CN107693788B - Pharmaceutical composition for preventing or treating hepatitis B and application thereof - Google Patents
Pharmaceutical composition for preventing or treating hepatitis B and application thereof Download PDFInfo
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- CN107693788B CN107693788B CN201710718894.1A CN201710718894A CN107693788B CN 107693788 B CN107693788 B CN 107693788B CN 201710718894 A CN201710718894 A CN 201710718894A CN 107693788 B CN107693788 B CN 107693788B
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- hepatitis
- hbcag
- antigen
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- A—HUMAN NECESSITIES
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- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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- 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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Abstract
The present invention relates to a pharmaceutical composition for preventing or treating hepatitis b and its use, comprising: i) HBsAg, a fragment of the antigen, a functional variant of the antigen or the fragment of the antigen, or a mixture of at least two thereof, ii) HBcAg, a fragment of the antigen, a functional variant of the antigen or the fragment of the antigen, or a mixture of at least two thereof, iii) MONTANIDE ISA adjuvant. The invention also relates to the use of the composition for the treatment of HBV infection and HBV mediated diseases, and to a method of treating HBV infection and HBV mediated diseases. The composition can mediate stronger immune response, including anti-HBs antibodies and anti-HBc antibodies, particularly mediate Th1 cell immune response, generate cytokine IFN-gamma related to virus elimination, promote the differentiation and maturation of anti-HBs-IgG 2a antibody subtypes, and realize the balance of humoral immunity and cellular immunity.
Description
Technical Field
The invention belongs to the field of immunology, and relates to a pharmaceutical composition for preventing and/or treating hepatitis B and application thereof in preparation of a medicament.
Background
Hepatitis B Virus (HBV) infection is one of the serious public health problems worldwide. HBV infection is an important cause of chronic hepatitis b, cirrhosis and hepatocellular carcinoma (Fattovich G.J Hepatol 2008. The main common drugs for clinical treatment of chronic HBV infection are nucleoside analogues and interferon. Nucleoside analogs do not completely eliminate cccDNA in hepatocytes and long-term use is prone to the emergence of drug-resistant mutants and rebound after drug withdrawal (Kwon H, lok as. Nat Rev Gastroenterol hepatol.2011; 8. Interferons are not suitable for asymptomatic HBV carriers, and in chronic HBV patients the incidence of serological switch of HBeAg after half a year of use is only 33%, and their use is limited by the large side effects of interferons (Tang SX, yu gl. Lancet 1990 (8684): 302).
The hepatitis B protein vaccine widely used at present generates protective neutralizing antibody by inducing humoral immunity, thus achieving the purpose of prevention. A great deal of research finds that the neutralizing antibody can only eliminate extracellular virus particles, while the elimination of intracellular infected viruses mainly depends on specific cellular immune response, helper T cells and CD4 + Th 1-type cytokines such as IFN-. Gamma.produced by T cells, especially virus-specific Cytotoxic T Lymphocytes (CTL), are eliminated (Chin R, lacamine S.Rev Med Viorl.2003:13 (4): 255-72). The intensity of cellular immune response directly determines the prognosis of hepatitis B. Therefore, the ideal therapeutic hepatitis B vaccine needs to induce specific humoral immunity and cellular immunity at the same time, and break through the immune tolerance of hepatitis B.
At present, most of the research and development ideas of hepatitis B therapeutic vaccines in China surround HBsAg, and achieve the effect of immune clearance by generating anti-HBsAg antibodies (anti-HBs), such as HBsAg-immunoglobulin complex (Xu D Z, ZHao K, et al plos ONE,2008, e2565) of Wen Yumei and high-dose hepatitis B surface antigen vaccines (Chunhuang, zhang Yijun and the like, guangdong medicine, 24 vol.07 in 2003, pages 740-706) of Zhang Yijun, and the like, and the therapeutic effects of the two vaccines are not ideal from the latest clinical data.
Research shows that the subtype of anti-HBs of chronic hepatitis B infected patients is mainly IgG4, while the subtype of anti-HBs of hepatitis B infection cured patients is IgG1IgG4, which shows that Th1 antibody subtype IgG1 plays an important role in the elimination process of hepatitis B infection, and whether the Th1 antibody subtype is higher than or equal to Th2 antibody subtype is evaluated to possibly suggest the effect of hepatitis B treatment (S.Rath, et al. Clin.exp. Immunol. (1988) 72, 164-167). Meanwhile, the subtype of anti-HBc antibody (anti-HBc) of a patient infected with hepatitis B is IgG1 & IgG3 & IgG4, while the subtype of anti-HBc of a patient cured by hepatitis B infection is changed into IgG3 & IgG1 & IgG4, which indicates that the change of the subtype of anti-HBc antibody, particularly anti-HBc antibody, is possibly closely related to the treatment of hepatitis B (Chien-Fu Huang, et al. Cellular & Molecular immunology.2006;3 (2): 97-106.).
In addition, studies have shown that the MONTANIDE ISA water-in-oil adjuvant can generate an inflammatory response and promote recruitment of Antigen Presenting Cells (APC) (such as macrophages and lymphocytes) besides having a slow release effect on antigen, antigen is endocytosed into APC through interaction of a surfactant and a cell membrane, and promotion of MHC class II molecule expression and cross presentation can induce strong MHC class I molecule presentation, so that antigen-specific CD8+ and CD4+ cellular immune responses and B cell activation can be simultaneously induced to generate antibodies (Jerome Akio Akira. Phil. Trans. R.Soc.B (2011) 366, 2748-2755). At present, tumor vaccines CIMAvax applying MONTANIDE ISA type adjuvants are on the market, and a large number of clinical tests applying the adjuvants are in progress, so that the clinical safety and the effectiveness of the adjuvants are fully proved.
Patent US4547367a uses HBc particles for the treatment/prevention of HBV infection and HBV mediated diseases, and immunization of chimpanzees with HBc particles can protect chimpanzees from HBV infection. Furthermore, the HBc particles are combined with HBs particles to immunize newborn infants produced by mothers of hepatitis B carriers, so that high titers of anti-HBs and anti-HBc antibodies are produced, and HBV infection is not observed in 18 months of monitoring. However, this patent does not specifically suggest evidence of subtype shift of anti-HBc antibodies and there is no direct evidence for treatment of HBV infection and HBV-mediated diseases.
Patent WO2007/031334A2 discloses hepatitis b therapeutic vaccine components comprising HBsAg, HBcAg and a saponin adjuvant, and CpG-ODN can be used as a co-adjuvant, however, the hepatitis b therapeutic vaccine requires combination therapy with nucleoside analogs in clinical practice to break through the immune tolerance of hepatitis b, and the e antigen turns negative by only 25%.
Patent CN201310080863.X discloses a therapeutic hepatitis B vaccine, which comprises HBsAg, HBcAg and a CpG-ODN adjuvant with a specific sequence, and the vaccine for hepatitis B can break through the immune tolerance of HBV transgenic mice and generate strong antigen-specific humoral and cellular immune response. However, no vaccine using CpG-ODN adjuvant is currently on the market, and there is a possible risk in view of clinical safety.
Disclosure of Invention
An object of the present invention is to overcome the drawbacks of the drugs for treating hepatitis b infection known in the prior art, and to provide a pharmaceutical composition which can generate a strong immune response in patients with chronic HBV infection, promote the differentiation of anti-HBs-IgG2a antibody subtypes, and bring IgG2a and IgG1 to equilibrium; induce the anti-HBc antibody subtype to change and/or break through the immune tolerance of HBV infected patients.
It is another object of the present invention to provide the 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.
To achieve the above object, the present invention provides a pharmaceutical composition comprising:
i) Hepatitis B surface antigen (HBsAg), a fragment of the antigen, a functional variant of the antigen or of the fragment of the antigen, or a mixture of at least two thereof,
ii) hepatitis B core antigen (HBcAg), a fragment of the antigen, a functional variant of the antigen or of the fragment of the antigen, or a mixture of at least two thereof,
iii) A MONTANIDE ISA adjuvant comprising an oil and a surfactant, preferably MONTANIDE ISA51VG or MONTANIDE ISA720. Wherein MONTANIDE ISA51VG is a mixture comprising mineral oil (Drakeol 6 VR) and surfactant mannide monooleate; MONTANIDE ISA720 is a mixture comprising squalene, squalane, and the surfactants mannide monooleate and polyoxyethylene 40 hydrogenated castor oil.
The present invention also provides a pharmaceutical composition comprising:
i) Hepatitis B surface antigen (HBsAg), a fragment of the antigen, a functional variant of the antigen or of the fragment of the antigen, or a mixture of at least two thereof,
ii) hepatitis B core antigen (HBcAg), a fragment of the antigen, a functional variant of the antigen or of the fragment of the antigen, or a mixture of at least two thereof,
iii) MONTANIDE ISA adjuvant, and
iv) a pharmaceutically acceptable carrier.
In particular, the pharmaceutical composition of the present invention is useful as a therapeutic vaccine for hepatitis b.
In some embodiments of the invention, the HBsAg has the sequence shown in SEQ ID NO. 1.
In other embodiments of the invention, the HBcAg has the sequence shown in SEQ ID NO 2.
In still further embodiments of the invention, the montainide ISA adjuvant comprises an oil and a surfactant, preferably montainide ISA 51VG or montainide ISA 720.
In still further embodiments of the invention, the pharmaceutical composition wherein the mass ratio of components i) and ii) is 0.5 to 5:1, the ratio of the volume of the MONTANIDE ISA 51VG to the sum of the volumes of components i) and ii) is 1:1, preferably aqueous phase volume ratio 1:1; the ratio of the volume of MONTANIDE ISA 720 to the sum of the volumes of components i) and ii) is 7:3, preferably aqueous phase volume ratio 7:3.
The invention also relates to a kit comprising a pharmaceutical composition according to the invention or a hepatitis b vaccine containing a pharmaceutical composition according to the invention, and instructions for its use.
In one aspect, the present invention relates to the use of a pharmaceutical composition according to the present invention for the preparation of a medicament for the treatment of HBV infection and/or HBV mediated disease in a subject, preferably said HBV infection and/or HBV mediated disease is selected from hepatitis b, cirrhosis and liver cancer.
In a further aspect, the present invention relates to the use of a pharmaceutical composition according to the invention for the manufacture of a medicament for generating an immune response against HBV, preferably inducing a Th1 and Th2 type immune response in a subject.
In another aspect, the invention relates to the use of a pharmaceutical composition according to the invention for the preparation of a medicament for the isotyping of an anti-HBc antibody in a subject.
In a further aspect, the present invention relates to the use of a pharmaceutical composition according to the present invention for the preparation of a medicament for breaking the immune tolerance of hepatitis b virus in a subject.
In a further aspect, the invention relates to the use of a pharmaceutical composition according to the invention in the manufacture of a medicament for achieving a Th1/Th2 immune response balance of the hepatitis b surface antigen in a subject (e.g. inducing a Th1 and Th2 type immune response approximately equally).
In yet another aspect, the present invention relates to a pharmaceutical composition comprising: i) A hepatitis b surface antigen (HBsAg), a fragment of the antigen, a functional variant of the antigen or of the fragment of the antigen, or a mixture of at least two thereof, ii) a hepatitis b core antigen (HBcAg), a fragment of the antigen, a functional variant of the antigen or of the fragment of the antigen, or a mixture of at least two thereof, and iii) a montnide ISA adjuvant for use in the treatment of HBV infection and/or HBV-mediated disease in a subject, for generating an immune response against HBV in a subject (preferably inducing a Th1 and Th2 type immune response), for effecting a subtype shift of anti-HBc antibodies in a subject, and/or for breaking hepatitis b virus immune tolerance in a subject.
In yet another aspect, the present invention relates to a method of treating HBV infection and/or HBV-mediated disease in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention.
In a further aspect, the present invention relates to a method of generating an immune response against HBV, preferably inducing a Th1 and Th2 type immune response, in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the invention.
In yet another aspect, the present invention relates to a method of isotyping an anti-HBc antibody in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention.
In a further aspect, the present invention relates to a method of breaking the immune tolerance of hepatitis b virus in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present invention.
The compositions of the present invention achieve unexpected technical results. The vaccine is matched with the existing commercial hepatitis B prevention vaccine (HBsAg + Al (OH) 3 ) Compared with HBsAg + CpG, HBsAg + MONTANIDE ISA vaccine and HBsAg + HBcAg + CpG vaccine, the hepatitis B enhanced vaccine can mediate stronger immune response in-vivo tests of mice, including anti-HBs antibodies and anti-HBc antibodies, particularly mediating Th1 cell immune response, generating cytokine IFN-gamma related to virus elimination, promoting the differentiation and maturation of anti-HBs-IgG 2a antibody subtype, and realizing the balance of humoral immunity and cellular immunity.
The composition HBsAg + CpG has been disclosed in the prior art (see patent CN101492672 a), while the present inventors have surprisingly found that the pharmaceutical composition according to the present invention (combination of HBsAg, HBcAg and montainide ISA adjuvant) can produce anti-HBs specific antibodies significantly better than the HBsAg + CpG composition, showing a surprising synergistic effect.
Compositions HBsAg + HBcAg + CpG have been disclosed in the prior art (see patent 201310080863. X), while the present inventors have surprisingly found that pharmaceutical compositions according to the present invention (combination of HBsAg, HBcAg and montainide ISA adjuvants) can produce anti-HBs specific antibodies significantly better than HBsAg + HBcAg + CpG compositions, and stronger cellular immune responses.
In addition, the pharmaceutical composition provided by the invention can mediate strong anti-HBc immune response on a C57BL/6 mouse, and realizes anti-HBc antibody subtype conversion, namely, the displayed anti-HBc antibody subtype relation is that the IgG2a antibody level is higher than that of IgG1, and the relation is consistent with the antibody subtype relation recovered by hepatitis B infected patients. This encouraging result indicates that the composition according to the invention can be used as a prophylactic and/or therapeutic vaccine for hepatitis B, thus solving the problem which has long been troubled.
Drawings
FIG. 1 shows a composition of the invention in combination with HBsAg + Al (OH) 3 HBsAg + CpG, HBsAg + MONTANIDE ISA and HBsAg + HBcAg + CpG groups are shown in the graph of enhancing the immune response of mice to hepatitis B surface antigen IgG.
FIG. 2 shows a composition of the invention in combination with HBsAg + Al (OH) 3 HBsAg + CpG, HBsAg + MONTANIDE ISA and HBsAg + HBcAg + CpG groups are compared with the graph of enhancing the Th2 immune response of mice to hepatitis B surface antigen.
FIG. 3 shows a composition of the invention in combination with HBsAg + Al (OH) 3 HBsAg + CpG, HBsAg + MONTANIDE ISA and HBsAg + HBcAg + CpG groups are compared with the graph of enhancing the Th1 immune response of mice to hepatitis B surface antigen.
FIG. 4 shows a composition of the invention in combination with HBsAg + Al (OH) 3 HBsAg + CpG, HBsAg + MONTANIDE ISA and HBsAg + HBcAg + CpG groups are shown to enhance the level of neutralizing antibodies to hepatitis B surface antigen in mice.
FIG. 5 is a graph showing that the anti-HBc antibody subtype produced by the composition of the present invention is IgG2a > IgG 1.
FIG. 6 shows a composition of the invention and HBsAg + Al (OH) 3 HBsAg + CpG, HBsAg + MONTANIDE ISA and HBsAg + HBcAg + CpG groups have a trend towards promoting differentiation of HBsAg Th1 cells at the cellular immune level.
FIG. 7 is a graph showing that compositions of the invention produce higher levels of protective antibody levels with the HBsAg + HBcAg + CpG groups than with replication-competent HBV transgenic mice.
FIG. 8 is a graph showing that the compositions of the invention produce a serological conversion of HBeAg/HBeAb with the HBsAg + HBcAg + CpG group more than with replication-competent HBV transgenic mice.
Detailed Description
The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
Notwithstanding that the numerical ranges and parameter approximations set forth the broad scope of the invention, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.
It should also be noted that, as used in this specification, the term "or" may be used interchangeably with the term "and/or" unless the context clearly dictates otherwise.
The term "polypeptide" as used herein refers to a polymer of amino acids and is not particularly limited by the minimum number of amino acids. It therefore also includes peptides, oligopeptides, dimers, trimers, oligomers, particles and the like. Furthermore, 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, thioation, etc.).
The term "fragment of an antigen" as used herein refers to a fragment of a natural or synthetic polypeptide which retains the antigenic properties of said natural or synthetic polypeptide, i.e. which is capable of eliciting an immune response against said natural or synthetic polypeptide.
The term "hepatitis b surface antigens (HBsAg, HBs)" as used herein is intended to encompass native HBsAg, fragments of HBsAg antigen, functional variants of HBsAg and any combination thereof. In particular, the native HBsAg is a native HBsAg polypeptide comprising 226 amino acids. More particularly, the HBsAg is a native HBsAg polypeptide derived from the HBV standard genotype A, B, C, D, E, F, G and/or H known today. In certain embodiments, the HBsAg has the sequence shown in SEQ ID NO. 1.
The term "fragment of the HBsAg antigen" as used herein is intended to mean a polypeptide which has a continuous or discontinuous fragment of less than 226 amino acids of the native HBsAg and which retains the antigenicity of the native HBsAg.
The term "HBsAg functional variant" as used herein is intended to denote a polypeptide which has at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, at most 2, at most 1 amino acid deletion, insertion, addition or substitution relative to the native HBsAg or HBsAg fragment and which retains the function (e.g. antigenicity) of the native HBsAg.
The term "hepatitis b core antigen (HBcAg, HBc)" as used herein is intended to encompass native HBcAg, fragments of HBcAg antigen, functional variants of HBcAg and any combination thereof. In particular, the native HBcAg is a native HBcAg polypeptide comprising 183 amino acids. More particularly, the native HBcAg is native HBcAg derived from the HBV standard genotype A, B, C, D, E, F, G and/or H known today. In some embodiments, the HBcAg is selected from HBcAg 1-x A polypeptide representing a fragment of natural HBcAg of amino acid 1-X, in particular X is 149 to 183. In other embodiments, the HBcAg is selected from the group consisting of a polypeptide having the sequence: amino acids 1-149 of SEQ ID NO:2, amino acids 1-150 of SEQ ID NO:2, amino acids 1-151 of SEQ ID NO:2, amino acids 1-152 of SEQ ID NO:2, amino acids 1-153 of SEQ ID NO:2, amino acids 1-154 of SEQ ID NO:2, amino acids 1-155 of SEQ ID NO:2, amino acids 1-156 of SEQ ID NO:2, amino acids 1-157 of SEQ ID NO:2, and ammonia 1-158 of SEQ ID NO:2 Amino acids 1 to 159 of SEQ ID NO. 2, amino acids 1 to 160 of SEQ ID NO. 2, amino acids 1 to 161 of SEQ ID NO. 2, amino acids 1 to 162 of SEQ ID NO. 2, amino acids 1 to 163 of SEQ ID NO. 2, amino acids 1 to 164 of SEQ ID NO. 2, amino acids 1 to 165 of SEQ ID NO. 2, amino acids 1 to 166 of SEQ ID NO. 2, amino acids 1 to 167 of SEQ ID NO. 2, amino acids 1 to 168 of SEQ ID NO. 2, amino acids 1 to 169 of SEQ ID NO. 2, amino acids 1 to 170 of SEQ ID NO. 2, amino acids 1 to 171 of SEQ ID NO. 2, amino acids 1 to 172 of SEQ ID NO. 2, amino acids 1 to 173 of SEQ ID NO. 2, amino acids 1 to 174 of SEQ ID NO. 2, amino acids 1 to 175 of SEQ ID NO. 2, amino acids 1 to 176, amino acids 1 to 177 of SEQ ID NO. 2, amino acids 1 to 178 of SEQ ID NO. 2, amino acids 1 to 178, and SEQ ID NO. 2. In certain embodiments, the HBcAg has the sequence shown in SEQ ID NO 2.
The term "fragment of HBcAg antigen" as used herein is intended to mean a polypeptide which has a continuous or discontinuous fragment of less than 183 amino acids of native HBcAg and which retains the antigenicity of native HBcAg.
The term "functional variant of HBcAg" as used herein is intended to mean a polypeptide which has at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, at most 2, at most 1 amino acid deletion, insertion, addition or substitution relative to native HBcAg or HBcAg fragment and which retains the function (e.g. antigenicity) of native HBcAg.
Preferably, in the present invention, both HBcAg and HBsAg are present in the form of particles in the composition according to the invention.
As used herein, the terms "pharmaceutical composition," "combination drug," and "drug combination" are used interchangeably and refer to a combination of at least one drug and optionally a pharmaceutically acceptable excipient or adjuvant, combined together to achieve a particular purpose. In certain embodiments, the pharmaceutical compositions include temporally and/or spatially separated combinations, so long as they are capable of acting together to achieve the objectives of the present invention. For example, the components contained in the pharmaceutical composition (e.g., HBsAg, HBcAg, and montainide ISA adjuvant) may be administered to a subject in bulk, or separately. When the ingredients contained in the pharmaceutical composition are administered separately to a subject, the ingredients may be administered to the subject simultaneously or sequentially.
The term MONTANIDE ISA adjuvant as used herein is an adjuvant comprising an oil and a surfactant, preferably MONTANIDE ISA 51VG and MONTANIDE ISA 720. Wherein the MONTANIDE ISA 51VG is a mixture comprising high purity mineral oil (Drakeol 6 VR) and surfactant diglycolic monooleate; wherein the MONTANIDE ISA 720 is a mixture comprising high purity squalene, squalane, and surfactants (mannide monooleate and polyoxyethylene 40 hydrogenated castor oil) (Jerome. Aucouurier, et al. MONTANIDE ISA 720and 51. MONTANIDE ISA 51VG and MONTANIDE ISA 720 are both purchased from Saybolt, france.
As used herein, "therapeutically effective amount" or "effective amount" refers to a dosage sufficient to show its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the subject's own condition and severity. Prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and depends on general practitioners and other physicians, often taking into account the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to physicians.
The term "HBV-mediated disease" as used herein is intended to mean a disease caused by, induced by, aggravated by, increased in risk for developing, and/or associated with Hepatitis B Virus (HBV), such as hepatitis b, hepatitis d, cirrhosis, hepatic ascites, liver cancer, and the like.
The term "anti-HBc antibody subtype switch" as used herein is intended to mean that the anti-HBc antibody subtype relationship in a subject has switched to be consistent with the antibody subtype relationship in a hepatitis b cured patient, i.e., with the antibody subtype relationship in a hepatitis b infected patient recovering after administration of the pharmaceutical composition of the present invention. Particularly, in mice, the anti-HBc antibody subtype is converted from IgG1> IgG2a to IgG2a > IgG2b > IgG1 or IgG2b > IgG2a > IgG1, for example IgG2a > IgG1, in humans, the anti-HBc antibody subtype is converted from IgG1> IgG3> IgG4 to IgG3> IgG1> IgG4.
The term "subject" as used herein refers to a mammal, such as a human, but may also be another animal, such as a domestic animal (e.g., dog, cat, etc.), a domestic animal (e.g., cow, sheep, pig, horse, etc.) or a laboratory animal (e.g., monkey, rat, mouse, rabbit, guinea pig, etc.).
Another embodiment of the composition of the invention comprises i) HBsAg or a variant of this antigen, ii) HBcAg 1-183 And iii) MONTANIDE ISA adjuvants, including MONTANIDE ISA 51VG and MONTANIDE ISA 720.
In some embodiments, the pharmaceutical composition of the invention wherein the mass ratio of components i) and ii) is from 0.5 to 5:1, the ratio of the volume of the montainide ISA 51VG to the sum of the volumes of components i) and ii) is 1:1, preferably aqueous phase volume ratio 1:1; the ratio of the volume ratio of MONTANIDE ISA 720 to the sum of the volumes of components i) and ii) is 7:3, preferably aqueous phase volume ratio 7:3.
In other embodiments, the composition of the invention may further comprise additional additives, such as pharmaceutical carriers or additives, especially when it is present in the form of a pharmaceutical formulation.
Preferred pharmaceutical carriers are especially water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides and the like. The type of pharmaceutical carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.
The pharmaceutical composition, vaccine or pharmaceutical preparation according to the invention may be administered by any suitable route, e.g. orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
Example 1.Hepatitis B surface antigen (HBsAg) in combination with hepatitis B core antigen (HBcAg) and MONTANIDE ISA51 enhances the immune response to total IgG from hepatitis B surface antigen.
The HBsAg amino acid sequence used in this example is SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
in order to detect the hepatitis B surface antigen total IgG immune response of the HBsAg + HBcAg + MONTANIDE ISA51 composition, the inventors separately labeled HBsAg, HBcAg and MONTANIDE ISA51, HBsAg, HBcAg and CpG-ODG, HBsAg and MONTANIDE ISA51, HBsAg and Al (OH) 3 The adjuvants were mixed well and used to immunize mice, and HBsAg-specific IgG levels in serum were determined and statistically analyzed to evaluate the HBsAg, HBcAg and CpG-ODG, HBsAg and MONTANIDE ISA51, HBsAg and Al (OH) 3 Combined use of HBsAg, HBcAg and MONTANIDE ISA51 has effect on HBsAg total IgG immune response.
C57BL/6 mice, female, 6-8 weeks, purchased from Shanghai Spikel company were used in this example. The HBsAg antigen used in the embodiment is prepared by the inventor, is a natural HBsAg expressed by Hansenula polymorpha, is an adw subtype, has a purity of more than 95%, is stored in a refrigerator at 4 ℃ for later use, and has a Purification preparation process shown in reports 301-310 of Protein Expression and Purification 56 (2007), and the specific steps are as follows: collecting thallus, homogenizing with a high pressure homogenizer, centrifuging at 4000g and 4 deg.C for 30min, and removing cell debris; clarification by microfiltration, 14% ammonium sulfate precipitation and 12% PEG10000 (w/v) precipitation; adjusting pH of the clarified supernatant to 7.0, performing DEAE ion exchange chromatography and hydrophobic chromatography, and collecting protein peak; after ultrafiltration and concentration, carrying out gel filtration chromatography, and collecting a target protein peak; storing in a refrigerator at 4 ℃ for later use. The HBcAg antigen used in this example is prepared by the present inventors, is a natural hepatitis B core protein expressed by Escherichia coli, and the purification and preparation process is reported in Li Jilai, xu Jingdeng, journal of Chinese biologicals 2011, volume 24, pages 1121-1125, and specifically comprises the following steps: after collecting the thalli, resuspending the thalli by using 10mmol/L sodium phosphate buffer solution, carrying out ultrasonication, centrifuging, collecting supernatant, adding saturated ammonium sulfate to ensure that the final concentration is 33 percent, fully mixing uniformly, and standing overnight at 4 ℃; the next day, centrifuging, resuspending the precipitate with 10mmol/L sodium phosphate buffer solution, placing into a dialysis bag, and dialyzing in 10mmol/L sodium phosphate buffer solution at 4 deg.C for 24h; performing CHT chromatography on the dialyzed solution, collecting a protein peak, concentrating, performing Sephacryl S-400HR gel filtration chromatography, and collecting a target protein peak; storing in a refrigerator at 4 ℃ for later use. The CpG-ODN sequence used in this example is 5'-TCG TTC GTT CGT TCG TTC GTT-3', prepared by chemical synthesis by the solid phase phosphoramidite triester method described in CN200810004736.0, starting from the 3' end, 1) deprotecting: removing a protective group DMT (dimethoxytrityl) of the nucleotide connected with the CpG by trichloroacetic acid to obtain free 5' hydroxyl for the next condensation reaction; 2) Activation: mixing the nucleotide monomer protected by phosphoramidite and tetrazole activator, and feeding the mixture into a synthesis column to form a phosphoramidite tetrazole active intermediate, wherein the intermediate and the nucleotide of which the protective group is removed from CpG undergo a condensation reaction; 3) Connecting: when meeting the nucleotide with the deprotected group on CpG, the intermediate of the tetrazole phosphoramidite activity will have affinity reaction with the 5' hydroxyl group, condense and remove tetrazole, and at this time, the oligonucleotide chain extends a base forward; 4) And (3) oxidation: during condensation reaction, the nucleotide monomer is connected with the oligonucleotide connected with CpG through a phosphorous ester bond which is unstable and easy to be hydrolyzed by acid or alkali, and then the phosphorous amide is oxidized into phosphotriester of sulfur-phosphorous double bond by using a thioreagent, so as to obtain the stable oligonucleotide; 5) And (3) sealing: to prevent unreacted 5' hydroxyl groups attached to CpG from being extended in subsequent cycles after the condensation reaction, this terminal hydroxyl group is usually blocked by acetylation; after the five steps, one deoxynucleotide is connected to the CpG nucleotide; repeating the processes of removing the protecting group, activating, connecting, oxidizing and sealing to obtain a crude DNA fragment; finally, the CpG-ODN is subjected to synthetic post-treatment such as cutting, protecting group removal, purification, quantification and the like to obtain the CpG-ODN; storing in a refrigerator at-20 deg.C for use.
HBsAg was directly diluted with PBS (Gibco Co.) or adsorbed to 1mg/ml Al (OH) 3 (fromBeijing Temple Bio-products Co., ltd.), the final protein concentration is 10. Mu.g/ml; diluting HBsAg to 20 mu g/ml by PBS, diluting CpG-ODN to 40 mu g/ml by PBS, and uniformly mixing 1:1 until the final HBsAg concentration is 10 mu g/ml and the final CpG-ODN concentration is 20 mu g/ml; diluting HBsAg to 20 μ g/ml with PBS, mixing with MONTANIDE ISA51 (purchased from Spirobeck corporation) adjuvant at a volume ratio of 1:1, and making HBsAg concentration to 10 μ g/ml; diluting HBsAg and HBcAg to 20 mu g/ml by PBS, diluting CpG-ODN to 40 mu g/ml by PBS, and uniformly mixing 1; HBsAg and HBcAg were diluted to 20. Mu.g/ml with PBS, and mixed with MONTANIDE ISA51 (purchased from Spirasci corporation) adjuvant 1:1 by volume ratio until the HBsAg concentration was 10. Mu.g/ml and the HBcAg concentration was 10. Mu.g/ml. C57BL/6 mice were immunized via left hind limb gastrocnemius muscle in a total volume of 100. Mu.l, with 10 mice per group. HBsAg + Al (OH) 3 Mice were injected with 1. Mu.g of menses Al (OH) 3 Adsorbed HBsAg; HBsAg + CpG group 1 μ g HBsAg and 2 μ g CpG were injected per mouse; HBsAg + MONTANIDE ISA51 group each mouse injected with 1 μ g HBsAg and equal volume of MONTANIDE ISA51 adjuvant; HBsAg + HBcAg + CpG-ODN groups 1. Mu.g HBsAg, 1. Mu.g HBcAg and 2. Mu.g CpG-ODN are injected into each mouse; HBsAg + HBcAg + MONTANIDE ISA51 groups each mouse injected with 1 u g HBsAg, 1 u g HBcAg and equal volume of MONTANIDE ISA51 adjuvant. Immunizations were performed every three weeks, and blood was collected ten days after the three-immunization and serum was isolated, which was started with 2% skim milk at a dilution of 1.
HBsAg was used to coat a 96-well microplate (from Nunc), 25ng per well, overnight at 4 ℃; washing the plate for 2 times, and sealing with 5% skimmed milk at 37 deg.C for 1 hr; washing the plate for 2 times, adding the serum to be detected which is diluted by 3 times in series, and acting for 1 hour at 37 ℃; after 3 washes, the plate was washed with 1; the plates were washed 3 times and developed with TMB (purchased from Thermo, USA) for 15 minutes at 100. Mu.l per well; the reaction was stopped with 2M sulfuric acid, 100. Mu.l per well, and the absorbance OD450 at 450nm (corrected for OD 630) was measured with a microplate reader, and the end point titer was determined. The results are shown in FIG. 1.
As can be seen from fig. 1, the immune response of the HBsAg + HBcAg + montainide ISA 51 group is significantly enhanced, the specific antibody titer can reach 5.4 logarithmic values, the specific antibody titer generated by the HBsAg + HBcAg + CpG-ODN group is 5.1 logarithmic values, and the specific antibody titer (titer) generated by the HBsAg + HBcAg + montainide ISA 51 group can be increased by more than 1 time compared with the HBsAg + HBcAg + CpG-ODN group; at the same time, the HBsAg + HBcAg + MONTANIDE ISA 51 group and the two-component HBsAg + MONTANIDE ISA 51 group, HBsAg + CpG-ODN group and HBsAg + Al (OH) 3 Compared with the titer of the specific antibodies generated by the groups, the antibody titers have significant differences (P respectively) <0.05、P<0.001、P<0.001 Specific antibody titers (titers) increased 31-fold, 25-fold, and 10-fold, respectively. The results show that the HBsAg + HBcAg + MONTANIDE ISA 51 composition vaccine of the invention not only adds CpG-ODN and Al (OH) to the two components 3 The hepatitis B vaccine with the MONTANIDE ISA 51 adjuvant can obviously enhance the immune response of the total IgG of the hepatitis B surface antigen, and can also enhance the immune response of the total IgG of the hepatitis B surface antigen compared with the three-component HBsAg + HBcAg + CpG-ODN vaccine.
Example 2.The combination of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and MONTANIDE ISA 51 enhances the Th2 immune response of hepatitis B surface antigen at the humoral immune level.
The HBsAg amino acid sequence used in this example is SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
the effect of the HBsAg + HBcAg + montainide ISA 51 composition of the present invention on the Th 2-class immune response of mice was determined according to the method described in example 1, wherein the method of example 2 differs from the method of example 1 in that the enzyme-labeled antibody used in the detection is horseradish peroxidase-labeled goat anti-mouse IgG1 (purchased from southern biotech, usa) and the dilution factor is 1 20000. The results are shown in fig. 2.
Antigen-specific immune responses are divided into two types, th1 and Th2, with Th 2-type responses corresponding to high levels of antigen-specific IgG1 antibody titers. Al (OH) 3 The vaccine is a strong Th2 vaccine adjuvant, can inhibit Th1 immune response, and is shown to induce high-level specific IgG1 antibody after immunization. CpG-ODG is a strong Th1 classThe vaccine adjuvant can generate stronger Th1 immune response. MONTANIDE ISA 51 can produce strong Th1 and Th2 immune responses. In this example, the antibody titer of the antigen-specific IgG1 produced by the HBsAg + HBcAg + montainide ISA 51 composition was up to 6.0 log values, the antibody titer of the specific IgG1 produced by the HBsAg + HBcAg + CpG-ODN group was 4.2 log values, and the antibody titer (titer) of the specific IgG1 produced by the HBsAg + HBcAg + montainide ISA 51 group was 63 times greater than that of the specific IgG1 produced by the HBsAg + HBcAg + CpG-ODN group, which showed significant differences (P + hcag + CpG-ODN group)<0.001 ); meanwhile, the HBsAg + HBcAg + MONTANIDE ISA 51 group and the two-component HBsAg + MONTANIDE ISA 51 group, HBsAg + CpG-ODN group and HBsAg + Al (OH) 3 Compared with the titer of the specific IgG1 antibody generated by the group, the group has very significant difference (all are P)<0.001 Specific antibody titers (titers) increased 79-fold, 50-fold, and 63-fold, respectively. The results show that the HBsAg + HBcAg + MONTANIDE ISA 51 composition vaccine of the invention not only adds CpG-ODN and Al (OH) to two components 3 And the MONTANIDE ISA 51 adjuvant hepatitis B vaccine can obviously enhance the specific IgG1 immune response of the hepatitis B surface antigen, and can also obviously enhance the specific IgG1 antibody of the hepatitis B surface antigen compared with three-component HBsAg, HBcAg and CpG-ODN groups, which indicates that the immune response of the hepatitis B surface antigen Th2 can be obviously enhanced.
Example 3.The combination of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and MONTANIDE ISA 51 enhances the Th1 immune response of hepatitis B surface antigen at the humoral immune level.
The HBsAg amino acid sequence used in this example is SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
the effect of the HBsAg + HBcAg + montainide ISA 51 composition of the present invention on the Th1 class immune response of mice was determined according to the method described in example 1, wherein the method of example 3 differs from the method of example 1 in that the enzyme-labeled antibody used in the detection is horseradish peroxidase-labeled goat anti-mouse IgG2a (purchased from southern biotech, usa) and the dilution factor is 1. The results are shown in fig. 3.
As mentioned in example 2, al (OH) 3 Is a strong Th2 vaccine adjuvant, can inhibit Th1 immune response, and is characterized by induction after immunizationVery low levels of specific IgG2a antibodies. CpG-ODG is a strong Th1 vaccine adjuvant, can enhance Th1 immune response, and is shown by high-level specific IgG2a antibody after immunization. MONTANIDE ISA 51 is capable of producing strong Th1 and Th2 class immune responses, manifested by induction of high levels of specific IgG1 and IgG2a antibodies following immunization. In this example, al (OH) 3 The specific IgG2a antibody titer induced as HBsAg adjuvant was only 1.57 log values, whereas the specific IgG2a antibody titer induced by montainide ISA51 as HBsAg adjuvant was up to 3.4 log values. When the composition of the invention, HBsAg, HBcAg and MONTANIDE ISA51 is used for immunization, the titer of the generated IgG2a antibody reaches 4.7 logarithmic values, the titer of the specific IgG1 antibody generated by the group of HBsAg, HBcAg and CpG-ODN is 4.2 logarithmic values, and the titer (titer) of the specific IgG2a antibody generated by the group of HBsAg, HBcAg and MONTANIDE ISA51 can be increased by 1.6 times compared with the titer (titer) of the specific IgG2a antibody generated by the group of HBsAg, HBcAg and CpG-ODN; at the same time, the HBsAg + HBcAg + MONTANIDE ISA51 group and two-component HBsAg + Al (OH) 3 The groups had very significant differences (P)<0.001 ); the antibody titers (titers) of the generated specific IgG1 antibodies were 4-fold and 20-fold higher than those of the HBsAg + CpG-ODN group and the HBsAg + MONTANIDE ISA51 group, respectively. The results show that the combination of the HBsAg, the HBcAg and the MONTANIDE ISA51 strongly stimulates the Th1 immune response aiming at the hepatitis B surface antigen.
Example 4.The combination of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and MONTANIDE ISA51 can enhance the neutralizing antibody level of mice to hepatitis B surface antigen.
The HBsAg amino acid sequence used in this example is SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
The detection of hepatitis B virus surface antibodies serves to monitor whether the vaccination with the hepatitis B vaccine is successful. Hepatitis b vaccines stimulate the immune system to produce hepatitis b virus surface antibodies equivalent to neutralizing antibodies, the titer of which is directly related to the protective power of the vaccine, the production of which has a significant effect on preventing HBV infection. Therefore, the inventor selects the international ARCHITECT international unit test system for hepatitis B virus surface antibody (CMIA) for detecting hepatitis B virus in the serum of immunized miceConcentration of surface antibody (anti-HBsAg) and statistical analysis were performed to evaluate the combination of HBsAg + HBcAg + MONTANIDE ISA 51 versus HBsAg + MONTANIDE ISA 51, HBsAg + CpG-ODN, HBsAg + Al (OH) 3 The protective effect of combining HBsAg + HBcAg + CpG-ODN on enhancing the level of protective antibodies.
C57BL/6 mice, female, 6-8 weeks, purchased from Shanghai Spikel company were used in this example. HBsAg antigen, HBcAg, cpG-ODN, al (OH) used in this example 3 And montnide ISA 51 adjuvant as described in example 1.
HBsAg was diluted directly with PBS or adsorbed to 1mg/ml Al (OH) 3 In the above, the final protein concentration is 10. Mu.g/ml; diluting HBsAg to 20 mu g/ml with PBS, diluting CpG-ODN to 40 mu g/ml with PBS, mixing 1:1 uniformly until the final HBsAg concentration is 10 mu g/ml and the final CpG-ODN concentration is 20 mu g/ml; diluting HBsAg and HBcAg to 20 mu g/ml by PBS, diluting CpG-ODN to 40 mu g/ml by PBS, and uniformly mixing 1; diluting HBsAg and HBcAg to 20 μ g/ml with PBS, mixing with MONTANIDE ISA 51 adjuvant 1:1 by volume ratio until HBsAg concentration is 10 μ g/ml and HBcAg concentration is 10 μ g/ml. C57BL/6 mice were immunized via left hind limb gastrocnemius muscle in a total volume of 100. Mu.l, with 10 mice per group. HBsAg + Al (OH) 3 Mice were injected with 1. Mu.g of menses Al (OH) 3 Adsorbed HBsAg; HBsAg + CpG group 1 μ g HBsAg and 2 μ g CpG were injected per mouse; HBsAg + HBcAg + CpG-ODN groups 1. Mu.g HBsAg, 1. Mu.g HBcAg and 2. Mu.g CpG-ODN are injected into each mouse; HBsAg + HBcAg + MONTANIDE ISA 51 groups each mouse injected with 1 u g HBsAg, 1 u g HBcAg and equal volume of MONTANIDE ISA 51 adjuvant. Immunizations were performed every three weeks, and blood was collected ten days after the three-immunization and serum was isolated. Serum from each mouse was diluted with PBS (for 250)<IU<1000 500-fold dilution and blood sampling; for IU>1000 5000-fold dilution of each mouse blood sample) was sent to the second subsidiary hospital of southeast university and tested using ARCHITECT international unit test system for hepatitis b virus surface antibody (chemiluminescence microparticle immunoassay, CMIA) in serum of immunized mice (ABBOTT ARCHITECT system test), the results of which are shown in fig. 4.
As can be seen from fig. 4, the HBsAg + HBcAg + montainide ISA 51 group can significantly enhance the level of the protective antibody, the specific protective antibody titer can reach 4.4 log values, the protective antibody titer generated by the HBsAg + HBcAg + CpG-ODN group is 4.3 log values, and the protective antibody titer (titer) generated by the HBsAg + HBcAg + montainide ISA 51 group can be increased by 1.2 times compared with the protective antibody titer (titer) generated by the HBsAg + HBcAg + CpG-ODN group; at the same time, the HBsAg + HBcAg + MONTANIDE ISA 51 group and the two-component HBsAg + Al (OH) 3 The groups, HBsAg + MONTANIDE ISA51 group and HBsAg + CpG-ODN group, produced protective antibody titers (titers) that were increased 10-fold, 2.5-fold, and 2.5-fold, respectively. The results show that the HBsAg + HBcAg + MONTANIDE ISA51 composition vaccine can obviously enhance the protective antibody level of hepatitis B surface antigen and enhance the protective power of the vaccine.
Example 5.anti-HBc antibody subtype IgG2a generated by combining hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and MONTANIDE ISA51>IgG1。
The HBsAg amino acid sequence used in this example was SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
the anti-HBc antibody subtype of the population cured by the hepatitis B infected patient is IgG3> IgG1> IgG4, and the corresponding antibody subtype relationship in a mouse is IgG2a > IgG2b > IgG1 or IgG2b > IgG2a > IgG1. The HBsAg + HBcAg + MONTANIDE ISA51 composition of the invention was assayed for IgG2a and IgG1 titers of mouse anti-HBc antibody subtypes according to the methods described in examples 2 and 3, and it was examined whether the composition could promote conversion of mouse anti-HBc antibody subtypes to IgG2a > IgG1, except that the coating antigen used in the assay was 1. Mu.g/ml HBcAg. The results are shown in fig. 5. The results in FIG. 5 show that the composition of HBsAg + HBcAg + MONTANIDE ISA51 of the invention produces anti-HBc antibody subtype IgG2a > IgG1 with significant differences (P < 0.01). The composition can promote the conversion of the mouse anti-HBc antibody subtype into the antibody subtype of a patient cured from hepatitis B infection.
Example 6.The combination of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and MONTANIDE ISA51 can significantly promote the Th1 cell differentiation of HBsAg in the aspect of cellular immunity.
The HBsAg amino acid sequence used in this example was SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
to elucidate the role of the combination of HBsAg + HBcAg + MONTANIDE ISA51 compositions in cellular immunity, the inventors prepared HBsAg, HBcAg and MONTANIDE ISA51, HBsAg, HBcAg and CpG-ODN, HBsAg and MONTANIDE ISA51, HBsAg and CpG-ODN, HBsAg and Al (OH) respectively 3 Mixing adjuvants, immunizing mouse, detecting IFN-gamma and IL-4 secretion level of spleen cell of immunized mouse by ELISPOT experiment, and performing statistical analysis to evaluate HBsAg, HBcAg, combined with MONTANIDE ISA51, HBsAg and CpG-ODN, HBsAg and MONTANIDE ISA51, HBsAg and Al (OH) 3 The combined effect of promoting Th1 cell differentiation.
C57BL/6 mice, female, 6-8 weeks, purchased from Shanghai Spikey company, were used in this example; the used HBsAg antigen, HBcAg antigen, MONTANIDE ISA51, cpG-ODN and Al (OH) 3 As described in example 1.
HBsAg was directly diluted with PBS (Gibco Co.) or adsorbed to 1mg/ml Al (OH) 3 (purchased from Beijing Temple of biologicals Co., ltd.) and the final protein concentration is 10. Mu.g/ml; diluting HBsAg to 20 mu g/ml with PBS, diluting CpG-ODN to 40 mu g/ml with PBS, mixing 1:1 uniformly until the final HBsAg concentration is 10 mu g/ml and the final CpG-ODN concentration is 20 mu g/ml; diluting HBsAg to 20 μ g/ml with PBS, mixing with MONTANIDE ISA 51 (purchased from Spirobeck corporation) adjuvant at a volume ratio of 1:1, and making HBsAg concentration to 10 μ g/ml; diluting HBsAg and HBcAg to 20 mu g/ml by PBS, diluting CpG-ODN to 40 mu g/ml by PBS, and uniformly mixing 1; HBsAg and HBcAg were diluted to 20. Mu.g/ml with PBS, and mixed with MONTANIDE ISA 51 (purchased from Spirasci corporation) adjuvant 1:1 by volume ratio until the HBsAg concentration was 10. Mu.g/ml and the HBcAg concentration was 10. Mu.g/ml. C57BL/6 mice were immunized via left hind limb gastrocnemius muscle in a total volume of 100. Mu.l, with 5 mice per group. HBsAg + Al (OH) 3 Mice were injected with 1. Mu.g of menses Al (OH) 3 Adsorbed HBsAg; HBsAg + CpG group 1 μ g HBsAg and 2 μ g CpG were injected per mouse; HBsAg + MONTANIDE ISA 51 group injected 1 mu per mousegHBsAg and equal volume of MONTANIDE ISA 51 adjuvant; HBsAg + HBcAg + CpG-ODN groups 1. Mu.g HBsAg, 1. Mu.g HBcAg and 2. Mu.g CpG-ODN are injected into each mouse; HBsAg + HBcAg + MONTANIDE ISA 51 groups each mouse injected with 1 u g HBsAg, 1 u g HBcAg and equal volume of MONTANIDE ISA 51 adjuvant. Immunizing once every three weeks, taking the spleen ten days after three-week immunization, and preparing spleen lymphocytes according to a conventional method, wherein the specific steps are as follows: taking spleen in sterile operation: the spleen was cut with sterile forceps and scissors, placed on a 70 μm nylon mesh (from BD Co.), and placed in a dish containing 5ml of precooled 2% FBS (from GIBCO Co) -PBS; spleen was ground with a grinding bar, spleen cells were passed through a mesh into a plate to obtain a cell suspension, which was placed in a 50ml sterile centrifuge tube filtered through a 40 μm nylon mesh screen (purchased from BD corporation) using a pasteur pipette; centrifuging at 300 Xg for 10 min at 4 ℃; the supernatant was discarded, and 5ml of 1 Xfurcellarant (purchased from BD Co.) was added to resuspend the cells, and allowed to act at room temperature for 5 minutes to break the red blood cells; the broken red reaction was terminated by adding 5ml of 2% FBS-PBS; centrifugation at 300 Xg for 5 minutes at 4 ℃; the supernatant was discarded, and 2ml of 2% FBS-PBS was added to resuspend the cells for use. IFN-. Gamma.and IL-4 were detected using Mouse IFN-. Gamma./IL-4 ELISPOT kit (BD Co.) and HBsAg as a peptide library of HBsAg as a mixture of the sequences shown in Table 1, and the number of spots was read on an ImmunoSPOT Series 3 automatic plate reader.
Mouse IFN-. Gamma./IL-4 was diluted with PBS (1, 200 dilution, BD Co.), 100. Mu.l/well was added to the ELISPOT plate and coated overnight at 4 ℃; discarding the coated antibody, washing the well with blocking solution (containing 10% FBS RPMI-1640 culture solution) for 1 time, adding blocking solution 200. Mu.l/well, and incubating at room temperature for 2h; diluting the peptide to 10. Mu.g/ml using 10% FBS-1640 medium; conA was diluted to 20. Mu.g/ml using 10% FBS-1640 medium; discarding the blocking solution, and mixing 1X 10 7 The spleen lymphocyte suspension of cells/ml and the prepared stimulant are respectively added into a 96-well plate according to 100 mul/well, and the steps are repeated in duplicate wells; 5% CO at 37 ℃ 2 Incubating in an incubator for 24h; discard the cell suspension, wash the plate 2 times, 3-5 m/time with deionized water, wash 3 times with PBST, 200. Mu.l/well, add Mouse IFN-. Gamma./IL-4 ELISPOT detection Antibody (1 diluted 250, BD Co.) diluted with 10% FBS PBS, 100. Mu.l/well, incubate for 2h at room temperature; discard detection antibody, wash plate 4 times with PBST, 200. Mu.l/well, add 10% FBS PBS dilution Streptavidian-HRP (1 dilution 100, BD Co.), 100. Mu.l/well, incubation for 1h at room temperature; discarding the enzyme conjugate, washing with PBST for 4 times, washing with PBS for 3 times, adding AEC substrate 100 μ l/Kong Xianse, observing spot formation with naked eye, and adding deionized water to terminate the reaction; spot numbers were read on an ImmunoSPOT Series 3 automatic plate reader. The results are shown in fig. 6.
Th1 cell mainly secretes IL-2, IL-12, IFN-gamma and TNF-beta, etc., mediates the immune response related to cell toxicity and local inflammation, participates in cellular immunity and formation of delayed type hypersensitivity inflammation, th2 cell mainly secretes IL-4, IL-5, IL-6 and IL-10, its main function is to stimulate B cell proliferation and produce antibody, and is related to humoral immunity. IFN-gamma can induce Th1 cell differentiation, but inhibit Th2 cell proliferation; IL-4 induces Th2 cell differentiation. In this example, the level of secretion of IFN-. Gamma.and IL-4 by splenocytes from immunized mice was measured by ELISPOT, and as a result, al (OH) 3 As HBsAg adjuvant, IL-4 is secreted at a level higher than IFN-gamma, indicating that Al (OH) 3 The adjuvant mainly stimulates B cells to proliferate and generate antibodies; cpG-ODN is used as HBsAg adjuvant to secrete IFN-gamma higher than IL-4 level, which indicates that CpG-ODN promotes Th1 differentiation; IFN-gamma was higher than IL-4 levels when MONTANIDE ISA51 was used as HBsAg adjuvant, indicating that MONTANIDE ISA51 promotes Th1 differentiation. The level of IFN-gamma secreted by the HBsAg, HBcAg and MONTANIDE ISA51 composition is far higher than that of IL-4, and the level of IFN-gamma can reach 600SFC/10 6 The level of IFN-gamma secreted by the group of HBsAg + HBcAg + CpG-ODN is about 250 SFC/splenocyte (spot-forming cells, SFC for short), and statistical analysis shows that the level of IFN-gamma secreted by the composition of HBsAg + HBcAg + MONTANIDE ISA51 and the level of IFN-gamma secreted by the composition of HBsAg + HBcAg + CpG-ODN have significant difference (p is the level of IFN-gamma) and the level secreted by the composition of HBsAg + HBcAg + CpG-ODN (p is the level of IFN-gamma) is the same as the level secreted by the composition of HBsAg + HBcAg + CpG-ODN (p is the level of the IFN-gamma of the HBcAg + CpG-ODN and the composition <0.001 ); meanwhile, the IFN-gamma level secreted by the HBsAg, HBcAg and MONTANIDE ISA51 components is obviously higher than that of the HBsAg, al (OH) of the two components 3 The group and the HBsAg + CpG-ODN group have significant difference (p)<0.001 ); this example further illustrates that the compositions of the present invention are primarily involved in cellular immunity and promote Th1 cell differentiation, and thus have the potential to kill HBV-infected hepatocytes.
Table 1: sequence information of the HBsAg peptide library used
| Peptide fragment numbering | Sequence of |
| 1 | FFLLTRILTI(SEQ ID NO：3) |
| 2 | FIIFLFIL(SEQ ID NO：4) |
| 3 | LVLLDYQGML(SEQ ID NO：5) |
| 4 | FLFILLLCLIFLLVLLD(SEQ ID NO：6) |
| 5 | SSWAFAKYL(SEQ ID NO：7) |
| 6 | ASVRFSWL(SEQ ID NO：8) |
| 7 | FAKYLWEWASVR(SEQ ID NO：9) |
| 8 | FVQWFVGL(SEQ ID NO：10) |
| 9 | WLSLLVPFVQWFVGLSPTVW(SEQ ID NO：11) |
| 10 | MWYWGPSL(SEQ ID NO：12) |
| 11 | IVSPFIPLL(SEQ ID NO：13) |
| 12 | WGPSLYSIVSPF(SEQ ID NO：14) |
Example 7.The combination of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and MONTANIDE ISA51 can break through the immune tolerance of replication-type HBV transgenic mice.
The HBsAg amino acid sequence used in this example is SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
the detection of hepatitis B virus surface antibodies serves to monitor whether the vaccination with the hepatitis B vaccine is successful. Hepatitis b vaccines stimulate the immune system to produce hepatitis b virus surface antibodies equivalent to neutralizing antibodies, the titer of which is directly related to the protective power of the vaccine, the production of which has a significant effect on preventing HBV infection. Therefore, the inventor selects international ARCHITECT international unit testing system for hepatitis b virus surface antibody (CMIA) to detect the concentration of hepatitis b virus surface antibody (anti-HBsAg) in the serum of immunized HBV transgenic mice, and performs statistical analysis to evaluate the protective effect of the combination of HBsAg, HBcAg and montainide ISA51 on enhancing the level of protective antibody relative to the combination of HBsAg, HBcAg and CpG.
The replication type HBV transgenic mouse used in the embodiment is an HBV whole gene transgenic mouse, contains 1.3 times of HBV genome, can be replicated, has a D type genotype, is half female and half male, is 6-8 weeks, and is purchased from Guangzhou 458 hospital; the HBsAg, HBcAg and MONTANIDE ISA51 used were as described in example 1.
Diluting HBsAg and HBcAg to 20 μ g/ml with PBS, mixing with MONTANIDE ISA51 (purchased from Saybox francisco) adjuvant at a volume ratio of 1:1 until the HBsAg concentration is 10 μ g/ml and the HBcAg concentration is 10 μ g/ml; HBsAg and HBcAg were diluted to 10. Mu.g/ml with PBS; cpG-ODN were diluted to 20. Mu.g/ml with PBS. 9 mice per group with a total volume of 100 mul were immunized with replication-competent HBV transgenic mice in the left hind limb gastrocnemius; PBS was used as a control group for immunization, 100 ul/mouse. Immunization was performed once every three weeks, blood was collected two weeks after each immunization for 6 times, and serum was subjected to prime mixing or blood dilution with PBS solution (for 250-straw IU-straw 1000, 500-fold dilution of individual sera from each mouse, for IU >1000, 5000-fold dilution of individual sera from each mouse) to be tested at the second subsidiary hospital of southeast university, and hepatitis b virus surface antibody in serum of immunized mice (ABBOTT corporation ARCHITECT systematic test) was tested with ARCHITECT international unit test system for hepatitis b virus surface antibody (chemiluminescence microparticle immunoassay, CMIA), with the results shown in fig. 7.
As can be seen from FIG. 7, the HBsAg + HBcAg + MONTANIDE ISA51 composition group can break through the immune tolerance of replication-type HBV transgenic mice, and the protective antibody level can reach 40000IU/ml; the protective antibody produced by the HBsAg + HBcAg + CpG composition is about 30000 IU/ml; the results show that the HBsAg + HBcAg + MONTANIDE ISA51 composition group vaccine of the invention generates higher protective antibody level on an immune replication type HBV transgenic mouse than the HBsAg + HBcAg + CpG composition, and can effectively break immune tolerance.
Example 8.Hepatitis b surface antigen (HBsAg), hepatitis b core antigen (HBcAg) in combination with montainide ISA51 resulted in HBeAg/HBeAb conversion in replication-competent HBV transgenic mice.
The HBsAg amino acid sequence used in this example is SEQ ID NO:1,HBcAg has the amino acid sequence of SEQ ID NO:2.
for patients with HBeAg positive chronic hepatitis B, serological conversion of HBeAg/HBeAb, namely disappearance of HBeAg and conversion of anti-HBe to positive, is an important clinical observation index, and the appearance of serological conversion of HBeAg/HBeAb indicates long-term prognosis improvement, such as reduction of liver cirrhosis incidence and slowing of disease progression. Therefore, the inventor uses internationally recognized PEI (Paul-Ehrlich-institute) HBeAg and HBeAb standard, selects the international ARCHITECT hepatitis B virus test system (chemiluminescence microparticle immunoassay, CMIA) to detect the concentration of HBeAg and HBeAb in the serum of the immune HBV transgenic mice, and carries out statistical analysis, thereby evaluating the influence of the combination of HBsAg, HBcAg and MONTANIDE ISA51 on the serum conversion of HBeAg/HBeAb relative to the combination of HBsAg, HBcAg and CpG.
The replication type HBV transgenic mouse used in the embodiment is an HBV whole gene transgenic mouse, contains 1.3 times of HBV genome, can be replicated, has a D type genotype, is half female and half male, is 6-8 weeks, and is purchased from Guangzhou 458 hospital; the HBsAg, HBcAg and MONTANIDE ISA51 used were as described in example 1.
Diluting HBsAg and HBcAg to 20 μ g/ml with PBS, mixing with MONTANIDE ISA51 (purchased from French Saybik Co.) adjuvant at a volume ratio of 1:1, and making HBsAg concentration 10 μ g/ml and HBcAg concentration 10 μ g/ml; HBsAg and HBcAg were diluted to 10. Mu.g/ml with PBS; cpG-ODN were diluted to 20. Mu.g/ml with PBS. 9 mice per group with a total volume of 100 mul were immunized with replication-competent HBV transgenic mice in the left hind limb gastrocnemius; PBS was used as a control group for immunization, 100 ul/mouse. The immunization is carried out once every three weeks for 6 times, blood is collected two weeks after the end point of the immunization, serum is diluted by PBS solution, HBeAg and HBeAb PEI standard products are subjected to gradient dilution, the serum is sent to a second subsidiary hospital of southeast university for detection, the HBeAg and HBeAb levels in the serum of immunized mice are detected by ARCHITECT hepatitis B virus international unit test system (chemiluminescence microparticle immunoassay, CMIA) (ARCHITECT system detection of ABBOTT company), and the result is shown in FIG. 8.
As shown in FIG. 8, the HBsAg + HBcAg + MONTANIDE ISA51 composition group has significantly decreased HBeAg levels (P < 0.05) relative to the PBS group, and the HBeAb antibody levels (P < 0.001) relative to the PBS group, and compared with the HBsAg + HBcAg + CpG composition group, the HBsAg + HBcAg + MONTANIDE ISA51 composition group has lower HBeAg levels, which indicates that the composition of the present invention has HBeAg/HBeAb serological conversion, inhibits HBV virus replication, and has better effect than the HBsAg + HBcAg + CpG composition. The composition is shown to have the tendency of inhibiting virus replication and converting HBeAg/HBeAb serology, and lays a solid foundation for the therapeutic vaccine of chronic hepatitis B.
Throughout this application, various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
While the present invention has been described in terms of the disclosed embodiments, it is to be understood that the specific examples and studies detailed above are merely illustrative of the invention. It will be understood that various modifications may be made without departing from the spirit of the invention. Accordingly, the invention is not to be restricted except in light of the attached claims.
Claims (7)
1. A pharmaceutical composition, comprising:
i) The hepatitis B virus surface antigen, the amino acid sequence of the hepatitis B virus surface antigen is shown in SEQ ID NO 1;
ii) hepatitis B virus core antigen, wherein the amino acid sequence of the hepatitis B virus core antigen is shown as SEQ ID NO. 2;
iii) MONTANIDE ISA adjuvant, and
iv) a pharmaceutically acceptable carrier;
wherein the MONTANIDE ISA adjuvant is MONTANIDE ISA 51 VG;
wherein the mass ratio of the components i) and ii) is 1:1, the ratio of the volume of MONTANIDE ISA 51 VG to the sum of the volumes of components i) and ii) is 1:1, and the concentrations of the components i) and ii) are both 10 μ g/ml.
2. The pharmaceutical composition of claim 1, wherein the MONTANIDE ISA 51 VG is a mixture comprising mineral oil and mannide monooleate.
3. Use of a pharmaceutical composition according to claim 1 or 2 in the manufacture of a medicament for treating a hepatitis b virus infection and/or a hepatitis b virus mediated disease in a subject.
4. The use according to claim 3, wherein the hepatitis B virus infection and/or hepatitis B virus mediated disease is selected from hepatitis B, cirrhosis and liver cancer.
5. Use of a pharmaceutical composition according to claim 1 or 2 for the preparation of a medicament for generating a humoral and cellular immune response against hepatitis b virus in a subject.
6. Use of a pharmaceutical composition according to claim 1 or 2 in the manufacture of a medicament for subtyping an anti-hepatitis b virus core antibody in a subject.
7. Use of a pharmaceutical composition according to claim 1 or 2 in the manufacture of a medicament for use in converting hepatitis b virus e antigen/hepatitis b virus e antibody in a subject.
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