WO2011047531A1

WO2011047531A1 - Novel immunological adjuvant and vaccines comprising said adjuvant

Info

Publication number: WO2011047531A1
Application number: PCT/CN2010/001488
Authority: WO
Inventors: 魏群
Original assignee: 北京师范大学
Priority date: 2009-10-21
Filing date: 2010-09-26
Publication date: 2011-04-28
Also published as: CN102038950B; CN102038950A

Abstract

The present invention provides an immunological adjuvant and vaccines comprising said adjuvant, wherein the major component of the adjuvant is recombinant human calcineurin B subunit.

Description

Novel immunoadjuvant and vaccine containing the same

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, 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.

In the past 20 years, the development of new vaccines has promoted the research of new adjuvants due to advances in immunology. Compared to traditional inactivated or live vaccines, modern vaccines consisting of genetically engineered recombinant antigens or chemically synthesized peptides often have problems such as weak immunogenicity and require new immunoadjuvants to enhance their effects. Although the traditional aluminum salt adjuvant is currently the only globally recognized human adjuvant, its co-immunization with many recombinant or synthetic peptide vaccine antigens does not trigger an effective immune response, making it difficult to meet the needs of new vaccine development. Therefore, there is a need to develop safer and more effective new adjuvants for humans, especially safe and non-toxic adjuvants that stimulate strong cellular immune responses, and immunoadjuvants suitable for mucosal, DNA and cancer vaccines.

There are a wide variety of new types of immunoadjuvants, and more recent studies include the following:

(1) 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;

(2) Nucleic acid adjuvants. Such as CpG oligodeoxynucleotides (CpG-0DN), in anti-allergic diseases, tumors And important applications in infectious diseases;

(3) Immunostimulating complex adjuvant. 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;

(4) Liposomal adjuvants. 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;

(5) Heat shock protein adjuvant. Heat shock proteins are 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.

Most of the existing adjuvants are exogenous substances, and in addition to the required immunostimulatory effects, adjuvants can cause adverse reactions. Among them, 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. In order to solve these problems, 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 . Summary of the invention

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. The research results, as well as the good prospects of small peptides as adjuvants, the inventors used genetic engineering methods to express and purify the C-terminal domain (DC) and N-terminal domain (DN) proteins of CNB domain fragment-CNB, further Its function is studied.

First, the construction and expression of the adjuvant plasmid of the present invention are carried out. 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

By 5, 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:

1. Ultrasonic disruption of the cells: 1/10-1/20 volume of 1 liter of the culture medium is added to the buffer (20 mmol/L Tr is, pH 7.4, 1 mmol/L EDTA, 0.2 mmol/L PMSF, Lmmol/L β-mercaptoethanol), then disrupt the cells at an output of 40% for 2 to 3 minutes.

2. Separation and purification: After crushing, the cells are passed through 70-100. C boiling water bath for 30-60 minutes, centrifuged at 12000 rpm for 20 minutes, and then the supernatant was taken, which was a crude extract. Add 3mmol / L CaCl ₂ , lmmol / L β-mercaptoethanol and 0.5 mol / L NaCl followed by buffer X20mmol / L Tri s, pH 7.4, 0.5 mmol / LCaCl ₂ , 1 ol / Lp巯乙醇乙醇乙醇巯巯 phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl After elution with /L DTT, the obtained product was subjected to gel filtration chromatography after lyophilization, and the lyophilized powder was dissolved in a buffer solution, and previously buffered (PBS, 20 Å/L Na2HP04-NaH2P04, pH 6. 5) 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. 15mol / L NaCl elution of the protein of interest, the resulting product was adjusted to pH 7.4 with buffer (20 瞧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.

Then, the quality control and use method of the adjuvant of the invention is:

1. Purity analysis: SDS-PAGE identification, purity is 98% or more.

2. Identification of physical and chemical properties: isoelectric point 4. 8.

1

3. Determination of concentration: using ultraviolet spectrophotometry.

4. Identification experiment: the adjuvant antibody is immunoblot positive, ultraviolet absorption light.

5. Biological activity: activation of calmodulin phosphatase A subunit, activation of macrophage proliferation.

6. Endotoxin determination: Refer to the 2005 edition of the Pharmacopoeia, sputum reagent method.

7. Properties and methods of use: It is water-soluble, has high solubility in water, and the aqueous solution is colorless and clarified. It can be stored in physiological saline or neutral pH buffer for 2 years or more, and can be dissolved in a small amount of physiological saline or buffer before use. When immunizing animals, they can be used together with sustained release agents (such as Freund's incomplete adjuvant). An advantage of the present invention is that since the immunoadjuvant of the present invention is an endogenous protein widely present in an organism, it is highly conservative in evolution and thus has low side effects. Compared with the existing protein adjuvants such as heat shock proteins with similar principles, the adjuvant has its remarkable advantages, mainly in the following aspects:

(1) 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;

(2) Strong stability, high heat resistance and long shelf life.

(3) It can be directly and effectively applied to animals and humans after simple mixing with antigen. 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.

(4) The gene sequence of the adjuvant in humans and animals (e.g., rats, mice, etc.) is substantially identical and has high homology. The conclusions of animal experiments and human experiments are more consistent. However, the heat shock protein has low gene homology in animals and humans, and the data of animal experiments and human data are poorly comparable, and there is a certain risk for the final treatment of human body. This is the safety advantage of this adjuvant.

After years of research on the mechanism of action of calcineurin Β subunit (CNB), we verified that CNB can stimulate the proliferation of macrophages and spleen cells, and initially identified that CNB can be involved in a single nucleus involved in innate immune regulation. The surface receptor Tol l-like receptor 4 of the cell binds to each other, activates the nuclear transcription factor NF KB in the cytoplasm and translocates into the nucleus, activates the NF KB pathway, and causes the expression of many related cytokines, chemokines and other genes and proteins. The level rises and the amount of secretion increases, thereby activating the response of the innate immune system. 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 μl cell suspension per well to 96-well cell culture plate. After 6.5 μl of the vaccine was diluted 1000-fold with RPMI 1640, 100 μl per well was added to the sputum group; 100 μl of RPMI 1640 was added to each well as a blank group. Five replicate wells were set in the experimental group and the blank group, cultured at 37 °C, 5% C02 for 48-72 h, 20 μl 5 g/ml MTT was added to each well, and culture was continued for 4 h. At 3000 rpm, centrifuge for 10 min, discard the supernatant, add 150 μl DMS0 per well to each well for ⁵ min, and dissolve the whole pellet. 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. We examined the in vitro proliferation ability of spleen lymphocytes of each group of mice. The results showed that the spleen cell proliferation ability of the adjuvant group (adjuvant group) was higher than that of the control group, and there was a significant difference. The results are shown in the table below.

Splenocyte proliferation in mice immunized with DTP vaccine

Adjuvant group saline group

Vaccine + adjuvant 0. 466 ± 0. 044 0. 152 ± 0. 005 Cell control 0. 278 + 0. 013 0. 132 ± 0. 003

P < 0.05, compared with saline group

2. Splenocyte proliferation in mice immunized with pneumolysin (PN)

In the same method, Ba lb/c mice were co-immunized with pneumococcal lysozyme antigen 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 antigen (PN). After stimulation, the cells were activated and proliferated. We examined the in vitro proliferation ability of spleen lymphocytes of each group of mice. The results showed that the spleen cell proliferation ability of the hemolysin antigen + adjuvant group mice was higher than that of the control group, and there was a significant difference. The results are shown in the table below. Splenocyte proliferation in mice immunized with pneumolysin (PN)

Hemolysin antigen group hemolysin antigen + adjuvant group control group hemolysin antigen + adjuvant 0. 218 ± 0. 036 0. 311 ± 0. 026 0. 133 ± 0. 004 cells 0. 160 ± 0. 027 0. 178 + 0. 038 0. 123+0. 015

P < 0.05, compared with the control group

3. Model antigen chicken egg albumin-immunized mouse spleen cell proliferation

In the same method, Bal6/c: 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

P < 0.05, compared with the control group

Example 2. Effect of this adjuvant on T cell subtype differentiation

BALB/C 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. The number, flow test results can be seen in Figure 2; the ratio of CD47CD8+ in the adjuvant experimental group is significantly different from that in the antigen (PN) group P < 0.05, indicating that the adjuvant is suitable for peripheral blood T cell subsets in normal mice. Have a certain influences. Proportion of peripheral blood T cell subsets in mice

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

*P < 0 ·. 05,. compared with the control (PBS) group; # P < 0.05, compared with the antigen (PN) group

Example 3. Effect of the adjuvant on the content of IFN-γ in serum

BALB/C 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-γ. The results showed that the serum levels of IFN-γ in the adjuvant group were significantly higher than those in the antigen group. IFN-γ content in mouse serum (pg/ml) time pneumolysin + adjuvant group pneumolysin antigen group control group

16. 59784 16. 30866 12. 07844 1 0d Plus Strong

25. 62908 19. 51234 15. 1 3637 After the epidemic

Example 4. The adjuvant effect of the adjuvant on dendritic cells

30 μg/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

CD83 PE 89. 47 4. 59 96. 37

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.

The above-described embodiments are merely illustrative of the invention, and are not intended to limit the invention, and various modifications and changes can be made by those skilled in the art without departing from the scope of the invention. The technical solutions are also intended to fall within the scope of the invention, and the scope of the invention should be defined by the claims.

Claims

Rights request

1. A novel immunological adjuvant whose main component is recombinant human calcineurin B subunit.

The immunological adjuvant according to claim 1, wherein the adjuvant comprises a preparation containing a recombinant human calcineurin B subunit.

The immunoadjuvant according to claim 2, wherein the preparation comprises a physiologically acceptable liquid preparation, an emulsion preparation or a lyophilized preparation.

4. Application of recombinant human calcineurin B subunit in the preparation of immunological adjuvants.

5. Use of an immunological adjuvant according to claim 1 in immunotherapeutic drugs and immunovaccines.

An immunological vaccine, wherein the adjuvant comprises at least the immunological adjuvant of claim 1.