CN101347616A

CN101347616A - Composing prescription of novel safe JY immunoadjuvant system and uses thereof

Info

Publication number: CN101347616A
Application number: CNA200710107526XA
Authority: CN
Inventors: 侯云德; 赵静; 张丽兰; 吴建华; 张利萍; 舒跃龙; 段招军; 郑丽舒
Original assignee: Beijing Yuance Pharmaceuticals Co Ltd; BEIJING JINDIKE BIOLOGICAL TECHNOLOGY INST
Current assignee: Beijing jindike biotechnology research institute
Priority date: 2007-05-18
Filing date: 2007-05-18
Publication date: 2009-01-21
Anticipated expiration: 2027-05-18
Also published as: CN101347616B

Abstract

The invention relates to a formula for a novel and safe JY immunity adjuvant system and the application, belonging to the field of pertinent, preventive and therapeutic vaccines. The formula which consists of polysaccharide with positive charges and cytokine (protein) with minus charges and stimulating immunity reaction, namely the JY adjuvant system, is used for mucosal immunity and system immunity of preventive vaccines and therapeutic vaccines to enhance the immunity effect of the vaccines. The JY adjuvant system with a reasonable formula has no obvious stimulation to mucosa tissues and the safety conforms to the requirements of medicines. The JY adjuvant system with the reasonable formula has generality and universality, can improve the effect of current vaccines and enhance immune response of new vaccines or availability of peptide or protein medicines in the JY adjuvant system.

Description

A kind of prescription and application thereof of JY immunoadjuvant system of new type of safe

Technical field:

The present invention relates to a kind of prescription and application thereof of JY immunoadjuvant system of new type of safe, belong to pointed preventative and therapy vaccine preparation field.

Background technology:

1. the research and development situation of global vaccine

The definition of classical vaccine is " deactivation or the attenuated pathogens that produce immunity at the infectious disease disease ", develop rapidly along with biotechnology, the definition of modern new generation vaccine is expanded far away: " adopt pathogen or relevant with pathogen; or target protein (polypeptide, peptide), polysaccharide or the nucleic acid relevant with disease (as cancer); with one or more compositions; directly or by certain carrier, can produce the antigen-specific immune responses reaction after entering body, reach prevention and treat the purpose of disease ".So vaccine is not only the main means of prevention and control infectious disease, still treats cancer, the biological preparation of multiple disease such as metabolic disease.The purposes of vaccine more and more expands to a plurality of medical domains (table 1-3).

Table 1 pair human health can cause the infectious disease pathogens and the vaccine prevention situation of serious harm

Annotate: mainly according to KAPER J, RAPPUOLI R AND BUCKLEY M:VACCINE DEVELOPMENT:CURRENT STATUS AND FUTURENEEDS, U.S. microorganism association data (2005) WWW.PENSAREDESIGN.COM; The author deletes to some extent and adds.

Table 2 is because unartificial (" nature threat ") has the infectious disease pathogens of serious threat to human health

What table 3 bio-terrorism person was easy to discharge has the infectious disease pathogens of serious harm to human health

Annotate: mainly according to KAPER J, RAPPUOLI R AND BUCKLEY M:VACCINE DEVELOPMENT:CURRENT STATUS AND FUTURENEEDS, U.S. microorganism association data (2005) WWW.PENSAREDESIGN.COM, the U.S. CDC biological weapons that the terrorist is possible are divided into A, B, C three class authors delete to some extent and add.

1.4 reporting, World Health Organization (WHO) will develop in 2015 and improved vaccine:

1. rotavirus (Rotavirus)

2. human papillomavirus's (Human papilloma virus) (going on the market)

3. streptococcus pneumoniae (Pneumococcus) (improved associating or based on proteinic vaccine) *

4. Japanese B encephalitis vaccine (Japanese B encephalitis) (improvement) *

5. meningococcus (Meningococcus A) (multivalence in conjunction with) *

6. measles (Measles) (aerosol) *

7. poliomyelitis (based on the inactivated vaccine of Sabin strain) *

8. poliomyelitis (unit price OPV 1 type) * *

9. dengue fever * *

10.A group B streptococcus (Group A Streptococcus) * *

11. respiratory syncytial virus (RSV) * *

12. dysentery (Shigella) * *

13.ETEC**

14. typhoid fever (Typhoid) (combination) * *

15. west Nile fever (West Nile Fever) * *

16.SARS**

17. malaria * *

(18.DTaP with 2 P antigens) * *

19. the new combination * * of existing vaccine

20. the pandemic vaccine of flu-prevention

Annotate: the * * in late period that * has been in exploitation will go on the market in the 2010-2015 approval.According to WHO data (2005)

The demand of this explanation vaccine is more and more urgent, and many vaccines still can not be put on market through the research of many decades, and many reasons are arranged certainly, and except that the reasons such as character of pathogen, the most important thing is does not also have suitable immunological adjuvant safely and effectively so far.

2. immunological adjuvant

The use of vaccine immunity adjuvant (Immunoadjuvant) is very important, and whether the use of immunological adjuvant often can determine the effect that certain vaccine uses.The immunological adjuvant of researching and developing at present is varied, comprise aluminium adjuvant (Alum Adjuvant), MF59 (agent of water cornerite zamene oil microemulsion), Loxoribine, corynebacterium granulosum (Corynebacterium granulosum), tryrosinase peptide (Tyrosinase Peptide), incomplete Fei Shi adjuvant (Incomplete Freund ' s Adjuvant), GPI-0100, CEL-1000, SCV-07, BCG, QS21, Trp-Lys-Tyr-Met-Val-d-Met, the SB-AS02B adjuvant, lipopeptide MALP-2, PADRE 965 10 Theramide, Corynebacterium Parvum, TherapeuticCrotoxin, non-methylated CpG dinucleotide (CpG ODN), dinitro benzene (Dinitrophenyl), HER Peptides, Ethylchlorformate, and the Fei Shi adjuvant (Freund ' s Adjuvant), tetanus toxoid (Tetanus Toxoid), HER-2neu Intracellular Domain Protein, CRL 1005, Ubenimex, immunostimulant Ampligen

(PolyI:C), telomerase 540-548 peptide (Telomerase 540-548 Peptide), AS02A, AS04, TLR etc.

But, through the general immunological adjuvant of countries in the world vaccine approving authority approval the aluminium adjuvant that has used many decades only being arranged still, the MF59 adjuvant only limits to influenza vaccines and uses.Therefore, suddenly wait to develop the immunological adjuvant or the adjuvant system of safety general.

3. chitosan

3.1 the structure of chitosan

The chemistry of chitosan (Chitosan) is called (1,4)-2-amino-2-deoxidation-β-D glucosan; Molecular formula: [C ₈H ₁₃NO ₅] _N1[C ₆H ₁₁NO ₄] _N2N2/ (n1+n2)＞60%.

Chitosan be called again chitosan (Deacetylated chitin),

Flonac N, chitosan, soluble chitin, chitan, chitosan, soluble chitin, viscosity chitin etc.In fact, adopt chitosan comparatively accurate.It takes off acetyl and can comprise all and it should be noted that with partially deacetylated present commercially available product generally all is partially deacetylated, seldom the seeing of deacetylation 100%, therefore, with (C ₆H ₁₁O ₄N) _nAs molecular formula is improper.

Chitosan is produced by chitin (chitin), and it is present in the crustacean shell, forms by deacetylation, and a series of acetyl degree and the different molecular weight of taking off can be arranged.The structure of chitosan is seen Fig. 1.

3.2 the biological activity of chitosan and in medically application

Chitosan is a kind of positively charged linear polysaccharide, and it is a kind of bioadhesion material, can play strong effect with nasal epithelial cells and rete malpighii thereof, to guarantee making medicine carrying passing through nasal mucosa before by mucociliary clearance.In addition, prove that chitosan also can increase the ability of polar medicine cell bypass carrying by the temporary transient opening of tight junction between the epithelial cell.Being used for the common chitosan type that the nasal cavity medicine sends is chitosan glutamate salt, and about its mean molecule quantity 250kDa, taking off the acetyl degree is more than 80%.Chitosan salt is water miscible (pH 6.5).

Influence two main barriers that snuffing receives and be carrying by epithelial membrane and medicament composing prescription by the quick removing of mucomembranous cilium.Present strategy is to adopt bioadhesion nasal delivery system, guarantees the time of contact at prolong drug prescription and nasal absorption position, to postpone the scavenging action of mucomembranous cilium.Such bioadhesion nasal delivery system can be powder and water preparation, or hydrogel system.Powder can lyophilization or dried granule or microspheres form, and chitosan is optimum to this system.Develop bioadhesion polysaccharide chitosan (Chitosan) recently, developed a series of delivery systems simultaneously based on this material.Chitosans can be used for medicine and biomedical industries, and the salt of recommendation is chitosan chloride and chitosan glutamate.

As above said, the mechanism of action of Chitosan has 2 aspects: the first, and cationic polymer can be combined in the mucous negatively charged sialic acid residues of nasal epithelial cells layer, thereby slows down by the removing speed of nasal cavity.Second, chitosan can act between the epithelial cell junction closely, the combination of chitosan can cause that tight junction is temporarily open, thereby increase the paracytic carrying of polar molecule, immunogen is entered in the mucous epithelium, directly contact, give full play to immunization with dendritic cell.

General polar medicine can not pass through Nasal Mucosa Absorption well, and these medicines comprise low molecular chemical compound and biological product, as polypeptide and protein.The molecular weight of solution or powder is more than 100kD, and to a certain degree deacetylated chitosans can improve the degree of absorption of polypeptide drugs, as insulin, calcitonin, LHRH analog, growth hormone and the polar compound that is used to press down painful non-peptide.

Chitosan has biological degradability, bioadhesive and short osmosis, and have no side effect, can improve the absorption of multiple route of administration such as biopharmaceutical macromolecular drug (genomic medicine, vaccine, protein medicaments, hormone medicine) is oral, nose usefulness, pulmonary, have wide practical use.Kas shows that chitosan is used widely in preparations such as bovine serum albumin (BSA), aminoacid, antithrombotic agent (water Ji Su, antithrombase and heparin), trofermin, diphtheria toxin, diphtherotoxin, monoclonal antibody, insulin, salmon calcitonin, highland nine hormones.Davis studies have shown that in sheep and human experimentation chitosan can improve the bioavailability of tetanus toxin, Desmopressin, insulin, leuprorelin, calcitonin, parathyroid hormone (PTH), cholecystokinin multiclass pulmonary administrations such as (CCK-8); Illum L and McNeela EA adopt chitosan to try out several bacterin preparation nasal-cavity administrations at human body, as influenza, diphtheria vaccine and morphine, can improve the chitosan that immunne response (being reflected in IgG antibody, IgA titre) and proof are lower than 0.25% concentration, administration in continuous 7 days is harmless to nasal cavity.

3.3 the safety of chitosan

Chitosan is the most inert material in the human body, and researcher proves " chitosan has hypotoxicity, and is few to this allergy sufferers, and medium degree immunostimulation is arranged ".Study even the toxicity of chitosan and the side effect of salt or sugar are mentioned in the same breath for one.

One after the clinical research reference that Helsinki carries out takes, have acute, directly reduce the effect of body weight.This research and other studies show that the people takes behind the chitosan not only weight loss, and blood pressure also descends.The volunteer that researcher will be participated in test is divided into two groups: use chitosan group and blank group capsule, the result shows that blank group has some to feel sick and headache, and experimental group does not then have.

Another side effect is can make the feces deliquescing after taking chitosan, and convenient effect is arranged.Chitosan also can promote the absorption of mineral and fatsoluble vitamin, but does not have the associated health problem therewith of reporting.

As far back as nineteen ninety-five, Hwang D studies show that, " Chitosan at some developed country's approved as food additive; some toxicologic study proves nontoxic " (Hwang D, Department of Food Science, University ofWisconsin, Madison.Journal?of?Agricultural?and?Food?Chemistry，1995，43(1)：33-37)。Chitosan used safely 30 years.Research shows that also rat is used 10%chitosan every day, does not see that toxic reaction does not have allergy yet.Taking vitamin C can increase the effect of Chitosan, and taking Chitosan is safe to health not only, and to environment also safety, convenient is useful to most of people.

List of references

1.Aspden，T.J.Illum?L.&Skaugrud

Eur.J.Pharm.Sci.，4，23-31，1996.

2.Chandy，T.Sharma，C.P.″Chitosan?-as?a?biomaterial?biosurface?technology?division，″Sree?ChitaTriunal?Institute?for?Medical?Sciences?and?Technology，Poojandrum，India.1990

3.Chobot，V.″Physiotherapeutic?aspects?of?diseases?of?the?circulatory?system，″Ceska?a?SlovenskaFarmacie，Aug?44：190-5，1995

4.Davis?SS.Delivery?of?peptide?and?non-peptide?drugs?through?the?respiratory?tract.J.Pharm?SciTechToday，1999，U(2)：450-456.

5.Kas?HS.Chitosan：propenies，preparations?and?application?to?microparticulate?systems.jMicro-capsulation，1997，14(6)：689

6.Illum?L.，Farraj?N.F.&Davis?S.S.，Pharmaceutical?Res.11，1186-1189，1994.

7.P.Artursson，T.Lindmark，S.S.Davis?&L.Illum，Pharmaceutical?Res.11，1358-1361，1994.

8.Illum?L.Nasal?drug?delivery?possibility，problem?and?solutions.J?Controlled?Release?87(2003)：187-198

9.Illum?L.Intranasal?delivery?of?morphine.J?Pharmacol?Exp?Ther.2002?Apr；301(1)：391-400

10.Illum?L.Chitosan?as?a?nasal?delivery?system：the?effect?of?chitosan?solutions?on?in?vitro?and?invivo?mucociliary?transport?rates?in?human?turbinates?and?volunteers.J?Pharm?Sci.1997Apr；86(4)：509-13

11.Illum?L.Chitosan?as?a?novel?nasal?delivery?system?for?vaccines.Advanced?Drug?DeliveryReviews?51(2001)81-96

12.McNeela?EA.Intranasal?immunization?with?genetically?detoxified?diphtheria?toxin?induces?T?cellresponses?in?humans：enhancement?of?Th2?responses?and?toxin-neutralizing?antibodies?byformulation?with?chitosan.Vaccine.2004?Feb?25；22(8)：909-14

4. Human Inter Leukin-2 (IL-2)

4.1IL-2 overview

IL-2 once adopted multiple title in history: embryo source sex factor (blastogenic factor, BF); Costimulator; Bite Yihong cell differentiation factor (eosinophil differentiation factor, EDF); KCHF (killer cell helper factor, KHF); Lymphocyte mitogenic factor (lymphocyte mitogenic Factor, LMF); Conditionity lymphocyte medium (lymphocyte-conditioned medium factor, LCM Factor); The lymphopoiesis factor (lymphocyte proliferation factor, LPF); Macrophage activation Cytotoxic factor (macrophage-activating factor for cytotoxicity, IMAF-C I); Plaque forms carefully Born of the same parents' enhancer (plaque forming cell enhancing factor, PFC-EA); Second cytotoxic T cell lures Lead the factor (secondary cytotoxic T-cell inducing factor, SCIF); SCIF (T-cell Growth factor, TCGF); Thymocyte differentiation factor (T colony-promoting activityTCPA); Chest Gland cell differentiation factor (thymocyte differentiation factor, TDF); The thymocyte factor,mitogenic (thymocyte mitogenic factor, TMF); TGP-3; Eosinophil differentiation factor (T-cell maturation Factor, TMF); T-cell growth factor (T-cell mitogenic factor, TMF); The T cell replacement The factor (T-cell replacing factor-3, TRF-3); Thymocyte stimulating factor (thymocyte stimulating Factor, TSF).

Under physiological condition, IL-2 is being subjected to mitogenesis element or allos thing to stimulate one of rear secretion by CD4 (+) T cell Plant 15kD albumen, it is essential to keeping normal immunoreaction; Along with wearing out of age, antigen and mitogenesis are plain to stimulate The ability of synthetic IL-2 descends, thereby the cell immune response ability reduces. Although only have T emiocytosis IL-2, it can Stimulate all quasi-lymphocytes, by with the combination of the specificity IL-2 acceptor of cell surface, performance impel cell proliferation and The function of differentiation. The NK cell can be expressed these acceptors in composition ground, and the expression of the IL-2 acceptor of most of T and B cell Then depend on the stimulation of antigen. No matter IL-2 is to cellular immunity and humoral immunity, still original and secondary immunoresponse all is very Important.

4.2IL-2 protein structure

The IL-2 total length is 153 amino acid, and 20 amino acid of N end are signal peptide. Ripe IL-2 molecule is by 133 ammonia Base acid forms, and molecular weight is 15.4kDa, the pI meta-alkali. The cytokine of it and other does not have homology in sequence. The IL-2 branch Son has single disulfide bond, is positioned at Cys58/105, and it is essential to the performance of its BA. IL-2 is a kind of sphere Albumen is made up of 4 main both sexes α spirals, and the latter is antiparallel manner and arranges, and its hydrophobic surface consists of a hydrophobic core. IL-2 has an O glycosylation site at 3 threonines. The difference of glycosylation degree can affect its molecular weight and electric charge.

The recombinant il-2 that Escherichia coli produce does not have glycosylation, but has the same BA of natural molecule. Glycosylated work With being to promote liver cell to its removing. The IL-2 molecule is very stable, and is acidproof and hot.

4.3IL-2 BA

IL-2 can efficiently stimulate the propagation of T cell of the same race. The human IL-2 can stimulate the propagation of mouse T cell under same concentration, But mouse IL-2 stimulates human T-cell's ability much lower, and approximately low 60-170 doubly. IL-2 is the growth factor of all T cell subsets. It is the nonspecific T cell proliferation of the antigen factor, to the propagation in akinete inducing cell cycle, causes activation T lymph thin Born of the same parents' clonal expansion. IL-2 also promotes the propagation of the B cell that activated. IL-2 amplification LAK cell (lymphokine-Activated killer cells). With external, in the situation of no any antigen, IL-2 can be optionally in vivo Stimulate the NK cell, the NK cell is considered to the immunocyte of virus infections particularly of microorganism in the cell. Antigenic stimulus is being arranged Lower, T, B cell generation cellular immunity and body fluid are exempted from. IL-2 can strengthen the secretion capacity of NK cell simultaneously, as mentioned above Activate and release various kinds of cell element, comprise IFN-γ, GM-CSF and TNF-α, β, then stimulate monocyte and huge biting Cell, secretion IL-1, IL-6, IL-8, TNF-α etc.

This shows that IL-2 plays central role in cell immune response, optionally damaged IL-2 can cause serious combining Close immunodeficiency disease (SCID). At present recombinant human interleukin--1 (rhIL-2) is by China SFDA, U.S. FDA, and Europe is common Body reaches the in the world careful mechanism of many national medicines approval listing treatment melanoma, kidney etc.

Summary of the invention

The present invention will have the polysaccharide of positive charge and have the compound recipe that cytokine negative charge, that have immune response stimulating (protein) is formed, it is the JY adjuvant system, mucosal immunity (the nasal cavity that is used for preventative vaccine and therapeutic vaccine, vagina, anus, oropharynx, the Sublingual) and systemic immunity (intramuscular injection, subcutaneous injection, intradermal injection etc.), improve the immune effect of vaccine or the drug utilization degree of raising peptide or pharmaceutical grade protein.The summary of the invention of JY adjuvant system is as follows:

1. with cytokine report is arranged all with immune response stimulating though have the polysaccharide of positive charge, they all have certain immunoadjuvant function, but our invention is: the two has intensive synergism, can stimulate and improve the immunne response of body significantly.The polysaccharide that has positive charge comprises the chitosan of different degree of deacetylation and other positively charged polysaccharide molecule; Cytokine with immune response stimulating comprises natural, reorganization, the interleukin II of modified, I type interferon-ALPHA 1a, b, c, d, e, α 2a, b, c, β 1a, 1b, interferon gamma, interferon-δ, interferon-κ, interferon-

λ

1,2,3, interferon ω, interferon, interferon-ε, Limitin etc., GM-CSF, G-CSF, M-CSG, stem cell factor, TLR _X, all kinds of thymosins, and other cytokine with immunoregulation effect.

2.JY the reasonable formula of adjuvant system: a kind of polysaccharide that has positive charge; a kind ofly have cytokine negative charge, that have immune response stimulating; one or more immunizing antigens; the pathogen that comprises deactivation or attenuation; the protease inhibitor that a kind of protected protein matter is difficult for being degraded; the buffer system of a kind of stable pH fits in prescription with rational dose ratio.

3. the JY adjuvant system of reasonable formula does not have tangible stimulation to mucosal tissue; Its safety meets the requirement of medicine.

4. the JY adjuvant system of reasonable formula has universality and versatility: can improve the effect of present already used vaccine, and improve immunne response or the peptide in the raising JY adjuvant system or the drug utilization degree of pharmaceutical grade protein of novel vaccine.

JY adjuvant system mucosal vaccine mechanism of action as shown in Figure 2.

Description of drawings

Fig. 1: chitin, chitosan, cellulosic structure figure

Fig. 2: JY adjuvant system mucosal immunity mechanism of action

Fig. 3: squamous epithelial tissue of matched group vestibule of nose portion, no abnormality seen.HE dyeing, 40 * 10

Fig. 4: squamous epithelial tissue of administration group vestibule of nose portion, no abnormality seen.HE dyeing, 40 * 10

Fig. 5: matched group nasal cavity olfactory region olfactory epithelium tissue, no abnormality seen.HE dyeing, 40 * 10

Fig. 6: experimental group nasal cavity olfactory region olfactory epithelium tissue, no abnormality seen.HE dyeing, 40 * 10

Fig. 7: matched group nose breathing portion cilium columnar epithelium tissue, no abnormality seen.HE dyeing, 40 * 10

Fig. 8: experimental group nose breathing portion cilium columnar epithelium tissue, no abnormality seen.HE dyeing, 40 * 10

Fig. 9: administration group nasal cavity nasal septum respiratory region cilium columnar epithelium is organized the infiltration of endolymph cell kitchen range, HE dyeing, 40 * 10

Figure 10: the variable concentrations chitosan to trivalent inactivated influenza vaccine nasal cavity immunity after influence (every treated animal: n=30 of positive rate of rotation of serum HI antibody; Hemagglutinin content: 4.5 μ g/ strain/agent, IL-2:20 ten thousand IU/ml)

Figure 11: the variable concentrations chitosan to trivalent inactivated influenza vaccine nasal cavity immunity after influence (every treated animal: n=30 of positive rate of rotation of serum HI antibody; Hemagglutinin content: 4.5 μ g/ strain/agent, IL-2:7.5 ten thousand IU/ml)

Figure 12: (ten thousand units/ml) are to influence (every treated animal: n=30 of serum HI antibody male rotary rate behind the trivalent inactivated influenza vaccine nasal cavity immunity for variable concentrations IL-2; Hemagglutinin content: 4.5 μ g/ strain/agent, chitosan: 0.5%)

Figure 13: (ten thousand units/ml) are to influence (every treated animal: n=30 of serum HI antibody male rotary rate behind the trivalent inactivated influenza vaccine nasal cavity immunity for variable concentrations IL-2; Hemagglutinin content: 4.5 μ g/ strain/agent, chitosan: 0.1%)

Figure 14: add with do not add 0.1% human albumin to trivalent inactivated influenza vaccine nasal cavity immunity after the influence (every treated animal: n=30, hemagglutinin content: 4.5 μ g/ strain/agent, chitosan: 0.1%, IL-2:20 ten thousand units per ml) of serum HI antibody male rotary rate

Figure 15: add with do not add 0.1% human albumin to trivalent inactivated influenza vaccine nasal cavity immunity after the influence (every treated animal: n=30, hemagglutinin content: 4.5 μ g/ strain/agent, chitosan: 0.1%, IL-2:7.5 ten thousand units per ml) of serum HI antibody male rotary rate

Figure 16: (vaccine is formed: IL-2,0.1% chitosan of 75000 units/ml content add the totivirus influenza vaccines to trivalent inactivated influenza vaccine nasal cavity immunity program to the influence of serum HI antibody male rotary rate; Wherein 1:1 immunity; 2:2 immunity (0,3 day); 3:3 immunity (0,3,7 days); 4:2 immunity (0,7 day))

Figure 17: the forward and backward serum antibody of the different classes of vaccine immune mouse of influenza intranasal inoculation increases multiple, and (1: the influenza all-virus vaccine adds IL-2 and chitosan; 2: the influenza all-virus vaccine adds chitosan; 3: the influenza all-virus vaccine adds IL-2; 4: the influenza all-virus vaccine; 5: chitosan adds IL-2:6:IL-2 and adds PBS; 7: not immune negative control)

Figure 18: (1: the influenza all-virus vaccine adds IL-2 and chitosan to the forward and backward serum antibody GMT of the different classes of vaccine immune mouse of intranasal inoculation; 2: the influenza all-virus vaccine adds chitosan; 3: the influenza all-virus vaccine adds IL-2; 4: the influenza all-virus vaccine; 5: chitosan adds IL-2:6:IL-2 and adds PBS; 7: not immune negative control)

Figure 19: different time serum HI antibody is compared with matched group and is increased multiple comparison (1: immunity blood sampling in back 21 days behind the influenza all-virus nasal spray vaccine immune mouse; 2: immunity blood sampling in back 28 days; 3: immunity blood sampling in back 35 days; Matched group HI titre is＜2.5)

Figure 20: different time serum HI antibody GMT relatively (1: took a blood sample in back 21 days by immunity behind the influenza all-virus nasal spray vaccine immune mouse; 2: immunity blood sampling in back 28 days; 3: immunity blood sampling in back 35 days; Matched group HI titre is＜2.5)

Figure 21: in the influenza all-virus nasal spray inactivated vaccine immune mouse average serum and titre (every group of 10 mices)

The specific embodiment:

Embodiment 1: nasal spray or oropharynx spraying vaccine/general prescription of JY adjuvant system:

Every milliliter contains:

One or more immunogens: according to different immunogen actual dose differences

Chitosan (or other positively charged polysaccharide): 1-10mg

IL-2 (or other have the cytokine of immunoloregulation function): 5-50 million international units

EDTA：0.05-2.0mg

Sodium hydrogen phosphate (Shi Ershui): 2.52mg

Sodium dihydrogen phosphate (two water): 2.02mg

Sodium chloride: 7mg

PH is 6.0-6.5

Embodiment 2: vagina spraying vaccine/general prescription of JY adjuvant system:

Every milliliter contains:

One or more immunogens are as HPV 16 and HPV 18VLP etc.: according to different immunogen actual dose differences

Chitosan (or other positively charged polysaccharide): 1-10mg

Interferon-ALPHA: 100-500 million international units

EDTA：0.05-2.0mg

Sodium hydrogen phosphate (Shi Ershui): 2.52mg

Sodium dihydrogen phosphate (two water): 2.02mg

Sodium chloride: 7mg

PH is 4.0-4.5

Embodiment 3: muscle or subcutaneous injection vaccine/general prescription of JY adjuvant system:

Every milliliter contains:

Chitosan (or other positively charged polysaccharide): 1-10mg

EDTA：0.05-2.0mg

Sodium hydrogen phosphate (Shi Ershui): 2.52mg

Sodium dihydrogen phosphate (two water): 2.02mg

Sodium chloride: 7mg

PH is 6.5-7.0

Embodiment 4: the safety and the effect of nasal spray trivalent inactivated influenza virus vaccine/JY adjuvant system

4.1JY adjuvant system safety testing

4.1.1JY the abnormal toxicity test of adjuvant system

Method by nasal spray has been carried out undue toxicity's reaction to white mice, Cavia porcellus.Be divided into 11 groups, wherein, 1 group is no JY adjuvant system vaccine group; The 2-11 group is different prescription JY adjuvant systems; IL-2:5-40 ten thousand IU/ml; Chitosan: 0.5-4.0mg/ml's is combined.2 vaccines of every white mice nasal spray, 20 vaccines of every nasal spray of Cavia porcellus (concentrating the back spraying).Experimental result shows that JY Adjuvanted vaccines system does not produce undue toxicity's reaction to mice and Cavia porcellus, and good safety is arranged.

The grouping of table 4 toxicity test

4.1.2.JY the Adjuvanted vaccines system is to rat nasal mucosa irritant test

Influenza all-virus nasal spray inactivated vaccine nasal cavity splashes into and gives Wistar rat, administration every day 1 time, and 0.1/ nasal cavity/time, continuous 7 days, other established the PBS matched group, and 6 every group, animal is killed in work in 24 hours after the last administration, draws portion of officina mucosa and carries out pathological examination.Duration of test, matched group and test sample treated animal overview index show no obvious abnormalities, and do not have red and swollen, the reaction of festering around vestibule of nose and the nostril, and obvious damage is not seen in the administration part of histopathological examination animal.Accompanying drawing 3-9 as a result.The result shows, the JY adjuvant system vaccine bronchia mucosal irritant test of collunarium repeatedly is negative.

4.2. the immune effect of nasal spray trivalent inactivated influenza virus vaccine/JY adjuvant system

Experimental result shows that the immune effect of the effect of nasal spray vaccine and equal vaccinate is suitable, but easy to use, and compliance is good, and side reaction is low, and old people and child all can use.The results are shown in accompanying drawing 10-21 and table 5.

The antibody result relatively behind two kinds of approach immune mouses of table 5 influenza nasal cavity totivirus inactivated vaccine and conventional injection inactivated vaccine

(test spraying vaccine: this institute develops; Spray nose matched group: PBS adds IL-2 and chitosan; Conventional vaccinate: the totivirus injected current influenza vaccine that long-living company produces; Injection matched group: the injection normal saline)

Embodiment 5: nasal spray measles/attenuated rubella live vaccine/JY adjuvant system

5.1. nasal spray Measles Vaccine,Live/JY adjuvant system

Before relatively exempting from after 20 people's immunity with exempted from back 21 days and the difference of 28 days average P/N values, two groups there were significant differences (P＜0.05).The ELISA measurement result sees Table 6.

The community immunity effect of table 6 nasal spray Measles Vaccine,Live/JY adjuvant system

5.2. nasal spray lepid attenuated live vaccine/JY adjuvant system

Before relatively exempting from after 20 people's immunity with exempted from back 21 days and the difference of 28 days average P/N values, two groups there were significant differences.Serum blood clotting inhibition titre on average increases multiple and the results are shown in Table 7.

The community immunity effect of table 7 nasal spray attenuated rubella live vaccine/JY adjuvant system

Claims

1. compound recipe of being formed by polysaccharide that has positive charge and the cytokine with immune response stimulating (protein) that has a negative charge, be the JY adjuvant system, be used to improve mucosal immunity and the effect of systemic immunity and the drug utilization degree that improves peptide or pharmaceutical grade protein of preventative vaccine and therapeutic vaccine;

2. above-mentioned 1 described a kind of polysaccharide that has positive charge is meant chitosan and other positively charged polysaccharide molecule of different degree of deacetylation;

3. the above-mentioned 1 described cytokine with immune response stimulating (protein) that has negative charge is meant and comprises natural or interleukin II reorganization or modified, I type interferon-ALPHA 1a, b, c, d, e, α 2a, b, c, β 1a, 1b, interferon gamma, interferon-δ, interferon-κ interferon-λ 1,2,3, interferon ω, interferon, interferon-ε, Limitin etc., GM-CSF, G-CSF, M-CSG, stem cell factor, TLR _X, all kinds of thymosins and other cytokine with immunoregulation effect;

4. above-mentioned 1 described preventative vaccine is meant that this vaccine of inoculation can prevent the biological preparation of multiple infectious disease or other disease;

5. above-mentioned 1 described therapeutic vaccine is meant inoculates the biological preparation that this vaccine can be treated cancer or other disease;

6. above-mentioned 1 described mucosal immunity is meant by nasal cavity, vagina, and anus, oropharynx, the immunne response that mucosal route vaccinations such as Sublingual produce comprises humoral immunization and cellular immunization;

7. above-mentioned 1 described systemic immunity is meant by intramuscular injection, subcutaneous injection, and the systemic immunne response that approach vaccinations such as intradermal injection produce comprises humoral immunization and cellular immunization;

8. above-mentioned 1 described JY adjuvant system is meant a kind of polysaccharide that has positive charge, a kind of cytokine with immune response stimulating, one or more immunizing antigens, the pathogen that comprises deactivation or attenuation, the protease inhibitor that a kind of protected protein matter is difficult for being degraded, the buffer system of a kind of stable pH; Fit in prescription with rational dose ratio;

9. the JY adjuvant system of reasonable formula does not have tangible stimulation to mucosal tissue, and its safety meets the requirement of medicine;

10. the JY adjuvant system of reasonable formula has universality and versatility, can improve the effect of present already used vaccine, and improves immunne response or the peptide in the raising JY adjuvant system or the drug utilization degree of pharmaceutical grade protein of novel vaccine.