CN102038950B - Novel immune adjuvant and vaccine containing same - Google Patents
Novel immune adjuvant and vaccine containing same Download PDFInfo
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- CN102038950B CN102038950B CN2009102364228A CN200910236422A CN102038950B CN 102038950 B CN102038950 B CN 102038950B CN 2009102364228 A CN2009102364228 A CN 2009102364228A CN 200910236422 A CN200910236422 A CN 200910236422A CN 102038950 B CN102038950 B CN 102038950B
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Classifications
-
- 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
Abstract
The invention relates to a novel immune adjuvant and a vaccine containing the same. The main ingredient of the immune adjuvant is recombinant human calcineurin B subunit. The immune vaccine at least contains the immune adjuvant. The purification and preparation method of the immune adjuvant is simple, easy to implement, high in yield and low in cost. And the immune adjuvant is very stable and thermal resistant and therefore can be stored for a long time. After being simply mixed with antigen, the immune adjuvant can directly and effectively act on animals and people. In addition, the gene sequence of the immune adjuvant is basically consistent with that of human and animals (such as rats and mice) and thus has high homology with that of human and animals. The coherence between the conclusion of animal experiments and the conclusion of human body experiments is high.
Description
Technical field
The present invention relates to a kind of novel immunological adjuvant and the vaccine that contains this immunological adjuvant.
Background technology
Immunological adjuvant is called for short adjuvant, refers to prior to antigen or with antigen and applies simultaneously, can non-specifically strengthen or change the specific immune response ability of body for antigen, strengthens the immunogenic material of corresponding antigens.
The research of adjuvant is apart from existing longer history of the present.Can plant for the adjuvant of application existing more than 100 at present, traditional adjuvant generally can be divided into four classes: 1. inorganic adjuvant, and as aluminium hydroxide, Alumen etc.2. organic adjuvant, microorganism and product thereof are as mycobacteria (tubercule bacillus, bacillus calmette-guerin vaccine), bacillus pumilis, bordetella pertussis, endotoxin, bacterial extract (muramyldipeptide) etc.; 3. synthetic adjuvant, as the double-stranded polynucleotide (double-stranded polyadenylic acid, uridylic acid) of synthetic, levamisole, inosine pranobex etc.; 4. oil preparation, as Freund adjuvant, adjuvant 65, mineral oil, plant wet goods.Freund adjuvant is the most frequently used in laboratory animal at present, uses but be unwell to the mankind, and also often adjuvant disease can occur after the animal multiple injection.
Over nearly 20 years, due to immunologic progress, the exploitation of new generation vaccine, promoted the research of novel adjuvant.With traditional deactivation or live body vaccine, compare, the modern vaccination be comprised of genetic engineering recombinant antigen or chemically synthesized polypeptide such as often exists a little less than immunogenicity at the problem, needs novel immunological adjuvant strengthen its effect.Although being the current generally acknowledged people in unique whole world, traditional Alum adjuvant uses adjuvant, but fail to excite effective immunne response when itself and many restructuring or synthetic polypeptide vaccine antigen co-immunization, make it to be difficult to meet the needs of new generation vaccine development, therefore, need the safer effective people of research and development to use novel adjuvant, especially safety non-toxic, can stimulate the adjuvant of stronger cellullar immunologic response and the immunological adjuvant of applicable mucosal vaccine, DNA vaccination and cancer vaccine.
Immunologic adjuvant is of a great variety, and comprising that recent research is more is following several:
(1) Cytokine adjuvant.As interleukin-11, interleukin-22, interferon and interleukin 12 etc., there is obvious immunological adjuvant effect, but the protective effect of enhanced virus, antibacterial and parasite vaccine excites the immunoreation to tumor antigen;
(2) Nuclec acid adjuvants.As CpG oligodeoxynucleotide (CpG-ODN), at aspects such as antianaphylaxis disease, tumor and infectious disease, important application is arranged;
(3) immunostimulating complex adjuvant.Immunostimulating complex is the spontaneous a kind of lipid vesicle had than high immunological activity formed after being mixed by 1: 1: 1 with cholesterol with a kind of glucosides extracted in soapbark by the antigen thing, be applied to the vaccine of various bacteria, virus and parasitic disease, effect with generation " comprehensively " immunne response, can strengthen for a long time specific antibody and reply.And can effectively pass through mucosal drug delivery, for preventing respiratory, infect;
(4) Liposome Adjuvant.Liposome is the lipid folliculus with single or multiple lift unit membrane spline structure of synthetic, the lipoid bimolecular of one or more similar cell unit membranes parcel aqueous media, is consisted of, and has the adjuvant carrier effect of holding concurrently.The structure of liposome is conducive to antigen presentation is processed to cell or other immunologically competent cells to antigen.Phagocyte is engulfed liposome and is destroyed its membrane structure, and released antigen also forms immune complex, is conducive to maintain long high-titer antibody and produces immunological memory.Liposome can biodegradation in host, avirulence own, and can reduce the toxicity of antigen, without local injection, react.Liposome can mix use with Freund adjuvant or aluminium hydroxide gel, and effect is better;
(5) heat shock protein adjuvant.Heat shock protein is that a class extensively is present in the protein in protokaryon and eukaryote, is the induced product of biological cell stress.The research discovery, HSP can become a kind of novel adjuvant, is present immunological adjuvant research, the especially focus of immunotherapy of tumors research.
Existing adjuvant majority is exogenous material, and except needed immunostimulation, adjuvant can also cause untoward reaction.Wherein local untoward reaction has inflammation, tuberosity, abscess etc., and the untoward reaction of whole body has allergy, heating, immunosuppressant, and the danger such as teratogenesis, carcinogenic, mutagenesis are even arranged.These untoward reaction may cause by the interaction of adjuvant and antigen itself, may be also adjuvant cause to the replying of specific antigen, or due to the cytokine that causes of vaccine adjuvant.In order to address these problems, desirable adjuvant should have following characteristics: can promote body fluid and cell-mediated immunne response, can act on weak immunity antigen, and not cause harmful side effect; Can, with the different approaches immunity, can be used for not synantigen; Can in immunosuppressed individuals, play a role; Be applied to edible animal and should do not leave the toxin remains; Can effectively affect immunoreation quality (control of the control of type, local immunity and cell type); Stable; Cheaply and easily produce immunne response.
Summary of the invention
The object of the invention is to, overcome the defect of above-mentioned prior art and a kind of novel immunological adjuvant and the vaccine that contains this immunological adjuvant are provided.
A kind of novel immunological adjuvant of the present invention, its main component is Recombinant Human Calcineurin B subunit.
More specifically, described adjuvant comprises the preparation that contains Recombinant Human Calcineurin B Subunit.Preferably, described preparation comprises physiologically acceptable liquid preparation, emulsion formulations or lyophilized formulations.
Another object of the present invention is to the application of Recombinant Human Calcineurin B Subunit in preparing immunological adjuvant.
Another purpose of the present invention is the application of above-mentioned immunological adjuvant in immunotherapy medicaments and immune vaccine.
The present invention also provides a kind of immune vaccine, and adjuvant wherein at least comprises above-mentioned immunological adjuvant.
The present inventor has built the gene engineering colibacillus of expressing CNB by the method for gene recombinaton, pass through fermentation technology, the research of the aspects such as purifying process, obtained high expressed, high-purity and meet the genetically engineered drug target level of product quality the CNB protein sample, for the industrialization of CNB is laid a good foundation.
The inventor has built the N end (1-84) of CNB and the expression system of C end (85-169) domain by the method for PCR, and structure and the character of two functional domains is studied, pharmacological research achievement in view of CNB, and small-molecular peptides is as the good prospect of adjuvant, the inventor has utilized gene engineering method expression and purification C end structure territory (DC) and N end structure territory (DN) albumen of CNB domain segment-CNB, further studied its function.
At first, carry out structure and the expression of adjuvant plasmid of the present invention.The cDNA of adjuvant of the present invention from Wistar Rat Brain cDNA Library (Perrino B et al, J.Biol.Chem.., 1996,270:340).
By 5 ' primer: 5 '-CCGCCATATGGGAAATGAGGCGAGTT-3 ' and 3 ' primer: 5 '-CGCGGGATCCTCACACATCTACCACCA-3 ' is through pcr amplification, separate the PCR product on agarose gel electrophoresis, after NdeI and BamHI enzyme action, through the T4DNA ligase pET-21a expression vector of packing into, be transformed in competence escherichia coli E.coli bacterial strain BL21 (DE3) plysS, be stored in the LB agar culture plate containing ampicillin 50ug/ml, picking list bacterium colony preculture, preculture liquid is added containing 50ug/ml containing in the TM culture medium of ampicillin, 37 ℃ of shaking tables are with 250rpm heat insulating culture 5-6 hour, 4500rpm, 20 minutes centrifugal, abandon supernatant, the results thalline.
Secondly, carry out the preparation of adjuvant of the present invention:
1, ultrasonication thalline: the 1/10-1/20 volume by 1 liter of bacterial culture fluid adds buffer (20mmol/L Tris, pH7.4,1mmol/L EDTA, 0.2mmol/L PMSF, the 1mmol/L beta-mercaptoethanol), then with output 40% smudge cells, the time is 2-3 minute.
2, separation and purification: after broken, thalline is through 70-100 ℃ of boiling water bath 30-60 minute, and 12000rpm gets supernatant after centrifugal 20 minutes, and this is crude extract.By volume add 3mmol/L CaCl
2, after 1mmol/L beta-mercaptoethanol and 0.5mol/LNaCl in advance through buffer (20mmol/LTris, pH7.4,0.5mmol/LCaCl
2The 1mmol/L beta-mercaptoethanol) the phenyl-Sepharose CL-4B chromatographic column of balance, wash most foreign protein with same buffer again, finally use buffer 20mmol/LTris, pH7.4,1mmol/L EGTA, 0.5mmol/L DTT eluting, products therefrom carries out gel permeation chromatography after lyophilization, after lyophilized powder is dissolved with buffer, in advance through buffer (PBS, 20mmol/L Na2HPO4-NaH2PO4, pH6.5) the Sephadex G-25 chromatographic column of balance, be replaced by buffer system the PBS system that is applicable to Physiological Experiment.Subsequently by products therefrom in advance through buffer (20mmol/LNa2HPO4-NaH2PO4, pH6.5) the DEAE Sepharose FF chromatographic column of balance, further wash away foreign protein through same buffer, finally use buffer 20mmol/LNa2HPO4-NaH2PO4+0.15mol/L NaCl eluting destination protein, (the 20mmol/L Na2HPO4-NaH2PO4 of buffer for products therefrom, pH7.4) after being adjusted to pH7.4, adjusting concentration is 1mg/ml, 0.22um after the sterilizing filter filtration sterilization, sealed packaging is preserved in-20 degree after lyophilization.
Then, the quality control of adjuvant of the present invention and using method are:
1. purity analysis: SDS-PAGE identifies, purity is more than 98%.
2. physicochemical properties are identified: isoelectric point, IP 4.8, absorbance value ε 277nml%=3.1
3. concentration determination: use ultraviolet spectrophotometry.
4. identification experiment: this adjuvant antibody immunoblotting positive, ultra-violet absorption spectrum.
5. biological activity: activate Calcineurin A subunit, activating macrophage propagation.
6. endotoxin measurement: with reference to version pharmacopeia in 2005, limulus reagent test.
7. character and using method: be water solublity, in water, dissolubility is large, and aqueous solution is colourless, clarification.Can in the buffer of normal saline or neutral pH, lyophilization preserve more than 2 years, dissolve and get final product with a small amount of normal saline or buffer before use.During immune animal, can with common use of slow releasing agent (as incomplete Freunds adjuvant etc.).
Advantage of the present invention is: because immunological adjuvant of the present invention is the endogenous protein extensively existed in a kind of organism, and high conservative on evolving, so toxic and side effects is very low.The existing protide adjuvant similar than principle is as heat shock protein, and this adjuvant has its significant superiority, mainly contains following several respects:
(1) method for preparing purified is simple and easy to do, and output is large, and cost is low.This is to tag the heat shock protein of purification in the advantage aspect preparation than needs;
(2) stability is strong, and heat resistance is high, long shelf-life.
(3) can simply mix rear directly useful effect in the animal and human with antigen.And heat shock protein can only with the antigen amalgamation and expression after, effect is arranged just now, independent heat shock protein with do not promote antigen reactive effect after antigen mixes.This adjuvant also has flexile advantage on occupation mode.
(4) gene order of this adjuvant in humans and animals (as rat, mice etc.) is basically identical, and homology is high.The continuity of zooperal conclusion and human experimentation is stronger.And the DNA homolog of heat shock protein in animal body and human body is lower, zooperal data and somatic data comparability are poor, for finally for human body therapy, certain risk being arranged.This is the advantage of this adjuvant in safety.
Through for many years for the research of calcineurin B subunit (CNB) mechanism of action, we have verified, but the propagation of CNB stimulating expression of macrophage and splenocyte etc., and Preliminary Identification goes out, CNB can mutually combine with the monocytic surface receptor Toll-sample receptor 4 that participates in the innate immunity adjusting, activate intracytoplasmic nuclear factor NF κ B transposition and enter core, activate NF κ B path, the gene and the protein expression level that cause many relevant cell factors, chemotactic factor etc. raise, secretory volume increases, thereby activates replying of innate immune system.Innate immune system is the first line of defence of body defence pathogen invasion, is also basis and the regulation and control person that the acquired immunity systems response activates.Our existing experimental data confirms that CNB can activate innate immune system, and this is the theoretical basis that CNB becomes a kind of immunologic adjuvant.
The specific embodiment
Embodiment 1: the impact of this adjuvant on mice spleen lymphocytes proliferation
1. the splenocyte proliferation experiment of DPT vaccine immune mouse
By people, Mice Body surface area and dosage commutation table, determine mice vaccine injection amount, one exempts from rear two weeks booster immunizations once, two kill the aseptic spleen of getting of mice in two weeks after exempting from prepares single splenocyte suspension, trypan blue detects cell survival rate more than 9 5%, adjust cell concentration to 1 * 106/ml, every hole adds on 100 μ l cell suspension to 96 porocyte culture plates.By 6.5 μ l vaccines, with after 10 00 times of RPMI 1640 dilutions, it is experimental group that every hole adds 100 μ l; Every hole adds 100 μ l RPMI 1640 to be blank group.Experimental group and blank group are respectively established 5 repeating holes, and 37 ℃, 5%CO2 cultivates 4 8～7 2h, and every hole adds 20 μ l 5 μ g/ml MTT, continue to cultivate 4h.300 0rpm, centrifugal 1 0min, abandon supernatant, and every hole adds 150 μ l DMSO concussion 15 min that precipitation is dissolved fully, and cell plates read absorbance (A) value on microplate reader, detect wavelength 57 0nm, reference wavelength 63 0nm.Stimulation index SI=dosing group OD value/matched group OD value.The splenocyte of the mice of DPT vaccine immunity is activated propagation again after antigenic stimulus, and we have detected the in-vitro multiplication ability of the splenocyte of respectively organizing mice.Result shows, the splenocyte multiplication capacity of adjuvant group (adjuvant group) mice is higher than matched group, and significant difference is arranged.Result is as shown in the table.
The splenocyte propagation of DPT vaccine immune mouse
P<0.05, compare with the normal saline group
2. the splenocyte of pneumolysin (PN) immune mouse is bred
The same method is used streptococcus pneumoniae bacteriolysin antigen and this adjuvant co-immunization Balb/c mice, and set up independent immunizing antigen group and inject the PBS matched group, prepare splenocyte suspension, the splenocyte of immune mouse again is activated and breeds after antigen (PN) stimulates, and we have detected the in-vitro multiplication ability of the splenocyte of respectively organizing mice.The result demonstration, the splenocyte multiplication capacity of hemolysin antigen+adjuvant group mice, higher than the matched group antigen group, and have significant difference.Result is as shown in the table.
The splenocyte propagation of pneumolysin (PN) immune mouse
P<0.05, compare with matched group
3. the splenocyte of model antigen chicken egg white immune mouse is bred
The same chicken egg white (OVA) and this adjuvant co-immunization Balb/c mice for method, and set up independent immunizing antigen group and inject the PBS matched group, prepare splenocyte suspension, the splenocyte of immune mouse again is activated and breeds after antigen (OVA) stimulates.See shown in accompanying drawing one.The result demonstration, the splenocyte multiplication capacity of ovalbumin+adjuvant group mice, be significantly higher than matched group and antigen group.Result is as follows.
The splenocyte propagation of ovalbumin antigen immune mice
P<0.05, compare with matched group
The impact of 2. adjuvants of embodiment on the differentiation of T cell subsets
BALB/C mice, male, 20 ± 1g., medication group mouse peritoneal is injected respectively pneumolysin antigen 20ug and this adjuvant 100ug/0.2ml/ mice, 20ug/0.2ml/ mice of control group mice injection pneumolysin antigen, 0.2ml/ days/mices of blank injection PBS, this adjuvant and antigen combined effect are to CD8
+The quantity of cell is significantly impact not, but can improve CD4
+The quantity of cell, the visible accompanying drawing two of flow cytometer detection result; This adjuvant experimental group CD4
+/ CD8
+Ratio compared notable difference P<0.05 with antigen (PN) group, illustrate that this adjuvant has a certain impact to normal mouse peripheral blood T cell subsets.
Mouse peripheral blood T cell subsets ratio
*P<0.05, and contrast (PBS) group and compare; #P<0.05, compare with antigen (PN) group
The impact of 3. adjuvants of embodiment on the content of IFN-γ in serum
BALB/C mice, male, 20 ± 1g, antigen used is pneumolysin antigen (PN), and immunization ways is the same, and two exempt from eye socket blood sampling in latter 7 days collects blood, centrifugal collection serum after blood coagulation, the serum content of Th1 cytokines IFN-γ in detection serum.The result demonstration, in interpolation adjuvant group serum, IFN-γ content is apparently higher than antigen group.
The content of IFN-γ (pg/ml) in mice serum
4. adjuvants of embodiment are to the ripe effect of facilitating of dendritic cell
30 μ g/mLCNB can promote surface of dendritic cells high expressed HLA-DR molecule, and highly express its specificity marker molecule CD83, promote that dendritic cell is transformed to maturity state by immaturity.
CNB promotes the expression of surface of dendritic cells molecule
Obviously, those of ordinary skill in the art, can, with a kind of novel immunological adjuvant of the present invention, prepare various types of immunotherapy medicaments and immune vaccine.
Above-described embodiment is used for illustrative purposes only; and be not limitation of the present invention; the those of ordinary skill in relevant technologies field; without departing from the present invention; can also make various variations and modification; therefore all technical schemes that are equal to also should belong to category of the present invention, and scope of patent protection of the present invention should be limited by each claim.
Claims (1)
1. the application of Recombinant Human Calcineurin B Subunit in preparing immunological adjuvant.
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