WO2011047531A1 - Nouvel adjuvant immunologique, et vaccins comprenant ledit adjuvant - Google Patents
Nouvel adjuvant immunologique, et vaccins comprenant ledit adjuvant Download PDFInfo
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- WO2011047531A1 WO2011047531A1 PCT/CN2010/001488 CN2010001488W WO2011047531A1 WO 2011047531 A1 WO2011047531 A1 WO 2011047531A1 CN 2010001488 W CN2010001488 W CN 2010001488W WO 2011047531 A1 WO2011047531 A1 WO 2011047531A1
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
Definitions
- the present invention relates to a novel immunological adjuvant and a vaccine containing the same. Background technique
- the immunoadjuvant abbreviated as an adjuvant refers to a substance which is used in advance of an antigen or in combination with an antigen, which can non-specifically enhance or change the specific immune response of the body to an antigen, and enhance the immunogenicity of the corresponding antigen.
- Adjuvant research has a long history. There are more than 100 kinds of adjuvants available at present.
- Traditional adjuvants can be generally divided into four categories: 1 inorganic adjuvants, such as aluminum hydroxide, alum and the like. 2 organic adjuvants, organisms and their products such as mycobacteria (Mycobacterium tuberculosis, BCG), Bacillus brevis, B.
- pertussis pertussis, endotoxin, bacterial extract (muramyl dipeptide), etc.
- 3 synthetic adjuvants such as synthetic Double-stranded polynucleotide (double-stranded poly-adenic acid, uridine), levamisole, isoproterenol, etc.
- 4 oils such as Freund's adjuvant, peanut oil emulsification, adjuvant, mineral oil, vegetable oil Wait. Freund's adjuvant is currently the most commonly used in experimental animals, but it is not suitable for human use, and adjuvant diseases often occur after multiple injections of animals.
- Cytokine adjuvant Such as interleukin 1, interleukin 2, interferon and interleukin 12, etc., have obvious immunoadjuvant effects, can enhance the protective effect of viral, bacterial and parasitic vaccines, and stimulate immune response to tumor antigens;
- CpG-0DN CpG oligodeoxynucleotides
- the immunostimulating complex is a kind of lipid vesicle with high immunological activity spontaneously formed by mixing a glycoside and cholesterol extracted from the antigen and the bark bark with 1:1:1, and is applied to various bacteria, Vaccines for viruses and parasitic diseases have the effect of producing a "comprehensive, immune response that enhances specific antibody responses over a long period of time and can be effectively administered through the mucosa for anti-respiratory infections;
- Liposomes are artificially synthesized lipid mites with a single or multi-layer unit membrane-like structure composed of one or more cell-unit membrane-like lipid-encapsulated aqueous media with adjuvant and carrier. effect.
- the structure of the liposome facilitates the presentation of antigen to antigen-treated cells or other immunocompetent cells. Phagocytic cells phagocytose liposomes and destroy their membrane structure, releasing antigens and forming immune complexes, which are beneficial for maintaining long-term high-valent antibodies and generating immune memory.
- Liposomes are biodegradable in the host, are not toxic by themselves, and can reduce the toxicity of the antigen without local injection reactions. Liposomes can be mixed with Freund's adjuvant or aluminum hydroxide gel for better results;
- Heat shock protein adjuvant is a class of proteins widely found in prokaryotic and eukaryotic organisms and are products of stress response in biological cells. The study found that HSP can be a new type of adjuvant, and is now a hot spot in immunoadjuvant research, especially in tumor immunotherapy.
- adjuvants are exogenous substances, and in addition to the required immunostimulatory effects, adjuvants can cause adverse reactions.
- local adverse reactions include inflammation, nodules, abscesses, etc.
- the adverse reactions of the whole body include allergies, fever, immunosuppression, and even teratogenic, carcinogenic, and mutagenic risks.
- These adverse effects may be caused by the interaction of the adjuvant and the antigen itself, or by the response to the specific antigen caused by the adjuvant, or by the cytokine caused by the vaccine adjuvant.
- the ideal adjuvant should have the following characteristics: It can promote humoral and cell-mediated immune responses, can act on weak immune antigens, and does not cause harmful side effects; can be immunized by different routes, can be used for different Antigen; can play a role in immunosuppressed individuals; should not be left with toxin residues in food animals; can effectively affect the quality of immune response (type control, local immunity and cell type control); stable; cheap and easy to produce immune response .
- the object of the present invention is to provide a novel immunological adjuvant by overcoming the above-mentioned drawbacks of the prior art. And a vaccine containing the same.
- a novel immunoadjuvant of the present invention whose main component is a recombinant human phosphatase B subunit. More specifically, the adjuvant comprises a formulation comprising a recombinant human calcitonase B subunit. Preferably, the formulation comprises a physiologically acceptable liquid formulation, emulsion formulation or lyophilized formulation. Another object of the invention is the use of a recombinant human calcineurin B subunit in the preparation of an immunological adjuvant. Still another object of the present invention is the use of the above immunoadjuvant in immunotherapeutic drugs and immunovaccines. The present invention also provides an immunizing vaccine, wherein the adjuvant comprises at least the above-mentioned immunoadjuvant.
- the inventors of the present invention constructed a genetically engineered Escherichia coli expressing CNB by genetic recombination, and obtained a CNB protein with high expression, high purity and conforming to the quality standard of genetic engineering drug products through research on fermentation process and purification process.
- the sample laid the foundation for the industrialization of CNB.
- the inventors constructed the expression systems of the N-terminal ( 1-84 ) and C-terminal ( 85-169 ) domains of CNB by PCR, and studied the structure and properties of the two functional domains, in view of the pharmacology of CNB.
- Adjuvants derived from cDNA of the present invention a library of rat brain cDM (Perr ino B et al, J. Bio l Chem, 1996, 270:... 340) 0
- Primer 5 -CCGCCATATGGGAAATGAGGCGAGTT-3' and 3
- Primer 5 -CGCGGGATCCTCACACATCTACCACCA-3' PCR amplification, separation of PCR products on agarose gel electrophoresis, digestion with Ndel and BamHI, T4DM
- the ligase was loaded into the pET-21a expression vector, transformed into competent E. coli strain BL21 (DE3) plysS, and stored in an LB agar plate containing ampicillin 50 ug/ml, and single colonies were picked for preculture.
- the preculture solution was added to a TM medium containing 50 ug/ml ampicillin, 37.
- the C shaker was incubated at 250 rpm for 5-6 hours, centrifuged at 4500 rpm for 20 minutes, the supernatant was discarded, and the cells were harvested. Secondly, the preparation of the adjuvant of the invention is carried out:
- a balanced Sephadex G-25 column was used to replace the buffer system with a PBS system suitable for physiological experiments. Subsequently, the obtained product was subjected to a DEAE Sepharose FF column previously equilibrated with a buffer (20 ol/L Na2HP04-NaH2P04, pH 6.5), and the heteroprotein was further washed away by the same buffer, and finally a buffer of 20 mmol/ L Na2HP04-NaH2P04+0.
- the resulting product was adjusted to pH 7.4 with buffer (20 ⁇ ol / L Na2HP04_NaH2P04, pH 7.4), adjusted concentration of lmg / ml, 0. After filtration and sterilization by a 22um sterile filter, the mixture was sealed and lyophilized and stored at -20 degrees.
- the adjuvant antibody is immunoblot positive, ultraviolet absorption light.
- Endotoxin determination Refer to the 2005 edition of the Pharmacopoeia, sputum reagent method.
- the purification preparation method is simple and easy, the output is large, and the cost is low. This is an advantage in preparation compared to heat shock proteins that require labeling purification;
- the heat shock protein can only be expressed by fusion with the antigen, and the heat shock protein alone does not promote the antigen reaction after mixing with the antigen.
- the adjuvant also has the advantages of flexibility in use.
- CNB calcineurin ⁇ subunit
- the innate immune system is the first line of defense against the invasion of pathogens, and is also the regulator of the activation of the acquired immune system.
- Our existing experimental data confirm that CNB can activate the innate immune system, which is the theoretical basis for CNB to become a novel immunological adjuvant. detailed description
- Example 1 Effect of the adjuvant on the proliferation of mouse spleen lymphocytes 1.
- the spleen cell proliferation experiment of the mice immunized with DTP vaccine According to the human and mouse body surface area and the dosage amount conversion table, the injection amount of the mouse vaccine was determined.
- One week after the first immunization the immunization was once performed.
- Two weeks after the second immunization the mice were sacrificed and the spleen was aseptically prepared to prepare a single spleen cell suspension. Trypan blue assay cell survival rate above 95%, adjust the cell concentration to 1 X 106 / ml, add 100 ⁇ l cell suspension per well to 96-well cell culture plate.
- the cell plate reads the absorbance (A) on the microplate reader with a detection wavelength of 57 Gnm and a reference wavelength of 63 Gnm.
- Stimulus index SI 0D value of the dosing group / 0D value of the control group.
- the spleen cells of mice immunized with DTP vaccine were activated and proliferated by antigen stimulation.
- mice were co-immunized with chicken ovalbumin (OVA) and the adjuvant, and a separate immune antigen group and a PBS control group were prepared to prepare a spleen cell suspension, and the spleen cells of the immunized mouse were again subjected to the antigen. (OVA) is stimulated to proliferate after stimulation. See Figure 1 for details. The results showed that the spleen cell proliferation ability of the ovalbumin + adjuvant group mice was significantly higher than that of the control group and the antigen group. The result is as follows.
- the spleen cell proliferation group of the mice immunized with ovalbumin antigen was compared with Ag Ag+CNB absorbance 0. 208 ⁇ 0. 015 0. 377 ⁇ 0. 0335 0. 502 ⁇ 0. 044 Stimulus index (SI ) 1. 000 ⁇ 0 015 1. 300 ⁇ 0. 046 2. 567 ⁇ 0. 026
- mice male, 20 ⁇ lg., mice in the drug group were injected with pneumolysin antigen 20 ug and the adjuvant l OOug/0. 2ml / mouse, and the control group was injected with pneumococcal hemolysis.
- the antigen was 20 ug/0. 2 ml/mouse, and the blank control was injected with PBS 0.2 ml/day/mouse.
- the adjuvant and the antigen had no significant effect on the number of CD8+ cells, but could increase the CD4+ cells.
- CD4+ CD8+ CD4+/CD8+ pneumolysin antigen 27.4% soil 5. 12% 9. 84% soil 1. 14% 2. 81 ⁇ 0. 22 pneumolysin antigen + adjuvant 33. 68% ⁇ 4. 83% 10. 08% ⁇ 1. 20% 3. 35 ⁇ 0. 30*# Control group 24. 82% soil 9 ⁇ 1 0% 9. 63% soil 2. 05% 2. 55 + 0. 33
- mice male, 20 ⁇ lg
- the antigen used was pneumolysin antigen (PN).
- the immune method was the same as above. Blood was collected from the eyelids 7 days after the second immunization. After blood coagulation, the serum was collected by centrifugation and the serum Thl cells were detected. Serum content of factor IFN- ⁇ . The results showed that the serum levels of IFN- ⁇ in the adjuvant group were significantly higher than those in the antigen group. IFN- ⁇ content in mouse serum (pg/ml) time pneumolysin + adjuvant group pneumolysin antigen group control group
- CNB 30 ⁇ g/mL CNB promotes high expression of HLA-DR molecules on dendritic cells and is highly expressed Its specific marker molecule, CD83, promotes the transformation of dendritic cells from immature to mature.
- CNB promotes the expression of surface molecules on dendritic cells Group (%) CNB Negative control protein LPS
- HLA-DR FITC 60. 6 3. 60 66. 5 It will be apparent to those skilled in the art that various types of immunotherapeutic drugs as well as immunizing vaccines can be prepared using a novel immunoadjuvant of the present invention.
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Abstract
La présente invention concerne un nouvel adjuvant immunologique et des vaccins comprenant ledit adjuvant. Le composant principal de cet adjuvant est la sous-unité B de la calcineurine de recombinaison humaine.
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WO1993003364A1 (fr) * | 1991-08-05 | 1993-02-18 | President And Fellows Of Harvard College | Detection d'immunosuppresseurs |
WO1996012806A1 (fr) * | 1994-10-24 | 1996-05-02 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions de proteines ayant une interaction avec la calcineurine et procedes |
CN1245720A (zh) * | 1998-08-26 | 2000-03-01 | 北京师范大学 | 含有钙调神经磷酸酶b亚基的药物组合物 |
WO2001096361A1 (fr) * | 2000-06-12 | 2001-12-20 | University Of Maryland Biotechnology Institute | Procede permettant de commander la liaison de la calmyrine a la preseniline |
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WO1993003364A1 (fr) * | 1991-08-05 | 1993-02-18 | President And Fellows Of Harvard College | Detection d'immunosuppresseurs |
WO1996012806A1 (fr) * | 1994-10-24 | 1996-05-02 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions de proteines ayant une interaction avec la calcineurine et procedes |
CN1245720A (zh) * | 1998-08-26 | 2000-03-01 | 北京师范大学 | 含有钙调神经磷酸酶b亚基的药物组合物 |
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