical carrier, preferably in an aqueous salt solution, in a concentration which is at least double, particularly preferably at least three times, yet more preferably at least four times and most preferably at least six times as great as the concentration of the HBcAg or of the HBsAg in the final targeted composition according to the invention (thus, should, for example, the concentration of HBcAg in the composition according to the invention amount to 10 ⁇ g/ml, it is preferred in the production of the composition that the HBcAg is first provided in an aqueous salt solution in a concentration of at least 20 ⁇ g/ml, particularly preferably at least 30 ⁇ g/ml, yet more preferably at least 40 ⁇ g/ml and most preferably at least 60 ⁇ g/ml).
  • HBcAg and HBsAg be comprised in the composition ac- cording to the invention, it is preferred to present these together in one and the same salt solution in the above-mentioned concentrations (at least two times, at least three times, at least four times or at least six times over-concentrated). It is further preferred according to the invention that these over-concentrated antigen- solutions, before the bringing into contact with the adjuvant in process step IV), are incubated for a time period of 10 minutes to 4 hours, particularly preferably from 20 minutes to 2 hours and yet more preferably from 30 minutes to 1 hour at a temperature within a range from 30 to 60 0 C, particularly preferably 35 to 55 0 C and yet more preferably 37 to 50 0 C.
  • the thus-obtained composition is incubated for a time period from 15 minutes to 5 hours, particularly preferably from 30 minutes to 3 hours and yet more preferably from 45 minutes to 2 hours at a temperature from 10 to 40 0 C, particularly preferably from 15 to 30 0 C, yet more preferably from 20 to 25 0 C and most preferably at room temperature.
  • the provision of the adjuvant in process step II) preferably comprises the following process steps:
  • PBS solution Li with a pH value in a range from 7.2 to 7.6, particularly preferably 7.3 to 7.5 and most preferably with a pH value of about 7.4; lib) provision of a solution L 2 of 5 to 15 mg/1, particularly preferably 7.5 to 12.5 mg/ml and most preferably about 10 mg/ml cholesterol and 5 to 15 mg/ml, particularly preferably 7.5 to 12.5 mg/ml and most preferably about 10 mg/ml phosphatidylcholine in 10 to 30 wt.%, particularly pref- erably 15 to 25 wt.% and most preferably about 20 wt.% aqueous de- canoyl-N-methylglucamide so lution; lie) provision of a PBS solution L 3 comprising 50 to 150 mg/ml, preferably 75 to 125 mg/ml and most preferably about 100 mg/ml of the mixture of the saponins from the fractions A and C with a pH value in a range from 6.0 to 6.4;
  • Hf dialysis of the solution L 4 against PBS solution with a pH value in a range from 6.0 to 6.4, particularly preferably 6.1 to 6.3 and most preferably with a pH value of about 6.2, whereby this dialysis preferably occurs at a tem- perature in a range from 4 to 30 0 C, particularly preferably 15 to 25 0 C and most preferably at ambient temperature and over a period from 0.5 to 24 hours, particularly preferably 1 to 12 hours and most preferably 2 to 6 hours;
  • Hg optionally repeated dialysis of the solution L 4 against PBS solution with a pH value in a range from 6.0 to 6.4, particularly preferably 6.1 to 6.3 and most preferably with a pH value of about 6.2, whereby this dialysis preferably occurs at a temperature in a range from 0 to 10 0 C, particularly preferably 2 to 6 0 C and most preferably at 4 0 C.
  • complexes from saponins are used as adjuvant (ISCO-matrix)
  • the complexes are prepared in sterile form and/or obtained commercially from appropriate suppliers and then the saponin complex or the saponin derivatives complex is brought into contact with the sterilised, aqueous HBcAg solution and optionally with the sterilised HBsAg solution or with a mixture of these two solutions.
  • the formation of a classic ISCOM-complex, in which the antigens are incorporated into the interior of the ISCO-matrix does not occur.
  • compositions can then be further combined with further addi- tives, such as, for example, further pharmaceutical carriers and additives, depending on whether the composition should be administered orally, intranasally, in- tradermally, subcutaneously, intramuscularly or intravenously.
  • further pharmaceutical carriers and additives such as, for example, further pharmaceutical carriers and additives, depending on whether the composition should be administered orally, intranasally, in- tradermally, subcutaneously, intramuscularly or intravenously.
  • pharmaceutical carrier and additive those compounds are in turn preferred which have already been mentioned as preferred pharmaceutical carriers and preferred additives in connection with the composition according to the invention.
  • compositions obtainable by the above-described process, whereby these compositions preferably have the same properties as the above-described compo- sition according to the invention.
  • a contribution to the solution of the above-mentioned objects is also provided by a pharmaceutical formulation comprising the composition according to the invention as well as at least one of the above-mentioned additives iv).
  • composition according to the invention or of the pharmaceutical formulation according to the invention for therapy and/or for prophylaxes of HBV-infections and HBV-mediated diseases.
  • the composition accord- ing to the invention or the pharmaceutical formulation according to the invention is used for administration in mammals, in particular human.
  • this serves in particular for therapy and/or for prophylaxes, preferably for therapy, of acute and/or chronic, preferably chronic HBV-infections.
  • prophylaxis in the context of an HBV-infection in humans comprises preferably the treatment of a human who has not yet any contact with HBV, with the aim of preventing the occurrence of an infection with HBV, the treatment of a human who has already had contact with HBV and who is in the immunotolerant chronic phase, with the aim of preventing this human from entering into the immunoactive chronic phase, as well as the treatment of a human who has already had contact with HBV and which is in the inactive carrier stage, with the aim of preventing this person from newly entering into the immunoactive phase.
  • the term "therapy” in connection with a HBV-infection in humans comprises preferably
  • composition according to the invention or the pharmaceutical formulation according to the invention is administered to patients who are already infected with HBV.
  • compositions according to the invention People in the acute infection phase, in the immunotolerant chronic phase, in the immunoactive chronic phase or in the inactive carrier stage can be treated with the composition according to the invention or with the pharmaceutical formulation according to the invention, separately or in combination with other therapies as necessary.
  • the composition or the pharmaceutical formulation respectively is administered to the patient in an amount which is sufficient in order to cause an effective CTL-response against HBV antigens and heals or at least partially stops HBV-associated symptoms and/or complications.
  • the aim of the therapy is a reduction or, in the ideal case, an elimination of virus-infected cells.
  • HBcAgi_ x and optionally HBsAg are defined as "therapeutically effective doses”.
  • the dose which is therapeutically effective for the respective application case depends, for example, on the exact composition of the composition according to the invention or of the pharmaceutical formulation according to the invention respectively, on the method of administration, on the stage and the severity of the treated disease, on the weight and general state of health of the patient as well as on the judgement of the prescribing doctor, but generally lies preferably within a range from around 0.1 to 2,000 ⁇ g (total amount of adjuvants, HBcAgi_ x and optionally HBsAg) for a patient weighing 70 kg, particularly preferably within a range from 0.5 to 1,500 ⁇ g, yet more preferably within a range from 1 to 1,000 ⁇ g and most preferably within a range from 10 to 500 ⁇ g, followed by renewed doses within a range from 0.1 to 2,000 ⁇ g over weeks to months, depending on the CTL-response of a patient, which is determined by measurement of HBV-specific CTL-activity in the peripheral blood lymphocytes (PBL) of the patient.
  • the administration should be continued for a length of time until at least the clinic symptoms or the laboratory indicators indicate that the HBV-infection has been eliminated or is at least under the control of the immune system, optionally for longer.
  • composition according to the invention or the pharmaceutical formulation according to the invention can, in principal, be administered orally, intranasally, intradermally, subcutaneously, intramuscularly or intravenously.
  • a contribution to the solution of the above-mentioned object is, furthermore, provided by the use of the composition according to the invention for production of a pharmaceutical formulation for therapy and/or prophylaxis of HBV-infections and HBV-mediated diseases.
  • composition according to the invention comprising
  • the adjuvant comprising a saponin
  • a pharmaceutical formulation for therapy of HBV-infections and HBV-mediated diseases preferably for therapy of HBV-infections and HBV-mediated diseases
  • the HBV-infection or the HBV-mediated disease is caused by an HB-virus of the genotype A
  • a composition according to the invention which uses, in addition to components i) and ii), as component iii) an HBsAg of the genotype B, C, D, E, F, G or mixtures of HBsAg of these genotypes, whereby an HBsAg of genotype A can also be comprised.
  • HBV-mediated diseases in which a human who has not yet come into contact with HBV or a human who has already come into contact with HBV, preferably a
  • HBV-infected person particularly preferably a patient suffering from chronic hepatitis B, is administered a therapeutically effected dose of the composition according to the invention or of the pharmaceutical formulation according to the invention, preferably in the above-described art and way.
  • compositions which also comprises, in addition to the HBcAgi_ x and the adjuvant, an HBsAg which is derived from a HBV-genotype or HBV-subtype, preferably HBV genotype, which differs from the genotype or subtype, preferably genotype, of that HB-virus which is causal for the HBV-infection or for the HBV-mediated disease of the patient.
  • Figure 1 shows the HBsAg208-2i5-specific T cell response in HBsAg transgenic mice with the use of compositions according to the invention which comprise HBcAg and HBsAg, with ISCOMATRIX ® as adjuvant, respectively determined after 12 days after the second immunisation.
  • Figure 2 shows the B cell response with the use of compositions according to the invention when immunising HBsAg transgenic mice, illustrated by the AUSAB- anti-HBsAg titre, respectively determine after 12 days after the second immunisa- tion.
  • Figure 3 shows the HBcAg9 3 _ioo-specific T cell response of HBV transgenic mice with the use of compositions according to the invention which comprise HBcAg and HBsAg, with ISCOMATRIX ® as adjuvant, respectively determined after 14 days after the second immunisation.
  • Figure 4 shows the HBsAgi9o_i97-specific T cell response of HBV transgenic mice with the use of compositions according to the invention which comprise HBcAg and HBsAg, with ISCOMATRIX ® as adjuvant, respectively determined after 14 days after the second immunisation.
  • Figure 5 shows the HBsAg content in the serum of HBV transgenic mice with the use of compositions according to the invention which comprise HBcAg and HBsAg, with ISCOMATRIX ® as adjuvant, respectively determined after 14 days after the second immunisation.
  • mice which express the HBsAg of the subtype ayw (coded by HBV-sequence with the Accession No. VO 1460 J02203), were obtained from Jackson Laborator, Bar Harbor, ME, USA). Male and female animals aged 8-16 weeks were used for immunizations.
  • C57Bl /6J-TgN(AIbI HB V)44Bri - transgenic mice were immunised with the amounts of HBcAg, HBsAg and adjuvant in the amounts given in table 1 in respectively 100 ⁇ l PBS, whereby the respective amounts given were injected intramuscularly respectively twice at a separation of 21 days.
  • the plasmid pCI/HBsAg a d w in amounts of 2x50 ⁇ g in respectively 50 ⁇ l was injected into each Tibialis anterior muscle.
  • Tg(HB V ayw 1.3genome)Chi32 (lineage 1.3.32) - transgenic mice (described in Guidotti L, Matzke B, Schaller H, and Chisari FV, "High-level hepatitis B virus replication in transgenic mice” J Virol. 1995; 69:6158-69) were bred and kept under standard pathogen-free conditions. Male and female animals aged from 8- 16 weeks are used for immunization. Immunization of HBV(1.3.32) - transgenic mice
  • HBV(1.3.32) - transgenic mice were immunised with the amounts of HBcAg, HBsAg and adjuvant in the amounts given in table 2 in respectively 100 ⁇ l PBS, whereby the respective amounts given were injected subcutaneously respectively twice at a separation of 21 days.
  • the plasmid pCI/HBsAgadw was injected in amounts of 2 x 50 ⁇ g in respectively 50 ⁇ l into each Tibialis anterior muscle.
  • the spleen was removed from the ani- mals and cell suspensions produced from the spleen cells.
  • the production of spleen cell suspensions is described in Schirmbeck R., B ⁇ hm W., Fissolo N., MeI- ber K., and Reimann J.: "Different imunogenicity ofH-2 K ⁇ -restricted epitopes in natural variants of the hepatitis B surface antigen ", Europ. J. Immunol. 2003, 33: 2429-38.
  • the spleen cells were incubated (re-stimulation) for four hours in RPMI- 1640 medium with 0.25 ⁇ M HBcAg-specific peptide (MGLKFRQL; Jerini BioTools, Berlin, DE, SEQ ID No. 01) or with 0.25 ⁇ M HBsAg-specif ⁇ c peptide (VWLSVIWM; Jerini BioTools, Berlin, DE, SEQ ID No. 02) and respectively 5 ⁇ g/ml Brefeldin A (BFA) (catalogue no. 15870, Sigma) at 37 0 C.
  • BFA Brefeldin A
  • the re- stimulated cells were harvested and the surface was colored with anti-CD8 mAb (anti-mouse CD8a-PE-conjugate; catalogue no.
  • 96-well-microtiter plates (Nunc, Wiesbaden, DE) were coated with the HBsAg (2 ⁇ g/mL in carbonate buffer) used for immunization overnight at 4 0 C (100 ⁇ L per well).
  • the coated plates were washed three times with PBST (phosphate buffered salt solution with 0.5 % Tween 20). The wells were blocked with 0.2 % gelatine for 1 hour at 37 0 C.
  • the mouse plasmas/serums to be determined were diluted in two steps in PBS (within the range 1 :250 to 1 :32,000) and respectively 100 ⁇ L of the dilutions added to the appropriate wells (in dupli- cate) and incubated for 1 hour at 37 0 C. Serum from non-immunised mice was used as control.
  • microtiter plate was then washed five times with PBST and incubated with an isotype-specific second antibody (peroxidase-conjugated).
  • an isotype-specific second antibody peroxidase-conjugated.
  • gamlgl catalogue no. GAM/IgGl/PO
  • IgG2b gamlg2b
  • Nordic Immunological Laboratories, Tilburg, Netherlands was used.
  • the colour reaction was then carried out with o- phenylenediamine (oPD) (Sigma; catalogue no. P2903) as substrate (1 mg/ml oPD with 1 ⁇ L/mL 30 % H 2 O 2 ) (100 ⁇ L per well).
  • oPD o- phenylenediamine
  • the anti-HBs antibody count in the serums of the immunized mice was determined in the IMX Automated Immunoassay Analyzer (Abbott Diagnostics, Wiesbaden, Germany) using the IMX AUS AB ® test kit (reference no. 2226-21, Ab- bott, Wiesbaden, Germany). The assay was carried out as described by the manufacturer.
  • the HBsAg content in the serums of the immunised mice was determined in the IMXTM Automated Immunoassay Analyzer (Abbott Diagnostics, Wiesbaden, Germany) using the IMX HBsAg V2 reagent (catalogue number 222822, Abbott, Wiesbaden, Germany). The assays were carried out as described by the manufacturer. Since it is a non-quantitative test, the adsorption values were compared with a recombinant HBsAg of known concentration. The HBsAg amount was given accordingly in arbitrary units.
  • the plasmid pHB320 (7,543 base pairs), which carriers the complete HBV genome, was constructed from the vector pBR322 (Bolivar F., Rodriguez R.L., Greene P.J., Betlach M.C., Heyneker H. L., Boyer H. W.: "Construction and Characterization of new cloning vehicles. II. A multipurpose Cloning System," Gene.
  • This vector had been cut beforehand with BamUl/Sall and the projecting ends filled with DNA polymerase.
  • the resulting plasmid pGC74 (6,046 base pairs) carries the tryptophan promoter and the ⁇ Bc-gene in opposing orientation. This orientation facilitates the following cloning steps by the reconstitution of singular restriction sites for optimisation of the ⁇ BcAg-expression cassette.
  • pGC74 was restricted with BamHl, the projecting ends filled with DNA-polymerase and post-restricted with Sail. This fragment was ligated with the vector pBR322 Ptrp. This vec- tor had been linearized beforehand with Aval, the ends filled with DNA- polymerase and post-restricted with Sail.
  • the thus-constructed plasmid pGC2 (5,552 base pairs) carries the tryptophan promoter and the ⁇ Bc-gene in the same orientation.
  • Plasmid p ⁇ BclO (4,825 base pairs) resulted, with a shortened sequence between tryptophan- promotor and HBc-gene to obtain an optimal Pfrp-Shine-Dalgarno-HBc structure or distance.
  • a 1,044 base pair-H/z ⁇ l-HBc- fragment was digested from pHBclO after restriction with Hhall with Sl- nuclease and cloned into the vector ⁇ pBR322-trp-I, a modified pBR322- plasmid. This construction resulted in pHBc3 (7,108 base pairs), with the optimised expression sequence of the HBc-gene under the control of the tryptophan promoter.
  • Plasmid pHBcl615 (7,129 base pairs) resulted therefrom.
  • PHBcl615 was linearised with Clal and a synthetic polylinker with an additional iso leucine codon at position 145 and with a termination codon incorporated. Plasmid pHBc2-7 was obtained.
  • HBcAg ⁇ s expression vector (p327cl45) a PCR fragment of the HBcAg gene was cut out of plasmid pHBc3 with the enzymes EcoBl and BamHI and cloned into a pBR322 vector cut with EcoKI/BamHl.
  • EcoBl and BamHI a 290 bp long fragment of the tetracycline-resistance gene is eliminated.
  • the thus resulting plasmid pBRC (4,608 bp) now carries an ampicilin resistence as sole resistance marker.
  • a second PCR fragment was generated from the tryptophan gene region of the plasmid pHBc3, cut with the enzymes EcoKl and Ncol and then ligated with EcoTI/NcoI restricted pBRC.
  • the resulting plasmid pCAK (4,755 bp) carries a 552 bp HBcAg fragment under control of the tryptophan promoter and an ampicillin resistance gene as marker.
  • the ampicillin resistance gene was in turn exchanged for a Kanamycin resistance gene (as Sspl/Nrul fragment after Klenow fill reac- tion), which came from a commercially obtainable plasmid pREP-4
  • the resulting plasmid pCAK-NEO5 carries the 552 bp HBcAg fragment under control of the tryptophan promoter with a Kanamycin resistance marker.
  • Hindlll/BamHl a fragment with the 552 bp HBcAg as well as the Kanamycin resistance gene was obtained from pCAK-NEO5 and cloned into a pBR327 vector likewise cut with Hindlll/BamHl.
  • the thus resulting plasmid pHBc-1719 (5,038 bp) now carries two resistance markers (ampicillin and Kanamycin) as well as an HBcAg (complete gene coding for 183 amino acids) under control of the tryptophan promoter.
  • the above-described plasmid pHBc2-7 carries a C-terminal shortened HBcAg fragment with an additional isoleucine at amino acid position 145 (HBcAgi_i 44+ i).
  • a 375 bp fragment was isolated from this sequence by restriction with XballHpal.
  • Plasmid pHBc-1719 was digested with the en- zymes Xbal and Nrul in order to remove the C-terminal part of the HBcAg gene as well as a part of the vector sequence. The remaining part of the pHBc-1719 was ligated with the above-described 375 bp XballHpal fragment.
  • the resulting vector is pHBc-11721 (4,287 bp) with a shortened HBcAg gene (HBcAgi 44+ i) under control of the tryptophan promoter.
  • the C-terminal amino acid isoleucine of the HBcAg i . 144+1 is replaced by glutamic acid by means of PCR mutagenesis of the expression vector pHBC- 11721.
  • the resulting expression vector has the designation p327cl45 and codes for HBcAg I45 . Competent E.
  • coli cells (Stem BL21 : Genotype B F ' dcm ompT hsdS (r B ⁇ ni B ⁇ )gal) were transformed with plasmid p327cl45 and obtained after plating on LB Agar with ampicillin overnight at 37 0 C. With respectively one individual colony, 300 ml M9 minimal medium (with addition of 1 % Casamino acids, Difco catalogue no. 223050, Becton-Dickinson, Heidelberg) and 0.2 % glucose are inoculated. The cultivation occurred overnight at 37 0 C on a round shaker in 1 1-Erlenmeyer flask. The cultures generally reached an optical density (OD5 4 o nm ) between 2 and 5.
  • the cells were centrifuged and the pellet re-suspended in lysis buffer
  • HBsAg HBsAg of the genotype A (subtype adw 2 ), of the genotype C (subtype adr), or of the genotype D (subtype ayw 4 ) respectively was prepared as described in: Brocke P, Schaefer S., Melber K., Jenzelewski V., M ⁇ ller F., Dahlems U., Bartelsen O., Park K.N., Janowicz Z.A., Gellissen G.: Recombinant hepatitis B vaccines: disease characterization and vaccine production"
  • the saponin complex which is commercially obtainable under the product description ISCOMATRIX ® (CSL Limited, Parkville, Australia) was used as adjuvant.
  • HBsAg a d W 2-plasmid DNA was used as positive control, pure PBS as negative control.
  • the composition according to the invention comprised PBS, HBcAgi_i 45 , HBsAg adw2 as well as ISCOMATRIX ® .
  • the amounts of the individual components are given in table 1.
  • the plasmid pCI/HBV a d W 2 core was prepared as described in Schirmbeck R., Dikopoulos N., Kwissa M., Leithauser F., Lamberth K. Buus S., Melber K., and Reimann J. (2003): , ⁇ reaking tolerance in hepatitis B surface antigen transgenic mice by vaccination with cross-reactive, natural HBsAg variants", Eur. J. Immunol. 3342-52, and isolated with QIAGEN-tip 500 (catalogue no. 12162; Qiagen, Hilden, Germany) as described by the manufacturer.
  • HBsAg a d W 2 and HBcAgi_i45 were first combined under aseptic conditions, diluted with PBS to four times the concentration of the final product and then incubated for 30 min- utes at 37 0 C with shaking.
  • ISCOMATRIX as delivered by the producer as aqueous dispersion, was then added and the end concentration of the antigen as given in table 1 was adjusted with PBS.
  • the thus-prepared composition was incubated for an hour at room temperature (20-25 0 C), then stored overnight (12-18 hours) at 2-8 0 C and used the following day for immunisation of HBsAg- transgenic mice.
  • the immunotolerance against HBsAg in HBsAg-transgenic mice can be penetrated with the composition according to the invention.
  • the saponin complex which is commercially obtainable under the product description ISCOMATRIX ® was used as adjuvant.
  • As negative control a composition comprising PBS and ISCOMATRIX ® was used.
  • the composition according to the invention comprised PBS, HBcAgi_i 45 ISCOMATRIX ® and HBsAg a dr. The amounts of the individual components are given in table 2.
  • compositions according to the invention were carried out according to the procedure described in connection with example 1 , whereby here too, the end concentrations of the antigens given in table 2 were adjusted by addition of PBS.
  • the thus-produced compositions were incubated for one hour at room temperature (20-25 0 C), then stored overnight (12-18 hours) at 2-8 0 C and used the following day for immunisation of HBV-transgenic mice.
  • both the immuno tolerance against HBsAg as well as the immuno tolerance against HBcAg can be penetrated with a composition according to the invention which comprises a heterologous HBsAg-genotype or -subtype respectively (i.e. a HBsAg which is based on a genotype which differs from the HBV-transgenic mouse).
  • a composition according to the invention of this type leads to a decrease of HBsAg in the serum of the HBV-transgenic mouse
  • the effect described in figure 5 would indicate a healing of a patient suffering from chronic hepatitis B.

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Abstract

Composition comprenant: i) HBcAg1-x, où x est un entier ente 100 et 160, un fragment de cet antigène, un variant de cet antigène ou le fragment de l'antigène considéré ou au moins deux de ces éléments, ii) un adjuvant comprenant une saponine, et éventuellement iii) un HBsAg, un fragment de cet antigène, un variant de cet antigène ou le fragment de l'antigène considéré ou au moins deux de ces éléments, l'adjuvant comprenant comme saponine entre 50 et 90 %, en poids, de la fraction A obtenue par le biais du procédé d'essai décrit, à partir d'extrait de Quillaja Saponaria Molina, et entre 10 et 50 %, en poids, de la fraction C obtenue par le procédés d'essai décrit, à partir d'extrait de Quillaja Saponaria Molina, le total des saponines de la fraction A et de la fraction C étant de 100 %, en poids; l'adjuvant comprend, outre la saponine: cholestérol, phosphatidylcholine et décanoyl-N-méthylglucamide. L'invention concerne aussi un procédé de production de composition, la composition résultante, une formulation pharmaceutique, l'utilisation de la composition ou de la formulation pour le traitement et/ou la prévention des infections au VHB et des maladies dont la médiation est assurée par le VHB, l'utilisation de la composition ou de la formulation pour la production d'un médicament pour le traitement et/ou la prévention de des infections au VHB et des maladies dont la médiation est assurée par le VHB, et un procédé pour leur traitement et/ou leur prévention.
PCT/EP2007/059672 2006-09-15 2007-09-13 Composition pour la thérapie et/ou pour la prophylaxie d'infections au vhb et de maladies dont la médiation est assurée par le vhb WO2008031878A1 (fr)

Applications Claiming Priority (4)

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EPPCT/EP2006/009014 2006-09-15
PCT/EP2006/009014 WO2007031334A2 (fr) 2005-09-16 2006-09-15 Composition destinee a traiter et/ou a prevenir des infections par le hbv et des maladies induites par le hbv
EP06019910.6 2006-09-22
EP06019910A EP1902727A1 (fr) 2006-09-22 2006-09-22 Vaccins comprenant la protéine du noyau du HBV tronquée ainsi qu'un adjuvant à base de saponine

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101942013A (zh) * 2010-07-07 2011-01-12 中国人民解放军第三军医大学 乙肝病毒核心抗原和e抗原的HLA-DR9限制性调节性T细胞表位及其应用
WO2019115817A3 (fr) * 2017-12-15 2019-08-08 Glaxosmithkline Biologicals Sa Régime et compositions pour l'immunisation contre l'hépatite b

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0859695A (ja) * 1985-10-03 1996-03-05 Takeda Chem Ind Ltd 新規ｄｎａおよびポリペプチド
WO1996011711A1 (fr) * 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
WO2004004762A1 (fr) * 2002-07-05 2004-01-15 Isconova Ab Preparation d'iscom et son utilisation
WO2005002620A1 (fr) * 2003-07-07 2005-01-13 Isconova Ab Fraction de quil a a faible toxicite, utilisation de cette derniere

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0859695A (ja) * 1985-10-03 1996-03-05 Takeda Chem Ind Ltd 新規ｄｎａおよびポリペプチド
WO1996011711A1 (fr) * 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
WO2004004762A1 (fr) * 2002-07-05 2004-01-15 Isconova Ab Preparation d'iscom et son utilisation
WO2005002620A1 (fr) * 2003-07-07 2005-01-13 Isconova Ab Fraction de quil a a faible toxicite, utilisation de cette derniere

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
AGUILAR J C ET AL: "Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen", IMMUNOLOGY AND CELL BIOLOGY, vol. 82, no. 5, October 2004 (2004-10-01), pages 539 - 546, XP002354413, ISSN: 0818-9641 *
BOULTER N R ET AL: "Theileria annulata sporozoite antigen fused to hepatitis B core antigen used in a vaccination trial", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 13, no. 13, 1995, pages 1152 - 1160, XP004057464, ISSN: 0264-410X *
CHEN XINCHUN ET AL: "Recombinant hepatitis B core antigen carrying preS1 epitopes induce immune response against chronic HBV infection.", VACCINE, vol. 22, no. 3-4, 2 January 2004 (2004-01-02), pages 439 - 446, XP004479365, ISSN: 0264-410X *
DAVIS H L ET AL: "DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 15, no. 8, June 1997 (1997-06-01), pages 849 - 852, XP004075668, ISSN: 0264-410X *
GATHURU ET AL: "Identification of DHBcAg as a potent carrier protein comparable to KLH for augmenting MUC1 antigenicity", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 23, no. 39, 15 September 2005 (2005-09-15), pages 4727 - 4733, XP005040630, ISSN: 0264-410X *
GUAN XIAO-JU ET AL: "Construction and characterization of an experimental ISCOMS-based hepatitis B polypeptide vaccine.", WORLD JOURNAL OF GASTROENTEROLOGY : WJG. APR 2002, vol. 8, no. 2, April 2002 (2002-04-01), pages 294 - 297, XP002354578, ISSN: 1007-9327 *
HOWARD C R ET AL: "PREPARATION AND PROPERTIES OF IMMUNE-STIMULATING COMPLEXES CONTAINING HEPATITIS B VIRUS SURFACE ANTIGEN", JOURNAL OF GENERAL VIROLOGY, vol. 68, no. 9, 1987, pages 2281 - 2289, XP009057159, ISSN: 0022-1317 *
MAGNIUS L O ET AL: "SUBTYPES, GENOTYPES AND MOLECULAR EPIDEMIOLOGY OF THE HEPATITIS B VIRUS AS REFLECTED BY SEQUENCE VARIABILITY OF THE S-GENE.ATITIS B", INTERVIROLOGY, vol. 38, no. 1/2, 1995, pages 24 - 34, XP000197124, ISSN: 0300-5526 *
MANCINI M ET AL: "INDUCTION OF ANTI-HEPATITIS B SURFACE ANTIGEN (HBSAG) ANTIBODIES IN HBAAG PRODUCING TRANSGENIC MICE: A POSSIBLE WAY OF CIRCUMVENTING NONRESPONSE TO HBSAG", JOURNAL OF MEDICAL VIROLOGY, ALAN R. LISS, NEW YORK, NY, US, vol. 39, no. 1, 1993, pages 67 - 74, XP009040798, ISSN: 0146-6615 *
MOREIN BROR ET AL: "Current status and potential application of ISCOMs in veterinary medicine", ADVANCED DRUG DELIVERY REVIEWS, vol. 56, no. 10, 23 June 2004 (2004-06-23), pages 1367 - 1382, XP002354382, ISSN: 0169-409X *
NEWMAN MARGARET ET AL: "Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins.", JOURNAL OF VIROLOGY, vol. 77, no. 24, December 2003 (2003-12-01), pages 12950 - 12960, XP002354380, ISSN: 0022-538X *
PEARSE MARTIN J ET AL: "ISCOMATRIX(R) adjuvant for antigen delivery", ADVANCED DRUG DELIVERY REVIEWS, vol. 57, no. 3, 10 January 2005 (2005-01-10), pages 465 - 474, XP004653216, ISSN: 0169-409X *
POLAKOS N K ET AL: "Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 1 MAR 2001, vol. 166, no. 5, 1 March 2001 (2001-03-01), pages 3589 - 3598, XP001146112, ISSN: 0022-1767 *
PUMPENS P ET AL: "HBV CORE PARTICLES AS A CARRIER FOR B CELL/T CELL EPITOPES", INTERVIROLOGY, vol. 44, no. 2-3, 2001, pages 98 - 114, XP009080591, ISSN: 0300-5526 *
RIEDL PETRA ET AL: "Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain", JOURNAL OF IMMUNOLOGY, vol. 168, no. 10, 15 May 2002 (2002-05-15), pages 4951 - 4959, XP002354381, ISSN: 0022-1767 *
SCHAEFER-S ET AL.: "Recombinant hepatitis B Vaccines - Disease Characterization and Vaccine Production", 28 January 2005, IN: HANSENULA POLYMORPHA; ED: GERD GELLISSEN; PRINT ISBN-10: 3527303413; ONLINE ISBN-10: 3527602356; P 175-210, XP002411804 *
SCHIRMBECK R ET AL: "BREAKING TOLERANCE IN HEPATITIS B SURFACE ANTIGEN (HBSAG) TRANSGENIC MICE BY VACCINATION WITH CROSS-REACTIVE, NATURAL HBSAG VARIANTS", EUROPEAN JOURNAL OF IMMUNOLOGY, WEINHEIM, DE, vol. 33, no. 12, December 2003 (2003-12-01), pages 3342 - 3352, XP009040725, ISSN: 0014-2980 *
SCHIRMBECK R ET AL: "DIFFERENT IMMUNOGENCITIY OF H-2 KB-RESTRICTED EPITOPES IN NATURAL VARIANTS OF THE HEPATITIS B SURFACE ANTIGEN", EUROPEAN JOURNAL OF IMMUNOLOGY, WEINHEIM, DE, vol. 33, no. 9, September 2003 (2003-09-01), pages 2429 - 2438, XP009040727, ISSN: 0014-2980 *
SO H S ET AL: "Effect of a novel saponin adjuvant derived from Quillaja saponaria on the immune response to recombinant hepatitis B surface antigen.", MOLECULES AND CELLS 30 APR 1997, vol. 7, no. 2, 30 April 1997 (1997-04-30), pages 178 - 186, XP002128520, ISSN: 1016-8478 *
TSAI S L: "Immunopathogenesis of viral hepatitis B and C.", CHANGGENG YI XUE ZA ZHI / CHANGGENG JI NIAN YI YUAN = CHANG GUNG MEDICAL JOURNAL / CHANG GUNG MEMORIAL HOSPITAL. JUN 1999, vol. 22, no. 2, June 1999 (1999-06-01), pages 159 - 170, XP009057239 *

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CN101942013B (zh) * 2010-07-07 2013-01-30 中国人民解放军第三军医大学 乙肝病毒核心抗原和e抗原的HLA-DR9限制性调节性T细胞表位及其应用
WO2019115817A3 (fr) * 2017-12-15 2019-08-08 Glaxosmithkline Biologicals Sa Régime et compositions pour l'immunisation contre l'hépatite b
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