CN103405762A

CN103405762A - Polyinosinic acid-polycytidylic acid dominated adjuvant

Info

Publication number: CN103405762A
Application number: CN2013102362483A
Authority: CN
Inventors: 林海祥
Original assignee: Newbiomed PIKA Pte Ltd
Current assignee: Newbiomed PIKA Pte Ltd
Priority date: 2005-06-08
Filing date: 2005-06-08
Publication date: 2013-11-27

Abstract

The invention provides a polyinosinic acid-polycytidylic acid dominated adjuvant. The invention provides an oligonucleotide adjuvant composition and an immunoreaction initiated application method thereof. The invention also provides an immunogen composition containing the oligonucleotide adjuvant composition and an antigen, for example, the two components are in a vaccine. The adjuvant composition disclosed by the invention has specific physical properties such as molecular weight, concentration and pH, and the properties can meet a required effective and safe adjuvant capable of inducing and strengthening immunoreaction and changing the type of immunoreaction. The invention further relates to application methods of the adjuvant compositions, and particularly relates to a method for inducing the immunoreaction for antigen compositions.

Description

The polyinosinic acid-polycytidylicacid of take is main adjuvant

The application is that application number is 200580015097.2, and the applying date is on June 8th, 2005, and denomination of invention is divided an application for the Chinese patent application of " polyinosinic acid-polycytidylicacid of take is main adjuvant ".

Technical field

The method that the present invention relates generally to adjunvant composition and carrys out the Promote immunity reaction with these adjunvant compositions, its specific compositions about the immunogenicity in order to promote antigen (immunogenicity), vaccine and method.It more specific about the polynucleotide adjunvant composition, contain the vaccine of this polynucleotide adjunvant composition, and utilize described polynucleotide adjunvant composition and vaccine to promote the immunoreactive method in host.

Background technology

The narration of correlation technique

Immune system has the specificity of generation and non-specific immunity.Generally speaking, B and T lymphocyte have the special receptor for specific antigen on their cell surface, can produce specific immunity.This immune system can be to each antigen with two kinds of mode Immunel responses: 1) humoral immune reaction, relate to and stimulate the B cell to produce antibody or immunoglobulin, antigen presenting cell (APCs) function and helper T lymphocyte (Th1 and Th2) function, and 2) cell immune response, the generation that relate generally to the T cell, comprise cytotoxic T lymphocyte (CTLs) and other relates to ctl response for example, Th1 and/or Th2 cell and APCs.

Nonspecific immunity comprises the various kinds of cell mechanism of action; for example the phagocytosis (phagocytosis) of macrophage (macrophages) or granular cell (granulocytes) to be to engulf foreign particle or antigen, and natural killer cell (NK cell) is active etc.It is original immunologic mechanism that non-specific immunity relies on, and can not show the acquired character of specificity and Memorability, and this acquired character is the typical feature of specific immune response.Key difference between specificity and nonspecific immunity be the former by cell mediated identification specificity antigen motif, have memory function; And the latter does not have this function.Therefore vaccination (relating to immunologic opsonin and Memorability) is that a kind of body of protecting is to resist the effective mechanism of harmful pathogen.

Adjuvant is the material of enhancing body to the immunoreation of antigen or change immunoreation type, when it gives jointly with antigen, (with this antigen, mix mutually, or with front or rear, give giving this antigen) and can promote or change immunoreation and the immunoreactive type for this specific antigen.

The typical adjuvant of common Promote immunity reaction comprises: aluminium compound (this adjuvant often is commonly referred to as aluminium adjuvant " Alum "), (complete Freund's adjuvant CFA is a kind of oil in water emulsion to oil in water emulsion, contain the tubercule bacillus that drying and heat kill go out), Saponin (separates from soap matter tree Quillaja Saponoria skin, this adjuvanticity composition is called as Quile A), CpG ODN(synthesizes few deoxyribonucleotide, CpG bis-polynucleotides that contain demethylation), MPL(is derivative from the lipopolysaccharide of Minnesota Re595 Sharpe bacillus (Salmonella minnesota Re595)), liposome (usually by making as biodegradable materials such as phospholipid) and biodegradable polymeric microspheres are (by as PLGA, the multiple polymers such as poly phosphazene (polyphosphazene) and polyanhydride are made).The adjuvant character of these materials respectively has pluses and minuses through assessment.

Adjuvant is applied in to vaccine for man, and particularly the child is that with the ultimate challenge of conventional vaccine most of adjuvant prescriptions generation toxicity and untoward reaction or adjuvant effect are not remarkable.The new technique used in the vaccine research and development just develops towards purification, subunit or synthetic antigen, and these antigens itself have weak immunogenicity.Developing new adjuvant is the main challenge that the vaccine research and development are faced to improve immunogenicity/effectiveness and to lower adverse side effect.

Polynucleotide complex is on testing as multiple uses such as adjuvants.Double-stranded RNA s(dsRNAs) be the very effective bio-modification factor, can under nanomole concentration, cause obvious effect to cell.The regulating effect of dsRNA is included in effect widely on molecule and cellular level.

On molecular level, dsRNAs can induce as the generation of synthetic, the protein kinase of interferon (interferon), promote and organize compatible antigen and suppress the biological effect such as metabolism.On cellular level, dsRNA can excite as biological effects such as pyrogen (pyrogenicity), cell division (mitogenicity), macrophage activation, activating cell immunity and initiation antiviral.The huge prospect of dsRNAs is the immunoregulation effect on antimicrobial therapy.United States Patent (USP) the 4th, 124, No. 702 proposition dsRNAs inducing interferon in the zooblast of living produces.United States Patent (USP) the 3rd, the adjuvant type vaccine antibody that 906, No. 092 propositions contain polynucleotide or polynucleotide complex produces enhancing.The people such as Houston set up PICLC(poly I: C poly-L-lysine-carboxymethyl cellulose complex) make a kind of effective adjuvant, increase by this primary antibody reaction and assisting (referring to people such as Houston without other adjuvant, Infection and Immunity, 14:318-9,1976C).Mould virus type dsRNA(Mycoviral dsRNA) be found to promote significantly blood coagulation antibody for the reaction of sheep red blood cell (referring to Wright and Adler-Moore, Biochemical and Biophysical Research Communications, 131:949-45,1985).

During the PIC(polyinosinic acid-polycytidylicacid) for animal, can cause serious toxic reaction under doses, for example, the people such as Phillips are reported in the inferior chronic Canis familiaris L. PIC that gives under 2.0mg/kg dosage can cause serious toxic action.The voluntary activity that shows as of toxicity lowers, coordinates bad, anorexia, vomiting, weight loss, the blood variation is reflected in the sinusoid capillary expansion of lobule center, hepatic necrosis, liver structural collapse and the general arthritis of the active rising of reduction hemopoietic function, alkali phosphatase and transaminase, involution of thymus, bone marrow destruction, liver (referring to people such as Phillips, Toxicology and Applied Pharmacology, 18:220-30,1971).

A kind of as in studied maximum polynucleotide complex, PIC is ineffective for monkey and human body the time, this be because PIC during entering primates (comprising the people) body after, understand very soon by the nuclease degradation in the above animal body of primates.Therefore many modes are used for improving the deficiency of PIC, for example, for high approximately 5 to 15 times of the comparatively former PIC of the repellence of pancreas ribonucleic acid enzyme hydrolysis, for example PIC and polyL-lysin carboxymethylcellulose complex (abbreviation PICLC) are found to be very effective antiviral or antitumor agent again by PIC and poly-L-lysine hydrobromide complex.PICLC is a kind of synthetic dsRNA that includes polynucleotide and polyC.Although PICLC is a kind of promising immunoregulation agent, at antimicrobial and anticancer therapy, fine potential is arranged, in human trial, produce serious adverse side effect, particularly repeatedly during the high dose medication.There is the adverse side effect of report to comprise heating, hypotension, leukopenia (leucopenia), the tired pain of limbs (myalgia), thrombocytopenia (thrombocytopenia) and Polyarthralgia (polyarthralgia).Therefore this product can not be for human body, and the toxicity of this product and stability problem must be overcome.So that PICLC can make to be used safely in to use human body.The treatment effectiveness of PICLC is subject to its stability in vivo in addition.

A kind of antiviral drugs that includes polyinosinic acid-polycytidylicacid (PIC), kanamycin (kanamycin) and calcium ion (this complex is called for short Av-PICKCa) is used to treat viral infection.Confirmed that Av-PICKCa induces the generation of sending out interferon and interleukin-2 at body.Giving separately Av-PICKCa can stimulate nonspecific immune reaction as antiviral drugs, and for example Av-PICKCa directly stimulates lower non-generation for the specific antigen interferon.This kind antiviral response is different from the antigen specific immune produced when adjuvant gives jointly with antigen and reacts very much.

The more important thing is that this case inventor is found to the character that Av-PICKCa has adjuvant, namely when with antigen, jointly giving, strengthen the ability of specific immune response.This case inventor finds that Av-PICKCa is a kind of effective adjuvant when with rabies and hemorrhagic fever virus antigen co-immunization.

The people such as Lin narration, Av-PICKCa can be used as a kind of adjuvant and (referring to Lin, waits the people; A new immunostimulatory complex (PICKCa) in experimental rabies:antiviral and adjuvant effects; Arch Virol, 131:307-19,1993; No. 93105862.7, Chinese patent).Chinese patent provide for No. 93105862.7 the complex that formed by PIC, kanamycin and calcium ion (being called for short PICKCa) people and mammal as the vaccine adjuvant purposes.

The Av-PICKCa sample is heterogeneous on molecular size and weight.In the literature, Av-PICKCa narrates with meansigma methods or the scope of the represented sedimentation coefficient value of SF (Svedbergs) S.In one embodiment, antiviral agent Av-PICKCa is present in agent between 5S to 8S (source A), refer to Zhung J.C., Research recollection of polyinosinic-polycytidylic acid (PIC) .The paper of fifth Chinese interferon conference in clinical application and theory, Xian1985, pp23-28.In other embodiments; the sedimentation coefficient of Av-PICKCa is 4S to 12S; having a meansigma methods is 6S agent (source B); perhaps sedimentation coefficient is 5S to 12S, and having a meansigma methods is 7S agent (source C), or sedimentation coefficient is 8S to 10S agent (source D); refer to Hu Q.G.; Tianjin Av-PICKCa ' slaboratory research and clinical application, Fujian Medical Journal, 1983.12; (6): 28-30 and Hu Q.G.Chinese Medical and Pharmaceutical Industry Journal, 1983 (9) 3134.

Utilize reduction formula mw=1,100xS ^2.2(referring to people such as Su B.X.; Introduction of Biochemical Technology, 1st Edition, Zhongshan University, 1978,356-357), the sedimentation coefficient of the heterogeneous molecule in Av-PICKCa can be converted into reciprocity molecular weight (mw means with dalton Daltons).Lower Table A demonstration is converted into daltonian result:

The characteristic of Table A: Av-PICKCa

* in list of references, there is no the data that propose

The people such as Lin are used for usining the original research of Av-PICKCa as adjuvant by a sample, the characteristic that the molecule of this sample has and the A that originates is similar, namely its sedimentation coefficient is 5S to 8S, this sedimentation coefficient is to equaling position 38,000 dalton to 107, the molecular weight of the molecule in 000 dalton's scope.(referring to people such as Lin., the same).

Generally believe, the PICKCa of form of ownership is all safe and effective on a 50-50 basis, because Av-PICKCa is a kind of form of PICKCa basically, and Av-PICKCa has been used as antiviral agent in the past.But the fact is not like this.What this case inventor carried out studies show that, while when the combination of PICKCa and antigen, being used as an adjuvant, the effectiveness of PICKCa and toxicity in fact can change along with the difference of molecular weight.When this case inventor finds Av-PICKCa as adjuvant, can not provide best effectiveness/safety state, and PICKCa can cause really and make us unacceptable adverse side effect in some cases.Therefore, industry still needs a kind of mankind of being more suitable for use and required immunoreactive safe and effective adjuvant can be provided.The present invention can meet this demand and other advantage is provided, and describes in detail and can understand with reference to aftermentioned.

Document

The coherent reference data is shown in down:

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United States Patent (USP) the 4th, 124, No. 702

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Summary of the invention

A kind of method that the invention provides polynucleotide adjunvant composition and react in order to induction of immunity.The present invention also provides a kind of immunogen constituent, comprises this polynucleotide adjunvant composition and antigen (for example these two kinds of compositions are positioned at a vaccine).Adjunvant composition of the present invention has specific physical property (for example molecular weight, concentration and pH), and these character can accord with provides a kind of effective and safe adjuvant that strengthens immunoreation and change the immunoreation type of inducing.The present invention more provides the using method of these adjunvant compositions, particularly brings out the immunoreation for the antigen thing.

In an embodiment, the invention provides a kind of polynucleotide adjunvant composition, comprise PIC (polyriboinosinic-polyribocytidylic acid) (PIC), an antibiotic and a cation, wherein this antibiotic can be kanamycin, and this cation can be divalent ion, for example a calcium.The present invention also provides a kind of immunogen constituent, comprises this polynucleotide adjunvant composition and antigen or vaccine.

The present invention, by defining a kind of novel composition that can securely and effectively be used as an adjuvant, to promote and/or to change the immunoreation in an animals or humans host, thereby promotes the lifting of knowledge.Although prior art shows antiviral agent Av-PICKCa and can, in order to as a kind of adjuvant, when the PICKCa of this kind form and antigen give jointly, only be observed and can cause limited specific immune response.In addition, PICKCa is found can cause in some cases and makes us unacceptable adverse side effect.

The present invention is by providing a kind of adjunvant composition to come in response to these problems, and said composition is at this referred to as " PIKA ", and it can be used as an adjuvant and the most effectively and safely gives animal, comprises the mankind.

PIKA is a kind of compositions, comprises a polynucleotide, an antibiotic and a cation, and said composition is become a kind of adjuvant by specifically research and development.The present invention includes several compositions, these compositionss have special product property, make these compositionss be particularly suitable for being used as the adjuvant in the immunogen constituent, to give animal and/or the mankind.

Concrete upper saying, the invention provides a kind of polynucleotide adjunvant composition, comprises a polynucleotide, an antibiotic and a cation, and wherein this polynucleotide can be PIC (PIC), this antibiotic is kanamycin, anthracycline (Anthracycline), butirosin sulfate (butirosin sulfate), gentamycin (gentamicin), hygromycin (hygromycin), amikacin (amikacin), dibekacin (dibekacin), nebramycin (nebramycin), beautiful its amide (metrzamide), neomycin (neomycin), puromycin (puromycin), streptomycin or streptozotocin (streptozocin), and this ion is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.

In detail, the invention provides the specification that includes a polynucleotide, an antibiotic and a cationic compositions, comprise molecular weight, concentration and pH, said composition can meet for the needs that cause maximum required immunoreation safe adjuvant.

The invention provides a kind of immunogen constituent, comprise this polynucleotide adjunvant composition and antigen or vaccine.

In certain embodiments, the present invention presents the form of a kind of combined complete (KIT), comprises this polynucleotide adjuvant and an immunogenic substance.

In addition, the invention provides method, by by this immunogen constituent, giving a host, to promote the immunoreation for antigenic substance.This host can be the mankind or animal.This gives process can be via as injections such as intramuscular, intraperitoneal, intravenous or subcutaneous injections or by through respiratory tract, having sucked.In other embodiments, but this immunogen constituent per rectum, vagina, nose, mouth, eye, part, transdermal or Intradermal transmit.

Accordingly, the invention provides a kind of adjuvant and a kind of immunogen constituent that can be used in safely human body or animal body.

Accordingly, on the one hand, characteristics of the present invention are a kind of polynucleotide adjunvant compositions, comprise a PIC (PIC), an antibiotic and a cation, wherein said composition contains on molecular weight for heterogeneous adjuvant molecules, and the molecular weight of this molecule is approximately 66,000 to 1,200,000 dalton.

In related embodiment, polynucleotide adjunvant composition molecule in said composition is heterogeneous on molecular weight, wherein this molecular weight is approximately 300, 000 to 1, 200, 000 dalton, or approximately 66, 000 to 660, 000 dalton, or approximately 300, 000 to 660, 000 dalton, or approximately 300, 000 to 2, 000, 000 dalton, or approximately 300, 000 to to4, 000, 000 dalton, or approximately 500, 000 to 1, 000, 000 dalton, or approximately 1, 000, 000 to 1, 500, 000 dalton, or approximately 1, 500, 000 to 2, 000, 000 dalton, or approximately 2, 000, 000 to 2, 500, 000 dalton, or approximately 2, 500, 000 to 3, 000, 000 dalton, or approximately 3, 000, 000 to 3, 500, 000 dalton, or approximately 3, 500, 000 to 4, 000, 000 dalton, or approximately 4, 000, 000 to 4, 500, 000 dalton, or approximately 4, 500, 000 to 5, 000, 000 dalton.

In related embodiment, polynucleotide adjunvant composition molecule in said composition has a mean molecule quantity, this mean molecule quantity is equal to or higher than 150, 000 dalton, or be equal to or higher than 250, 000 dalton, or be equal to or higher than 350, 000 dalton, or be equal to or higher than 500, 000 dalton, or be equal to or higher than 650, 000 dalton, or be equal to or higher than 750, 000 dalton, or be equal to or higher than 1, 000, 000 dalton, or be equal to or higher than 1, 200, 000 dalton, or be equal to or higher than 1, 500, 000 dalton, or be equal to or higher than 2, 000, 000 dalton.

Accordingly, in an aspect, characteristics of the present invention are a kind of polynucleotide adjunvant compositions, comprise a PIC (PIC), an antibiotic and a cation, wherein said composition contains the adjuvant molecules that is heterogeneous on molecular size, and the SF of these adjuvant molecules (Svedbergs) is about 6.43S to 24.03S.

In related embodiment, polynucleotide adjunvant composition molecule in said composition is heterogeneous on molecular size, wherein this molecular size is about 12.8S to 24.03S, or approximately 6.43 to 18.31S, or approximately 12.8 to 18.31S, or about 12.8S to 30.31S, or about 12.8S to 41.54S, or about 13.5S to 18.31S, or about 13.5S to 24.03S, or approximately 16.14 to 22.12S, or about 22.12S to 26.6S, or about 26.6S to 30.31S, or about 30.31S to 33.55S, or about 33.55S to 36.45S, or about 36.45S to 39.1S, or about 39.1S to 41.54S, or about 41.54S to 43.83S, or about 43.83S to 45.95S.

In other related embodiment, this polynucleotide adjunvant composition has an average settlement coefficient unit (Svedbergs), and this average settlement coefficient is higher than 9, or higher than 12, or higher than 13.5, or higher than 15, or higher than 16, or higher than 17, or higher than 18, or higher than 19, or higher than 20, or higher than 21, or higher than 22, or higher than 25, or higher than 30.

In a related embodiment, the antibiotic in said composition is kanamycin, neomycin, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide, puromycin, streptomycin or streptozotocin.

In another related embodiment, this adjunvant composition more comprises a kind of calcium ion.

In another related embodiment, the cation in said composition is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.This cation can be the form of inorganic salt or organic double compound.

The calcium ion source can be for example calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.

Special related fields, the invention provides a kind of polynucleotide adjunvant composition, comprise PIC (PIC), kanamycin and calcium, wherein said composition is included on molecular weight the adjuvant molecules that is heterogeneous, the molecular weight of these adjuvant molecules is approximately 66,000 to 1,200,000 dalton.

In related embodiment, this PIC (PIC), the molecular weight of kanamycin and calcium molecule is approximately 300, 000 to 1, 200, 000 dalton, or approximately 66, 000 to 660, 000 dalton, or approximately 300, 000 to 660, 000 dalton, or approximately 300, 000 to 2, 000, 000 dalton, or approximately 300, 000 to 4, 000, 000 dalton, or approximately 500, 000 to 1, 000, 000 dalton, or approximately 1, 000, 000 to 1, 500, 000 dalton, or approximately 1, 500, 000 to 2, 000, 000 dalton, or approximately 2, 000, 000 to 2, 500, 000 dalton, or approximately 2, 500, 000 to 3, 000, 000 dalton, or approximately 3, 000, 000 to 3, 500, 000 dalton, or approximately 3, 500, 000 to 4, 000, 000 dalton, or approximately 4, 000, 000 to 4, 500, 000 dalton, or approximately 4, 500, 000 to 5, 000, 000 dalton.

In other related embodiment, the PIC of this adjuvant (PIC), kanamycin and calcium molecular composition are heterogeneous on molecular weight, these molecules have a mean molecule quantity, this mean molecule quantity is equal to or higher than 150, 000 dalton, be equal to or higher than 250, 000 dalton, or be equal to or higher than 350, 000 dalton, or be equal to or higher than 500, 000 dalton, or be equal to or higher than 650, 000 dalton, or be equal to or higher than 750, 000 dalton, or be equal to or higher than 1, 000, 000 dalton, or be equal to or higher than 1, 200, 000 dalton, or be equal to or higher than 1, 500, 000 dalton, or be equal to or higher than 1, 500, 000 dalton.

Special related fields, the invention provides a kind of polynucleotide adjunvant composition, comprise PIC (PIC), kanamycin and calcium, wherein said composition is included on molecular size the adjuvant molecules that is heterogeneous, and the SF of these adjuvant molecules (Svedbergs) is about 6.43S to 24.03S.

In other related embodiment, this PIC (PIC), kanamycin and calcium composition have an average settlement coefficient, and this average settlement coefficient is higher than 9, or higher than 12, or higher than 13.5, or higher than 15, or higher than 16, or higher than 17, or higher than 18, or higher than 19, or higher than 20, or higher than 21, or higher than 22, or higher than 25, or higher than 30.

In certain embodiments, the present invention is for a kind of polynucleotide adjunvant composition, comprise PIC (PIC), kanamycin and calcium, wherein said composition is preferably and gets rid of some compositions molecule, particularly at these molecules that are excluded, do not have on the degree of significant immunogen effect, the molecular weight of the compositions molecule wherein be excluded is for approximately or lower than 30, 000 dalton, approximately or lower than 40, 000 dalton, approximately or lower than 50, 000 dalton, approximately or lower than 60, 000 dalton, approximately or lower than 70, 000 dalton, approximately or lower than 80, 000 dalton, approximately or lower than 90, 000 dalton, approximately or lower than 100, 000 dalton, approximately or lower than 150, 000 dalton, approximately or lower than 200, 000 dalton, approximately or lower than 250, 000 dalton, approximately or lower than 300, 000 dalton, approximately or lower than 350, 000 dalton, approximately or lower than 400, 000 dalton, approximately or lower than 450, 000 dalton, approximately or lower than 500, 000 dalton, approximately or lower than 600, 000 dalton, approximately or lower than 700, 000 dalton, approximately or lower than 800, 000 dalton, approximately or lower than 900, 000 dalton, approximately or lower than 1, 000, 000 dalton.

In certain embodiments, the invention provides a kind of polynucleotide adjunvant composition, comprise PIC (PIC), kanamycin and calcium, wherein be preferably and get rid of some molecule of said composition, particularly at these molecules that are excluded, do not have the immunogen effect of significance degree, the molecular size of the molecule wherein be excluded is for approximately or lower than 4.49S, approximately or lower than 5.12S, approximately or lower than 5.67S, approximately or lower than 6.16S, approximately or lower than 6.6S, approximately or lower than 7.02S, approximately or lower than 7.4S, approximately or lower than 7.77S, approximately or lower than 9.34S, approximately or lower than 10.64S, approximately or lower than 11.78S, approximately or lower than 12.8S, approximately or lower than 13.73S, approximately or lower than 14.59S, approximately or lower than 15.39S, approximately or lower than 16.14S, approximately or lower than 17.54S, approximately or lower than 18.81S, approximately or lower than 19.99S, approximately or lower than 21.09S, approximately or lower than 22.12S.

Special related fields, the invention provides a kind ofly in order to promote antigenic immunogenic composition of antigen thing, comprise the compositions of this polynucleotide adjuvant.

In related embodiment, this immunogen constituent comprises this polynucleotide adjuvant and antigen.

In related embodiment, the antigen source is a kind of mankind's antigen, a kind of non-human animal's antigen, a kind of plant antigen, a kind of bacterial antigens, a kind of fungal antigen, a kind of virus antigen, a kind of parasite antigen or a kind of cancerous protuberance antigen.

In related embodiment, this immunogen constituent comprises this polynucleotide adjunvant composition and a rabies antigen.

In certain embodiments, antigen can be purified into from a natural origin, synthesizes or can obtain by the genetic recombination technology by solid-phase synthesis.Antigen can comprise a protein fragments, and this protein fragments contains one or more immunogen district of this molecule.Antigen also can by live, intact cell attenuation or truncate or killed or microorganism (for example complete virion) provide.

In other embodiments, antigen comprises one or more composition, other mankind's antigen that stems from infectious agent, plant antigen, cancerous protuberance, anaphylaxis composition and for example can cause autoimmune disease.In other embodiments, antigen comprises one or more infectious agent, stems from any in virus, antibacterial, mycobacteria (Mycobacterium), fungus and parasite.

Polynucleotide adjunvant composition of the present invention also can be used for the Promote immunity reaction, and this immunoreation is for the antigen that uses DNA vaccination to produce.DNA sequence in order to coding for antigens in these vaccines can be " exposed ", or is accommodated in a carrier system as lipophore.

In other related embodiment, rabies antigen is to be selected from mankind's double somatocyte vaccine (HDCV), or hamster nephrocyte deactivation purification semple type rabies vaccine (HKC-IPRV), or the rough semple type rabies vaccine of hamster nephrocyte deactivation (HKC-ICRV), or purification type Vero cell rabies (PVRV), or purification type chick-embryo cell (PCEC), or purification type duck embryo vaccine (PDEV), or hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) or the rough rabies antigen of hamster nephrocyte deactivation (HKC-ICRA).

Special related fields, the invention provides a kind of in order to promote antigenic immunogen constituent of antigen thing, comprise can inducing antigen-specific the polynucleotide adjunvant composition of cell immune response.

Special related fields, the invention provides a kind of in order to promote antigenic immunogen constituent of antigen thing, comprise can inducing antigen-specific the polynucleotide adjunvant composition of B cell immune response.

Special related fields, the invention provides a kind ofly in order to promote antigenic immunogen constituent of antigen thing, comprise simultaneously inducing T cell and the B cell polynucleotide adjunvant composition to the antigen specific immune response.

Aspect special related fields, the invention provides a kind of in order to promote antigenic immunogen constituent of a material, comprise this polynucleotide adjunvant composition and a hamster nephrocyte deactivation purification type rabies antigen, wherein the existence of this rabies antigen should reach a minimum flow, for example surpasses 1 iu (IU).

In related embodiment, this immunogen constituent comprises this polynucleotide adjunvant composition and hamster nephrocyte deactivation purification type rabies antigen, wherein the existence of this rabies antigen should reach a minimum flow, for example surpass 0.25 iu, surpass 0.5 iu, surpass 1.2 ius, surpass 1.4 ius, surpass 1.6 ius, surpass 1.8 ius, surpass 2.0 ius, surpass 2.2 ius, surpass 2.4 ius, surpass 2.6 ius, surpass 2.8 ius, surpass 3.0 ius, surpass 3.2 ius, surpass 3.4 ius, surpass 3.6 ius, surpass 3.8 ius, or surpass 4.0 ius.

Special related fields, the invention provides a kind of in order to promote antigenic immunogen constituent of a material, it comprises this polynucleotide adjunvant composition and hamster nephrocyte deactivation purification type rabies antigen, and wherein this adjuvant and rabies antigen present approximately 1 to 1 ratio.

In related embodiment, this immunogen constituent comprises this polynucleotide adjunvant composition and hamster nephrocyte deactivation purification type rabies antigen, and wherein this adjuvant and rabies antigen present lower than 1 to 10, and approximately 1 to 9, approximately 1 to 8, approximately 1 to 7, approximately 1 to 5, approximately 1 to 4, approximately 1 to 3, approximately 1 to 2, approximately 2 to 1, approximately 3 to 1, approximately 4 to 1, approximately 5 to 1, approximately 6 to 1, approximately 7 to 1, approximately 8 to 1, approximately 9 to 1, approximately 10 to 1, or be greater than 10 to 1 ratio.

Special related fields, the invention provides a kind of adjunvant composition or immunogen constituent, this immunogen constituent wherein, or be comprised in the adjunvant composition of this immunogen constituent, be present a solid or liquid form or be positioned at solution or suspension.

Special related fields, the invention provides a kind of adjunvant composition or comprise the immunogen constituent of an adjunvant composition, wherein this adjunvant composition or immunogen constituent can be frozen drying.

In related embodiment, the invention provides a kind of combined complete (KIT), comprise this adjunvant composition and antigenic substance.

Special related fields, the invention provides a kind of purposes of polynucleotide adjunvant composition, with preparation, promote host's immunoreactive medicament.

Special related fields, the invention provides a kind of in order to promote the immunoreactive method for the antigen thing, comprise and in order to the antigenic immunogen constituent that promotes the antigen thing, give the host by one, this immunogen constituent comprises this polynucleotide adjunvant composition.

In related embodiment, the method that this immunogen constituent is given to a host can be selected from any mode in following method, and the method includes: the outer injection of gastrointestinal tract, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, through respiratory tract suction, rectal delivery, vagina transmission, per nasal transmission, through port transmission, through eye transmission, local transmission, transdermal transmission or Intradermal transmission.

Special related fields, the invention provides a kind of in order to promote the immunoreactive method for the antigen thing, comprise and in order to the antigenic immunogen constituent that promotes the antigen thing, give a host by one, this immunogen constituent comprises this polynucleotide adjunvant composition, and wherein this host is animal.

Special related fields, the invention provides a kind of in order to promote the immunoreactive method for the antigen thing, comprise and in order to the antigenic immunogen constituent that promotes the antigen thing, give a host by one, this immunogen constituent comprises this polynucleotide adjunvant composition, and wherein this host is the mankind.

By the follow-up content and appended graphic that describes in detail, These characteristics of the present invention and other advantage will become obvious.

The accompanying drawing explanation

Fig. 1 shows the relative molecular weight of Av-PICKCa and PIKA sample.

After Fig. 2 showed immune PIKA vaccine, the dose dependent that the vaccine-induced specificity of PIKA produces the interferon gamma cytokine generated.

The specific embodiment

The present invention can by follow-up for some specific descriptions of the present invention and wherein included embodiment detailed content and more easily understood.

The application's case is with reference to open source literature in the entire chapter content, and the content of these open source literatures is merged in the application's case as a reference, to narrate fully the level of the technical field of the invention.

Before further narrating the present invention, should bright Liao the present invention can not be limited in described specific embodiment, because these embodiment must be various.Should bright Liao term as used in this specification be also only in order to set forth specific embodiment, but not as restriction, because scope of the present invention will be defined in appended claim.

Unless define separately, all technology as used in this specification and scientific words all with this case under have in technical field common knowledge the personage the same meaning of general bright Liao.Just implement now preferred approach of the present invention and material and narrated, but and any method of method and materials similar described in this description or equivalence and material all can be in order to implement or test the present invention.All open source literatures of mentioning in this description all are incorporated in this as a reference, to disclose and to illustrate method and/or the material in described open source literature.

It should be noted that the term used as in this case description and claim, unless the front and back literary composition means bright other meaning is arranged separately, otherwise odd number shape term " a ", " and " and " the " comprise plural shape term.Therefore, for example mention that " sentence " namely comprises the plural form of this sentence, mention that " this fragment " namely comprises to mention one or more fragment and have the knack of equivalent of knowing in the art technology personage etc.It should be noted that in addition claim can be formulated as any alternative composition of eliminating.Therefore, this explanation is will be as using removing property terms such as " (solely) only arranged ", " only having (only) " when claim constitutive requirements relevant refers content or the aforementioned basis (antecedent basis) during use " negative sense " restriction (" negative " limitation).

Nominal definition

Before statement detailed content of the present invention, should understand the several terms that are used in this description.

" adjuvant " this term be used in herein refers to increase or changes immunoreactive any material or the mixture of substances of host for the antigen thing.Specifically say:

1. " PICKCa " this term is to censure in general manner a compositions consisted of PIC, kanamycin and calcium, and compositions is without specific physics and immunogenicity characteristic.

2. " Av-PICKCa " refers to that PICKCa commercially is used the form as antiviral agent.

3. " PIKA " refers to the present composition, comprise PIC, an antibiotic (for example kanamycin) and a cation (for example calcium), wherein said PIKA is characterised in that physical characteristic (such as the molecular weight of narrating in this description, size etc.), so that PIKA can show the characteristic of an adjuvant after giving, and have compared to take PICKCa and be the higher effect intensity of example (for example inducing the immunoreation of enhancement) for example, as the lower adverse side effect of example (toxicity reduction) and compared to take Av-PICKCa.

" contain the PIC molecule " or " containing the PIC thing " is to censure without limitation PIC, it is optionally for example, for example, by compound or combine an antibiotic (kanamycin) in the above compositions that contains the PIC molecule and at least one or the two in a cation (calcium).

The composition (for example containing the PIC molecule) that refers to said composition at front " heterogeneous " this term used hereinafter of adjunvant composition of the present invention is not homogeneous on the two physical characteristic of molecular weight, size or this.

" animal " this term comprises the mankind and all raising and wild animal and avian, and it comprises cattle, horse, milk cattle, pig, sheep, goat, Canis familiaris L., cat, rabbit, deer, ermine, chicken, duck, goose, turkey, cockfighting etc. without limitation.

" antibody " this term comprises the antigen thing binding fragment of many strains and monoclonal antibody and these antibody, comprises Fab, F (ab ') 2, Fd, Fv fragment, and the strand derivant of these antibody and fragment.In addition, " antibody " this term comprises the antibody of natural generation and the antibody that non-natural occurs, comprise for example mosaic type (chimeric), difunctionality type (bifunctional) and (humanized) antibody that personalizes, and relevant synthesising different structure form (isoforms).

As used in this specification, refer to can be under suitable situation for example, by any material of immune system institute identification (being bonded to antibody or processed, with the inducing cell immunoreation) for " antigen thing " this term.

" antigen " refers to a kind of material, comprise the constituent that presents the vaccine form, wherein this vaccine itself comprises the antigen thing, and can comprise or can not comprise the adjuvant except PIKA, when for example, via suitable approach (outside gastrointestinal tract) while giving, this antigen can cause such as forming the immunoreation such as antibody, comprises specifically the antibody in conjunction with this antigen.Two characteristics of antigen are their immunogenicity and their antigenicity, and namely they cause immunoreactive ability in vivo to immunogenicity, and antigenicity is their selectivity identification capabilities that antibody had of being produced by antigen induction namely.

The term such as " cellular immunization (cell-mediated immunity) " and " cell immune response (cell-mediated immune response) " refers to the immune defence power that lymphocyte provides, and for example the T lymphocyte is at the phylactic power defensive power provided when being injured cell.A cell immune response generally includes lymphocytic propagation.When measuring " lymphocytic propagation ", can measure the multiplication capacity of lymphocyte in response to a specific antigen.Lymphopoiesis refers to the cell proliferation of B cell, T-accessory cell or cytotoxic t-lymphocyte (CTL).

" antigen thing effective dose " refers to that the consumption of antigen thing will cause the individual specific immune response produced for this antigen thing, this antigen thing optionally with one adjuvant combination.

The meaning of " increase immunoreation " this expression way or similar expression way is, compared to previous immunoreation state, immunoreation is enhanced, improves or rises, and favourable to the host, described previous immunoreation state is for example to give immunogen constituent of the present invention immunoreation state before.

The term such as " humoral immunity (humoral immunity) " and " humoral immune reaction (humoral immune response) " refers to the immune form that produces antibody molecule in response to antigenic stimulus.

" immunoreation " this term refers to any reaction of the immune system of a vertebrates individuality for the antigen thing.Typical immunoreation includes but not limited to cell and local and systemic humoral immune reaction, for example comprise the antigenic specificity inducing action of CD8+CTLs ctl response, comprise that T-cell proliferative response and cytokine disengage the auxiliary type T-cell effect in acting on, and the B-cell immune response that comprises antibody response.

" induction of immunity reaction " this term that this description is used is to comprise in general manner immunoreactive inducing and/or potentiation (induction and/or potentiation).

" generation immunoreation " this term refers to stimulation, initial or cause an immunoreation.

" strengthen (potentiating) immunoreation " and refer to that an immunoreation of both having deposited is enhanced, encourages, supplements, increases, promotes, increases or extends.

" poly-I:C " or terms such as " PIC " refer to a compositions that contains polyI and poly-ribose cytidine nucleic acid, also are referred to as respectively polyinosinic acid-polycytidylicacid.

" immune commercial weight " this term refers to, viewed immunoreation when there is no the polynucleotide adjuvant, and the antigen thing is enough to immune response stimulating antigen consumption while adding compositions of the present invention jointly to give.

" immunopotentiation amount (immunopotentiating amount) " this term refers to, viewed antibody titer and/or cell immune response when there is no the polynucleotide adjuvant, when adjuvant and antigen thing give jointly in the present composition, cause antibody titer and/or cell immune response to increase required adjuvant amount.

As used in this specification, " mixing " this term comprises forming any method of the composition of compositions; These methods include but not limited to fusion, distribution, dissolving, emulsifying, coagulation, suspension or other method that composition part of compositions is reasonably combined.

It is medicinal acceptable that the chemicals of " pharmaceutically acceptable salt " mean this salt, and has the required pharmacologically active of parent compound.These salt comprise: the acid of (1) synthetic salt, and mineral acids such as hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid forms salt jointly, or and for example acetic acid, propanoic acid, acid, cyclopentyl propionic acid, ethanedioic acid, acetone acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxy benzenes anilide) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethyl sulfonic acid, 1, the 2-ethionic acid, the 2-ethylenehydrinsulfonic acid, benzenesulfonic acid, the 4-chlorobenzenesulfonic acid, the 2-LOMAR PWA EINECS 246-676-2, the 4-toluenesulfonic acid, camphorsulfonic acid, glucose formic acid, 4, 4'-di-2-ethylhexylphosphine oxide-(3-hydroxyl-2-alkene-1-carboxylic acid), the 3-phenylpropionic acid, trimethylace tonitric, three grades of butyl lactic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, the common salt that forms such as the organic acid such as muconic acid, or (2) existing acid proton in parent compound is replaced just like metal ions such as alkali metal group metal ion, alkaline earth metal ion or aluminium ions, or coordination is during just like organic bases such as ethanolamine, diethanolamine, triethanolamine, amido butantriol (tromethamine), meglumines (N-methylglucamine), formed salt.

Any processing for the disease in vertebrates (the particularly mankind) body contained in " treatment " this term, and comprise: (i) prevention one may have the tendency of falling ill but individual diseases that NYD goes out to fall ill; (ii) contain disease, for example stop this advancing of disease; Or (iii) releive disease, for example cause this disease to disappear.

" unit dosage forms " as used in this specification this term refers to several units that are separation on entity, be suitable as the unit dose that is used in human and animal's individuality, each unit contains the present composition of a scheduled volume, this scheduled volume present afterwards as calculated one be enough to and pharmacy/pharmacology on acceptable diluent, supporting agent or the common consumption that produces required effect of carrier.

The invention introduction

The present invention is about being used in compositions and the method for Promote immunity reaction in the mankind, animal or cell culture, and this immunoreation can be body fluid and/or cell immune response.Generally speaking, this constituent comprises a kind of immunogen constituent that contains an adjuvant.The existence of this adjuvant can promote or change immunoreation.Therefore, this body fluid and/or cell immune response are more effective because of the existence of this adjuvant.In addition, this adjuvant can change immunoreactive quality by the generation of several hypotypes (heterogeneous) that affect immunoglobulin and cytokine.

The key characteristic of this adjuvant is that it can induce required immunoreation level and kind and can not cause the ability of corresponding adverse side effect.Only there is at present minority for the mankind's adjuvant, to have this specific character combination through ratifying.The safety criterion of immunogenic substances (referring in particular to vaccine) is strict and severe the execution.Therefore, the major limitation of developing a kind of successful adjuvant is to develop and a kind ofly is enough to induce a suitable immunoreactive effective product, and can not cause corresponding adverse side effect.

Preferred embodiment of the present invention is a kind of polynucleotide adjuvant, and wherein this polynucleotide is PIC (PIC).PIC is shown as a kind of effective adjuvant individually, makes us unacceptable safety speciality but can show, and unstable in the mankind and primate body.The invention provides a kind ofly by PIC and an antibiotic and the combined compositions formed of cation, it can promote the required immunogen characteristic of an adjuvant, and improves the safety and stability speciality.

Further basis of the present invention is that the physics and the biological nature that come from the PIKA molecule of finding adjunvant composition can affect immunoreation and adverse side effect.This case the inventor be surprised to find that during researching and developing, in the aftermentioned mode, adjust some characteristic of this polynucleotide adjuvant, and it will become effectively and/or the person becomes more or less and has toxicity more or less.Therefore, define this adjunvant composition from the angle of physical characteristic, namely likely more correctly describe this adjunvant composition in the characteristic provided on better immunogenic response and better safety/stability speciality.

Therefore, at this adjuvant of the present invention that is called the PIKA adjuvant for short, fully by its chemical composition, added that the combination of the Basic Physical Properties of the molecule that forms this adjuvant defines.Therefore, show superior immunogen character and can use safely simultaneously in the particular form of the PIKA of animals or humans and the most suitably by one or more specific speciality that normally is combination, be defined, comprising composition, molecular weight, molecular size, concentration and pH.

PIKA comprises a polynucleotide usually, one antibiotic and a cation, wherein this polynucleotide can be PIC (PIC), and this antibiotic is amido glucosides (kanamycin for example, streptomycin, safe hundred mycins (tobramycin), neomycin, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide, puromycin or streptozotocin), and this ion is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.

" amido glucosides " antibiotic refers to that this antibiotic structurally contains the amine sugar that is engaged to an amido cyclic alcohol ring (hexose core) by glycosidic bond.Amido glucosides antibiotic is derivative from multiple streptomycete (Streptomyces) and micromonospora (Micromonospora), or synthetic production.For example, kanamycin is a kind of amido glucosides antibiotic that is obtained from soil bacteria Steptomyces Kanamycetics, is used for the treatment of multi-infection, the infection particularly caused by Glan formula negative bacteria (Gram-negative bacteria).

The PIKA compositions is in the sodium chloride/phosphate buffer via the pH value that has a pH6 to pH8 at, and polyinosinic acid, poly, an antibiotic and a cation source are mixed and make.The concentration of polyinosinic acid and poly is generally 0.1 to 10mg/ml, is preferably 0.5 to 5mg/ml and be more preferred from 0.5to2.5mg/ml.Hyperchromic value (hyperchromicity value) should, higher than 10%, be preferably higher than 15% and be more preferred from higher than 20%.The preparation of PIC and and the combination of kanamycin and calcium preferably under the quality standard that accords with international good processing procedure standard, carry out.

In certain embodiments of the present invention, kanamycin in the polynucleotide adjunvant composition can be used or be replaced by one or more antibiotic with one or more antibiotic is common, and these antibiotic are selected from and comprise in the group that safe hundred mycins, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide, neomycin, puromycin, streptomycin and streptozotocin form.The concentration of the antibiotic in polynucleotide adjunvant composition of the present invention (such as kanamycin etc.) is generally approximately 10 to 100,000 units/ml, is preferably approximately 100 to 10,000 units/ml, and is more preferred from approximately 500 to 5,000 units/ml.

In certain embodiments of the present invention, this polynucleotide adjunvant composition more comprises a cation (cation), bivalent cation normally, and a cation of an alkali metal group metal normally.In compositions of the present invention, this cation generally is used as a cationic source, for example a kind of salt or complex, for example a kind of organic or inorganic salt or complex, and normally a kind of inorganic salt or organic double compound.Typical cation comprises but must not be limited to calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.

In compositions of the present invention, can be furnished with cation (for example calcium), this cationic concentration position, in the scope of about 10umol to 10mmol/ml, is preferably about 50umol to 5mmol/ml, and is more preferred from about 100umol to 1mmol/ml.

As previously mentioned, cation can be any suitable salt or the form of organic double compound, comprises but must not be limited to chloride, fluoride, hydroxide, phosphate or sulfate.For example, when this cation was calcium, this ion can be the form of calcium carbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.

When the cation in adjunvant composition of the present invention is calcium, it is can be with other cation combined or replaced by other cation, these cationes comprise cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc, and wherein these ions can be the form of inorganic salt or organic double compound.

The compositions of gained further is transformed into PIKA via an extra manufacture process, and this extra manufacture process relates to separating and has the molecular size that defines and/or the molecule of weight.The similar approach of utilizing filtration, chromatography, heat treatment, centrifugalize, electrophoresis and having become standard procedure is isolated the polynucleotide molecule with particular characteristics, is to have the knack of in the art technology personage to know.

In certain embodiments of the present invention, this polynucleotide adjunvant composition further defines by the physical characteristic of molecular weight.During researching and developing, this case inventor is surprised to find that between the molecular weight of polynucleotide adjunvant composition and effectiveness and has positive correlation.The effect intensity level that the immunogen constituent that contains this polynucleotide adjunvant composition is observed, comprise the ability that induction of immunity globulin and cytokine produce, and can increase and rise along with the molecular weight of polynucleotide adjunvant composition.The molecular weight of polynucleotide adjuvant can be measured by the described agarose gel electrophoresis of embodiment 1.

As described in the subsequent embodiment part, this case the inventor find, and the vaccine constituent that contains the PIKA adjuvant of a diversified molecular weight can show direct association (referring to embodiment 2) between molecular weight and antigenic specificity protection effect intensity.Similarly, this case the inventor find, when the combination of PIKA adjunvant composition and a rabies antigen gives the host, has directly associated (referring to embodiment 3) between the molecular weight of PIKA adjunvant composition and the ability of inducing the gamma type interferon to produce.

This case inventor in 1996 during the human trial that China carries out, utilize that a kind of this adjuvant comprises the PICKCa of ultra high molecular weight specification with the rabies vaccine of adjuvant, this case the inventor further identify, and resulting composition unexpectedly demonstrates has the unacceptable adverse side effect level of making us.The resulting result of clinical trial in 1996, before be not disclosed, be shown in now in embodiment 4.Research for molecular weight is shown in embodiment 5 and 6.This test is to carry out under the administration of ministry of Health of China food medication management department.Therefore, if can anticipate these adverse side effects based on knowledge at that time, this adjuvant will can not give human experimentation in a clinical setting of being supervised.

This case the inventor find, and molecular weight reaches 1.0x10 in preclinical test ⁶PIKA adjunvant composition of the present invention, and contain molecular weight and reach 5.5x10 ⁵The vaccine constituent of PIKA adjunvant composition, in specific toxicity test, demonstrate broad safety scope (referring to embodiment 7).Having highest weight is 1.2x10 ⁶PIKA successfully used in preclinical study (referring to embodiment 3).This case inventor carries out further studies show that the safety (referring to embodiment 8) when PIKA and an antigen thing are merged into the vaccine form.

The subsequent experimental result that this case inventor carried out in China in 2002 also shows, application PIKA can provide a kind of safe and efficient people to use adjuvant.The result of this experiment before was not disclosed, and was presented at now in embodiment 9.

Therefore, with the aforementioned basis that is viewed as, the molecule comprised in the preferred embodiment of PIKA has physical characteristic on molecular weight and/or size, these physical characteristics can increase the benefit of effect intensity and effectiveness, provides simultaneously a suitable safety scope not cause any adverse side effect.Existing molecule in Av-PICKCa, position molecular weight ranges than low side, can be effective as a kind of antiviral synthetic, but while in an immunogen constituent, being used as adjuvant, significantly than the molecular composition poor efficiency of PIKA.In addition, PIKA has shown to have the safety performance better than PICKCa.

Therefore, a molecular weight that critical aspects is present composition PIKA of the present invention.

The creativity of PIKA forms and generally comprises one group or a group molecule, wherein these molecules have physical characteristic on for example molecular weight and/or size, these physical characteristics provide required effect in induction of immunity reaction, and lower better or avoid adverse side effect (for example, while giving PICKCa concurrent adverse side effect).Generally speaking, the molecule of PIKA is heterogeneous on molecular weight and/or size.

Except as otherwise noted, otherwise the adjunvant composition PIKA of the present invention be generally called in this description comprises PIC, and this PIC can be for example, for example, with an antibiotic (kanamycin) and a cation (calcium) mutually compound.Molecule in PIKA for example, for example, is heterogeneous on molecular weight (estimating with dalton) or size (estimating with sedimentation coefficient).

For example, when by a scope, describing the heterogeneous character (molecular weight or size) of PIKA molecule, the description of this scope refer in this manual the PIKA molecule in compositions molecular weight or the summary of size under and the upper limit, rather than hint or point out that said composition has each molecular weight of representing in this scope or the PIKA molecule of size.Therefore, for example molecular weight ranges is approximately 66,000 to 1,200,000 dalton refer to that approximately 66,000 dalton are to approximately 1,200,000 daltonian PIKA molecule is comprised in compositions, and 88,000 daltonian PIKA molecules must not be present in compositions (although this molecule can exist really).

When with molecular weight ranges, defining the physical characteristic of the PIKA molecule in the present composition, these PIKA molecules are heterogeneous on molecular weight, and wherein this molecular weight ranges is approximately 300,000 to 660,000 dalton, approximately 300,000 to 1,200,000 dalton, about 66,000 to 660,000 dalton, or approximately 66,000 to 1,200,000 dalton.

The present invention also relates to the compositions with the PIKA molecule that is heterogeneous on molecular weight, wherein this molecular weight ranges is approximately 300, 000 to 2, 000, 000 dalton, approximately 300, 000 to 4, 000, 000 dalton, approximately 500, 000 to 1, 000, 000 dalton, approximately 1, 000, 000 to 1, 500, 000 dalton, approximately 1, 500, 000 to 2, 000, 000 dalton, approximately 2, 000, 000 to 2, 500, 000 dalton, approximately 2, 500, 000 to 3, 000, 000 dalton, approximately 3, 000, 000 to 3, 500, 000 dalton, approximately 3, 500, 000 to 4, 000, 000 dalton, approximately 4, 000, 000 to 4, 500, 000 dalton, or approximately 4, 500, 000 to 5, 000, 000 dalton.Position all is present in compositions at the upper and lower limit place and position of these scopes PIKA molecule in these scopes.

When with mean molecule quantity, defining the physical characteristic of the PIKA molecule in the present composition, the mean molecule quantity of this PIKA molecule can be equal to or higher than 150,000 dalton, be equal to or higher than 250,000 dalton, be equal to or higher than 350,000 dalton, be equal to or higher than 500,000 dalton, be equal to or higher than 650,000 dalton, be equal to or higher than 750,000 dalton, be equal to or higher than 1,000,000 dalton, be equal to or higher than 1,200,000 dalton, be equal to or higher than 1,500,000 dalton, or be equal to or higher than 2,000,000 dalton.

When with sedimentation coefficient, defining the physical characteristic of the PIKA molecule in the present composition, sedimentation coefficient is a tolerance of molecular weight and size, and the sedimentation coefficient of this PIKA molecule (S) can be higher than 9S or higher than about 12S or higher than about 13.5S, or higher than 15S, or higher than 16S, or higher than 17S, or higher than 18S, or higher than 19S, or higher than 20S, or higher than 21S, or higher than 22S, or higher than 25S, or higher than 30S.

In certain embodiments, the invention provides a kind of polynucleotide adjunvant composition, comprise PIC (PIC), kanamycin and calcium, wherein said composition eliminating one can record the molecule of quantity, the molecular weight of these molecules is approximately or lower than 30, 000 dalton, approximately or lower than 40, 000 dalton, approximately or lower than 50, 000 dalton, approximately or lower than 60, 000 dalton, approximately or lower than 70, 000 dalton, approximately or lower than 80, 000 dalton, approximately or lower than 90, 000 dalton, approximately or lower than 100, 000 dalton, approximately or lower than 150, 000 dalton, approximately or lower than 200, 000 dalton, approximately or lower than 250, 000 dalton, approximately or lower than 300, 000 dalton, approximately or lower than 350, 000 dalton, approximately or lower than 400, 000 dalton, approximately or lower than 450, 000 dalton, approximately or lower than 500, 000 dalton, approximately or lower than 600, 000 dalton, approximately or lower than 700, 000 dalton, approximately or lower than 800, 000 dalton, approximately or lower than 900, 000 dalton, or approximately or lower than 1, 000, 000 dalton.In this embodiment, do not have at these molecules that are excluded that on the degree of significant immunogen effect, to get rid of these lower molecular weight molecules be particularly advantageous.

This case inventor shows in specific toxotest, comprise molecular weight and reach 1.0x10 ⁶The PIKA of daltonian molecule is safe (referring to embodiment 7) for animal.Including highest weight is 1.2x10 ⁶The PIKA of daltonian molecule is used in clinical front test (referring to embodiment 3) safely.When PIKA is used in an immunogen constituent, also show and have safety (referring to embodiment 8).The composition of PIKA provides advantage on effectiveness.Include molecular weight and be low to moderate 6.6x10 ⁵The PIKA of daltonian molecule also can induce effective immunoreation when being used in the mankind and animal, and has the safety of wide wide region.The molecular weight of needed smallest molecule is increased to 6.6x10 ⁵Dalton, and be preferably and be promoted to 3.0x10 ⁵Dalton, can improve the effectiveness of adjuvant, and without on safety standards, compromising.

This case inventor further finds, the concentration of polynucleotide adjunvant composition may have influence on the molecular weight of contained molecule in said composition.The molecular weight of PICKCa has shown that meeting increases (referring to embodiment 5) along with the rising of the concentration of adjunvant composition.This case the inventor observe, and the rising of polynucleotide adjuvant concentration can cause PICKCa molecule coalescent (or claiming coagulation), and makes molecule have higher molecular weight.This process has been shown as irreversible.Therefore, follow-up being diluted in a suitable medium of polynucleotide adjunvant composition can not caused the reduction of adjuvant molecules molecular weight.If observed in embodiment 6, when combined, result obtains a compositions that remains with the high molecular scope when the concentrated macromole form of polynucleotide adjunvant composition and rabies antigen.With the formed a kind of rabies vaccine of this mode, in the human clinical trial, demonstrate adverse side effect (referring to embodiment 4).

PIKA compositions of the present invention can be allocated in any physiologically acceptable buffer, but take phosphate buffer as good.Also can use as the replacement product of other acceptable buffer such as acetate, Tris (tris), bicarbonate, carbonate as phosphate buffer.

The pH value of aqueous solution composition is better between 4.0 and 10.0, but better be that the pH value of this system is adjusted to 6 to 8.5, this pH value can significantly not reduce other stability formed part, and is unlikely to incompatible on physiology.In certain embodiments, the moisture of this immunogen constituent is the normal saline of bufferingization.While wanting to give these constituents with the gastrointestinal tract external square type, be preferably that these solution is made to tension force (being osmotic pressure) is basically identical with normal physiological body fluid, to avoid constituent because of the tissue bulking or absorb fast after giving of the diversity ion concentration between constituent and physiological fluid.

The consumption of bufferingization normal saline in these constituents is that constituent numerical value is reached to the required consumption of integer.That is to say, enough buffering normal saline solution consumptions will mix mutually with other composition and reach 100%, so that constituent reaches volume required.

In certain embodiments, antigen can be purified and obtain, obtained by solid phase synthesis from a natural origin, or is obtained by gene recombination technology.Antigen can comprise a protein fragments, and this protein fragments contains one or more immunogen district of this molecule.Antigen also can from live, intact cell or microorganism (for example complete virion) attenuation or truncate or that be inactivated provide.

In other embodiments, antigen comprises one or more composition, other mankind's antigen that stems from infective micro-organisms, plant antigen, cancerous protuberance, anaphylaxis material and for example can cause autoimmune disease.In other embodiments, antigen comprises one or more infective micro-organisms, stems from any in virus, antibacterial, mycobacteria, fungus and parasite.

Polynucleotide adjunvant composition of the present invention also can be used for the Promote immunity reaction, and this immunoreation is for the antigen that uses DNA vaccination to produce.DNA sequence in order to antigen encoding in these vaccines can be " exposed ", or is accommodated in a carrier system as liposome.

In certain embodiments, this polynucleotide adjunvant composition can be used in combination with vaccine.Vaccine is can contain other adjuvants or not contain other adjuvants.Included vaccine kind is infection disease, anticancer, antiallergic, anti-autoimmune and immunological contraception.

The present invention also relates to the Combination application of polynucleotide adjuvant of the present invention and any applicable rabies antigen.

In certain embodiments, rabies antigen can be the rough rabies antigens of deactivation such as rabies antigen as rough as the deactivation of hamster nephrocyte (HKC-ICRA), or the deactivation purification type rabies antigen such as hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA).

In certain embodiments, this polynucleotide adjunvant composition can be used jointly with a rabies vaccine.The rabies vaccine be applicable to is commercial commercially available or still in research and development, comprise deactivation type, subunit type, gene recombinaton type and polypeptide type vaccine, mankind's double somatocyte vaccine (HDCV) for example, or hamster nephrocyte deactivation purification semple type rabies vaccine (HKC-IPRV), or the rough rabies vaccine of hamster nephrocyte deactivation (HKC-ICRV), or purification type Vero cell rabies (PVRV), or purification type chick-embryo cell (PCEC), or purification type duck embryo vaccine (PDEV).But these rabies vaccine not can both be induced before exposure and after exposing, have the cell immune response of importance in immunization.For example, when polynucleotide adjunvant composition (PIKA) and rabies vaccine give jointly, the immunoreation be caused comprises: non--specific reaction (for example macrophage function rising), humoral response (for example promoting the generation of specific antibody) and cell immune response (for example produce cytokine, comprise interferon and interleukin-2).

In certain embodiments, the invention provides a kind of combined complete (KIT), comprise this polynucleotide adjuvant and antigen thing.

The immunogen constituent of a kind of PIKA of including can cause specific immune response in two kinds of modes: i) humoral immune reaction, (other cell also relates to the antibody response process that produces to relate to stimulation B cell generation antibody or immunoglobulin, antigen-oblatio cell (APCs for example, comprise macrophage) and helper T lymphocyte (Th1 and Th2), and ii) cell immune response, the generation that relate generally to the T cell, comprise cytotoxic T lymphocyte (CTLs) and other relates to ctl response for example, Th1 and/or Th2 cell and APCs.Industry has known to assess the method (referring to embodiment 10,11,12 and 13) of an intraindividual body fluid and/or cell immune response.

In addition, this polynucleotide adjunvant composition can (be IgG1, IgG2, IgG3 and IgG4 for IgG s by affecting the immunoglobulin hypotype (heterogeneous) that produce; For mouse IgG s, be IgG1, IgG2a, IgG2b and IgG3) and their affinity, immunoreactive character changed.

In the Mus body, the reaction regulated and controled by the Thl cell can produce IgG1, IgG2a, IgG2b and less IgG3, also is conducive to produce cell immune response for antigen.If the IgG reaction for antigen is regulated and controled by Th2 type cell, it is mainly the generation of promoting IgGI and IgA.

The NIH potency test that utilizes a kind of constituent consisted of PIKA adjuvant and hamster nephrocyte deactivation purification type rabies antigen to carry out unexpectedly demonstrates, and the immunity intensity of this constituent needs exist (referring to the embodiment 14) of a minimum rabies antigen.The effect intensity of this constituent increases sharply with respect to the existence of the extra rabies antigen that surpasses 1IU antigen amount.Therefore, through observing, this constituent is to peak when there is the rabies antigen of the 1.5IU to 2.5IU that has an appointment in constituent advancing the speed on effect intensity.This NIH potency test is described in: Laboratory Techniques in Rabies, Edited by F X Meslin, M M Kaplan H Koprowski, 4th Edition, ISBN92415441.

Utilize the test that a kind of constituent consisted of PIKA adjuvant and hamster nephrocyte deactivation purification type rabies antigen carries out to show, the immunity intensity of this constituent surpasses the antigen volume along with the adjuvant volume and increases.When PIKA increased for the ratio of hamster nephrocyte deactivation purification type rabies antigen, this effect intensity can increase, and the preferred volume ratio is referring to embodiment 15 higher than 3:1().

The present invention relates to polynucleotide adjuvant of the present invention and the common method of using of antigen, for example to induce antigenic specificity humoral response and/or the Specific T cell immunity reaction in body.The immunoreation of inducing can be in there is no before immune individuality the reaction for antigen, or can for example, in order to existing immunoreation (Booster) before promoting.

In certain embodiments, PIKA adjunvant composition and a kind of immunogen constituent that comprises PIKA adjuvant and antigen thing can be by cold Frozen dryings, and the solid form long time is stable to be preserved to be.Cold Frozen seasoning is to have the knack of the art technology personage to know.The immunogen constituent lyophilized products that will contain PIKA and antigen thing is dissolved in water and shows that it maintains original effectiveness level (referring to embodiment 16).

This immunogen constituent can be prepared to as a kind of injectable type solution, suspension or Emulsion.The preparation of required immunogen constituent formula is described in New Trends and Developments in vaccines, the people such as edited by Voller in general manner., University Park Press, Baltimore, Md., USA, 1978.Immunogen constituent of the present invention can be used as following dosage form, forms such as capsule, solution, Emulsion, suspension or elixir comes gastrointestinal tract to give, any non-activity vehicle can be for preferred, normal saline solution for example, or the phosphate-buffered normal saline solution, or can suitably dissolve compound that the inventive method the used any vehicle for the inventive method application.

Immunogen constituent of the present invention can utilize the multiple method of knowing to give body.In certain embodiments, the immunogen constituent can give by the outer approach of gastrointestinal tract, injection systems such as muscle, abdominal cavity, vein, subcutaneous injection, or suck through respiratory tract.In other embodiments, but this immunogen constituent per rectum, vagina, nose, mouth, eye, transdermal or Intradermal part give.When the antigen thing be encapsulated is injection when the form of giving, can rests on injection site and reach for two weeks, thereby provide an antigen storage that produces continuous schedule release or pulsed release in vivo to release a little.This transmission system can be made single injection type immunogen formula for the antigen thing that needs the reaction of multiple injection ability induction of immunity.

For example, for aqueous solution, carrying out for gastrointestinal tract gives outward, this solution where necessary should be by bufferingization suitably, and first with enough normal saline solutions or glucose, dilutes so that it presents to wait opens.These specific aqueous solutions are specially adapted to vein and intraperitoneal gives.About this point, have the knack of the art technology personage and can know operational sterile aqueous matchmaker matchmaker thing with reference to this case disclosure.Can comprise the buffer that contains or do not contain dispersant and/or antiseptic for the present invention's typical injection Vehicle, and edible oil, mineral oil, cod liver oil, Squalene (squalene) ,-, two-or triglyceride, and the mixture of these compositions.

These form required accurate dosage is along with individual different, according to individual species, age, body weight and general state, and the seriousness of the disease that is treated or prevents, infection or the patient's condition, the predetermined substance used and its form that gives etc. and change to some extent.Have the knack of after the art technology personage learns the teaching content of this description, can only utilize routine experiment and determine suitable consumption.After giving for the first time, individuality can accept one or the supplementary immunization of appropriate intervals repeatedly again.

Above said content is to narrate in general manner the present invention.Below describe embodiment and can assist to understand the present invention.These embodiment only be take and are illustrated as purpose, but not intention is limited for scope of the present invention.When situation becomes or be giving treatment in accordance with seasonal conditions, replace when relating to the present invention's variation and equivalence in form.Although this description is used specific term, these terms, are intentions for explanation, rather than to be restricted to purpose.

Embodiment

Embodiment 1. measures the molecular weight of PIKA and AV-PICKCA

This embodiment illustrates the mensuration mode of PIKA adjuvant, and makes comparisons with Av-PICKCa.

Agarose gel electrophoresis is known by haveing the knack of in the art technology personage, so this description is only narrated characteristics of the present invention.Agarose gel used in the present invention has the concentration of 1.5% agarose.Molecular marker is the 100bp DNA ladder shape bands of a spectrum of 100bp to 1000bp, corresponding to 6.6x10 ⁴To 6.6x10 ⁵Daltonian molecular weight ranges.The concentration of 4ul load sample is 1mg/ml.Fig. 1 shows a representative graph with reference to the resulting agarose gel sample result of teaching content of this paragraph.The test of five (5) different batches shows the broad range of their molecular weight distribution.Their the molecular weight upper limit is that position is 2.3x10 at Av-PICKCa ⁵Dalton to PIKA be 5.28x10 ⁵In daltonian scope.

The comparison of the immune effectiveness of embodiment 2.PIKA and AV-PICKCA

This embodiment shows that highest weight is that 230,000 daltonian Av-PICKCa and highest weight are the differences of 528,000 daltonian PIKA samples on effect intensity.

By the PIKA adjuvant of three batches of different molecular weights, and a collection of molecule had corresponding to the molecular weight of Av-PIKA molecular weight, and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) are combined.Subsequently, make the gained constituent accept the NIH potency test.

This NIH potency test is a strict and lot of experiments comparative study, relatively to be examined rabies vaccine and standard type rabies vaccine.With a rabies virus strain alive, infect the mice after vaccination, and measure their survival rate.The rabies vaccine of different dilute strengths is given to the mice of different groups.The survival rate be exposed between the mice group of experimental and standard type vaccine is compared, and determine effect intensity (the Laboratory Techniques in Rabies of experimental vaccine, editor F X Meslin, M M Kaplan H Koprowski, the 4th edition, ISBN92415441).

By each combination of being checked the relative vaccine standard substance of effectiveness of vaccine of PIKA adjuvant standardized, wherein the effectiveness of vaccine standard substance is 1 by demarcation, and relative effectiveness is demarcated into to the multiple that the more non-combined standard substance of the effectiveness of being examined combined vaccine increase.These results of table 1 general introduction.As shown in table 1, the molecular weight of PICKCa adjuvant is higher, increases the tire effectiveness of gained of rabies vaccine just higher.

Table 1. molecular weight is for the effect of rabies vaccine effect intensity

Interferon between embodiment 3.PIKA and Av-PICKCA produces relatively

This embodiment shows, has on molecular weight to be limited to 230,000 daltonian Av-PICKCa samples and to have on molecular weight to be limited to the difference of 1,200,000 daltonian PIKA sample room on the ability that inducing interferon produces.

Two batches are had on molecular weight and are limited to 1.2x10 ⁶Dalton and 4.6x10 ⁵Daltonian PIKA, and a collection of have on molecular weight be limited to 2.3x10 ⁵Daltonian Av-PICKCa makes comparisons.

Make PIKA and Av-PICKCa compositions and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) combined.By these constituents through subcutaneous injection in mice.After two hours, measure content amount in the interferon serum of every mice.The general process of measurements interference element is to have the knack of in the art technology personage to know.In simple terms, in 96 well culture plates, under the amount in 0.15ml/ hole, be vaccinated approximately 30,000 L929 cells.After three days,, to the degree at the bottom of merging hole blood serum sample is diluted with the ratio of 1:20 to 1:640 when Growth of Cells, add in hand-hole with serum 0.1ml/ hole.Each dilute sample is all with three multiple hole tests.By these culture plates under 37 ℃, cultivate to the next day.Blood serum sample is washed away.With stomatitis herpesvirus VSV granule, detecting the generation of interferon tires.Table 2 shows is tired by the interferon of generation that these mixture are induced.As shown in table 2, the molecular weight of PIKA sample is higher, and the interferon that is induced to produce just the more.

Relation between table 2. molecular weight and interferon produce

Embodiment vaccine for man clinical trial (having toxic side effects) in 4.1996 years

This embodiment shows that PICKCa adjuvant and the combined meeting of a vaccine produce one and make us unacceptable adverse side effect level after giving human immunity.

The purpose of this research is to assess safety and the immunoreation of a kind of rabies vaccine, it is that 11.95mg/ml and molecular mass are 69 that this rabies vaccine comprises concentration, 700(please notes that molecular mass is not equal to dalton in this example, referring to embodiment 5) the PICKCa adjuvant, and the rough rabies antigen of hamster nephrocyte deactivation (HKC-ICRA).The result of above-mentioned clinical trial and conclusion are from unexposed.

The patient of 40 participation clinical trials is divided into two 20 people's group.Each group was the 1st day, the 3rd day, the 7th day, the 14th day and the dosage injection through five 2ml of muscle acceptance in the 30th day.One group of rabies antigen of accepting to have the PICKCa adjuvant, and another group accepts to have the rabies antigen of aluminium adjuvant.

From the viewpoint of safety, with regard to body temperature, part and general symptom, observed in latter 24 hours, 48 hours and 72 hours in each time injection.Following observation is:

Table 3. injection has the HKC-ICRA adverse side effect afterwards of aluminium adjuvant or PICKCa

The general adverse side effect comprises: heating (1 example), a rash (2 example), arthralgia (2 example), lymph node great (1 example), throat's swelling (1 example).The locality untoward reaction comprises: injection site erythrosis (6 example).

This case inventor's follow-up study is by molecular weight and the size (referring to embodiment 5 and 6) of viewed side effect owing to molecule in adjuvant.

Relation between embodiment 5.PICKCA concentration and its molecular weight

The concentration that increases the PICKCa adjuvant that shows this embodiment causes the molecular weight of constituent to increase.

PICKCa can be made and have different concentration.Suppose when PICKCa is made into variable concentrations it is a kind of polymer composites that can multi-form existence.Experiment purpose can be reached with laser scattering technology.Laser scattering technology is widely in order to gravimetry average molecular mass (Mw) and the radius of gyration (Rg).Instrument is commercial buys and measuring process is to have the knack of in the art technology personage to know.Table 4 demonstrates, relevant with its concentration by the viewed PICKCa molecular weight of laser scattering technology.

Table 4. is by the viewed molecular weight of laser scattering technology

The concentration of PICKCa (mg/ml)	The weight average molecular mass
		11.95	6.97x10 ⁴
2.00	7.30x10 ³
		1.00	2.00x10 ³

The front concentration of embodiment 6.PICKCA and the relation between the vaccine molecular weight

This embodiment shows the relation between the molecular weight of the increase of PICKCa adjuvant molecules amount and gained constituent, and this gained constituent contains PICKCa adjuvant and the rough rabies vaccine of hamster nephrocyte deactivation.

Before the combination of PICKCa sample, concentration is also under a cloud can affect the interior antigen of vaccine.Several PICKCa samples and the rough rabies vaccine of a kind of hamster nephrocyte deactivation are combined.Experiment purpose can be reached with laser scattering technology.Laser scattering technology is widely in order to gravimetry average molecular mass (Mw) and the radius of gyration (Rg).Instrument is commercial buys and measuring process is to have the knack of in the art technology personage to know.Table 5 shows that the front concentration increase of the combination of PICKCa causes the Mw of rabies vaccine to increase.

Relation before the combination of table 5.PICKCa between the Mw of concentration and rabies vaccine

The concentration of PICKCa (mg/ml)	The weight average molecular mass	The radius of gyration (Rg)
			11.95	29.6x10 ⁴	17.2x10 ²
4.00	22.2x10 ⁴	15.0x10 ²
			2.00	13.8x10 ⁴	11.8x10 ²

1.00	5.60x10 ⁴	7.55x10 ²
			1.00	5.29x10 ⁴	6.50x10 ²

Embodiment 7.PIKA chronic toxicity test

This experiment shows the safety speciality of PIKA adjuvant when the restriction highest weight.

Regulation (WS1-XG-050-2000) according to the China national drug standard is carried out a chronic toxicity test.In simple terms, with the amount of 0.5 milliliter/mice, will contain 0.3 milligram and have on molecular weight and be limited to approximately 525,000 to the about mice of sodium chloride solution intravenous injection to five an about 18-22 gram of (5) body weight of 1,000,000 daltonian PIKA adjuvant.The mice of observation through injecting 7 days, and weigh after observing end.Its result of table 6 general introduction, show that the molecular weight of PIKA adjuvant can be up to 1.0x10 ⁶Dalton, and do not have obvious toxicity.

Table 6.PIKA adjuvant chronic toxicity test

Embodiment 8: the toxicity research of the PIKA in the vaccine constituent

The purpose of this experiment is to confirm the safety of PIKA adjuvant.

By PIKA adjuvant (molecular weight 66,000 dalton to 660,000 dalton) and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) take PIKA:HKC-IPRA as the ratio of 4:1 combined.

To be made comparisons by vaccine constituent and a kind of commercial deactivation purification semple type rabies vaccine (IPRV) of buying that PIKA and HKC-IPRA form, this deactivation purification semple type rabies vaccine (IPRV) contains an aluminium adjuvant.

Five doses of (5) vaccine constituents are given to mice when the 0th day, the 3rd day, the 7th day, the 14th day and the 28th day.The dosage given is equivalent to carry out approximately 300 times of adult's dosage of normal human subject rabies immunization.

The result of Toxicity Observation is presented at following table 7:

Table 7: the safety given after rabies vaccine is observed

Lexical or textual analysis: (frequency of observing)/(sum)

Obtaining conclusion is that the more commercial commercially available IPRV of this PIKA/HKC-IPRA combination is safer.

Embodiment 9:PIKA adjuvant is in the safe handling of human body

In 2002, utilize a constituent formed by PIKA constituent (molecular weight 66,000 to 660,000 dalton) and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) to carry out immune five (5) volunteers.This vaccine constituent is given to the volunteer when the 0th day, the 3rd day, the 7th day, the 14th day and the 30th day.

Any patient, after each time vaccination, is not observed part or systemic side effect.

Utilize standard NIH potency test to measure the effect intensity of vaccine, result is presented at following table 8:

Table 8: the effect intensity of rabies vaccine is observed

Post-immunized day	ED50	Neutralizing antibody IU/ml
			0	0	0
14	>1.9	>1.84
			45	2.35	5.17

The above results is pointed out the vaccine constituent consisted of PIKA and HKC-IPRA inducing specific immunoreation and has been induced the generation of protectiveness neutralizing antibody.

Test (cell immune response) after embodiment 10. exposes

After exposing, test is that vaccine can be eradicated from the host's health after infected the most clearly proof of cause of disease.Therefore, it is an index of vaccine-induced cell immune response.

In after exposure, checking, carry out infecting mouse with the wild type strain of rabies virus, (molecular weight ranges is from 1.65x10 to inoculate subsequently to have combined the PIKA adjuvant ⁵To 1.2x10 ⁶Dalton) hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA), or combination has the HKC-IPRA of aluminum hydroxide adjuvant (aluminium adjuvant), a commercial commercially available rabies vaccine (PVRV), or phosphate buffered solution (PBS).Result demonstrates the PIKA adjuvant and has promoted survival rate, referring to table 9.

Counteracting toxic substances test after table 9. exposes

9.1 the mortality rate after treatment

Group	Predetermined 80% dead dosage	Predetermined 50% dead dosage
			PIKA adds HKC-IPRA	2/20	0/20
Aluminium adjuvant adds HKC-IPRA	10/20	9/20
			PVRV	16/20	3/20
Matched group (PBS)	15/20	14/20

9.2 the survival rate after vaccine therapy

Group	Predetermined 80% dead dosage	Predetermined 50% dead dosage
			PIKA adds HKC-IPRA	90.00%	100.00%
Aluminium adjuvant adds HKC-IPRA	50.00%	55.00%
			PVRV	20.00%	85.00%
Matched group (PBS)	25.00%	30.00%

By subcutaneous injection live mice that rabies virus infected after injection the 6th hour, the 1st day, the 2nd day and the 3rd day with vaccine therapy.

The generation of embodiment 11. antigenic specificity cell immune response gamma type interferon

The generation of gamma type interferon is an index of Cell-mediated Immunity.

In this experiment, from two bit test group patients and two matched group individualities, adopting to obtain blood sample.These aspiration patients have been vaccinated the rabies vaccine with PIKA, and this PIKA rabies vaccine contains PIKA(molecular weight ranges position 66,000 to 660,000 dalton) and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA).Vaccination in 2.5 years before collecting blood sample.

Result in Fig. 2 demonstrates, and compared to the matched group individuality, the gamma type that two bit test group patients produce disturbs and have significant difference.

To jointly cultivate from the identical HKC-IPRA that uses in the PBMCs that separates blood sample and original immunity.After 1 day, with the ELISPOT method, detection specificity produces the cell quantity of gamma type interferon.Observe a dosage dependence effect.

The resulting conclusion of above-mentioned observation is:

The rabies vaccine that contains PIKA adjuvant of the present invention has the ability that specificity causes that the gamma type interferon produces, and mean can the inducing specific cell immune response.(that is to say, this reaction is for rabies antigen), and be different from nonspecific reaction.

The efficiency assay of embodiment 12:PIKA

The purpose of this experiment is to show the ability of PIKA in the generation of inducing gamma type interferon and interleukin 12 (IL-12).

In clean environment, will take from splenocyte sample that the normal health mice separates in variable concentrations PIKA(molecular weight ranges position 66,000 to 660,000) existence under cultivate.Cultivate after three days, utilize IL-12(p40) and gamma type interferon specific antibody, with ELISA, test to analyze the cytokine levels in supernatant.Experimental result is presented at following table 10:

Table 10: the in vitro that cytokine produces

The resulting conclusion of above-mentioned experiment is that PIKA induction of lymphocyte dosage dependence produces gamma type interferon and IL-12 cytokine.

In further testing, four (4) mices are given the PIKA(molecular weight ranges position of 500ug/ mice 66,000 to 660,000 via lumbar injection).Use phosphate buffer to test as negative matched group.Injected latter 5 hours, and took out blood sample and prepare serum.Utilize IL-12(p40) and gamma type interferon specific antibody, with ELISA, test to detect the cytokine levels in serum.Experimental result is presented at following table 11:

Table 11: the in vivo test that cytokine produces

Above-mentioned experiment conclusion is that the PIKA prompting promotes antigen presenting cell function, inducing cell immunologic function.

The common use of embodiment 13:PIKA and deactivation purification type Vero cell rabies antigen

The purpose of this experiment is that assessment combination has the effectiveness of the PIKA of deactivation purification type Vero cell rabies (PVRV).

PIKA and the PVRV that will have 66,000 to 660,000 daltonian molecular weight ranges are combined, to form a kind of rabies vaccine.With the NIH potency test, assess the effect intensity of obtained vaccine constituent.Result is presented at following table 12:

Table 12: for the NIH result of the test of PIKA and deactivation purification type Vero cell rabies

The vaccine constituent	Antigen	Adjuvant	Constituent effect intensity
				PVRV	0.021IU/ml	Nothing	0.46
PVRV adds PIKA	0.021IU/ml	PIKA	3.68

The conclusion obtained is the effect intensity that PIKA can promote deactivation purification type Vero cell rabies.

Embodiment 14. antigen doses

This experiment shows the hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) need to have minimum flow and PIKA adjuvant, and (molecular weight ranges is from 66,000 to 660,000 dalton) jointly be present in constituent, to cause the specific immune response with essence enhancement level.

The PIKA adjuvant that the rabies antigen of incremental change is added to quantitative 0.1mg.With NIH standard rabies vaccine potency test, detect its effect intensity.After the measurable original effect strength increase arrived, and before as expection, reaching the effect intensity stabilization, along with an obvious and theatrical effect strength increase (referring to table 13) is observed in the increase of antigen amount.

Table 13: the vaccine potency intensity of the hamster nephrocyte deactivation purification type rabies antigen of increment

After HKC-IPRA antigen reaches an IU, observe and increase a small amount of HKC-IPRA, PIKA induces vaccine potency intensity obviously to increase.

The conclusion obtained is before significant immune response is induced, to have minimum antigen amount.Excessive antigen amount to certain a bit after, the increase of antigen amount only can the effect intensity of feedback slightly to increase.

The ratio of embodiment 15. antigen adjuvants

This experiment demonstrates the best combined amount of hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) and PIKA adjuvant (the molecular weight ranges position is 66,000 to 660,000).

Utilize various dose antigen to mix mutually with the various dose adjuvant, add PBS to guarantee the constant of cumulative volume.Utilize the NIH potency test to measure the effect intensity of obtained vaccine.Result is presented at table 14.

The comprehensive consideration of result of the test points out, to be PIKA drop in 3 to 1 scope for the ratio of antigen at least in best vaccine combination.

Table 14: the rabies vaccine effect intensity under various antigen and adjuvant ratio

Embodiment 16.PIKA and combination have the lyophilization of the PIKA of rabies vaccine to preserve

This experiment shows that PIKA is stable under freeze-dried.

Freeze Drying Technique has been used in the long preservation rabies vaccine, can reach more than 3 years.This case inventor wants to test PIKA(molecular weight ranges from 66,000 to 660,000) and the rabies vaccine that contains PIKA in lyophilization, whether favourablely preserve.Following constituent is used in lyophilization and preserves in test: PIKA i) be not frozen, add in the lyophilization type hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) through restoring, ii) through restoring PIKA, add the dry constituent of frozen type of HKC-IPRA, the iii) commercially available rabies vaccine of lyophilization type (not adding PIKA) through restoring, and the rabies vaccine standard substance that iv) are not frozen.Table 15 demonstrates, and for the long preservation rabies vaccine, is desirable through cryodesiccated PIKA and the rabies vaccine that contains PIKA.

The impact for rabies vaccine effect intensity is preserved in table 15. lyophilization

Although the present invention is described with reference to specific embodiment, should understand that these embodiment are used as illustrative, scope of the present invention is not limited only to this.For the personage who has in the technical field of the invention general knowledge, the concrete example of selection of the present invention is clearly.The embodiment of these selections drops in the spirit and scope of the present invention.Therefore, interest field of the present invention is that the scope by appended claims refers, and can be supported by aforementioned disclosure.

Claims

1. polynucleotide adjunvant composition comprises:

PIC, antibiotic and cation, wherein said composition contains the polynucleotide adjunvant composition molecule that is heterogeneous in molecular weight or size, wherein this molecular weight is between approximately 66,000 to 1,200, in 000 daltonian molecular weight ranges, and should size in the molecular size scope of about 6.4S to 24.0S.

2. polynucleotide adjunvant composition as claimed in claim 1, wherein the molecular weight ranges of this polynucleotide adjunvant composition is from approximately 300,000 to 1,200,000 dalton, or this molecular size scope is from about 12.8S to 24.0S.

3. polynucleotide adjunvant composition as claimed in claim 1, wherein the molecular weight ranges of this polynucleotide adjunvant composition is from about 66,000 to 660,000 dalton, or this molecular size scope is from about 6.4S to 18.3S.

4. polynucleotide adjunvant composition as claimed in claim 1, wherein the molecular weight ranges of this polynucleotide adjunvant composition is from about 300,000 to 660,000 dalton, or this molecular size scope is from about 12.8S to 18.3S.

5. polynucleotide adjunvant composition comprises:

PIC, antibiotic and cation, wherein this polynucleotide adjunvant composition has and is equal to or higher than 150,000 daltonian mean molecule quantities, or has the mean molecule size that is equal to or higher than 9.3S.

6. polynucleotide adjunvant composition as claimed in claim 5, wherein the mean molecule quantity of this polynucleotide adjunvant composition is equal to or higher than 300,000 dalton, or this mean molecule size is equal to or higher than 12.8S.

7. polynucleotide adjunvant composition as claimed in claim 5, wherein the mean molecule quantity of this polynucleotide adjunvant composition is equal to or higher than 350,000 dalton, or this mean molecule size is equal to or higher than 15.3S.

8. as the described polynucleotide adjunvant composition of any one in claim 1 to 7, wherein this antibiotic is kanamycin, neomycin, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide, puromycin, streptomycin or streptozotocin.

9. as the described polynucleotide adjunvant composition of any one in claim 1 to 7, wherein this cation is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc; And wherein this cation is the form of inorganic salt or organic double compound.

10. as the described polynucleotide adjunvant composition of any one in claim 1 to 7, wherein this cation source is calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.

11. as the described polynucleotide adjunvant composition of any one in claim 1 to 7, wherein this antibiotic is Kanamycin Sulfate, and this cation is provided by calcium chloride.

12., in a combined complete, comprise as the described polynucleotide adjunvant composition of any one and antigen thing in claim 1 to 7.

13. an immunogenic composition, comprise as the described polynucleotide adjunvant composition of any one and antigen in claim 1 to 7.

14. immunogenic composition as claimed in claim 13, wherein this antigen is mankind's antigen, non-human animal's antigen, plant antigen, bacterial antigens, fungal antigen, virus antigen, parasite antigen or cancerous protuberance antigen.

15. immunogenic composition as claimed in claim 14, wherein this antigen is the rabies antigens.

16. immunogenic composition as claimed in claim 15, wherein this antigen is deactivation purification type rabies antigens.

17. immunogenic composition as claimed in claim 16, the deactivation purification type rabies antigen of this hamster nephrocyte wherein existed is greater than 1 iu.

18. immunogenic composition as claimed in claim 16, wherein this polynucleotide adjunvant composition is greater than 3 to 1 with the ratio of the deactivation purification type rabies antigen of hamster nephrocyte.

19. immunogenic composition as claimed in claim 13, wherein this immunogenic composition comprises the adjuvant of the special body fluid that can cause simultaneously enhancing and cell-mediated immunne response.

20. immunogenic composition as claimed in claim 13, this adjunvant composition wherein comprised in this immunogenic composition or this immunogenic composition be solid form or liquid form be present in solution or suspension in.

21. immunogenic composition as claimed in claim 13, wherein this adjunvant composition and/or this immunogenic composition are frozen drying.

22. the method in order to the immune response of the enhancing antagonism original, the method comprises: this adjunvant composition comprised in immunogenic composition as claimed in claim 13 or this immunogenic composition is delivered medicine to the host.

23. as claimed in claim 22 in order to strengthen the method for the immune response of resisting the original, wherein this immunogenic composition can be by being selected from an administration in the group that comprises following approach, and this approach comprises the outer drug administration by injection of gastrointestinal tract, intramuscular injection, lumbar injection, intravenous injection, subcutaneous injection, through respiratory tract suction, rectally, vagina administration, nose administration, oral administration, administration through eye, topical, transdermal or intradermal administration.

24. the purposes of polynucleotide adjunvant composition as described as claim 1 to 7 any one in the medicine of the immune response for the preparation of strengthening the host.

25. as claimed in claim 22 in order to strengthen the method for the immune response of resisting the original, wherein this host is the mankind.

26. as claimed in claim 22 in order to strengthen the method for the immune response of resisting the original, wherein this host is animal.

27. a polynucleotide adjuvant constituent comprises:

PIC, antibiotic and cation, wherein the preparation concentration of this polynucleotide adjuvant constituent should be in the 0.1-10mg/ml scope.