CN101890161B - Oligonucleotide composition adjuvant - Google Patents
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Abstract
The invention relates to an oligonucleotide composition adjuvant, namely a polyribosome inosinic acid-polyribosome cytidylic acid and kanamycin composition adjuvant, belonging to the field of biological pharmacy. The adjuvant can reinforce specific humoral immunity and cell immunity of an antigen, can obviously improve the titer of a rabies vaccine and neutralize antibody ED50. The invention provides a safe, efficient, convenient and economic adjuvant and an immunogen composition, thus being used for preparation of prophylactic vaccines, therapeutic vaccines, immunotoxin and antiserum of humans and animals.
Description
Technical field
The invention belongs to biomedicine field, relate to oligonucleotide composition adjuvant, be i.e. PIC PIC, kanamycin composition adjuvant.Safety, is effectively provided, and cheap adjuvant and immunogenic composition, comprise the component of adjuvant, content, immunogenic substances, immunization route, immune object etc.For the preventative vaccine of humans and animals, therapeutic vaccine, antitoxin, sero-fast preparation.
Technical background
Immunological adjuvant grows up along with the research of vaccine.Nineteen twenty-six, Glenny is applied to diphtheria toxoid with aluminum salt as adjuvant; Nineteen thirty-seven, Freund has developed Fu Shi Freund's complete adjuvant.Adjuvant and antigen are applied simultaneously, can change or the specific immune response of enhancing body to antigen non-specificly, strengthen the immunogenicity of this antigen, and adjuvant itself no antigen.Adjuvant can improve vaccine quality, reduces costs, and improves output.Cytozoicus pathogen vaccines not only, the weak immunogenicity vaccine of restructuring needs adjuvant, and traditional purified vaccine needs adjuvant equally.Widely the aluminium adjuvant of application can only be induced humoral immunization, inducing cell immunity effectively, and the ability that the vaccine-induced antibody such as protein subunit are produced a little less than, therefore the research Showed Very Brisk of new adjuvant.At present, hundreds of natural and synthetic materials have been proved adjuvanticity, because safety, the problems such as cost, only have aluminium adjuvant to be ratified for people by FDA.European Union has ratified AS04 in recent years for Hepatitis B virus vaccine, and Italy has ratified MF59 for Infuenza subunit vaccine.
Immunne response is that body immune system gets rid of to take of antigenic stimulus generation the physiological process that antigen is object.Pathogen is invaded after human body, and what first start is natural nonspecific immune reaction; Along with immune identification, started acquired Immunel response: humoral immunization and cellular immunization.
B cell recognition antigen, produces specific antibody through the differentiation that stimulates proliferation, and neutralizes a toxin, and promotes macrophage phagocytic, humoral immunity.T cell recognition antigen presenting cell APC processing, the antigenic peptides of processing; APC by antigenic activation after, produce IL-12, IFN-α etc., stimulate NK cell to produce IFN-γ; Raise MHC and the costimulatory molecules on APC surface and express, submission antigen.CD4
+after T cell is activated by MHCII-antigenic peptides, differentiating into T h1 and Th2 cell subsets.The main secretion of gamma-IFN of Th1 cell, IL-2 etc., IFN-γ activated macrophage, mediated cell immunity; The auxiliary B cell of IL-2 produces Mus IgG2a, human IgG, the anti-intra-cellular pathogens immunity of induction human IgG; Th2 cell is mainly secreted the cytokines such as IL-4, stimulates B cell proliferation and eosinophilic granulocyte's activation, produces Mus IgG1, people IgE, induction people antiparasitic immunity, and main anti-born of the same parents infect outward.CD8
+t cell produces IL-2 after being activated by MHC I-antigenic peptides, irritation cell poison T cell CTL proliferation and differentiation.The IL-2 indirect stimulation CTL that Th1 produces, Th1 cytokine participates in the cellular immunization of CTL mediation.Specific cellular immunity is CD4
+th1 and CD8
+cTL cell, the former activated macrophage, induces immune inflammation; The latter's mat secretory cell toxin and cell death inducing, to kill the target cell with antigen, main anti-intracellular infection and antineoplastic immune.In Th cell differentiation procedure, IL-12, the cytokines such as IFN-α are impelled Th1 and CTL differentiation, and induce human IgG antibody to produce, and in induction humoral immunization and cellullar immunologic response, play an important role.Body, by immunne response, is removed " antigen foreign body ".
Immunological adjuvant can stimulator antigen presenting cell APC, and the release cells factor and chemoattracting cytoking can be raised these cells to local organization and lymph node, strengthen humoral immunization and cellular immunization.Desirable immunological adjuvant, except strengthening humoral immunoresponse(HI), can also strengthen by Th1 and CD8
+the cellullar immunologic response of T mediation.PIC can offer dendritic cell DC maturation by inducing antigen, maintains DC secreting high levels IL-12; Produce IFN-a, high expressed MHC and costimulatory molecules, inducing T cell propagation; And effectively offer MHC-I polypeptide antigen, induce stronger ctl response; Can activate NK cell, strengthen the generation of IgG2a and IgG1.
Immune function is identified " oneself " and " nonego " exactly.In recent years, Janeway etc. have proposed pattern recognition theory, by the natural immunity for main target molecule be called pathogen-associated molecular pattern PAMP, PAMP and pattern recognition receptors PRR interact, activate innate immunity, promote the generation of inflammatory cytokine, in innate immune defence, play an important role, and finally activate acquired immune system.PAMP mainly refers to extensively be present in the molecular marker on pathogen cells surface, is the very basic structure of pathogen existence, as LPS etc.The PRR of identification PAMP comprises Toll sample receptor TLR and non-TLR.Have at present more than 10 kind of TLR and non-TLR and be proved, most vaccine adjuvants are TLR parts, and adjuvant is enforcement effect by PRR.Part has the function of Expression of Activated PRR cell, especially has the function of activation DC, and after part and DC effect, DC activates cytokine and the chemotactic factors such as generation IFN, and can raise DC and submission antigen to T cells.Research shows: PIC on DC surface and endochylema have TLR3 receptor.PIC, under TLR3 and MDA5 mediation, by signal transduction path, causes IL-12, the releases such as I type IFN, and the costimulatory molecules on rise DC surface, induction DC differentiation and maturation, finally activates specific immunity, and induction Th1 polarization is replied, at the CD4 of antigenic specificity
+t cell and CD8
+in T cellullar immunologic response, strengthen humoral immunization and cellular immune function, finally cause the performance of antiviral effect and adjuvant effect.
As far back as the sixties, found PIC energy inducing interferon abroad, but people and primate, due to the Degradation of nuclease, and can not be for human body.After this PIC and lysine and cellulose are compounded to form to PICLC, PICLC is inducing interferon effectively, but toxicity is too large, has the serious side effects such as heating, hypotension in human trial, also can not be for human body.The end of the seventies, domestic discovery kanamycin can be stablized PIC double-spiral structure, had developed kanamycin, calcium ion polyinosini, had after this developed kanamycin polyinosini.The compositions such as PIC, kanamycin can be resisted the degraded of nuclease and be induced human interferon, and existing PIC, kanamycin compositions are widely used in people as anti-virus formulation Av-pick.Desirable adjuvant should be safety, effective, convenient, economic.Polynucleotide PIC, kanamycin composition adjuvant are provided for this reason.This adjuvant is compared and is had high concentration with anti-virus formulation Av-pick, low heat feature.PIC produces with macromolecule often making high concentration, easily produces untoward reaction, through exploratory experiment, has developed high concentration, stable, and molecular size is about 4S-13S, safety, effectively PIC, kanamycin composition adjuvant.
List of references: [1] PolyI:C used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12.Redouane Rouas etal.International immunology, 2004, 16 (5): 767-773.[2] Genetic susceptibility to polyI:C-induced IFN α-dependent accelerated disease in lupus-prone mice.TN Jorgensen et al.Immunity.2006, 7:555-567.[3] PolyI:C used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12.Redouane Rouas et al.
International?immunology，2004，16(5)：767-773.[4]Genes?and?Production?of?IFN-α；by?human?and?murine?mast?cells?induced?by?double-stranded?RNA?Evidence?for?activation?for?through?toll-like?receptor-3(TLR-3).
M.kulla.Journal?of?Allergy?and?Clinical?immunology.2003，113(2)：S48.[5].Stimulation?of?humoral?and?cellular?antibody?formation?in?mice?by?polyI：C.Turner，W?et?al.Proc.Soc.Exp.Biol.Med.1970，133：334-338.
The Th of take polarization is replied and pattern recognition theory is basis, and invention provides safety, effectively, and cheap oligonucleotide composition adjuvant.Said composition adjuvant is PIC, kanamycin compositions PICK (polyriboinosinic-polyribocytidylic acid, kanamycin), and this adjuvant can special humoral immunization and the cellular immunization of enhancement antigen.
It is heterogeneous that described oligonucleotide composition adjuvant molecular size and molecular weight are, and molecular size scope is about 4S to 13S sedimentation coefficient, and molecular weight ranges is about 23KD to 310KD (kilodalton).
Described PICK adjuvant is liquid, and content is PIC:2mg/ml-15mg/ml, kanamycin 500IU/ml-5000IU/ml, PH 6.0-8.0.
The preparation of PICK adjuvant relates to the technology such as heat treatment, filtration.The hyperchromic value of PICK is greater than 55%; 231,267nm has minimum and maximal ultraviolet absorption.
In described PICK adjuvant, kanamycin can be replaced by other aminoaglycon antibiotics.These antibiotic comprise Certomycin, tobramycin, ribostamycin, neomycin, hygromycin, amikacin, dibekacin, gentamycin, puromycin, streptomycin, streptozotocin, Sisomicin etc.
A kind of immunogenic composition is provided, described oligonucleotide composition adjuvant and antigen, consists of, this antigen is vero cell rabies virus purifying antigen, and this antigen can be replaced by other arbitrary pathogen antigen or toxoid, comprises single antigen or associating antigen; This pathogen antigen is selected from virus, antibacterial, and the antigens such as cancerous protuberance, comprise pathogen or purification composition or recombinant subunit or the synthetic polypeptide antigen of deactivation or attenuation.
Described immunogenic composition dosage form is liquid dosage form, or freeze-dried formulation or spray-type or peroral dosage form.Described immunogenic composition, immunization route is one of following; Intramuscular injection, lumbar injection, subcutaneous injection, intradermal injection, respiratory tract sucks, and gastrointestinal tract is oral etc.
Described immunogenic composition, immune to liking people, for preventative vaccine, therapeutic vaccine; This immunogenic composition immunity object is also animal, for preventative vaccine, and antitoxin, sero-fast preparation.
PICK adjuvant has the following advantages: the safety of 1.PICK is proven with the application of anti-virus formulation Av-pick through people.2.PICK is water solublity, and injection site good absorbing without immunopathogenesis, is better than aluminium adjuvant without scleroma.3. can enhancement antigen special humoral immunization and cellular immunization, effectiveness shows through animal test results, and: PICK can obviously improve that rabies vaccine is tired and neutralizing antibody ED
50, be better than aluminium adjuvant and without adjuvant.4.PICK is liquid, easy to use.5. the cost of a person-portion rabies vaccine PICK adjuvant 1mg is at about 2 jiaos, very cheap economy.The synthesis and preparation process of 6.PICK adjuvant, reproducible, difference between batch is little, is suitable for the large production of scale.
Accompanying drawing 1: deactivation Vero cell rabies virus infection liquid is at the elution profile of Sepharose-4FF column chromatography.
The elution profile of accompanying drawing 2:PICK adjuvant on SephacrylS-200.
Accompanying drawing 3:PICK adjuvant is at the scintigram of 200-300nm.
The PAGE electrophoretogram of accompanying drawing 4:PICK adjuvant.
The specific embodiment
Example 1. effect ED
50measure
Join Seedling: deactivation Vero cell infection liquid obtains mad dog stock solution through Sepharose-4FF column chromatography purification; PICK adjuvant adds rabies vaccine stock solution to make adjuvant Seedling PK-V with variable concentrations; Prepare aluminium adjuvant Seedling AL-V and without adjuvant Seedling IPRV simultaneously.Effect ED
50measure and use NIH method: each is tested Seedling and does 5,25,125 times of dilutions, 16 mices of every dilution factor, 14-16g, in 0,7 days peritoneal immunity secondaries, 0.5ml/, 14 days encephalocoele counteracting toxic substances, 0.03ml/ is only, and do counteracting toxic substances contrast, and record dead mouse number, within 28 days, judge, by Reed-Muench method, calculate ED
50and LD
50.
200 μ gPICK improve the rabies vaccines ED that tires
502 times, in Table 1.
Table 1. effect ED
50
The thermally-stabilised ED of example 2. effect
50measure
Aluminium adjuvant Seedling AL-V, without adjuvant Seedling IPRV, PICK adjuvant Seedling PK-V, each test Seedling place respectively simultaneously 4 ℃ two weeks, 37 ℃ two weeks, by NIH method, measure the thermally-stabilised ED of effect
50: each is tested Seedling and does 5,25,125 times of dilutions, 16 mices of every dilution factor, and in 0,7 day peritoneal immunity, 0.5ml/, 14 days encephalocoele counteracting toxic substances, 0.03ml/, and do counteracting toxic substances contrast, and record dead mouse number, within 28 days, judge, by Reed-Muench method, calculate ED
50and LD
50.0.2mg/mlPICK Seedling ED
50after 37 ℃ of 2W higher than 4 ℃ of 2W without one times of adjuvant Seedling.In Table 2, table 3.
The thermally-stabilised ED of table 2 effect
50measure (1)
The thermally-stabilised ED of table 3. effect
50measure (2)
Example 3. neutralizing antibody ED
50measure
Immunity and blood sampling: (1) aluminium adjuvant Seedling AL-V, without adjuvant Seedling IPRV, PICK adjuvant Seedling PK-V (300 μ gPICK), three groups of Seedling difference subcutaneous inoculation white mice, 8/group, by immunity in 0,3,7,14,30 day 5 times, 0.1ml/ only, in 7,14,30,60 days from mouse orbit venous blood collection, separation of serum, frozen after 56 ℃ of deactivations in 30 minutes.(2), without adjuvant Seedling IPRV, adjuvant Seedling PK-V (120 μ gPICK) difference subcutaneous inoculation white mice, 8/group, in immunity in 0,3,7,14,30 day 5 times, 0.2ml/ only; Two groups in 7,14,45 days from mouse orbit venous blood collection, and after separation of serum equal portions mix, deactivation is frozen.
Neutralizing antibody ED
50measure and carry out with mice neutralization test: within 7 days, serum is done 2,6,18,54,162 times of dilutions; Within 14 days, serum is done 40,80,160,320,640 times of dilutions; Within 30 days, 45 days, 60 days, serum is done 80,160,320,640,1280 times of dilutions, 6 mices of each dilution factor, 10-12g.In 37 ℃, the serum of serial dilution and CVS virus and mice encephalocoele injection after 1 hour, 0.03ml/ only, press Reed-Muench method calculating ED according to dead mouse number after 14 days
50and LD
50.
PICK adjuvant Seedling neutralizing antibody ED
50higher than aluminium adjuvant Seedling and without adjuvant Seedling.In Table 4, table 5.
Table 4 neutralizing antibody ED
50measure (1)
Table 5. neutralizing antibody ED
50measure (2)
Example 4. abnormal toxicity tests
Mice 18-22g, Cavia porcellus 250-350g, the PICK Seedling of various dose, only, 5/group, after 7 days, body weight is complete increases as qualified mouse peritoneal injection 0.5ml/, and only, 2/group, after 7 days, body weight is complete increases as qualified guinea pig intraperitoneal injection 5ml/.The final concentration of suggestion PICK is not higher than 0.2mg/ml.In Table 6.
Table 6. abnormal toxicity test
Example 5. hypersensitive tests
6 of mices, 18-22g, only, tail vein injection 0.2ml after two weeks, observes 15 minutes 0.2mg/mlPICK Seedling subcutaneous injection 0.2ml/.6 of Cavia porcelluss, 250-350g, 0.2mg/mlPICK Seedling abdominal cavity every other day sensitization three times, 0.5ml/ time, 3/group was respectively at the 14th day, and 21 days intravenous injection 1ml/ only, observe 15 minutes.Without anaphylaxis.In Table 7.
Table 7. hypersensitive test
Example 6. splenocyte conversion tests
Immunity and grouping: aluminium adjuvant Seedling AL-V, without adjuvant Seedling IPRV, PICK adjuvant Seedling PK-V (200 μ gPICK), three groups of Seedlings are subcutaneous inoculation white mice respectively, and 3/group, 0.5ml/ only, puts to death mice for 10 days, and the aseptic spleen of getting grinds, and filters washing.With RPMI-1640, adjust cell concentration to 1 * 10
6/ ml, 0.1ml/ hole adds 96 orifice plates.Transform and each 3 multiple hole of contrast, 0.1ml/ hole, ConA:100 μ g/ml, RPMI-1640 is control wells, 37 ℃, 5%CO
2hatch 48 hours, every hole adds MTT (5mg/ml) 10 μ l, continues to cultivate 4 hours, and centrifugal 5 minutes of 1000rpm, abandons and reset and add dimethyl sulfoxide, and 0.1ml/ hole shakes 10 minutes slowly, and microplate reader 570nm surveys trap A, with this, calculates lymphocytic multiplication capacity.ConA can stimulate T cell transformation propagation, and stimulation index SI=transforms three multiple hole A meansigma methods/contrast three multiple hole A meansigma methodss.The SI of PICK Seedling is apparently higher than without adjuvant Seedling and aluminium adjuvant Seedling, and PICK adjuvant can inducing cell immunity.In Table 8.
Table 8. splenocyte transformation experiment
PK-V and AL-V ratio
*p<0.01, with IPRV ratio
△ △p<0.01, AL-V and IPRV ratio: P>0.05.
Claims (7)
1. an oligonucleotide composition adjuvant, is characterized in that being comprised of PIC PIC, kanamycin.
2. oligonucleotide composition adjuvant described in claim 1, it is heterogeneous that its molecular size and molecular weight are, and molecular size is 4S-13S sedimentation coefficient, and molecular weight is 23kD-310kD.
3. oligonucleotide composition adjuvant described in claim 1, this adjuvant is liquid, PIC:2.0mg/ml-15mg/ml wherein, kanamycin 500IU/ml-5000IU/ml, pH6.0-8.0.
4. oligonucleotide composition adjuvant described in claim 1, hyperchromic value is greater than 55%, 231,267nm has minimum and maximal ultraviolet absorption.
5. an immunogenic composition, is comprised of oligonucleotide composition adjuvant described in claim 1 to 4 any one and antigen, and this antigen is Veto cell rabies virus purifying antigen.
6. immunogenic composition described in claim 5, immune to liking people, for preventative vaccine, and therapeutic vaccine.
7. immunogenic composition described in claim 5, immune to liking animal, for preventative vaccine, and antiserum preparation.
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| WO2018059404A1 (en) * | 2016-09-30 | 2018-04-05 | Sheng Ye | Use of polyinosinic–polycytidylic acid compositions in treatment of malignant effusion |
| CN109939228A (en) * | 2019-03-25 | 2019-06-28 | 苏州博特龙免疫技术有限公司 | A kind of new type water-solubility immunologic adjuvant and preparation method thereof |
| CN111150849A (en) * | 2020-04-02 | 2020-05-15 | 广州隽沐生物科技股份有限公司 | Composition and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1095951A (en) * | 1993-05-31 | 1994-12-07 | 林海祥 | Poly I: C compound immunologic adjuvant and contain the vaccine of this adjuvant |
| CN1997391A (en) * | 2005-06-08 | 2007-07-11 | 申益皮卡生物技术有限公司 | Polyinosinic acid-polycytidylic acid-based adjuvant |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1095951A (en) * | 1993-05-31 | 1994-12-07 | 林海祥 | Poly I: C compound immunologic adjuvant and contain the vaccine of this adjuvant |
| CN1997391A (en) * | 2005-06-08 | 2007-07-11 | 申益皮卡生物技术有限公司 | Polyinosinic acid-polycytidylic acid-based adjuvant |
Non-Patent Citations (2)
| Title |
|---|
| 聚肌胞生物学功能与应用;舒昌杰;《陕西医学杂志》;19891231;第18卷(第10期);40-43 * |
| 舒昌杰.聚肌胞生物学功能与应用.《陕西医学杂志》.1989,第18卷(第10期),40-43. |
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