CN101890161A

CN101890161A - Oligonucleotide composition adjuvant

Info

Publication number: CN101890161A
Application number: CN200810161680XA
Authority: CN
Inventors: 郝国荣; 戚风春; 袁延宝; 郭岩; 付博; 董雪林; 李宇辉; 隋红; 陈维金; 胡晓明; 于洪涛; 郑晓丽; 郭立君
Original assignee: 郝国荣
Priority date: 2008-10-07
Filing date: 2008-10-07
Publication date: 2010-11-24
Anticipated expiration: 2028-10-07
Also published as: CN101890161B

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

Oligonucleotide composition adjuvant

Technical field

The invention belongs to biomedicine field, relate to oligonucleotide composition adjuvant, promptly poly-ribose inosine-poly-ribose cytidylic acid PIC, kanamycin composition adjuvant.Safety, effectively is provided, and cheap adjuvant and immunogenic composition comprise the component of adjuvant, content, immunogenic substances, immunization route, immune object etc.The preventative vaccine that is used for 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 the Fu Shi Freund's complete adjuvant.Adjuvant and antigen are used simultaneously, and non-change specifically of energy or enhancing body strengthen this immunogenicity of antigens to antigenic specific immune response, and adjuvant itself and no antigen.Adjuvant can improve vaccine quality, reduces cost, and improves output.Cytozoicus pathogen vaccines not only, the weak immunogenicity vaccine of reorganization needs adjuvant, and traditional purified vaccine needs adjuvant equally.Widely the aluminium adjuvant of Ying Yonging can only be induced humoral immunization, inducing cell immunity effectively, and the ability that vaccine-induced antibody such as protein subunit is produced a little less than, so the research Showed Very Brisk of new adjuvant.At present, hundreds of natural and synthetic materials have been proved adjuvanticity, because safety, problems such as cost have only aluminium adjuvant to be used for the people by the FDA approval.European Union has ratified AS04 in recent years and has been used for Hepatitis B virus vaccine, and Italy has ratified MF59 and has been used for the influenza subunit vaccine.

To be body immune system produce antigenic stimulus immunne response is the physiological process of purpose to get rid of antigen, comprises that antigen presentation, lymphocyte activation, immune molecule form and series reaction such as immunological effect generation.Immune system is made up of thymus, spleen and lymphoid tissue etc., after pathogen is invaded human body, what at first start is natural immunity reaction, can attack and kills it it as natural killer cell, monokaryon one macrophage, and this immunoreation is nonspecific.Along with the identification of human immune system to pathogen, started immunoreation at pathogen, promptly acquired specific immune reaction comprises specific humoral immunity and specific cellular immunity.The former produces specific immunoglobulin, and the latter mainly produces some cytotoxic T lymphocytes and cytokine in order to kill the pathogen of invasion.

Bone-marrow-derived lymphocyte Direct Recognition antigen produces specific antibody through the cofactor differentiation that stimulates proliferation, and neutralizes a toxin, and promotes macrophage phagocytic, the mediation humoral immunization.The cell-mediated cellular immunization of T.The T cell can not Direct Recognition antigen, can only discern through antigen presenting cell APC processing, the antigenic peptides after handling.APC by antigenic activation after, produce IL-12, IFN-α etc. stimulate the NK cell to produce IFN-γ; Raise the MHC on APC surface and the expression of costimulatory molecules, submission antigen, proliferation and differentiation becomes Th effector lymphocyte behind the Th cell-stimulating.CD4 ⁺After the T cell is activated by the MHCII-antigenic peptides, differentiating into T h1 and Th2 cell subsets.The feature of Th1 reaction is secretion of gamma-IFN, IL-2 etc., IFN-γ activated macrophage, mediated cell immunity; The auxiliary B cell of IL-2 produces Mus IgG2a, human IgG, induces the anti-intra-cellular pathogens immunity of human IgG.The feature of Th2 reaction is secretion IL-4 etc., and auxiliary B cell produces Mus IgG1, people IgE, induces people IgE antiparasitic immunity.CD8 ⁺The T cell produces IL-2 after being activated by MHC I-antigenic peptides, and irritation cell poison T cell CTL proliferation and differentiation activates CTL; The IL-2 indirect stimulation CTL that Th1 produces.The effector lymphocyte of specific cellular immunity mainly is Th1 type CD4 ⁺T and CD8 ⁺The CTL cell, the former activated macrophage induces immune inflammation; Latter's mat secretory cell toxin and cell death inducing are with antigenic target cell to kill.Special cellullar immunologic response plays an important role in removing intra-cellular pathogens infection and anti tumor immune response.In the Th cell differentiation procedure, cytokine is to influence the polar key factor of Th1/Th2.The IL-12 that activation APC produces, IFN-α stimulate the NK cell to produce IFN-γ, induce generations such as Th1 differentiation and IFN-γ, and IFN-γ further activates M Φ cell and strengthens the ability that it produces IL-12; The Th1 cytokine promotes the CTL differentiation, and induces the human IgG antibody to produce.IL-12, cytokines such as IFN-α are impelled Th1 and CTL differentiation, play an important role in inducing humoral immunization and cellullar immunologic response.Body reaches the purpose of removing " antigen foreign body " by immunne response.

But immunological adjuvant stimulator antigen presenting cell APC discharges cytokine and chemoattracting cytoking, can raise these cells to local organization and lymph node, strengthens humoral immunization and cellular immunization.Ideal immunological adjuvant can also strengthen by Th1 and CD8 except strengthening the humoral immunoresponse(HI) ⁺The cellullar immunologic response of T mediation.PIC can offer dendritic cell DC maturation by inducing antigen, keeps DC secreting high levels IL-12 ^[1]Produce IFN-α ^[2], high expressed MHC and costimulatory molecules ^[1], inducing T cell propagation; And effectively offer the MHC-I polypeptide antigen, induce stronger ctl response ^[3]Can activate the NK cell, strengthen the generation of IgG2a and IgG1 ^[4]

Functions of immune system is discerned " oneself " and " nonego " exactly, immunogenicity how to understand some not-self antigens so a little less than.In recent years, Janeway etc. have proposed pattern recognition theory, with the natural immunity at main target molecule be called pathogen-associated molecular pattern PAMP (pathogen associated molecularpatterns), PAMP and pattern recognition receptor PRR interact, the activation natural immunity is replied, promote releasing and activity of inflammatory cytokines, 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, and they are tending towards conservative in evolution, is the structure very basic to pathogen existence, as LPS etc.The PRR of identification PAMP comprises Toll sample receptor TLR and non-Toll sample receptor.Have more than 10 kind of TLR at present and non-Toll sample receptor is proved, most vaccine adjuvants are TLR parts, adjuvant enforcement effect by PRR.Part has the function that the PRR cell is expressed in activation, and the function of activation DC is especially arranged, after part and the DC effect, and cytokine and chemotactic factors such as DC activation generation IFN, and can raise DC and submission antigen to T cells.Studies show that: PIC on the DC surface and endochylema TLR3 and non-Toll sample receptor MDA5 are arranged ^{[5] [6]}PIC relies on and the dependent/non-dependent signal transduction path by MyD88 under TLR3 and MDA5 mediation ^[7], activating NF-kB, MAPK and ISP-1 cause cytokine IL-12, releases such as I type IFN, the CD80 on rise DC surface, CD86, costimulatory moleculeses such as IFN are induced the DC differentiation and maturation, finally activate specific immunity ^[8], induce the Th1 polarization to reply, at the CD4 of antigenic specificity ⁺T cell and CD8 ⁺Strengthen humoral immunization and cellular immune function in the T cellullar immunologic response, finally cause the performance of antiviral effect and adjuvant effect ^[9]

Found PIC energy inducing interferon as far back as the sixties, but people and primate, because the Degradation of nuclease, and can not be used for human body.After this PIC and poly-L-lysine and carboxymethlycellulose are compounded to form PICLC, PICLC is inducing interferon effectively, but toxicity is too big, and serious side effects such as heating, hypotension are arranged in human trial, also can not be used for human body.After discover, compositionss such as PIC, kanamycin can be induced human interferon, and existing PIC is used for the people as anti-virus formulation Av-pick.Ideal adjuvant should be a safety, effective, convenient, economic.Polynucleotide PIC, kanamycin composition adjuvant are provided for this reason.This adjuvant is compared with anti-virus formulation Av-pick has high concentration, the characteristics of low heat.PIC easily produces anaphylaxis making high concentration often with the high molecular generation greater than 24S, through a large amount of exploratory experiments, has developed high concentration, and stable, molecular size is at the PIC of 4-13S ^[10], provide safety, effectively PIC, kanamycin composition adjuvant.

List of references: [1] PolyI:C used for human dendritic cell maturationpreserves their ability to secondarily secrete bioactive IL-12.RedouaneRouas et al.International immunology, 2004,16 (5): 767-773.[2] Geneticsusceptibility to polyI:C-induced IFN α-dependent accelerated diseasein lupus-prone mice.TN Jorgensen et al.Immunity.2006,7:555-567.[3] polyI:C induces stable maturation of functionally active human dendriticcells.Verdijk RM et al.J immunol1999,163 (1): 57-61.[4] The role of NKcells during in vive antigen-specific antibody responses.JA Wilder etal.The Journal of Immunology, 1996,156 (1): 146-152.[5] Genes andProduction of IFN-α; By human and murine mast cells induced bydouble-stranded RNA Evidence for activation for through toll-likereceptor-3 (TLR-3) .M.kulla.Journal of Allergyand Clinicalimmunology.2003,113 (2): S48.[6] .Essential role of mda-5in type I IFNresponse to polyI:C and encephalomyocarditis picornavirus.Leonid Gitlinet al.Proc Natl Acad Sci USA.2006,103 (22): 8459-8464.[7] .Recognitionof double-stranded RNA and activation of NF-kB by Toll-l ike receptor3.Alexopoulou L et al.Nature.2001,413:732-8.[8] .Toll-like receptor3mediators a more potent antiviral responses than Toll-like receptor4.Doyle SE et al.J Immunol.2003,170 (7): 3565-71.[9] .Stimulation ofhumoral and cellular antibody formation in mice by polyI:C.Turner, W etal.Proc.Soc.Exp.Biol.Med.1970,133:334-338.[10]. European patent JP1238597.

Summary of the invention

Reply and pattern recognition theory based on the Th polarization, invention provides safety, effectively, and cheap oligonucleotide composition adjuvant.The said composition adjuvant is poly-ribose inosine-poly-ribose cytidylic acid, kanamycin compositions PICK (polyriboinosinic-polyribocytidylic acid, kanamycin).But humoral immunization and cellular immunization that this PICK adjuvant enhancement antigen is special.

It is heterogeneous that described oligonucleotide composition adjuvant molecular size and molecular weight are, and molecular size is in about 4S to 13S sedimentation coefficient scope, and molecular weight is between about 23, and 000D to 310 is in the 000D dalton scope.

Described PICK adjuvant exists with liquid form.Content is PIC:2.0-15mg/ml, kanamycin: 500IU/ml-5000IU/ml, PH6.0-8.0.The suggestion of PICK adjuvant final concentration is not higher than 0.2mg/ml.

The preparation of PICK adjuvant relates to filtration, chromatography, heat treatment, technology such as electrophoresis.PICK adjuvant bacterial endotoxin≤100EU/ agent.Hyperchromic value is greater than 55%; 231,267nm has minimum and maximal ultraviolet absorption.

In the described PICK adjuvant, kanamycin can replace with the common use of one or more antibiotic or by one or more antibiotic.These antibiotic are that aminoaglycon antibiotics comprises micronomicin, Certomycin, tobramycin, ribostamycin, defeat utterly mycin, neomycin, hygromycin, amikacin, dibekacin, gentamycin, nebramycin, puromycin, streptomycin, streptozotocin, Sisomicin, etimicin, arbekacin, beautiful its amide, butirosin sulfate, anthracycline etc.

A kind of immunogenic composition is provided, forms by described oligonucleotide composition adjuvant and antigen.This antigen is vero cell rabies virus purifying antigen.This antigen can be replaced by other single antigen or associating antigen.These antigens are pathogen antigen or toxoid.Pathogen antigen can be antigens such as antibacterial, virus, cancerous protuberance; The pathogen or purification composition or recombinant subunit or the synthetic polypeptide antigen that comprise deactivation or attenuation.

Described immunogenic composition is a liquid dosage form, also or freeze-dried formulation or spray-type or peroral dosage form.Described immunogenic composition, immunization route be a kind of in the following cohort: intramuscular injection, lumbar injection, subcutaneous injection, intradermal injection; Respiratory tract sucks; Gastrointestinal tract is oral.

Described immunogenic composition, immunity is used for preventative vaccine, therapeutic vaccine to liking the people; This immunogenic composition immunity object is animal also, is used for preventative vaccine, antitoxin, sero-fast preparation.

The PICK adjuvant has the following advantages: the safety of 1.PICK has obtained proof through the application of Av-pick.2.PICK be water solublity, the injection site good absorbing does not have the no immunopathogenesis of scleroma, is better than aluminium adjuvant.3. can enhancement antigen special humoral immunization and cellular immunization, effectiveness shows through animal test results, and: PICK can improve obviously that rabies vaccine is tired and neutralizing antibody ED ₅₀, be better than aluminium adjuvant and do not have adjuvant.4.PICK be liquid dosage form, easy to use.5. the cost of a person-portion rabies vaccine PICK adjuvant 1mg is about 2 jiaos, very cheap economy.6.PICK the synthesis and preparation process of adjuvant, good reproducibility, difference between batch is little, is suitable for the big production of scale.

Description of drawings

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. is renderd a service ED ₅₀Measure

Join Seedling: deactivation Vero cell infection liquid obtains mad dog stock solution through the Sepharose-4FF column chromatography purification; The PICK adjuvant adds rabies vaccine stock solution with variable concentrations and makes adjuvant Seedling PK-V; Cofabrication aluminium adjuvant Seedling AL-V and do not have adjuvant Seedling IPRV.Render a service ED ₅₀Measure and use the NIH method: each is tested Seedling and does 5,25,125 times of dilutions, 16 mices of every dilution factor, and 14-16g is in 0,7 days peritoneal immunity secondaries, 0.5ml/, 14 days encephalocoele counteracting toxic substances, 0.03ml/ are only, and do the counteracting toxic substances contrast, record dead mouse number was judged in 28 days, pressed the Reed-Muench method and calculated ED ₅₀And LD ₅₀

200 μ gPICK improve the rabies vaccine ED that tires ₅₀2 times, see Table 1.

Table 1. is renderd a service ED ₅₀

Example 2. is renderd a service thermally-stabilised ED ₅₀Measure

Aluminium adjuvant Seedling AL-V, no adjuvant Seedling IPRV, PICK adjuvant Seedling PK-V, each is tested Seedling and places 4 ℃ of two week respectively simultaneously, in 37 ℃ of two week, measures the thermally-stabilised ED of effectiveness with the NIH method 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 the counteracting toxic substances contrast, record dead mouse number was judged in 28 days, pressed Reed-Muench method calculating ED ₅₀And LD ₅₀0.2mg/mlPICK Seedling ED ₅₀Be higher than 4 ℃ of 2W behind 37 ℃ of 2W and do not have one times of adjuvant Seedling.See Table 2, table 3.

Table 2 is renderd a service thermally-stabilised ED ₅₀Measure (1)

Table 3. is renderd a service thermally-stabilised ED ₅₀Measure (2)

Example 3. neutralizing antibody ED ₅₀Measure

Immunity and blood sampling: (1) aluminium adjuvant Seedling AL-V, no adjuvant Seedling IPRV, PICK adjuvant Seedling PK-V (300 μ gPICK), three groups of subcutaneous immune white mice of Seedling difference, 8/group, by immunity in 0,3,7,14,30 day 5 times, 0.1ml/ only, in 7,14,30,60 days from the mouse orbit venous blood collection, separation of serum, frozen after 56 ℃ of deactivations in 30 fens.(2) the subcutaneous immune white mice of no adjuvant Seedling IPRV, adjuvant Seedling PK-V (120 μ gPICK) difference, 8/group, in immunity in 0,3,7,14,30 day 5 times, 0.2ml/ is only; Two groups in 7,14,45 days from the mouse orbit venous blood collection, it is frozen that the separation of serum equal portions mix the back deactivation.

Neutralizing antibody ED ₅₀Measure and carry out with the mice neutralization test: serum was done 2,6,18,54,162 times of dilutions in 7 days; Serum was done 40,80,160,320,640 times of dilutions in 14 days; Serum was done 80,160,320,640,1280 times of dilutions in 30 days, 45 days, 60 days, 6 mices of each dilution factor, 10-12g.In 37 ℃ in the serum of serial dilution and the CVS virus and mice encephalocoele injection after 1 hour, 0.03ml/ only press Reed-Muench method calculating ED according to the dead mouse number after 14 days ₅₀And LD ₅₀

PICK adjuvant Seedling neutralizing antibody ED ₅₀Be higher than the aluminium adjuvant Seedling and do not have the adjuvant Seedling.See 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, 0.5ml/ of mouse peritoneal injection, 5/group, body weight is complete after 7 days increases to qualified, and guinea pig intraperitoneal injection 5ml/, 2/group, body weight is complete after 7 days increases to qualified.The final concentration of suggestion PICK is not higher than 0.2mg/ml.See Table 6.

Table 6. abnormal toxicity test

Example 5. hypersensitive tests

6 of mices, 18-22g, 0.2mg/mlPICK Seedling subcutaneous injection 0.2ml/, two week back tail vein injection 0.2ml observed 15 minutes.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 observed 15 minutes.There is not irritated reaction.See Table 7.

Table 7. hypersensitive test

Example 6. splenocyte conversion tests

Immunity and grouping: aluminium adjuvant Seedling AL-V, no adjuvant Seedling IPRV, PICK adjuvant Seedling PK-V (200 μ gPICK), three groups of subcutaneous immune white mice of Seedling difference, 3/group, 0.5ml/ only put to death mice in 10 days, and the aseptic spleen of getting grinds, filtration, washing.Transfer cell concentration to 1 * 10 with RPMI-1640 ⁶/ ml, the 0.1ml/ hole adds 96 orifice plates.Transform and each 3 multiple hole of contrast, the 0.1ml/ hole, ConA:100 μ g/ml, RPMI-1640 are control wells, 37 ℃, 5%CO ₂Hatched 48 hours, every hole adds MTT (5mg/ml) 10 μ l, continues to cultivate 4 hours, and centrifugal 5 minutes of 1000rpm abandons and resets and add dimethyl sulfoxide, and the 0.1ml/ hole shook 10 minutes slowly, and microplate reader 570nm surveys trap A, calculates lymphocytic multiplication capacity with this.ConA can stimulate T cell transformation propagation, and stimulation index SI=transforms three multiple hole A meansigma methodss/contrast three multiple hole A meansigma methodss.The SI of PICK Seedling is apparently higher than no adjuvant Seedling and aluminium adjuvant Seedling, but the immunity of PICK adjuvant inducing cell.See Table 8.

Table 8. splenocyte transformation experiment

PK-V and AL-V ratio ^*P＜0.01 is with the IPRV ratio ^{△ △}P＜0.01, AL-V and IPRV compare: P〉0.05.

Claims

1. an oligonucleotide composition adjuvant is made up of poly-ribose inosine-poly-ribose cytidylic acid P I C, kanamycin.

2. it is heterogeneous that the described oligonucleotide composition adjuvant of claim 1, its molecular size and molecular weight are, and molecular size is in about 4S to 13S sedimentation coefficient scope, and molecular weight is between about 23, and 000D to 310 is in the 000D dalton scope.

3. the described composition adjuvant of claim 1 to 2 exists with liquid form.P I C2.0-15mg/ml wherein, kanamycin 500IU/ml-5000IU/ml, PH6.0-8.0.The final concentration suggestion of this adjuvant is not higher than 0.2mg/ml.

4. the described composition adjuvant of claim 1 to 3, bacterial endotoxin≤100EU/ agent, hyperchromic value greater than 55%, 231,267nm has minimum and maximal ultraviolet absorption.

5. in the described adjuvant of claim 1 to 4, kanamycin can replace with the common use of one or more antibiotic or by one or more antibiotic, and these antibiotic are that aminoaglycon antibiotics comprises tobramycin, micronomicin, ribostamycin, spectinomycin, Sisomicin, netilmicin, etimicin, arbekacin, gentamycin, streptomycin, neomycin, hygromycin, amikacin, dibekacin, nebramycin, puromycin, streptozotocin etc.

6. an immunogenic composition is made up of each described oligonucleotide composition adjuvant of claim 1 to 5 and antigen.This antigen is vero cell rabies virus purifying antigen.This antigen can be replaced by other single antigen or associating antigen.These antigens are pathogen antigen or toxoid.Pathogen antigen can be a virus, antibacterial, antigens such as cancerous protuberance; The pathogen or purification composition or recombinant subunit or the synthetic polypeptide that comprise attenuation or deactivation.

7. the described immunogenic composition of claim 6 is a liquid dosage form, also or freeze-dried formulation or spray-type or peroral dosage form.

8. the described immunogenic composition of claim 6 to 7, immunization route be a kind of in the following cohort: intramuscular injection, lumbar injection, subcutaneous injection, intradermal injection; Respiratory tract sucks; Gastrointestinal tract is oral.

9. the described immunogenic composition of claim 6 to 8 is immune to liking the people, is used for preventative vaccine, therapeutic vaccine.

10. the described immunogenic composition of claim 6 to 8, immune object be animal also, is used for preventative vaccine, antitoxin, sero-fast preparation.