WO2014139359A1 - 乙型肝炎疫苗 - Google Patents
乙型肝炎疫苗 Download PDFInfo
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- WO2014139359A1 WO2014139359A1 PCT/CN2014/072570 CN2014072570W WO2014139359A1 WO 2014139359 A1 WO2014139359 A1 WO 2014139359A1 CN 2014072570 W CN2014072570 W CN 2014072570W WO 2014139359 A1 WO2014139359 A1 WO 2014139359A1
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- hbsag
- cpg
- hbcag
- odn
- hepatitis
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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
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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
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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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- 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
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
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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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- 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
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- 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
- C12N2730/10171—Demonstrated in vivo effect
Definitions
- the present invention relates to a hepatitis B vaccine.
- the present invention relates to a hepatitis B vaccine comprising a hepatitis B surface antigen, a hepatitis B core antigen, and a thiooligodeoxynucleotide having immunostimulatory activity.
- HBV infection is one of the world's serious public health problems. HBV infection is an important cause of chronic hepatitis B, cirrhosis and hepatocellular carcinoma (Fattovich G. J Hepatol 2008; 48: 335-352). Clinical treatments Commonly used drugs for chronic HBV infection are mainly nucleoside analogues and interferons. Nucleoside analogues do not completely eliminate cccDNA in hepatocytes, and long-term use can easily lead to the emergence of resistant mutants and rebound after drug withdrawal (Kwon H, Lok AS. Nat Rev Gastroenterol Hepatol. 2011; 8:275-284.) .
- Interferon is not suitable for asymptomatic HBV carriers.
- the incidence of HBeAg seroconversion is only 33% after half a year of use, and the side effects of interferon are also limited (Tang SX, Yu GL ⁇ ancet) 1990;335(8684): 302)
- the currently widely used hepatitis B protein vaccine induces humoral immunity and produces protective antibodies for prevention purposes.
- protective antibodies can only eliminate extracellular virus particles, while viruses that eliminate intracellular infection mainly rely on specific cellular immune responses, TH types such as IFN- ⁇ produced by helper T cells and CD 4+ T cells.
- Cytokines especially virus-specific cytotoxic T lymphocytes (CTL), are cleared (Chin R, Lacamini S. Rev Med Viorl. 2003: 13(4): 255-72).
- CTL cytotoxic T lymphocytes
- the strength of the cellular immune response directly determines the prognosis of hepatitis B. Therefore, the ideal therapeutic hepatitis B vaccine needs to induce specific humoral and cellular immunity at the same time, breaking through the immune tolerance of hepatitis B.
- anti-HBsAg antibody subtypes in patients with chronic hepatitis B infection are mainly IgG4, while anti-HBsAg antibody subtypes in patients with hepatitis B infection are IgGl ⁇ IgG4, indicating that Th1 type during hepatitis B infection clearance
- the antibody subtype IgG1 plays an important role. Evaluating whether a Thl type antibody subtype is higher than or equal to a Th2 type antibody subtype may suggest an effect of hepatitis B treatment (S. Rath, et al. Clin.exp. Immunol.
- the anti-HBcAg antibody subtype of patients with hepatitis B infection is IgGl > IgG3 > IgG4, and the anti-HBcAg subtype of patients with hepatitis B infection is changed to IgG3>IgGl>IgG4, indicating anti-HBcAg, especially anti-HBcAg
- the transformation of antibody subtypes may be closely related to the treatment of hepatitis B (Chien-Fu Huang, et al. Cellular & Molecular Immunology. 2006;3(2):97-106.) 0
- Patent US 4,547,367 utilizes HBcAg particles to treat/prevent HBV infection and HBV-mediated diseases, and HBcAg particle immunization of chimpanzees protects chimpanzees from HBV infection. Moreover, HBcAg particles combined with HBsAg particles immunized newborns born to mothers of hepatitis B carriers, producing high titers of anti-HBsAg and anti-HBcAg antibodies, and no HBV infection was observed during the 18-month monitoring. However, the patent does not explicitly provide evidence for a subtype of anti-HBcAg antibody and there is no direct evidence for treatment of HBV infection and HBV-mediated disease.
- Patent WO2007/031334 which protects a hepatitis B therapeutic vaccine component comprising HBsAg, HBcAg and a saponin adjuvant, and CpG-ODN can be used as a shared adjuvant, however, the hepatitis B therapeutic vaccine is in clinical use. It is necessary to combine nucleoside analogues for combination therapy in order to break through the immune tolerance of hepatitis B, and e antigen is only negative Summary of the invention
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising: i) hepatitis B surface antigen (HBsAg), ii) hepatitis B core antigen (HBcAg), iii) CpG oligo-oxynucleotide (CpG-ODN) and/or Optionally iv) a pharmaceutically acceptable carrier.
- the pharmaceutical compositions of the invention are useful as prophylactic or therapeutic vaccines.
- the pharmaceutical composition consists of the above components i) - iii) and optionally iv).
- the HBsAg has the sequence set forth in SEQ ID NO: 1.
- the HBcAg has the sequence set forth in SEQ ID NO:2.
- the CpG-ODN comprises a phosphorothioate linkage.
- the CpG-ODN is a thiooligooxydeoxynucleotide, preferably a per-thiooligooxydeoxynucleotide.
- the CpG-ODN comprises two or more 5,-NTCGTT-3, motifs.
- the CpG-ODN is 15 to 35 nucleotides in length, preferably 20 to 25 nucleotides.
- the CpG-ODN has a sequence selected from the group consisting of: 5, -TCG TTC GTT CGT TCG TTC GTT-3' (SEQ ID NO: 3), 5, -TCG TTC GTT CGT TCG TTC GTT CGT T-3' (SEQ ID NO: 4), 5, -TCG TCG TCG TCG TCG TCG-3' (SEQ ID NO: 5) and 5, -TCC ATG ACG TTC CTG ACG TT -3' (SEQ ID NO: 6), preferably the CpG-ODN has the sequence: 5, -TCG TTC GTT CGT TCG TTC GTT-3,.
- the relative weight ratio between components 0, ii) and iii) in the pharmaceutical composition ranges from 1: 0.2-5: 1-50, preferably 1 : 1-5: 2 ⁇ 15.
- the present invention also relates to a hepatitis B vaccine comprising: 0 hepatitis B surface antigen (HBsAg), ii) hepatitis B core antigen (HBcAg), iii) CpG oligodeoxygenation Nucleotide (CpG-ODN) and optionally iv) a pharmaceutically acceptable carrier.
- HBsAg hepatitis B surface antigen
- HBcAg hepatitis B core antigen
- CpG-ODN CpG oligodeoxygenation Nucleotide
- the invention also relates to a kit comprising a pharmaceutical composition or a hepatitis B vaccine according to the invention and optionally instructions for its use.
- the invention relates to the use of a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of a HBV infection and/or an HBV mediated disease in a subject, preferably said HBV infection and/or
- the HBV-mediated disease is selected from the group consisting of hepatitis B, cirrhosis and liver cancer.
- the invention relates to the use of a pharmaceutical composition according to the invention for the manufacture of a medicament for the production of an immune response against HBV (preferably, inducing a Thl and Th2 type immune response) in a subject.
- the invention relates to the use of a pharmaceutical composition according to the invention for the manufacture of a medicament for subtype transformation of an anti-HBcAg antibody in a subject.
- the invention relates to the use of a pharmaceutical composition according to the invention for the manufacture of a medicament for breaking through hepatitis B virus immune tolerance in a subject.
- the present invention relates to a pharmaceutical composition according to the present invention for preparing a balance of a Th1/Th2 immune response to a hepatitis B surface antigen in a subject (e.g., substantially equivalently inducing a Th1 and Th2 type immune response) Use in medicine.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising: 0 hepatitis B surface antigen (HBsAg), ii) hepatitis B core antigen (HBcAg), and iii) CpG oligodeoxynucleotide (CpG- ODN).
- the pharmaceutical composition of the present invention can be used for treating HBV infection and/or HBV-mediated diseases in a subject, for generating an immune response against HBV in a subject (preferably, inducing a Th1 and Th2 type immune response), For subtype transformation of an anti-HBcAg antibody in a subject, for eliciting antigen-specific CTL killing activity in a subject and/or for breaking through hepatitis B virus immune tolerance in a subject.
- the invention relates to a method of treating a HBV infection and/or a HBV mediated disease in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the invention.
- the present invention relates to generating an object for HBV in an object A method of immunizing (preferably, inducing a Th1 and Th2 type immune response) comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition according to the invention.
- the invention relates to a method of subtype transformation of an anti-HBcAg antibody in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the invention.
- the invention relates to a method of breaking through hepatitis B virus immune tolerance in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the invention.
- the invention relates to a method of eliciting antigen-specific CTL killing activity in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the invention.
- the present invention relates to a method of achieving a balance of a hepatitis B surface antigen Th1/Th2 immune response (eg, substantially equivalently inducing a Th1 and Th2 type immune response) in a subject, comprising administering to the subject a therapeutically effective amount A pharmaceutical composition according to the invention.
- a hepatitis B surface antigen Th1/Th2 immune response eg, substantially equivalently inducing a Th1 and Th2 type immune response
- the pharmaceutical compositions of the invention do not comprise a saponin adjuvant.
- FIG. 1 shows the compositions of the invention compared with the conventional vaccine components for specific total hepatitis B surface antigen I g G FIG enhanced immune response in mice.
- Figure 2 shows a graph of the composition of the invention enhancing the immune response of a protective antibody in mice compared to conventional vaccine components.
- Figure 3 is a graph showing that the composition of the present invention enhances the Th2 immune response of hepatitis B surface antigen at humoral immunity levels compared to conventional vaccine components.
- Figure 4 is a graph showing that the composition of the present invention enhances the hepatitis B surface antigen Thl immune response in humoral immunity levels compared to conventional vaccine components.
- Figure 5 shows a graph of the humoral immunity level of the composition of the present invention promoting the balance of the Thl/Th2 immune response of hepatitis B surface antigen compared to conventional vaccine components.
- Figure 6 shows that the anti-HBcAg antibody subtype produced by the composition of the present invention is Figure of IgG2a > IgGl.
- Figure 7 is a graph showing that the composition of the present invention promotes HBsAg CTL epitope-specific Thl cell differentiation and inhibits Th2 cell proliferation in cellular immunity as compared with the conventional vaccine component.
- Figure 8 is a graph showing the synergistic effect of HBcAg in combination with HBsAg + CpG in the composition of the invention on the production of antigen-specific I g G antibody levels.
- Figure 9 shows the ability of HBsAg to bind to HBsAg + CpG in the composition of the present invention.
- HBsAg + CpG has the ability to promote HBsAg-specific Thl cell differentiation at the cellular level.
- FIG. 10 shows the composition of the present invention breaks immune tolerance to FIG HBV transgenic mice and mice immune tolerance in I g G antigen-specific immune response.
- Figure 11 shows a diagram of the composition of the present invention breaking through the immune tolerance of HBV transgenic mice and immunotolerant mice in generating a neutralizing antibody immune response.
- Figure 12 shows the composition of the present invention breaks immune tolerance HBV transfected immune tolerance in mice and mice gene, to produce high-titer anti-HBcAg specific antibody I g G of FIG.
- Figure 13 shows a graph of the anti-HBcAg antibody subtype produced by the composition of the present invention in HBV transgenic mice and immunotolerant mice as IgG2a > IgGl.
- Figure 14 shows that the composition of the present invention has a greater ability to clear HBsAg antigen in HBV transgenic mice than conventional vaccine components.
- Figure 15 shows the in vivo killing activity of the composition of the present invention having HBsAg and HBcAg antigen-specific CTLs, demonstrating that the composition of the present invention can be used as a chronic hepatitis B therapeutic vaccine.
- An object of the present invention is to overcome the problems known in the prior art for the treatment of type B Defects in hepatitis-infected drugs.
- Another object of the present invention is to provide a use of the composition for the treatment of HBV infection and/or HBV mediated diseases, and a method of treating HBV infection and/or HBV mediated diseases.
- the present invention provides a pharmaceutical composition comprising:
- HBsAg a fragment of the antigen, a variant of the antigen, or a mixture of at least two thereof
- HBcAg a fragment of the antigen, a variant of the antigen or a fragment of the antigen, or a mixture of at least two thereof,
- the oligonucleotide is a per-thio modification, having two or more copies of 5,-NTCGTT-3 in its sequence, motif, 20 to 25 bases in length base.
- the oligonucleotide is preferably derived from the following base sequences: 5,-TCG TTC GTT CGT TCG TTC GTT-3,, 5,-TCG TTC GTT CGT TCG TTC GTT CGT T-3,, 5,-TCG TCG TCG TCG TCG TCG TCG TCG-3, or 5,-TCC ATG ACG TTC CTG ACG TT-3,. More preferably, it is 5,-TCG TTC GTT CGT TCG TTC GTT-3.
- composition of the present invention achieves an unexpected technical effect. Compared with the existing commercially available hepatitis B preventive vaccine, it can mediate a stronger immune response in vivo in mice, including anti-HBsAg antibodies, anti-HBcAg antibodies, anti-ads serotype neutralizing antibodies, and in particular, Thl
- the cellular immune response produces a cytokine IFN- ⁇ associated with viral clearance, promotes differentiation and maturation of anti-HBs-IgG2a antibody subtypes, and transforms against anti-HBcAg antibody subtypes to achieve a balance between humoral and cellular immunity.
- composition HBsAg+CpG has been disclosed in the prior art (see patent CN101492672), and the inventors have surprisingly found that the pharmaceutical composition according to the invention (combination of HBcAg, HBsAg and CpG ODN) can produce significantly better
- the anti-HBsAg-specific antibodies of the HBsAg+CpG composition exhibited an unexpected synergistic effect.
- composition according to the invention can break through the immune tolerance of transgenic mice, producing high titers of anti-HBsAg antibodies, anti-HBcAg antibodies, neutralizing antibodies, mediated Thl cellular immune response promotes differentiation and maturation of the I g G2a antibody subtype.
- the multiple immunization of the composition according to the present invention can significantly eliminate the hepatitis B virus in the transgenic mouse and decrease the expression level of the hepatitis B virus.
- composition of the present invention has also been shown to induce strong antigen-specific cytotoxic T lymphocyte (CTL) in vivo killing activity, particularly HBsAg CTL and HBcAg CTL in vivo killing activity, which further confirms that the composition of the present invention greatly exceeds
- CTL cytotoxic T lymphocyte
- both BALB/c mice and model mice can mediate a strong anti-HBcAg immune response, and achieve anti-HBcAg antibody subtype transformation, that is, the anti-HBcAg antibody subtype relationship is expressed as IgG2a antibody.
- the level is higher than I g Gl , which is consistent with the antibody subtype of Hepatitis B infected patients.
- polypeptide refers to a polymer of amino acids and has no specific minimum number of amino acids. Therefore, it also includes peptides, oligopeptides, dimers, trimers, oligomers, particles and the like. Further, the term “polypeptide” includes not only a pure amino acid polymer obtained after translation in a ribosome, but also a polypeptide obtained by post-translational modification (e.g., glycosylation, acetylation, phosphorylation, thiolation, etc.).
- post-translational modification e.g., glycosylation, acetylation, phosphorylation, thiolation, etc.
- antigenic fragment refers to a fragment of a natural or synthetic polypeptide that retains the antigenic properties of the natural or synthetic polypeptide, i.e., is capable of eliciting an immune response against the natural or synthetic polypeptide.
- hepatitis B surface antigen (HBsAg, HBs), as used herein, is intended to encompass native HBsA g , HBsAg antigenic fragments, HBsAg functional variants, and any combination thereof.
- the native HBsAg is contained A natural HBsAg polypeptide of 226 amino acids.
- the HBsAg is a native HBsAg polypeptide derived from the known HBV standard genotypes A, B, C, D, E, F, G and/or H.
- the HBsAg has the sequence shown in SEQ ID NO: 1.
- HBsAg antigenic fragment as used herein is intended to mean a polypeptide having a continuous or discontinuous fragment of less than 226 amino acids in native HBsAg and which retains the antigen of native HBsAg. Sex.
- a functional variant of HBsAg as used herein is intended to mean a polypeptide having up to 30, up to 25, up to 20, up to 15, at most for a native HBsAg or HBsAg fragment. 10, up to 5, up to 4, up to 3, up to 2, up to 1 dentate acid deletion, insertion, addition or substitution, and the polypeptide retains the function (eg, antigenicity) of native HBsAg.
- hepatitis B core antigen HBcAg, HBc
- HBc hepatitis B core antigen
- the native HBcAg contains 183 Natural aminoccAg polypeptides of amino acids. More particularly, the native HBcAgs are native HBcAgs derived from the known HBV standard genotypes A, B, C, D, E, F, G and/or H. In some embodiments In the above, the HBcAg is selected from the group consisting of HBcAg ⁇ polypeptides, which represent a fragment of 1-X-position acid of natural HBcAg, in particular, X is 149 to 183.
- the HBcAg is selected from the group consisting of Peptide: 1-149 of SEQ ID NO: 2, ⁇ acid, 1-150 of SEQ ID NO: 2, 1-151 of SEQ ID NO: 2, SEQ ID NO: 1-152 of 2: ⁇ acid, 1-153 of SEQ ID NO: 2, acid of 1-154 of SEQ ID NO: 2, 1-155 of SEQ ID NO: 2 Acid, 1-156 of SEQ ID NO: 2, 1-157 of SEQ ID NO: 2, acid of 1-158 of SEQ ID NO: 2, of SEQ ID NO: 2 -159-position ⁇ 3 ⁇ 4 ⁇ acid, 1-160-position of SEQ ID NO: 2, 1-1-1 of SEQ ID NO: 2 ⁇ Acid, 1-162-position acid of SEQ ID NO: 2, 1-163-position acid of SEQ ID NO: 2, 1-164-position acid of SEQ ID NO: 2, SEQ ID NO: 2 1-165 bit ⁇ ⁇ acid, 1-166 acid of SEQ ID NO: 2, 1-167 of SEQ ID NO: 2, 1-168 of SEQ ID NO: 2,
- the HBcAg has the sequence set forth in SEQ ID NO:2.
- HBcAg antigenic fragment is intended to mean a polypeptide having a continuous or discontinuous fragment of less than 183 amino acids in native HBcAg and which retains native HBcAg. Antigenicity.
- a functional variant of HBcAg as used herein is intended to mean a polypeptide having up to 30, up to 25, up to 20, up to 15, at most for a native HBcAg or HBcAg fragment. 10, up to 5, up to 4, up to 3, up to 2, up to 1 basal acid deletion, insertion, addition or substitution, and the polypeptide retains the function (eg, antigenicity) of native HBcAg.
- both HBcAg and HBsAg are present in the form of granules in the composition according to the present invention.
- compositions are used interchangeably and mean at least one drug that is combined together to achieve a particular purpose, and optionally Pharmaceutically acceptable excipients or combinations of excipients.
- the pharmaceutical compositions include combinations that are separated in time and/or space, as long as they are capable of acting together to achieve the objectives of the present invention.
- the components contained in the pharmaceutical composition may be administered to the subject as a whole or separately to the subject.
- the ingredients can be administered to the subject simultaneously or sequentially.
- CpG oligodeoxynucleotide refers to a short single-stranded synthetic DNA molecule containing one or more "CpG" units, wherein C represents cytosine. , G represents guanine, p represents a phosphodiester bond. In particular, the CpG oligodeoxynucleotide is unmethylated. In some embodiments, the CpG-ODN comprises a phosphorothioate linkage or sulfur Phosphoroester backbone.
- the CpG-ODN is a phosphorothioate oligodeoxynucleotide (ie, a thiooligooxydeoxynucleotide).
- the CpG- All internucleotide linkages in the ODN are phosphorothioate linkages, ie, the CpG-ODN is a per-thiooligooxydeoxynucleotide.
- the CpG-ODN comprises two or more Further, 5,-NTCGTT-3, motif.
- the CpG-ODN is 15 to 35 nucleotides in length, preferably 20 to 25 nucleotides.
- the CpG- ODN has a sequence selected from the group consisting of: 5, -TCG TTC GTT CGT TCG TTC GTT-3' (SEQ ID NO: 3), 5, -TCG TTC GTT CGT TCG TTC GTT CGT T-3 ' ( SEQ ID NO: 4 ), 5, -TCG TCG TCG TCG TCG TCG-3' (SEQ ID NO: 5 ) and 5, -TCC ATG ACG TTC CTG ACG TT-3' (SEQ ID NO: 6), more specifically the CpG-ODN has the sequence: 5, -TCG TTC GTT CGT TCG TTC GTT-3,.
- terapéuticaally effective amount refers to a dose sufficient to demonstrate its benefit to the subject to which it is administered.
- the actual amount administered, as well as the rate and timing of administration, will depend on the condition and severity of the subject being treated.
- the prescription for treatment eg, the determination of the dose, etc.
- the prescription for treatment is ultimately the responsibility of the GP and other physicians and depends on their decision, usually considering the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and the Other factors known.
- HBV-mediated disease is intended to mean a disease caused by, induced, aggravated, increased, and/or associated with hepatitis B virus (HBV), such as a hepatitis B virus carrier, Hepatitis B, cirrhosis, liver ascites, liver cancer, etc.
- HBV hepatitis B virus
- anti-HBcAg antibody subtype transition is intended to mean that the anti-HBcAg antibody subtype relationship in a subject is converted to an antibody subtype relationship in a hepatitis B cured patient after administration of the pharmaceutical composition of the present invention, ie, It is consistent with the antibody subtypes that healed in patients with hepatitis B infection.
- anti-HBcAg antibody subtypes are converted from IgG1>IgG2a to IgG2a > IgG2b > IgG1 or IgG2b > IgG2a > IgG1, eg IgG2a>IgGl, in humans, anti-HBc antibody subtypes are from IgGl > IgG3 > IgG4 was converted to IgG3>IgGl>IgG4.
- subject refers to a mammal, such as a human, but can also be other animals, such as wild animals (such as herons, donkeys, cranes, etc.), livestock (such as ducks, geese, etc.) or Experimental animals (such as raccoon, monkey, rat, mouse, rabbit, guinea pig, groundhog, ground squirrel, etc.).
- wild animals such as herons, donkeys, cranes, etc.
- livestock such as ducks, geese, etc.
- Experimental animals such as raccoon, monkey, rat, mouse, rabbit, guinea pig, groundhog, ground squirrel, etc.
- composition of the present invention which comprises i) HBsAg or a variant of the antigen, ii) HBcAg 1 183 , and iii ) a 21 base thiooligonucleotide CpG-ODN , 5,-NTCGTT-3, motif with two or more copies in its sequence.
- the relative weight ratio between components i), ii) and iii) in the pharmaceutical compositions of the invention is in the range of 1: 0.2-5: 1-50, preferably 1: 1-5 : 2-15, more preferably 1:1:2.
- compositions of the present invention may further comprise additional additives, such as pharmaceutical carriers or additives, especially when it is in the form of a pharmaceutical formulation.
- compositions of the invention do not comprise a saponin adjuvant.
- Preferred pharmaceutical carriers are especially water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffer), dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose , magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol or polyalkylene glycol such as polypropylene glycol, triglyceride and the like.
- the type of pharmaceutical carrier employed depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
- the composition according to the invention may comprise a wetting agent, an emulsifier or a buffer substance as an additive.
- compositions, vaccine or pharmaceutical preparation according to the present invention can be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
- suitable route for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
- Example 1 Hepatitis B surface antigen (HBsAg) was combined with hepatitis B core antigen (HBcAg) and CpG-ODN to enhance the immune response of hepatitis B surface antigen-specific total IgG.
- mice were immunized with HBsAg-specific I g G levels in serum and statistically analyzed to evaluate the combination of HBsAg and Al(OH) 3 with HBsAg, HBcAg and CpG-ODN for HBsAg.
- the HBsAg antigen used in this example was purchased from Prospec (batch number: 1111 PHADW22, sequence shown as SEQ ID: 1), and was an adw2 subtype expressed by Pichia pastoris, with a purity of 95% or more, 4. Save the spare in the C refrigerator.
- the HBcAg (sequence shown in SEQ ID NO: 2) antigen used in the present embodiment is prepared by the inventors, and is a natural hepatitis B core protein expressed by Escherichia coli, and the purification preparation process thereof See Li Jilai, Xu Jing, etc. in the Journal of Chinese Journal of Biological Products 2011, Vol.
- the specific steps are as follows: After collecting the cells, resuspend with 10 mM sodium phosphate buffer, ultrasound The supernatant was collected by centrifugation, and saturated ammonium sulfate was added to make a final concentration of 33%. After thorough mixing, it was allowed to stand overnight at 4 ° C; the next day, centrifugation, the pellet was resuspended in 10 mM sodium phosphate buffer and placed in a dialysis bag.
- the CpG-ODN sequence used in this example is 5,-TCG TTC GTT CGT TCG TTC GTT-3', which is prepared by the solid phase phosphoramidite triester chemical synthesis method described in the CN200810004736.0 patent, starting from the 3rd end.
- Deprotection group first remove the protecting group DMT (dimethoxytrityl) of the nucleotide attached to CpG with trichloroacetic acid, and obtain the free 5, hydroxyl group for the next condensation.
- activation mixing the phosphoramidite-protected nucleomonomer with the tetrazole activator and entering the synthesis column to form a phosphoramidite tetrazole active intermediate, which has been deprotected on CpG The nucleotide undergoes a condensation reaction; 3) linkage: the phosphoramidite tetrazole active intermediate encounters a deprotected nucleotide on CpG, and will affinity with its 5, hydroxyl group, condense and remove the tetrazole.
- DMT dimethyl methoxytrityl
- the oligonucleotide chain is extended by one base forward;
- Oxidation During the condensation reaction, the nucleotide monomer is linked to the oligonucleotide attached to CpG through a phosphorous ester bond, and the phosphorous ester bond Unstable, easily hydrolyzed by acid or alkali.
- the phosphite is used to oxidize the phosphoramidite to sulfur. Double-bonded phosphotriester to obtain a stable oligonucleotide;
- Blocking After the condensation reaction, in order to prevent the 5 which is not involved in the reaction on CpG, the hydroxyl group is extended in the subsequent cyclic reaction, often by acetylation.
- a deoxynucleotide is attached to the nucleotide of CpG; repeat the above deprotection, activation, ligation, oxidation, blocking process to obtain a crude DNA fragment ; ⁇ Cleavage, deprotection, purification, quantification and other synthetic post-treatment to obtain the corresponding CpG-ODN; -20* water tank for storage.
- HBsAg and HBcAg were released to 10 g/ml with PBS (Invitrogen); CpG-ODN was diluted to 20 g/ml with PBS.
- ⁇ 1 ( ⁇ ) 3 adjuvant was purchased from Tiantan Biological.
- BALB/c mice were immunized with left hind limbs and intestinal muscles, each in a volume of 100 ⁇ , with 10 mice per group.
- HBsAg+ Al(OH) group 3 was injected with lg per mouse Al(OH) 3 adsorbed HBsAg.
- Each mouse in the HBsAg+HBcAg+CpG-ODN group was injected with lg HBsAg, lg HBcAg and 2 g CpG-ODN. Immunize once every three weeks, blood is separated from the blood for ten days after the second exemption, and the serum is diluted 1:30 with 2% skim milk according to the conventional method, and then serially diluted by 3 times for detecting antigen specificity. IgG total antibody.
- the specific antigen-specific I g G total antibody detection procedure was as follows: 96-well microtiter plate (purchased from Nunc) was coated with HBsAg, 25 ng per well, 4 ° C overnight; 3 ⁇ 4 2 times, 5% degreased The milk was blocked at 37 ° C for 1 hour; after washing the plate twice, the above-mentioned 3 times serial dilution of the test serum was added, 37. C was applied for 1 hour; after washing 3 times, 1:30000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (purchased from SIGMA, USA) was added at 50 ⁇ l, 37 per well.
- Example 2 Hepatitis B surface antigen (HBsAg) in combination with hepatitis B core antigen (HBcAg) and CpG-ODN enhances the level of protective antibody against hepatitis B surface antigen.
- the role of hepatitis B virus surface antibody detection is to monitor the success of hepatitis B vaccine vaccination.
- the hepatitis B vaccine system produces a hepatitis B virus surface antibody equivalent to a neutralizing antibody, and its potency is directly related to the protective ability of the vaccine, and the production of the antibody has a remarkable effect on preventing HBV infection. Therefore, the present inventors selected the internationally accepted ARCHITECT Hepatitis B Virus Surface Antibody International Unit Test System (Chemiluminescence Microparticle Immunoassay, CMIA) to detect hepatitis B virus surface antibody (anti-HBsAg) in the serum of immunized mice. Concentration, and statistical analysis to evaluate the combination of HBsAg+HBcAg+CpG-ODN relative to The protective effect of HBsA g +Al(OH) 3 on enhancing the protective antibody level.
- mice Female, 6-8 weeks, were purchased from Shanghai Slack Company in this example.
- HBsAg and HBcAg were diluted to 10 g/ml with PBS; CpG-ODN was diluted to 20 g/ml with PBS.
- BALB/c mice were immunized with the left hind limb gastrocnemius muscle, each injection volume was 100 ⁇ , 10 mice per group.
- Each mouse in HBsAg+Al(OH) group 3 was injected with 1 ⁇ ⁇ ⁇ 1( ⁇ )3 adsorbed HBsAg, and HBsAg+HBcAg+CpG-ODN group was injected with lg HBsAg, lg HBcAg and 2 g CpG-ODN.
- the HBsAg+HBcAg+CpG-ODN group can significantly enhance the protective antibody level, and the specific protective antibody titer can reach 4.3 logarithmic values, which is significant compared with the HBsAg+Al(OH) 3 group.
- Sexual difference (PO.05), specific protection antibody titer (potency) can be increased by about 5 times.
- the above results indicate that the HBsAg+HBcAg+CpG-ODN composition vaccine of the present invention can significantly enhance the protective antibody level of hepatitis B surface antigen and enhance the protective ability of the vaccine than the ⁇ 1( ⁇ ) 3 adjuvant hepatitis B vaccine.
- Example 3 Hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and CpG-ODN were combined to enhance the Th2 immune response of hepatitis B surface antigen at the level of humoral immunity.
- HBsAg hepatitis B surface antigen
- HBcAg hepatitis B core antigen
- CpG-ODN CpG-ODN
- Th1 and Th2 Antigen-specific immune responses are classified into two types, Th1 and Th2, in which Th2 responses correspond to high levels of antigen-specific IgG1 antibody titers.
- ⁇ 1( ⁇ ) 3 is a highly potent Th2 vaccine adjuvant that inhibits the Th1 immune response and is characterized by high levels of specific IgG1 antibodies induced by immunization.
- the HBsAg+HBcAg+CpG-ODN composition produced significantly higher levels of IgG1 antibodies than the HBsAg+Al(OH) 3 group (PO.001), and the HBsAg-specific IgG1 antibody titer (potency) was The increase is close to 10 times.
- the composition of the present invention can produce a HBsAg-specific IgG1 antibody which is stronger than the Al(OH) 3 adjuvant group, and enhances the hepatitis B surface antigen Th2 immune response.
- Example 4 Hepatitis B surface antigen (1188 8 ), hepatitis B core antigen (1180 2 ) and CpG-ODN were combined to enhance the hepatitis B surface antigen Thl immune response at the level of humoral immunity.
- Al(OH) 3 is a very strong Th2 type vaccine adjuvant capable of inhibiting the Th1 type immune response, which is shown to induce very low levels of specific IgG2a antibodies after immunization.
- the specific IgG2a antibody titer induced by Al(OH) 3 as an HBsAg adjuvant was only 2.17 logarithmic values.
- HBsAg + HBcAg + CpG-ODN composition upon immunization produced HBsAg specific antibody titers increased I g G2a about two pairs of values, i.e., 100 times.
- the above results indicate that the combination of HBsAg+HBcAg+CpG-ODN of the present invention strongly stimulates the Th1 immune response against hepatitis B surface antigen.
- Example 5 Hepatitis B surface antigen (1188 8 ), hepatitis B core antigen (1180 2 ) In combination with CpG-ODN, it promotes the balance of Thl/Th2 immune response of hepatitis B surface antigen at the level of humoral immunity.
- the HBsAg+HBcAg+CpG-ODN composition of the present invention not only produces a higher level of Th2 type immune response in mice, but also produces a very high level of Th1 type immune response.
- Thl promotes CTL response, the so-called cellular immune tendency, while Th2 promotes antibody production, the so-called humoral immunity.
- One purpose of therapeutic hepatitis B vaccine is to eliminate HBsAg antigen in serum, which requires the production of effective protective antibodies, mainly Humoral immunity works; another purpose is to kill target cells that are infected with HBV, that is, CTL responses, which require cellular immunity to function, so both Th1 and Th2 immune responses are important.
- the antigen-specific IgG2a/IgG1 titer ratio (LoglO) will be analyzed. The results are shown in Fig. 5. If the ratio is less than 0, the immune response is biased toward Th2, and greater than 0 indicates that the immune response is biased toward Th1, and close to 0 indicates that the immune response tends to be balanced.
- Example 6 Anti-HBcAg antibody subtype IgG2a>IgG1 produced by the combination of hepatitis B surface antigen (1188 8 ), hepatitis B core antigen (1180 2 ) and CpG-ODN.
- the anti-HBcAg antibody subtype of a cured population of hepatitis B infected patients is IgG3>IgG1>IgG4, and its corresponding antibody subtype relationship in mice is IgG2a > IgG2b > IgG1 or IgG2b > IgG2a > IgGl pulp according to Example 3 and The method described in Example 4 was used to determine the titer of the anti-HBcAg antibody subtype IgG2a and IgG1 of the HBsAg+HBcAg+CpG-ODN composition of the present invention, and whether the composition can promote the conversion of the mouse anti-HBcAg antibody subtype to IgG2a>IgGl, except that the coating antigen used in the assay was lg/ml HBcAg.
- the results are shown in Figure 6.
- the results in Figure 6 show that the anti-HBcAg antibody produced by the HBsAg+HBcAg+CpG-ODN composition of the present invention Subtype IgG2a>IgGl, and significant Difference (PO.001).
- This composition is intended to promote the conversion of mouse anti-HBcAg antibody subtype into antibody subtypes in patients with hepatitis B infection.
- Example 7 Hepatitis B surface antigen, hepatitis B core antigen combined with CpG-ODN significantly promoted HBsAg CTL epitope-specific Thl cell differentiation and inhibited Th2 cell proliferation.
- mice used in this example female, 6-8 weeks, purchased from Shanghai Slack Company; used HBsAg antigen, HBcAg, CpG-ODN and ⁇ 1 ( ⁇ ) 3 adjuvant As described in Example 1.
- HBsAg and HBcAg were diluted to 10 g/ml with PBS; CpG-ODN was diluted to 20 g/ml with PBS.
- BALB/c mice were immunized with the left hind limb gastrocnemius muscle, each injection volume was 100 ⁇ , 5 mice per group.
- HBsAg+Al(OH) group 3 was injected with lg via ⁇ 1( ⁇ )3 adsorbed HBsAg, and HBsAg+HBcAg+CpG-ODN group was injected with lg HBsAg, lg HBcAg and 2 g CpG-ODN.
- spleen lymphocytes Immunize once every three weeks, take 10 days after the second excretion, prepare spleen lymphocytes according to the conventional method, as follows: Aseptically take the spleen: Cut the spleen with sterile forceps and scissors, place on a 70 ⁇ nylon mesh screen (purchased from BD), placed in a flat phoenix containing 2 ml of pre-cooled 2% FBS (purchased from GIBCO)-PBS; the spleen was ground with a grinding rod, and the spleen cells were passed through a sieve plate to obtain a cell suspension.
- Aseptically take the spleen Cut the spleen with sterile forceps and scissors, place on a 70 ⁇ nylon mesh screen (purchased from BD), placed in a flat phoenix containing 2 ml of pre-cooled 2% FBS (purchased from GIBCO)-PBS; the spleen was ground with a grinding rod, and the spleen
- the suspension was placed in a 50 ml sterile centrifuge tube (purchased from BD) via a 40 ⁇ m nylon mesh using a Pasteur pipette; 300 xg, 4. C centrifuge for 10 minutes; discard the supernatant, add 5 ml of lx redness agent (purchased from BD), resuspend the cells, and let the cells break at room temperature for 5 minutes to break the red blood cells; add 5 ml of 2% FBS-PBS to stop the red-breaking reaction; 300 Xg, 4. C centrifuge for 5 minutes; discard the supernatant and add 2 ml 2% Resuspend the cells in FBS-PBS for use.
- Antigen-specific IFN- ⁇ and IL-4 secretion was detected using a Mouse IFN- ⁇ /IL-4 ELISPOT kit (BD), and the stimulator was a peptide library of HBsAg. After the test was completed, the number of spots was read on an Immimo SPOT Series 3 automatic plate reader.
- BD Mouse IFN- ⁇ /IL-4 ELISPOT kit
- the HBsAg peptide library consists of 54 15-amino acid polypeptide fragments covering the entire HBsAg full-length sequence, with 11 amino acids overlapping each pair of adjacent polypeptides, representing all possible HBsAg CTL epitopes.
- the peptide design of the HBsAg peptide library is shown as SEQ ID NO: 7 to SEQ ID NO: 60. All peptides were synthesized, purified, dispensed and lyophilized by the Chinese Peptide Company.
- Specific antigen-specific IFN- ⁇ and IL-4 secretion assays are as follows: PBS # Mouse IFN- ⁇ /IL-4 (1:200 dilution, BD), 100 ⁇ /well to ELISPOT plate , overnight coating at 4 °C; discard the coated antibody, wash the well once with blocking solution (containing 10% FBS RPMI-1640 medium), add blocking solution 200 ⁇ /well, incubate for 2 h at room temperature; use 10% FBS -1640 medium diluted peptide library to 10 g / ml; ConA was diluted to 20 g / ml in 10% FBS-1640 medium as a positive control; discard the blocking solution, lxlO 7 cells / ml of spleen lymphocyte suspension and The configured peptide library or ConA control was added to the 96-well plate at 100 ⁇ /well, and repeated in duplicate; incubated at 37 ° C in a 5% CO 2 incubator for 24 h; discard the cell suspension and wash the plate with dei
- Thl cells mainly secrete IL-2, IL-12, IFN-y and TNF_p/, which mediate immune responses related to cytotoxicity and local inflammation, and participate in cellular immunity and delayed hypersensitivity inflammation.
- Th2 cells mainly secrete IL-4, IL-5, IL-6 and IL-10, and their main function is to stimulate B cell proliferation and produce antibodies.
- IFN- ⁇ can induce Th1 cell differentiation, but inhibits Th2 cell proliferation; IL-4 i leads to Th2 cell differentiation.
- ELISPOT was used to detect the secretion levels of antigen-specific IFN- ⁇ and IL-4 in spleen cells of immunized mice, and as a result, Al(OH) 3 as HBsAg adjuvant secreted HBsAg-specific IL-4 levels higher than IFN.
- composition of the invention mainly participates in HBsAg-specific cellular immunity, promotes the differentiation of Th1 cells, and achieves the proliferation balance of Th1/Th2 cells, and thus has the potential to kill liver cells infected with HBV and to clear free HBV virus.
- Example 8 ⁇ Combination of HBcAg, HBsAg and CpG-ODN has a synergistic effect on the production of antigen-specific I g G antibody levels
- [113] used in this example is BALB/c mice, female, 6-8 weeks, purchased from Shanghai Slack Company; used HBsAg antigen, HBcAg, CpG-ODN and ⁇ 1 ( ⁇ ) 3 adjuvant As described in Example 1.
- HBsAg and HBcAg were diluted to 10 g/ml with PBS; CpG-ODN was diluted to 20 g/ml with PBS.
- BALB/c mice were immunized with the left hind limb gastrocnemius muscle, each in a volume of 100 ⁇ l, with 10 mice per group.
- HBsAg+HBcAg+CpG-ODN group was injected with lg HBsAg, lg HBcAg and 2 g CpG-ODN. Immunize once every three weeks, and serum is separated from the blood ten days after the second exemption. The serum is diluted 1:30 with 2% skim milk according to the conventional method, and then serially diluted by 3 times for detection. Antigen-specific I g G total antibody. According to the method described in Example 1, the HBsAg-specific antibody titers produced by the HBsAg+HBcAg+CpG-ODN group and the HBsAg+CpG-ODN group of the present invention were determined. The results are shown in Figure 8.
- HBcAg, HBsAg and CpG-ODN combined with the HBsAg+CpG-ODN group produced higher levels of HBsAg-specific IgG, specific antibody titers up to 4.0 logarithmic values, significant Difference (PO.01), specific antibody titer (potency) can be increased by about 3 times.
- the above results indicate that the HBsAg+HBcAg+CpG-ODN composition of the present invention produces significantly higher HBsAg-specific IgG antibody levels than HBsAg+CpG-ODN after addition of HBcAg, demonstrating synergy of HBcAg, HBsAg and CpG-ODN. .
- Example 9 Combination of HBcAg, HBsAg and CpG-ODN Promotes HBsAg-specific Thl cell differentiation in cellular immunity compared to HBsAg+CpG-ODN.
- Example 8 HBcAg, HBsAg and CpG-ODN were synergistically examined in terms of humoral immunity, and the present inventors also analyzed whether HBcAg, HBsAg and CpG-ODN in combination promote Thl cell differentiation of HBsAg in cellular immunity. To further verify the synergistic effect of HBcAg, HBsAg and CpG-ODN.
- HBsAg, HBcAg and CpG-ODN, HBsAg and CpG-ODN adjuvant were mixed, and mice were immunized.
- the levels of IFN- ⁇ and IL-4 secreted by spleen cells of immunized mice were detected by ELISPOT assay.
- the combination of HBsAg and HBcAg, CpG-ODN in combination with HBsAg+CpG-ODN promoted the differentiation of Th1 cells in HBsAg.
- mice Female, 6-8 weeks, used in this example were purchased from Shanghai Slack Company; HBsAg, HBcAg and CpG-ODN used were as described in Example 1.
- HBsAg and HBcAg were diluted to 10 g/ml with PBS; CpG-ODN was diluted to 20 g/ml with PBS.
- BALB/c mice were immunized with the left hind limb gastrocnemius, each with a volume of 100 ⁇ l, 10 mice per group.
- HBsAg+CpG-ODN group Each mouse was injected with lg HBsAg and 2 g CpG-ODN, and each mouse in the HBsAg+HBcAg+CpG-ODN group was injected with lg HBsAg, lg HBcAg and 2 g CpG-ODN.
- the combination of HBcAg and HBsAg+CpG-ODN has higher levels of HBsAg-specific IFN- ⁇ secreted by the HBsAg+CpG-ODN group and lower HBsAg-specific IL-4 levels, indicating that the HBsAg of the present invention is present.
- the +HBcAg+CpG-ODN composition can promote the differentiation of HBsAg-specific Th1 cells in cellular immunity, indicating that HBcAg, HBsAg and CpG-ODN have synergistic effects.
- Example 10 ⁇ HBsAg+HBcAg+CpG-ODN composition broke through the immune tolerance of HBV transgenic mice (adr serotype) and immune tolerant mice (B10.M).
- mice There are two types of immunotolerant mice used in this example: the first one is HBV transgenic mice, the serotype is adr serotype, male, 10-12 weeks, purchased from Shanghai Southern Model Animal Center; The two are B10.M mice, which are histocompatibility mice. Immunologically tolerated, male, 6-8 weeks, purchased from The Jackson Laboratory; HBsAg, HBcAg and, CpG-ODN and ⁇ 1 ( ⁇ ) 3 adjuvants used as described in Example 1.
- HBsAg and HBcAg were diluted to 10 g/ml with PBS; CpG-ODN was diluted to 20 g/ml with PBS. Mice were immunized with the left hind limb gastrocnemius muscle, each in a volume of 100 ⁇ , with 4 mice in each group.
- HBsAg+Al(OH) group 3 was injected with lg via ⁇ 1( ⁇ ) 3 adsorbed HBsAg, HBsAg+HBcAg+CpG-ODN group was injected with lg HBsAg, lg HBcAg and lO g CpG- ODN. Immunization every three weeks, blood was collected two weeks after each immunization, and a total of 6 times were immunized, and HBsAg-specific I g G total antibody was detected according to the method described in Example 1. The results are shown in Fig. 10, as in Example 2. The method described was used to detect the produced HBsAg antibody, and the results are shown in Fig. 11.
- the HBsAg+HBcAg+CpG-ODN composition group can break the immune tolerance of the two model mice, and the HBsAg-specific I g G antibody titer can reach 4.0 log or more, with HBsAg+ Compared with the Al(OH) 3 group, there was a significant difference (P ⁇ 0.05), and the HBsAg-specific IgG antibody titer (titer) could be increased by 10-200 times.
- the protective antibody level can reach 2.5 pairs. Above the value, compared with the HBsAg+Al(OH) 3 group, the level of protective antibody produced can be increased by 5-20 times.
- HBsAg present invention + HBcAg + CpG-ODN vaccine composition ratio ⁇ 1 ( ⁇ ) 3 adjuvant hepatitis B vaccine in mice immune tolerance can significantly enhance the specificity of the total hepatitis B surface antigen I g G and protective antibodies The immune response is more effective in breaking through immune tolerance.
- Example 11 ⁇ HBsAg+HBcAg+CpG-ODN composition produced high titer anti-HBcAg specific IgG antibody in HBV transgenic mice (adr serotype) and immune tolerant mice (B10.M).
- HBsAg and HBcAg were diluted to 10 g/ml with PBS; CpG-ODN was diluted to 20 g/ml with PBS.
- Each mouse was injected with l g HBsAg, l g HBcAg and lO g CpG-ODN through the left hind limb gastrocnemius.
- the injection volume was ⁇ , 4 mice per group. Immunization every three weeks, blood was collected two weeks after each immunization, and immunization was performed 6 times.
- the HBcAg antigen-specific IgG total antibody was detected according to the method described in Example 1, except that the coating antigen was lg HBcAg antigen, and the result was Shown in Figure 12.
- the HBsAg+HBcAg+CpG-ODN composition produced high titers of anti-HBcAg-specific I g G antibodies on both model mice, and the antibody titer was obtained two weeks after the immunization. Up to 2.5 logarithmic values, after which the antibody titer can be above 4.0 logarithmic values. The above results indicate that the HBsAg+HBcAg+CpG-ODN composition of the present invention produces a high titer anti-HBcAg specific IgG antibody.
- Example 12 ⁇ HBsAg+HBcAg+CpG-ODN composition An anti-HBcAg antibody subtype IgG2a>IgG1 produced in HBV transgenic mice (adr serotype) and immunotolerant mice (B10.M).
- the present inventors mixed HBsAg, HBcAg and CpG-ODN for the combination of iiHBsAg+HBcAg+CpG-ODN in anti-HBcAg antibody subtype IgG2a>IgGl produced on HBV transgenic model mice and immunotolerant mice.
- the mice were immunized and the anti-HBcAg antibody subtypes IgG2a and IgG1 titers in the serum were determined.
- mice There were two types of immunotolerant mice used in this example, as described in Example 10; the HBsAg, HBcAg and CpG-ODN used were as described in Example 1.
- HBsAg and HBcAg were diluted to 10 g/ml with PBS; CpG-ODN was diluted to 20 g/ml with PBS.
- Each mouse was injected with 1 ⁇ ⁇ HBsAg, lg HBcAg and 10 g CpG-ODN through the left hind limb gastrocnemius.
- the injection volume was 100 ⁇ l, with 4 mice per group.
- Anti-HBcAg antibody was detected according to the method described in Example 6. Subtypes I g G2a and IgG1 titers, the results are shown in Figure 13.
- Figure 13 shows the anti-HBcAg antibody subtype I produced by the HBsAg+HBcAg+CpG-ODN composition of the present invention in HBV transgenic mice (adr serotype) and immunotolerant mice (B10.M).
- Example 13 ⁇ HBsAg+HBcAg+CpG-ODN composition has a tendency to clear HBsAg antigen in HBV transgenic mice.
- HBsAg+HBcAg+CpG-ODN composition breaks through the immune tolerance of HBV transgenic mice, and the mice producing antigen-specific antibodies are analyzed to see if the expression of HBsAg in the body can decline. The clearance of HBsAg antigen.
- HBV transgenic mice used in this example were serotyped adr serotype (reconstructed from C57 mice), male, 10-12 weeks, purchased from Shanghai Southern Model Animal Center; used HBsAg, HBcAg, CpG-ODN and ⁇ 1 ( ⁇ ) 3 adjuvants were as described in Example 1.
- HBsAg and HBcAg were diluted to 10 g/ml and 100 g/ml, respectively, with PBS; CpG-ODN was diluted with PBS to 20 g/ml and 200 g/ml, respectively.
- the mice were immunized with the left hind limb gastrocnemius muscle, each in a volume of 100 ⁇ l, with 8 mice per group.
- HBsAg+Al(OH) 3 mice were injected with 1 ⁇ 1( ⁇ ) 3 adsorbed HBsAg per mouse; HBsAg+HBcAg+CpG-ODN components, 1 group of mice injected with 1 HBsAg, 1 HBcAg and 10 CpG-ODN, another group of mice injected with 10 HBsAg, 10 HBcAg and 20 CpG-ODN.
- the serum of pre-immune and six-week two weeks is diluted 1:200 according to the conventional method with 2% skim milk.
- the HBsAg antigen was used as a standard, and the serum of C57 mice (purchased from Shanghai Slack Company) was 1000 ng/mK 500 ng/mK 250 ng/mK 125 ng/ml, 62.5 ng/ml, 31.25 ng/ml, 15.625 ng/ml, 7.8. Dilute to ng/ml as the starting concentration, then dilute 1:200 with 2% skim milk, then use the HBsAg antigen detection kit for diluted samples and standards (Shanghai Kehua Company) The HBsAg antigen concentration was measured.
- the concentration of HBsAg antigen contained in the serum of each mouse before immunization and after six weeks of immunization was calculated using the measured standard curve, and the % decrease of HBsAg antigen concentration after six weeks of six weeks was calculated. The results are shown in Figure 14.
- the specific HBsAg antigen concentration detection procedure is as follows: Take out the pre-coated anti-HBsAg reaction plate, add 75 ⁇ 1 diluted serum and negative, positive control in the reaction well; cover the reaction with cover paper ⁇ , set the reaction plate 37 Incubate for 1 h at °C; remove the reaction plate, tear off the seal, add 50 ⁇ M enzyme conjugate to the sample to be tested and negative, positive control well; shake on microwell shaker for 10 s; cover the reaction with cover paper The reaction plate was incubated at 37 e C for 30 m; the reaction plate was taken out, the sealing plate was removed, and the reaction plate was washed 5 times; immediately after the washing, the developer A and the developer B were added to each well 50 ⁇ 1, and mixed; The microporous oscillator was shaken for 10 s; the reaction was covered with a cover paper, and the reaction plate was incubated at 37 ° C for 30 m; 50 ⁇ l of the stop solution was added to all wells, and the reaction was
- the HBsAg+HBcAg+CpG-ODN composition group had a lower % HBsAg antigen concentration in HBV transgenic mice than the HBsAg+Al(OH) 3 group, with significant difference (P ⁇ 0.05).
- the % decrease of HBsAg antigen concentration is more obvious, with an average of more than 90%. This indicates that the composition has a tendency to scavenge HBsAg antigen and turn HBsAg antigen negative, laying a solid foundation for chronic hepatitis B therapeutic vaccine.
- Example 14 ⁇ The HBsAg+HBcAg+CpG-ODN composition has HBsAg and HBcAg antigen-specific CTL in vivo killing activity.
- Antigen-specific CTL in vivo killing is the most direct evidence for the efficacy of therapeutic vaccines.
- the detection of HBsAg and HBcAg antigen-specific CTL in vivo killing activity in mice immunized with HBsAg+HBcAg+CpG-ODN composition is 3 ⁇ 4 ⁇ CTL in vivo killing activity, CTL killing rate was calculated.
- mice used in this example were female, 6-8 weeks, purchased from Shanghai Slack Company; the HBsAg antigen, HBcAg and CpG-ODN adjuvant used were as in Example 1. Said. [149] HBsAg and HBcAg were diluted to 100 g/ml with PBS; CpG-ODN was diluted to 200 g/ml with PBS. The mice were immunized with the left hind limb gastrocnemius muscle, each in a volume of 100 ⁇ l, with 8 mice per group.
- Each mouse in the HBsAg+HBcAg+CpG-ODN group was injected with 10 HBsAg, lO g HBcAg and 20 CpG-ODN.
- the immunization was performed once every two weeks, and three times in total, and the killing activity of HBsAg and HBcAg antigen-specific CTL was measured ten days after the three exemptions, and the results are shown in Fig. 15.
- Specific antigen-specific CTL in vivo killing activity detection steps are as follows: Aseptically take the spleen of unimmunized mice, slides, 300xg, 4. Centrifuge for 5 min, discard the supernatant; resuspend the cells by adding 5 ml of red blood cell lysate (purchased from BD), lyse the red blood cells at room temperature for 5 min, wash twice with 10 ml PBS (purchased from GIBCO); CFSE with PBS (purified from Molecular Probes) diluted to 4 ⁇ and 0.4 ⁇ , mixed with an equal volume of cell suspension, and allowed to stand at room temperature for 7 min; 300 ⁇ g, 4.
- the prepared labeled cell mixture was injected into the mice immunized with HBsAg+HBcAg+CpG-ODN by eyelids, 100 ⁇ M per mouse; 15-17 h later, the mouse spleen cell suspension was prepared, using 2% FBS- The cells were resuspended in PBS and detected by flow cytometry; the percentage of antigen-specific CTL killing was calculated.
- the above HBcAg peptide library consists of 43 peptides of 15 J acid, covering the entire HBcAg full-length sequence, with 11 amino acids overlapping each pair of adjacent polypeptides, representing all possible HBcAg CTL epitopes, eg Fragments 1-9, 5-19, 9-23 of SEQ ID NO: 2, 169-183 acid.
- the peptides of the above HBcAg peptide library are designed as shown in the sequences SEQ ID NO: 61 to SEQ ID NO: 103. All peptides were synthesized, purified, dispensed and lyophilized by the Chinese Peptide Company.
- the HBsAg+HBcAg+CpG-ODN composition group has HBsAg and HBcAg antigen-specific CTL bodies on C57BL/6J mice.
- the internal killing activity has a CTL killing rate of about 30%. This indicates that the composition has HBsAg and HBcAg antigen-specific CTL in vivo killing activity, providing the most direct evidence for chronic hepatitis B therapeutic vaccine.
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EP14765662.3A EP2974740B1 (en) | 2013-03-13 | 2014-02-26 | Hepatitis b vaccine |
JP2015561921A JP6499592B2 (ja) | 2013-03-13 | 2014-02-26 | B型肝炎ワクチン |
US14/773,829 US9878035B2 (en) | 2013-03-13 | 2014-02-26 | Hepatitis B vaccine |
HK16106930.2A HK1218872A1 (zh) | 2013-03-13 | 2016-06-16 | 乙型肝炎疫苗 |
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CN201310080863.XA CN104043120B (zh) | 2013-03-13 | 2013-03-13 | 乙型肝炎疫苗 |
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US (1) | US9878035B2 (zh) |
EP (1) | EP2974740B1 (zh) |
JP (1) | JP6499592B2 (zh) |
CN (1) | CN104043120B (zh) |
HK (1) | HK1218872A1 (zh) |
WO (1) | WO2014139359A1 (zh) |
Cited By (3)
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CN113058033A (zh) * | 2019-12-16 | 2021-07-02 | 远大赛威信生命科学(南京)有限公司 | 一种用于预防和治疗乙型肝炎的药物组合物及其用途 |
CN114409802A (zh) * | 2020-12-31 | 2022-04-29 | 中国科学院微生物研究所 | 禽流感病毒三聚体亚单位疫苗及其应用 |
CN114437185A (zh) * | 2020-12-31 | 2022-05-06 | 中国科学院微生物研究所 | 冠状病毒三聚体亚单位疫苗及其应用 |
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CN104873969B (zh) * | 2015-04-16 | 2018-06-19 | 南京赛威信生物医药有限公司 | 基于HBV PreS-S、C抗原及新型佐剂CpG的治疗性乙型肝炎疫苗 |
CA3023022A1 (en) * | 2016-05-04 | 2017-11-09 | Transgene Sa | Combination therapy with cpg tlr9 ligand |
CN107693788B (zh) * | 2017-08-21 | 2022-10-11 | 远大赛威信生命科学(南京)有限公司 | 一种用于预防或治疗乙型肝炎的药物组合物及其用途 |
GB201721069D0 (en) | 2017-12-15 | 2018-01-31 | Glaxosmithkline Biologicals Sa | Hepatitis B Immunisation regimen and compositions |
WO2020134682A1 (zh) | 2018-12-24 | 2020-07-02 | 南京远大赛威信生物医药有限公司 | 用于治疗乙型肝炎的药物制剂及其制备方法和用途 |
BR112022005687A2 (pt) | 2019-09-30 | 2022-06-21 | Gilead Sciences Inc | Vacinas contra o hbv e métodos para tratar o hbv |
JP2023506439A (ja) | 2019-12-13 | 2023-02-16 | 遠大賽威信生命科学(南京)有限公司 | 医薬組成物及びその用途 |
CN111728955A (zh) * | 2020-06-19 | 2020-10-02 | 中山大学 | 一种用于乙肝治疗的纳米颗粒及其制备方法和治疗性疫苗 |
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CN113058033A (zh) * | 2019-12-16 | 2021-07-02 | 远大赛威信生命科学(南京)有限公司 | 一种用于预防和治疗乙型肝炎的药物组合物及其用途 |
CN114409802A (zh) * | 2020-12-31 | 2022-04-29 | 中国科学院微生物研究所 | 禽流感病毒三聚体亚单位疫苗及其应用 |
CN114437185A (zh) * | 2020-12-31 | 2022-05-06 | 中国科学院微生物研究所 | 冠状病毒三聚体亚单位疫苗及其应用 |
CN114409802B (zh) * | 2020-12-31 | 2023-10-20 | 中国科学院微生物研究所 | 禽流感病毒三聚体亚单位疫苗及其应用 |
Also Published As
Publication number | Publication date |
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EP2974740A4 (en) | 2016-08-24 |
EP2974740A1 (en) | 2016-01-20 |
JP2016512204A (ja) | 2016-04-25 |
US9878035B2 (en) | 2018-01-30 |
EP2974740B1 (en) | 2019-06-19 |
US20160136264A1 (en) | 2016-05-19 |
CN104043120A (zh) | 2014-09-17 |
JP6499592B2 (ja) | 2019-04-10 |
CN104043120B (zh) | 2017-05-31 |
HK1218872A1 (zh) | 2017-03-17 |
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