CN1285753A - Transdermal delivery of particulate vaccine compositions - Google Patents

Abstract

A method for enhancing the immune response to a selected antigen is disclosed. The method entails delivering a particulate adjuvant composition transdermally, preferably using a needleless syringe system. Also described are methods for forming crystalline particles from pharmaceutical compositions and then delivering the same to a subject. The crystallized compositions are particularly suitable for transdermal vaccine delivery using a needleless syringe system.

Description

Transdermal delivery of particulate vaccine compositions

The present invention relates to microparticle compositions.More particularly, the present invention relates to send the method for microparticle compositions, promptly form crystalline particle, then it is delivered to the method for main body by pharmaceutical composition.The suitable especially needleless injector system transdermal administration vaccine that uses of this microparticle compositions.

In non-intestinal delivery technique, deliver drugs in the skin surface and the ability by skin surface (transdermal administration) has many advantages.Particularly, compare with traditional drug-supplying system, transdermal administration safety, convenience and do not have injury, and the danger that infects as acupuncture pain, processed individuality, the pollution that is brought to the healthcare worker by shank and disposable pin once in a while or the danger of infection have been avoided sending together subject matter with non-intestinal.In addition, this blood concentration of sending also institute's administration has given highly control.

Recently, described a kind of novel transdermal administration drug system, it need use needleless injector with control dosage, thereby the microgranule that will contain solid drugs is injected not have and hindered skin and hinder skin by nothing.Particularly, the US5630796 that people such as Bellhouse have has described a kind of needleless injector, and it sends the drug particle that transmits with hypersonic air flow.This needleless injector (also being referred to as " PowderJect needleless injection device ") is used for transdermal administration powdered drug chemical compound and compositions, is used for the gene material being delivered to living cells (for example gene therapy) and being used for biologics is delivered to skin, muscle, blood or lymph.Also needleless injector and surgical knot can be share in to organ surface, solid tumor and/or operation chamber (for example cavity after tumor bed or the tumor resection) delivering drugs and biological preparation.Use this equipment can be safely and easily send and to suit to be roughly the pharmaceutical agent of solid, particulate form preparation.

A concrete needleless injector generally includes an elongated tubular product such nozzle, but it has the rupture disc that the air flue of originally sealing in this nozzle also roughly is placed on this nozzle upstream end place.Be positioned at this rupture disc place and use a launch device to transmit but medicament particle to be transmitted is treated, this launch device with a gas pressure in this diaphragm upstream one side, this air pressure be enough to break this diaphragm and in nozzle, produce hypersonic air flow (containing the medicine particle) to send from its downstream.Therefore the transfer rate of these particles with the 1-8 Mach number can be transmitted from needleless injector, but this transfer rate can easily obtain by the rupture disc that breaks.

The structure of another kind of needleless injector generally includes aforesaid similar elements, it is the drug particle that replacement transmits in hypersonic air flow, the nozzle downstream is equipped with a bistable state diaphragm, this diaphragm can move between static " oppositely " position (wherein diaphragm has a concave surface to contain the medicine particle on downstream face) and movable " turning up " position (wherein the result of the supersonic shock wave effect of diaphragm upstream face makes its downstream face outwards protrude).In this mode, drug particle contained in the diaphragm concave surface is shifted target skin or mucomembranous surface with ultrasonic initial velocity onto from transdermal administration equipment.

Use above-mentioned needleless injector structure that the particle that particle size range is about 0.1-250 μ m is usually carried out transdermal administration.Particle greater than about 250 μ m also can be transmitted by this equipment, and its upper limit is that particle size is brought the injury that things turn out contrary to one's wishes to Skin Cell.The actual range of the particle penetration that is transmitted depends on initial velocity and the skin density and the kinematic viscosity of particle size (for example nominal particle diameter is that the supposition particle is for spherical), particle density, particle impacting skin surface.The target particle density that is used for Needleless injection is generally about 0.1-250g/cm ³, injection speed is generally about 200-3000m/sec.

Unique especially characteristic of needleless injector is the strict control ability of the particle penetration degree of depth that transmits, and makes the target administration arrive diverse location thus.For example, can select particle characteristics and/or equipment operation parameter, thereby for example different intradermal or the length of penetration of subcutaneous transmission are provided.A kind of approach need select particle size, particle density and initial velocity so that about 2-10kg/sec/m to be provided, more preferably from about the momentum density of 4-7kg/sec/m (for example, particle momentum is divided by the particle front face area).Can reach the transmission accurate control, tissue selectivity of drug particle to this control of momentum density.

Said system provides a kind of unique apparatus that is sent to vaccine antigen in skin or the tissue or passes through skin or tissue.But, to render a service in order to increase antigen, many antigens need use immunological adjuvant.Immunological adjuvant plays the immunoreation that increases iuntercellular and body fluid.These adjuvants comprise store adjuvant, absorption and/or precipitation the antigen of giving and be used for keeping antigenic chemical compound here at injection position.The typical case stores adjuvant and comprises aluminium compound and water-in-oil emulsion.

Increase antigenicity although store adjuvant, when subcutaneous or intramuscular injection, often caused serious persistent local response, for example rheumatic granulomas, abscess and cicatrization.Other adjuvant, for example lipopolysaccharide and muramyldipeptide can cause pyrogenic reaction and/or conjunctivo-urethro-synovial syndrome (influenza class symptom, dispersivity joint do not accommodate anterior uveitis, arthritis and urethritis sometimes) by injection.Therefore, need one effectively and the method for safe and continuous transmission adjuvant, thereby improve given antigenic immunoreation.

The invention provides the unique system of unique adjuvant and vaccine combination and transmission drug particles compositions, this pharmaceutical composition comprises vaccine and other medicament.The present invention also provides the novel method of preparation drug particles compositions.

In one embodiment, provide a kind of method that improves selected antigen immune.This method comprises:

(a) antigen of effective dose is invested the vertebrates main body; With

(b) be enough to improve the particle adjuvant compositions of the amount of antigen immune, wherein adjuvant transmitted into or skin or tissue by the vertebrates main body, wherein also use the transdermal administration technology to carry out administration.

Antigen and adjuvant can appear in the identical or different compositions, and can be administered into the identical or different position of vertebrates main body.And, can be before or after adjunvant composition or give antigen with it simultaneously.

In particularly preferred embodiments, use the needleless injector transporter to give adjuvant and/or antigen.

In another embodiment, the present invention relates in the vertebrates main body, cause immunoreactive method.This method comprises the particulate vaccine compositions transdermal administration in the skin of vertebrates main body or tissue or by wherein.This particulate vaccine compositions comprises:

(a) the selected antigen of effective dose; With

(b) present in an amount at least sufficient to improve the adjuvant of antigen immune.

In another embodiment, the present invention relates to suitable use the transdermal administration technology transfer in vertebrates main body skin or tissue or the particle adjuvant compositions by wherein.Can with this particle adjuvant compositions by in the skin of vertebrates main body or tissue or the particle adjuvant compositions of the amount by wherein being enough to cause physiological effect be used to cause physiological effect.

In another embodiment, provide a kind of method that the conventional medicament preparation is transformed into the crystalline particle that preferably uses the needleless injector transdermal administration.Therefore, in one aspect of the invention, liquid drug preparation (for example, perhaps with moisture form, perhaps with the reorganization lyophilized products) is mixed with proper excipient is for example sugared, drying obtains crystal composition then.This excipient makes it have enough rigidity, structure and density in the gained crystallized product through selecting.Can directly the crystal composition that has sufficient density now be used for the Needleless injection medicine-feeding technology, perhaps can further process so that more segmentation and/or uniform crystal composition to be provided.

In an embodiment more of the present invention, the crystalline drug compositions is sent to main body, so that bring desirable processing.One concrete aspect, by Needleless injection the crystallization vaccine combination is sent to main body, so that in main body, have biological respinse.In a preferred embodiment, this crystallization vaccine combination is sent to main body, thereby in main body, causes the specific antigen immunoreation.

In another embodiment of the invention, provide the crystallization pharmaceutical composition.This crystalline drug has enough grain structures, rigidity and/or density feature, and this makes it suit to use the needleless injector system to be sent in skin or the mucosal tissue or passes through wherein.Can use method of the present invention to prepare crystalline drug compositions of the present invention, and therefore comprise vaccine combination.

According to content disclosed herein, those of ordinary skills can easily expect these and other embodiment of the present invention.

The accompanying drawing summary

Figure 1A and 1B have described and estimated the result of particle size to the embodiment 1 of IgG antibody response influence in the particulate vaccine preparations.

Fig. 2-4 has described enzyme-linked immunosorbent assay (ELISA) result of the serum that is obtained by the mouse of using needleless injector to transmit the immunity of crystallization Hib conjugation vaccine combination.In Fig. 2, with the PRP-CRM197 conjugate as catching phase, in Fig. 3, with diphtheria toxin, diphtherotoxin as catching phase, in Fig. 4, with the PRP-HSA conjugate as catching phase.

Fig. 5 has described the IgG antibody response of accepting the Hib conjugation vaccine combination of reduction dosage in the main body with microgranule or liquid form.

Fig. 6 A and 6B have described the immunologic process that Hib conjugation immune composition is provided with microgranule or liquid form.

Fig. 7 has described the antibody response of the inactivation influenza virus vaccine compositions that transmits with microgranule or liquid form.This data represented serum IgG titer geometric mean from pooled serum.

Fig. 8 A and 8B have described the result of influenza virus stimulation study in the main body of the inactivation influenza virus vaccine compositions immunity that transmits in order to microgranule or liquid form.Main body among Fig. 8 A is accepted 25 μ g inactivation viruses, yet the main body among Fig. 8 B is accepted the inactivation virus of 5 μ g.This data represented average percentage that originally loses weight.

Fig. 9 has described in the result who transmits and with aluminum be influenza virus stimulation study in the main body of inactivation influenza virus vaccine compositions immunity of adjuvant in order to microgranule or liquid form.These data represented 8 average percentage that originally animal loses weight.

Figure 10 has described in the result who transmits and with PCPP be influenza virus stimulation study in the main body of inactivation influenza virus vaccine compositions immunity of adjuvant in order to microgranule or liquid form.These data represented 8 average percentage that originally animal loses weight.

Figure 11 A and 11B have described in the result who transmits and with CpG be influenza virus stimulation study in the main body of inactivation influenza virus vaccine compositions immunity of adjuvant in order to microgranule (Figure 11 A) or liquid form (Figure 11 B).These data represented 8 average percentage that originally animal loses weight.

Figure 12 has described in the result who transmits and with MPL be influenza virus stimulation study in the main body of inactivation influenza virus vaccine compositions immunity of adjuvant in order to microgranule or liquid form.These data represented 8 average percentage that originally animal loses weight.

Before describing the present invention in detail, be interpreted as the present invention and be not limited in concrete pharmaceutical preparation or the machined parameters, nature can change. Also should understand term used herein only is in order to describe the specific embodiment of the invention, not to be intended for use to limit the present invention.

Must be noted that in this specification and the appended claims book singulative " " and " this " comprise plural indicant, unless clearly demonstrate in addition. Therefore, for example " a kind of medicament " comprises the mixture of two or more medicaments, and " a kind of antigen " comprises the mixture of two or more antigens, and " a kind of excipient " comprises the mixture of two or more excipient, etc. A. definition

Except as otherwise noted, common understand equivalent in meaning of those of ordinary skills under all scientific and technical terminologies used herein and the present invention. Although can be with many with described same or analogous method and material are for test of the present invention herein, described herein be preferred material and method.

When description is of the present invention, uses following term, and plan to be defined as follows.

" cutaneous penetration " meaning is that particle is sent to destination organization, thereby part, zone or general reaction are provided. This forms contrast with material is directly added in living cells and the design tissue that level is performed the operation in born of the same parents by cell membrane. Preferably, the particle size of the material of giving is greater than the cell size that occurs in the destination organization. Usually, for mammalian cell, will obtain this desirable effect greater than the particle of 10 μ m. Suitable particle size scope will be in following discussion.

Therefore, term " transdermal " the administration meaning is (for example entering corium or epidermis), transdermal (for example " through skin ") and transmucosal administration in the corium, namely is administered in skin or the mucosal tissue by reagent passage or by wherein. Referring to for example Transdermal Drug Delivery:Developmental Issues and Research Initiarives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery:Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987) and Transdermal Delivery of Drugs, Vols.1-3, Kydonieus and Berner (eds.), CRC Press, (1987).

" needleless injector " meaning is the apparatus of transdermal delivery microparticle compositions, does not insert traditionally the pin of skin. Discussion to the used needleless injector of the present invention will run through in full.

Term used herein " medicament " meaning is any compound or composition, will induce required pharmacology and/or physiological effect by part and/or general action when it being administered into organism (human body or animal body). Therefore this term comprises those compounds or the chemical reagent of thinking traditionally medicine and vaccine, and comprises for example peptide, hormone, nucleic acid, the equimolecular biological agent of gene structure.

" antigen " meaning is the molecule that contains one or several epi-position, and this epi-position produces the specific immune response of cellular antigens or humoral antibody reaction with the stimulation of host immune system. Therefore, antigen comprises protein, polypeptide, antigen protein fragment, compound sugar, polysaccharide etc. And this antigen can derive from any known viruse, bacterium, parasite, plant, protozoan or fungi, also can be whole organism. This term also comprises tumour antigen. Similarly, oligonucleotide or the polynucleotide of expression antigen are also included within this antigen scope in for example dna immunization is used. Also comprise synthetic antigen, such as multi-epitope, side epi-position and other restructuring thing or synthetic gained antigen (people (1993) Eur.J.Immunol.23:2777-2781 such as Bergmann; The people such as Bergmann (1996) J.Immunol. 157:3242-3249; Suhrbier, A. (1997) Immunol. and Cell Biol. 75:402-408; The people such as Gardner (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-Junly 3,1998).

Term " vaccine combination " meaning is the pharmaceutical composition that contains arbitrarily antigen, and said composition can be used for disease or the symptom of prevention or treatment main body. Therefore this term had both comprised subunit vaccine, the vaccine combination that namely contains antigen, this antigen is to separate and disperse at relevant with antigen in nature whole organism, also comprise contain wholely be killed, the composition of bacterium, virus, parasite or other microorganism of dilution or inactivation.

Viral vaccine composition used herein comprises, but be not limited to, contain or from the member of following section: thin RNA Viraceae (such as poliovirus etc.), Caliciviridae, Togaviridae (such as rubella virus, dengue fever virus etc.), flavine Viraceae, coronaviridae, Reoviridae, birnavirus section, Rhabdoviridae (such as hydrophobin etc.), filamentous virus section, Paramyxoviridae (such as mumps virus, measles virus, respiratory syncytial virus (RSV) etc.), orthomyxoviridae family's (such as first, second and influenza virus C etc.), Bunyaviridae, grains of sand sample Viraceae, Retroviridae (for example, HTLV-I, HTLV-II, HIV-1 and HIV-2), SIV (SIV) and other virus. In addition, viral antigen can be from papillomavirus (such as HPV), herpesviral, hepatitis viruse, for example hepatitis A virus (HAV), hepatitis type B virus (HBV), HCV (HCV), hepatits delta virus (MDV), E Hepatitis virus (HEV) and G Hepatitis virus (HGV) and Far East Russian encephalitis virus. Referring to, Virology for example, 3^rd Edition(W.K.Joklik ed.1988)，Fundamental Virology，2 ^ndEdition (B.N.Fields and D.M.Knipe, eds.1991) has described these and other virus. Mattress seedling composition used herein comprises, but be not limited to, contain or derive from cause following organic those: diphtheria, cholera, pulmonary tuberculosis, lockjaw, pertussis, meningitis and other pathogenic state, the example that comprises the helicobacter pylori of first type, B-mode and the third type diplococcus meningitidis, the bloodthirsty bar mattress of influenza B (HIB) and anti-parasitic vaccine combination comprise and derive from the organism that causes malaria and Lyme disease.

Contain selected antigen and adjuvant together composition or with the main body adjuvant vaccine combination of administration altogether, when it have than the equivalent antigen that does not give adjuvant larger cause immunoreactive ability the time, demonstrate " the immunogene performance of raising ". Since the stronger immunogenicity of antigen or since in the main body of giving antigen more low dosage or still less the antigen of dosage reach immune response, so vaccine combination can present " the immunogene performance of raising ". The immunogenicity of this raising can be by following mensuration: adjunvant composition and antigen controlling agent are administered into animal body, Application standard is measured relatively antigen titration degree and/or relatively the two iuntercellular immunity, this standard test such as the known radiommunoassay in capable territory, enzyme linked immunosorbent assay (ELISA), CTL mensuration etc. For the purposes of the present invention, " effective dose " of adjuvant should be to improve the altogether amount of administration antigen immune reaction, so that antigen presentation goes out the immunogenicity that improves as mentioned above.

Similarly, " effective dose " of antigen be stimulate immunoreactive amount in the main body to antigen. Immune response can be body fluid, intercellular and/or the protective immune response.

Term used herein " altogether administration " is as when adjuvant and the common administration of vaccine antigen, the meaning is or while or parallel adjuvant and the antigen of transporting, for example when both in same combination, occurs or both in while almost but administration in the composition that diverse location is separating, and adjuvant and antigen are carried at different time in separate compositions. For example, can before or after carrying antigen, identical or different position carry adjunvant composition. Carry adjuvant and carry between the antigen can the interval a few minutes, by several hours, by several days. And, although use Transdermal Delivery such as needleless injector that preparation compositions is administered into skin, can use traditional medicine-feeding technology, as passing through traditional syringe and traditional vaccine rifle administration of vaccines composition.

Term used herein " processing " comprises arbitrarily following: prevention infection or infect again reduces or eliminates symptom, and reduces or eliminate pathogen fully.Processing can play prevention (before the infection) or treatment (after the infection) effect.

" vertebrates main body " meaning is heart-shaped member, particularly mammal of any subphylum, includes, but not limited to human and other primates.The concrete age do not indicated in this term.Therefore, adult and newborn individuality all should be included.

Medicine separately or with other medicines or reagent, is typically made the pharmaceutical composition that can contain one or more additional materials such as carrier, carrier and/or excipient." carrier ", " carrier " and " excipient " typically refer to inertia, nontoxic and not with compositions in other component with harmful interactive material of mode.Can use these materials to increase the amount of solid in the drug particles compositions, for example use those of dispute mist drying or Freeze Drying Technique preparation.The example of used usually " excipient " or " carrier " comprises pharmaceutical grade glucose, sucrose, lactose, trehalose, mannitol, Sorbitol, inositol, glucosan, starch, cellulose, sodium phosphate or calcium, calcium sulfate, citric acid or tartaric acid (with its pharmaceutically acceptable salt), glycine, high molecular weight polyethylene glycol (PEG) and its combination.Illustration excipient with used as stabilizers comprises common obtainable cryoprotector and antioxidant.B. universal method

The invention provides delivery of particulate pharmaceutical composition, particularly vaccine combination.Can use transdermal needleless injector administration device to these microparticle compositions of body delivery.On the existing inoculation method basis that relies on conventional needle and injector to inject technology usually, be improved to the ability of tissue with microgranule (powder) form as skin transdermal administration vaccine combination.Thus, nearly all present vaccine is all by administered intramuscular.But the vaccine of being injected needs the partial draining lymph node, so that the beginning immunoreation.Most of vaccine combination of intramuscular injection will be diffused into surrounding tissue and circulation, therefore loss or diluted very soon.On the contrary, when skin transdermal administration vaccine combination for example, such loss will not take place.This is because the vascularity of upper layers of skin is poor, therefore has the antigenic ability of stronger reservation.Because the course of dissolution of particulate vaccine compositions in skin is slow, so it is retained in the skin better.Cellular component in the skin also can help strengthening the performance of the vaccine of transdermal administration.This is owing to there is the dense network of arborescent cell in the hypodermic layer of langerhans' cells and skin in the epidermal area.These cells are very important to beginning and maintenance immunoreation.By near these immunocytes, carrying vaccine combination, can reach the immunoreation stronger than traditional intramuscular injection.These immunocytes also can be picked up this vaccine and move on to the partial draining lymph node, thus the beginning immunoreation.

Microparticle compositions has also been strengthened the safety and the effect of immunomodulator commonly used such as adjuvant to the transdermal administration of skin or mucosal tissue.Immunomodulator is the important component of vaccine and immunization therapy normally.Immunomodulator has many functions, comprises for example reinforced immunological, inhibition immunity and regulates immunity.The effect of vaccine or immunization therapy has been strengthened in the reinforcement of immunity.The reinforcement of immunity also makes immune system react to more low dose of vaccine combination.For example, aluminum (Alum) adjuvant immunity regulator is used to prepare diphtheria and tetanus toxin vaccine, thereby strengthens their immunogenicity.Some have the immunomodulator that suppresses immune performance and also are used to handle some disease, as auto-immune disease and organ transplantation.Immunomodulator also can instruct immune system generation or Th1-or the reaction of Th2-type, perhaps a kind of reaction conversions of having set up is arrived another kind of.This immunity modulating performance is extremely important to immunotherapy.For example, have the immune main body of being partial to the reaction of Th2-type and be easy to the allergy.The immunomodulator that therefore can help to improve the reaction of Th1-type is used for immunotherapy so that these individual desensitizations.

With regard to the traditional vaccine compositions, typically carry immunomodulator by intramuscular injection.One of problem of this injecting method is that immunomodulator arrives body circulation toxicity afterwards.Reason for this reason, many immunomodulators all can not be used for human body.Because most of injected material is very fast from the injection position diffusion, therefore usually in order to enter circulation effectively, intramuscular injection also needs the immunomodulator (transdermal administration immunomodulator relatively) of high dose.Thus, may need an adjuvant with its activity of generation in injection position or partial draining lymph node, thereby improve immune performance.

According to the present invention, immunomodulator to the transdermal administration of skin or mucosal tissue because of following former thereby favourable.At first, skin and mucosa are the very effective positions of immune system.As mentioned above, the dense network (for example, the arborescent cell of langerhans' cells in the epidermal area and skin layer) that in each layer of skin, has immunocyte.Mucomembranous epithelial cell contains a large amount of Intradermal arborescent cell of going up.These cells are to beginning and keep immunoreation, make their immunity modulation cause target is very important.By carrying immunomodulator in the very near place of these cells, avoid simultaneously because diffusion and disappearing fast, reach than being feasible by the stronger immune modulation effect of intramuscular injection.The effective dose of immunomodulator also can reduce greatly.Help reducing the toxicity relevant than low dosage with many immunomodulators.

Therefore, in one embodiment, the present invention needs a step with traditional pharmacy formation crystalline particle (suitable transdermal administration).Although method of the present invention can be widely used for any pharmaceutical composition, this paper specifically consults and uses liquid state (moisture) or recombinates the method illustration the present invention as raw material of dry vaccine combination.

A kind of conventional method of preparation and storage vaccine medicine relates to freezes in method (lyophilization).Lyophilization relate to one from material dewatered technology, and relate to quick freezing under low-down temperature, then under condition of high vacuum degree by the distillation fast dewatering.This technological model ground produces the low density porous particle with open matrix structure.Be stable on these chemistry of particles, but when it being added in aqueous environment, just recombinate (decompose and/or enter solution) fast.

Other method by sensitivity or preparation of temperature-sensitive biomolecule and storage vaccine combination is a spray drying.Spray drying relates to uses a nozzle, spray webbing dish or miscellaneous equipment with one or more solid solution atomizations, then evaporating solvent from droplet.More particularly, spray drying relates to liquid pharmaceutical (for example solution, serosity, emulsion etc.) with high degree of dispersion and mixes with the hot-air of appropriate volume and evaporate and dry liquid droplet.The normally narrow meshed even spheroidal particle of the feature of spray-dried medicament.These Particle Density are low and dissolution velocity is fast.

In a method of the present invention, fluid composition (moisture or the spray-dried or freeze dried compositions of recombinating) is transformed into suitable drying, the crystalline powder that is conveyed into and/or passes through skin or mucosal tissue.Can be with this fluid composition and appropriate carrier or mixed with excipients with reinforcement crystallization formation, particle structure, rigidity and/or density feature.Preferred carrier or excipient comprise pharmaceutical grade sugar and analog, comprise for example trehalose.Then said composition is carried out drying under suitable evaporation conditions, obtain crystal composition.This crystallization can be shifted out from desiccated surface or container then, for example use mortar and pestle broken gently.The gained crystalline powder can be put into the kit of suitable administration then, thereby use needleless injector that it is administered into main body.

Although in the present invention without limits, above-mentioned method can be used for obtaining size at the about 250 μ m of about 0.1-, and about 250 μ m of preferably about 10-and particle density are the about 25g/cm of about 0.1- ³Crystalline particle.These crystalline particles can be used for handling or the prevention various diseases.

In another embodiment, the present invention relates to delivery of particulate compositions, particularly adjuvant and vaccine combination.As mentioned above, adjuvant and vaccine combination can be crystal form, perhaps can be with amorphous particulate form administration.

The used antigen of the present invention can use produced in several ways well known by persons skilled in the art.Particularly, can use the standard purification technique directly to separate antigen by natural source.Perhaps, can use known technology reorganization ground to produce antigen.Referring to, Sambrook for example, Fritsch﹠Maniatis, Molecular Cloning:ALaboratoryManual, Vols. I, II and III, SecondEdition (1989); DNA Cloning, Vols. I, and II (D.N.Glover ed.1985).Can described aminoacid sequence be that the basis is through the synthetic antigen used herein of the chemical polymerization thing synthetic method of for example solid phase method of peptide synthesis also.These methods are known to those skilled in the art.Referring to, J.M.Stewart and J.D.Young for example, Solid Phase Peptide Synthesis, 2 ^NdEd., Pierce Chemical Co., Rockferd, IL (1984) and G.Barany andR.B.Merrifield, The Peptides:Analysis, Synthesis, Biology, editors E.Gross and J.Meienhofer, Vol.2, Academic Press, NewYork, (1980), pp.3-254, for solide phase peptide synthesistechniques:and M.Bodansky, Principles of Peptide Synthesis, Spinger-Verlag, Berlin (1984) and E.Gross and J.Meienhofer, Eds., The Peptides:Analysis, Synthesis, Biology, supra, Vol.1, forclassical solution synthesis.

In case after obtaining, interested antigen is formed particulate vaccine compositions as described herein and it is administered into main body, usually with the immunological adjuvants that is used to improve the antigen immune reaction.As explained above, this adjuvant can appear at identical or the independent group compound in, can with vaccine combination administration, perhaps administration before or after the antigen administration simultaneously.In addition, can carry out the adjuvant administration in identical or different position.

Unfortunately, the toxicity height of most of known adjuvants.Therefore, the adjuvant that only suitable at present human body uses is Alum, the aluminum salt composite.

But many adjuvants are used for zooscopy and carry out clinical preceding and clinical research over against several adjuvants that human body uses.

Surprisingly, being considered to human body usually uses the too high adjuvant of toxicity can use this method administration., very unclear by concrete theoretical the constraint, use the transdermal administration method to the percutaneous drug delivery adjuvant, arborescent cell interaction in langerhans' cells and the cortex in the epidermal area of permission skin.These cells are important to beginning and maintenance immunoreation.Therefore, can be by aim at the adjuvant effect that administration obtains raising to these cells or near it.In addition, because the vascularization of upper layers of skin is poor, the amount that enters body circulation adjuvant reduces, and has therefore reduced toxic effect.And because Skin Cell constantly comes off, residual adjuvant is eliminated rather than is adsorbed.And, carry the amount of adjuvant to lack than the amount of using conventional art such as intramuscular injection to carry.Therefore, the present invention can use various adjuvants effectively, and does not have toxicity together.These adjuvants include, but not limited to the adjuvant that formed by for example aluminum salt (Alum) such as aluminium hydroxide, aluminum phosphate, aluminum sulfate; Oil-in-water and water in oil emulsion liquid formulation as Complete FreundsAdjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); By the adjuvant that the bacterial cell wall fraction forms, for example comprise the adjuvant of single phosphinylidyne lipoid A (MPL) (people (1985) Tet.Lett.26:1545-1548 such as Imoto), trehalose dimycolate (TDM) and cell wall skeleton (CMS); Derive from the bacteriotoxic adjuvant of ADP-ribosylation, the potent toxin of lineup's body comprises that diphtheria toxin, diphtherotoxin, pertussis toxin, PT (PT), cholera toxin (CT), escherichia coli heat-labile toxin (LT1 and LT2), false unit cell mattress belong to A type endotoxin, bacillus botulinus C2 and C3 toxin and from the toxin of bacillus perfringens, Clostridium spiroforme and clostridium difficile, particularly ADP-ribosylation bacteriotoxin mutant CRM for example ₁₉₇, avirulence diphtheria toxin mutation (referring to, people (1992) Vaccine such as people (1989) Adv.Exp.Med.Biol.251:175 such as Bixler and Constantino for example); The Saponin adjuvant of the particle that produces as Quil A (US5057540) or by the Saponin of for example ISCOMs (immunostimulating complex); Cytokine, for example interleukin (as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), interferon (as IFN-), macrophage clone stimulating factor (M-CSF), tumor necrosis factor (TNF) etc.; Muramyl peptide such as N-acetyl group-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl group-non-muramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl group muramyl-L-alanyl-D-isoglutamine base-L-alanine-2-(1 '-2 '-two palmityls-sn-glycerol-3 hydroxyl phosphorus acyloxy)-ethamine (MTP-PE) etc.; Derive from CpG family molecule, CpG dinucleotide and comprise the synthesis of oligonucleotides nucleotide of CpG composition adjuvant (referring to, people J.Immunol. (1998) 160:870-876 such as people Vature (1995) 374:546 such as Krieg and Davis for example), for example TCCATGACGTTCCTGATGCT (SEQ ID NO:1) and ATCGACTCTCGAGCGTTCTC (SEQ ID NO:2); And synthetic adjuvant, as PCPP people Vaccines (1998) 16:92-98 such as (poly-[two (carboxyl phenoxy groups) are phosphazine] () Payne.These adjuvants can be from many suppliers such as Accurate Chemicals, RibiImmunechemicals, Hamilton, MT, GIBCO, Sigma, and St.Louis, MO is commercially available.

In case after obtaining, will have or not have antigenic adjuvant interested to use any suitable granulating technique to form the particle of suitable transdermal administration, these technology for example have air-dry (crystallization), lyophilization (lyophilizing), spray drying or supercritical fluid technology.As mentioned above, also said composition can be made crystal composition.

After their form, use suitable transdermal administration technology with this particle transdermal administration to mammalian tissues.The various particle acceleration equipments of suitable transdermal administration substances of interest are known in the art, and can find use in enforcement of the present invention.The particle that particularly preferred transdermal drug delivery system uses needleless injector will contain solid-state drug is transmitted in intact skin and the tissue with control dosage or passes through wherein.Referring to, people's such as Bellhouse US5630796 for example, it has been described a kind of needleless injector and (also has been referred to as " PowderJect ^_Needleless injection device ").Other needleless injector structure is known in the art and description is arranged in this article.

To prepare the mode that is complementary particle is administered in the main body, and its amount is for reaching the effective dose of required physiological reaction with dosage.This reaction that produces should have the effect of preventing and/or treating.Therefore, for example, if just carrying antigen or vaccine combination, conveying capacity should enough produce immunoreation.If adjuvant and antigen are carried altogether, its conveying capacity should be enough to improve the antigenic immunoreation of common conveying so.Obviously, the amount for the treatment of administration composition depends on the concrete material carried, pending main body and waits the disease of preventing or handling.

Usually, the adjuvant of about 0.5 μ g-1000 μ g, more preferably the about 500 μ g of 1 μ g-adjuvant and most preferably from about the adjuvant of the about 300 μ g of 5 μ g-be effective to improving the antigenic immunoreation of giving.Therefore, for example, for CpG, the employed dosage range of this method is about 0.5-50 μ g, more preferably about 25 μ g of 1-and about 20 μ g of 5-most preferably.Similarly, concerning Alum or PCPP, the employed dosage of this paper is the about 500 μ g of about 25 μ g-, preferably about 250 μ g of about 50-and about 150 μ g of 75-most preferably from about.Concerning MPL, the employed dosage range of this method is about 10-250 μ g, preferably about 20-150 μ g and about 75 μ g of 40-most preferably from about.

Those skilled in the art use conventional method just can easily measure the dosage of other adjuvant.Dosage depends on and comprises common administration antigen, and this adjuvant is as many factors of the ability of immunostimulant.

Similarly, if transdermal with identical or different composition forms administration antigen, usually with 50ng-1mg and more preferably the about 50 μ g antigens of 1 μ g-be used to produce immunoreation.Required accurate amount is according to age of pending main body and conventional health status, treatment conditions and specific antigen or selected antigenic severity, administration position, and other factors and changing.Those skilled in the art can be by reading this description and easily measuring suitable effective dose by routine test.

It can be unit dose or multiple dose that dosage is handled.For vaccine combination, multiple dose is a kind of: the vaccinated phase I can be individual individually dosed for 1-10, then give other dosage at following interval, select to keep and/or the reinforced immunological reaction, for example second dosage was at 1-4 month, if necessary, after some months, give later dosage.Dosage mechanism also to small part, is determined and is relied on doctor's judgement by the needs of main body.And, if wish prophylactic words, before infecting for the first time, give said composition usually with interested pathogen.If wish treatment, for example reduce symptom or recurrence, after infecting for the first time, give said composition usually.C. test

Be the example of implementing the specific embodiment of the present invention below.These embodiment only are used for illustration purpose, in any case the scope that is not intended to limit the present invention.

Endeavour to ensure the degree of accuracy (for example, amount, temperature etc.) of used data to the greatest extent, but nature should allow certain test error and deviation.C.1 particle goods and particle form technology

Embodiment 1

Particulate vaccine compositions

Carrying out following research is in order to evaluate different excipient and prilling process the physical property of gained particulate vaccine compositions to be made the influence of vaccine immunogenicity again with these.Subunit protein antigen of select diphtheria toxin, diphtherotoxin (dT), purifying and the different excipient preparations that comprise mannitol (adding polyvinylpyrrolidone (PVP)), sucrose and trehalose.Also comprise two kinds of powder-making techniques, lyophilization and air-dry (evaporation drying).In order to test every kind of goods, using 3 " stainless steel sift of diameter partly classifies the gained microparticle compositions by following particle:＜20 μ m, 20-38 μ m, 38-53 μ m and 53-75 μ m.

The physical characteristic of particulate vaccine compositions comprises the evaluation to size distribution, osmotic energy, light microscopy, scanning electronic microscope examination, absolute density, pore size distribution, surface area analysis and X-ray powder diffraction.

In order to measure with needleless injection device (PowderJect for example ^_Needleless injection device) carries or the immunogenicity of making vaccine again by conventional syringe/pin injection, 0 and 4 weeks inoculation Balb/C mouse (female, 6-8 age in week).Use PowderJect ^_The mouse of needleless injection device inoculation accepts to be equipped with 5 μ g vaccines of 1mg excipient.With conventional needle and syringe peritoneal injection contrast mouse.Strengthen two weeks in the past after, from 8 mouse, collect serum and compile, measure dT antibody by ELISA.

The result of the physical characteristic of microparticle compositions is as follows: (1) air-dry method produces the high density crystalline particle, and produces the noncrystalline powder of low relatively density during lyophilization; (2) the particle size distribution demonstration does not rely on excipient and used granulation process.These particles through screening be divided into 5 classes (＜20 μ m, 20-38 μ m, 38-53 μ m, 53-75 μ m and＞75 μ m), and the Mass Distribution of each part is about equally.

The result of immunogenicity evaluation be described in following table 1 and 2 and Fig. 1 in.This shows the immunogenicity between the different particulate vaccine compositions closely similar (between the different big fraction of all goods and same article).All microparticle compositions by the needleless injector administration cause than the higher antigen titration degree of matched group (conventional needle and syringe administration).The trehalose excipient demonstrates better slightly than other goods, and 20-53 μ m partly demonstrates higher immunogenicity (referring to Fig. 1).But＜20 μ m and＞75 μ m particles part are all compared according to the immunogenicity of (Aquo-composition, conventional needle and syringe administration) and are wanted high.Also can find out the Needleless injection (PowderJect of crystallization vaccine combination particle ^_) administration generation comparison photograph (conventional needle and syringe) seroconversion rate (referring to table 2) that injecting method is high.Thus, at single PowderJect ^_The animal of 97% (having 157 in 162) produces serum antibody after the inoculation, yet the seroconversion rate of matched group is 37.5% (having 3 in 8).

Table 1
						The IgG antibody (pooled serum) of dT
Excipient	Granulation process	Particle diameter (μ m)	Medication	Cause	Strengthen
						Saline	Contrast	????-	Syringe and pin	????1220	?111600
Trehalose	Air-dry	???＜20	????PJ*	????1550	?616380
						Trehalose	Air-dry	??20-38	????PJ	????5460	?1390700
Trehalose	Air-dry	??38-53	????PJ	????7295	?1282100
						Trehalose	Air-dry	??53-75	????PJ	????2690	?577080
Trehalose	Air-dry	???＞75	????PJ	????2310	?620460
						Trehalose/mannitol	Lyophilization	??20-38	????PJ	????1590	?441140
Trehalose+mannitol	Lyophilization	??38-53	????PJ	????4590	?686540
						Trehalose+mannitol	Lyophilization	??53-75	????PJ	????2850	?657780
Trehalose+mannitol	Lyophilization	???＞75	????PJ	????1940	?282630
						Sucrose	Air-dry	??20-38	????PJ	????4650	?1160220
Sucrose	Air-dry	??38-53	????PJ	????3040	?515760
						Sucrose	Air-dry	??53-75	????PJ	????1750	?372160
Sucrose	Air-dry	???＞75	????PJ	????5230	?759240
						Sucrose	Lyophilization	??20-38	????PJ	????1525	?618100
Sucrose	Lyophilization	??38-53	????PJ	????2950	?614780
						Sucrose	Lyophilization	??53-75	????PJ	????7830	?802850
Sucrose	Lyophilization	???＞75	????PJ	????1800	?780710
						Sorbitan/PVP	Lyophilization	??20-38	????PJ	????3700	?494330
Sorbitan/PVP	Lyophilization	??38-53	????PJ	????2810	?609940
						Sorbitan/PVP	Lyophilization	??53-75	????PJ	????4580	?935700
Sorbitan/PVP	Lyophilization	???＞75	????PJ	????1590	?447630

PJ=PowderJect ^_Annotate: 0 week serum titer＜200.All dT dosages of inoculation are 5 μ g.

Table 2
							Seroconversion rate
Excipient	Granulation process	The animal that serum has transformed/all animals
							＜20	?20-38	?38-53	?53-75	??＞75
		Trehalose	Air-dry	?4/4	??7/8	??7/8						??7/8	??8/8
Trehalose+sorbitan	Lyophilization						??-	??8/8	??8/8	??8/8	??7/8
		Sucrose	Air-dry	??-	??7/8	??8/8						??7/7	??7/7
Sucrose	Lyophilization						??-	??8/8	??8/8	??8/8	??6/8
		Sorbitan/PVP	Lyophilization	??-	??8/8	??7/8						??8/8	??7/8
The DT/ injection	???-						?3/8

C.2 the formation of crystallization vaccine combination and evaluation

Embodiment 2

The preparation of crystallization vaccine combination

Use the many traditional vaccine compositionss of following method crystallization.

Obtain streptococcus pneumoniae capsule polysaccharide #14 (CP14) by ATCC with freeze-dried powder.The water for injection of 1ml volume is dispersed among the CP14 of 2mg bottle, the gained suspension is mixed under 4 ℃ whole night continuously by the description of manufacturer is described.Make 100 μ l aliquot mixture, before needs, keep freezing.Take by weighing trehalose (Sigma) powder of many 99.5mg and mix, obtain the CP14 of 500 μ g altogether with the aliquot CP14 suspension that thaws.The water for injection of about 1200 μ l is used to dissolve CP14/ trehalose mixture, and this solution is fully mixed.Then this five equilibrium sample solution of 100 μ l is dispersed on the surface of weighing boat, and is placed in the constant gas that fume hood provides.Then next in 1-2 days with the droplet evaporation drying, thereby form crystallized product.Remove this crystallization and use mortar and pestle is pulverized gently from the weighing boat.

Contain the adult bottle Engerix-B that uses of the standard that is adsorbed on the last 20 μ gHepB surface antigens of 0.5mg aluminium hydroxide (Alum) with one ^_(Smith Kline Beecham) mixes with the 10mg trehalose, and gained solution is fully mixed.100 μ l droplets are dispersed on the weighing boat, and through this paper drying recited above.After about 36 hours evaporation drying, use scraper carefully to remove crystallization vaccine residue from the weighing boat.Using mortar and pestle to pulverize this crystal composition gently then obviously reduces up to big crystallite size.1.25 ± 0.25mg aliquot is dispersed in the medicine box, obtains the hbs antigen of 2.5 μ g standard doses.

A large amount of hbs antigenes (HbsAg) that acquisition is purified from human plasma (BiodesignInternational).With this HbsAg and trehalose and dI aqueous solution.Gained solution is mixed gently, pour in the glass Petri dish and under fume hood air-dry 2 days.Then with in the exsiccator (Nalgene Plastic exsiccator) of nitrogen wash dry one day again.Collect this drying solid compositions by curettage, use mortar and pestle to pulverize then.The gained dried powder is weighed, the dried material total amount is obtained the amount of dry matter in each dosage divided by the quantity of need preparation dosage.The particle size distribution of the HbsAg vaccine combination of preparation changes in (1-100 μ m) very on a large scale.Typically, each dosage need about 1-2mg dried material, and weighing difference is about 10% or lower.

Acquisition contains the quantitative HibTITER of PRP-CRM197 conjugation vaccine combination ^_(WyethLederle).Said composition contains and the conjugated poly-ribosyl ribose phosphate of CPM197 mutant diphtheria toxin, diphtherotoxin carrier (from the polysaccharide of influenza B haemophilus), and this paper is referred to as " Hib conjugation vaccine combination ".This Hib conjugation vaccine combination is mixed with trehalose, and gained solution is fully mixed.Then this solution is dispersed on the weighing boat, through dry as mentioned above.After the evaporation drying, obtain crystallization vaccine residue, use mortar and pestle that this crystal composition is pulverized gently, claim its optimal dose and put into medicine box by the needleless injector administration from the weighing boat.

(Storrs CT) obtains a large amount of influenza virus, the PR8 bacterial strain by Spafas.In addition, from Dr.Yoshihero Kawaoka, Veterinary School, (Madison Wisconsin) obtains a large amount of influenza virus, the Aichi bacterial strain to University of Wisconsin.Every kind of virus is handled (1: 4000, following 48 hours at 4 ℃) inactivation through standard formalin.Then with inactivation virus (perhaps PR8 or Aichi) and trehalose and dI aqueous solution.Gained solution is mixed gently, and pour in the glass Petri dish, under fume hood air-dry 2 days.Then with in the exsiccator (Nalgene Plastic exsiccator) of nitrogen wash dry one day again.Collect this drying solid compositions by curettage, use mortar and pestle to pulverize then.The gained dried powder is weighed, the dried material total amount is obtained the amount of dry matter in each dosage divided by the quantity of need preparation dosage.The particle size distribution of the whole inactivation viral vaccine compositions of preparation changes in (1-100 μ m) very on a large scale.Typically, each dosage need about 1-2mg dried material, and weighing difference is about 10% or lower.

Obtain a large amount of diphtheria toxin, diphtherotoxins (Accurate Chemical﹠Scientific Corp., by Statems Serum Institute, Denmark produces).With this toxin and trehalose and dI aqueous solution.Gained solution is mixed gently, pour in the glass Petri dish and under fume hood air-dry 2 days.Then with in the exsiccator (Nalgene Plastic exsiccator) of nitrogen wash dry one day again.Collect this drying solid compositions by curettage, use mortar and pestle to pulverize then.The gained dried powder is weighed, the dried material total amount is obtained the amount of dry matter in each dosage divided by the quantity of need preparation dosage.The particle size distribution of the diphtheria toxin, diphtherotoxin vaccine combination of preparation changes in (1-100 μ m) very on a large scale.Typically, each dosage need about 1-2mg dried material, and weighing difference is about 10% or lower.

Embodiment 3

Inoculate with the crystallization vaccine combination

Equipment: except as otherwise noted, be used for needleless injector administration device (the perhaps PowderJect of percutaneous drug delivery research ^_ND equipment chain or Oral PowderJect ^_Equipment chain) be from PowderJect Technologies, Ltd., Oxford, UK obtains.This PowderJect ^_ND equipment is generally described among the US5630796.This Oral PowderJect ^_Equipment is generally described in the International Application No. WO 96/20022.Gas bomb in the employed equipment of this paper typically is filled with the helium of 40-60 bar pressure, although also can use the pressure of 30-80 bar.In operation, start this equipment, make the film rupture of the load medicine box that contains particle to discharge the compressed helium in the gas bomb.Produce ultrasound condition, and the gained high velocity air is propelled particle in the destination organization surface as bullet.The pressure that changes helium in the gas bomb can be controlled the degree of depth of infiltration.For conventional needle and syringe administration, disposable syringe is equipped with syringe needle No. 26.5.

Mouse and inoculation: female Balb/c mouse or Swiss Webster mouse from authorized seller (for example HSD) bought for 7 ages in week adapted to for 1 week in the mouse device before inoculation.The 100mg/kg ketamine that is mixed with the 10mg/kg xylazine by intraperitoneal (IP) injection is anaesthetized mouse, with target location peritoneum skin by picking the hair of pruning.The vaccination position is also mutually by pushing this needleless injector equipment gently relatively.Typical immunologic mechanism constitutes: twice vaccination, 4 weeks at interval are before each inoculation and strengthen after two weeks in the past the hemorrhage collection blood of process posterior orbit under anesthesia.

ELISA: usually, measure the antibody response of making vaccine again by ELISA.The 0.1 μ g that is used for PBS with each hole whole night under 4 ℃ measures the plate that antigen applies 96 holes.These plates with the TBS flushing that contains 0.1%Brij-35 3 times, were cultivated 1.5 hours with the test serum that is diluted in the PBS that contains 1%BSA.The titer standardization of adding to the serum titer that contains the high-level antibody of relative specific antigen on each plate and being used to make final data to analyze.Wash plate then and use goat antibody to the specific biotin mark of mouse immunoglobulin IgG or IgG subclass (1: 8000 in PBS, Southern Biotechnology) at room temperature to cultivate 1 hour.Then wash again three times, this plate was at room temperature cultivated 1 hour with Succ-PEG-DSPE-horseradish peroxidase conjugate (1: 8000 in PBS, Southern Biotechnology).At last, wash these plates and use tmb substrate medicine box (from Bio-Rad, Richmond, CA acquisition) to develop.Use Softmax Pro4.1 program (Molecular Devices) A that surpasses 0.1 times of average background ₄₅₀Endpoint titration degree as the highest dilution determination test serum of calibration.Measure the absorbance of average background by the hole of accepting all reagent except that test serum.

1. with crystallization streptococcus pneumoniae capsule polysaccharide #14 (CP14) inoculation.Preparation with test matrix shown in route of administration (intraperitoneal (IP) or intradermal (ID)) and the table 3 is that the basis forms Balb/c mouse test group at random.The 4th week of using above-mentioned crystallization CP14 vaccine combination to make these mouse be caused and cause is afterwards strengthened, and is hemorrhage after strengthening 10 days then.Should strengthen, injection mouse anesthetis is anaesthetized these mouse, makes it can posterior orbit before strengthening administration more hemorrhage.For the needleless injector administration, remove unhaired hide by pruning, use from PowderJect ^_60 bar air pressure of needleless injector administration device carry out administration.The serum analysis sample is to measure pneumococcal antigen titration degree then.

Table 3
						Group number	????n	Route of administration	Pressure (bar)	Preparation	CP dosage (μ g)
???Ⅰ	????3	????N/S-IP	???---	CP14+ trehalose and Alum	????5
						???Ⅱ	????3	????N/S-IP	???---	The CP14+ trehalose	????5
???Ⅲ	????3	????N/S-IP	???---	????CP14+Alum	????5
						???Ⅳ	????4	????PJ-ID	????60	The CP14+ trehalose	????5
???Ⅴ	????4	????PJ-ID	????60	The CP14+ trehalose	????10
						???Ⅵ	????4	????PJ-ID	????60	????CP14	????50
???Ⅶ	????2	????PJ-ID	????60	Trehalose only	????0

2. with the inoculation of crystallization hepatitis B (HepB) SAV compositions.Crystallization HepB vaccine combination (as mentioned above) is arrived mouse by following administration (with 2.5 μ g saccharide dosage).With the vaccine combination is that the basis is divided into 2 groups with 8 Balb/c mouse, and medicine-feeding technology uses: (1) uses pin-syringe intraperitoneal to carry tradition (liquid state) Engerix-B ^_Vaccine combination (n=2); (2) use PowderJect ^_The needleless injector intradermal is carried this crystallization vaccine combination (n=6).PowderJect ^_The conveying of equipment is pressed and is 40-60 bar.After causing for 3 weeks all animals are strengthened, strengthens 10 days afterwards hemorrhage.

The blood sample that uses administration to strengthen obtaining after 10 days is then measured the antigen titration degree of hbs antigen by ELISA.The result is described in the following table 4.Titer is considered to protect serum greater than about 10mIU/mL.This shows, pass through needleless injector and accept to have in 6 animals of crystal composition 5 by this crystallization vaccine combination protection.

Table 4
				The mouse numbering	Route of administration	???Po(bar)	Estimate
????1	Pin-syringe (IP)	????---	?????---
				????2	Pin-syringe (IP)	????---	?????---
????3	?PowderJect _(ID)	????40	Cause: administration is poor
				????4	?PowderJect _(ID)	????40	Cause: good
????5	?PowderJect _(ID)	????40	Cause: good
				????6	?PowderJect _(ID)	????60	Cause: good
????7	?PowderJect _(ID)	????60	Cause: good
				????8	?PowderJect _(ID)	????60	Cause: slight bleeding

Use enough all antigen titration degree (the process PowderJect that increases greatly in the animal of accepting Needleless injection of next reinforcement of phase syncrystallization HepB SAV compositions ^_Equipment), all 6 animals all obtain the protection of crystallization vaccine combination as a result.

3. with poly-ribosyl ribose phosphate conjugation vaccine combination (Hib conjugate) inoculation of crystallization hemophilus influenza.By following to the above-mentioned crystallization Hib of mouse administration conjugation vaccine combination.With medicine-feeding technology, dosage be formulated as the basis Swiss Webster mouse is divided into test group: (1 group, the liquid Hib conjugation vaccine combination of 2 μ g (PRP sugar) uses conventional needle and syringe through the IP administration, n=3); (2 groups, the crystallization Hib conjugation vaccine combination of 2.5 μ g (PRP sugar) uses PowderJect ^_Needleless injector is administered into skin, n=6); (3 groups, contrast (not making overtesting), n=3).All animals received one through inoculation cause, and then strengthen in the 4th week after causing.The 2nd week was collected blood serum sample after reinforcement, compiled and used the fixedly antigen titration degree of PRP-CRM197 of ELISA mensuration.

Because human body and the adherent observation difference of the anti-haemophilus polysaccharide of mouse (Hbps) antibody, so the described HbO-HAELISA of people (1990) J.Immunol.Methods 135:121-128 such as Phipps is suitable for the test mouse serum.The suitable appreciation condition of measuring the anti-HbPs antibody of mouse is annotated in following table 5.All other appreciation conditions are as described in the people such as Phipps.

Table 5
				Human body	Mouse
Antigen coating buffer agent	????PBS	????50mM?HEPES
			The agent of serum dilution buffer	PBS/0.3% polysorbas20/0.01M EDTA	???50mM?HEPES/0.1％Brij ?????35/1％FBS
Serum incubation time and temperature	60 minutes, room temperature	Whole night, 4 ℃
			The secondary antibody conjugate	Anti-Human's body Ig*AP	Anti--mouse IgG*AP
Hyperconjugation thing buffer agent	The PBS/0.05% polysorbas20	??50mM?HEPES/0.1％Brij ????35/1％FBS
			Dcq buffer liquid	The PBS/0.1% polysorbas20	?50mM?HEPES/0.1％Brij?35

* estimate to demonstrate and use this ELISA to measure quantitative value for antibody in mouse serum to be higher than and to use radiation antigen (RABA) measure in conjunction with estimating, particularly be lower than 10 μ g/mL's for serum titer in RABA.

ELISA the results are shown in the following table 6.This shows that this crystallization vaccine combination has with the traditional vaccine compositions compares comparable result.

In order further to characterize the immunoreation in the animal of accepting Hib conjugation vaccine combination, carry out following research.Compile three test group of one group of 6 mouse as follows: (1 group, the crystallization Hib conjugation vaccine combination of 2 μ g dosage (PRP sugar) uses PowderJect ^_The administration of needleless injector equipment intradermal); (2 groups, the conventional liquid Hib conjugation vaccine combination of 2 μ g dosage (PRP sugar) uses pin and syringe intraperitoneal (IP) administration); (3 groups, contrast).Animal via causes, and 4 weeks are after strengthen then.The serum of strengthening collecting after 2 weeks is compiled, use above-mentioned elisa technique to estimate serial dilution.In one-level ELISA, with the PRP-CRM197 conjugate as catching phase.The result of this one-level ELISA is reported in the following table 7, and is described among Fig. 2.This shows that crystal composition has the reaction roughly the same with the traditional vaccine administration.

Table 7 vaccine reaction
						Dilution	????IP*	??????PJ*	Do not make overtesting
??1	?100	???1.09(0.024)	??1.131(0.012)	??0.058(0.019)
					??2	?300	???0.934(0.053)	??0.989(0.003)	??0.02(0.016)
??3	?900	???0.8015(0.0445)	??0.774(0.004)	??0.02(0.014)
					??4	?2700	???0.449(0.071)	??0.502(0.002)	??0.015(0.013)
??5	?8100	???0.279(0.006)	??0.277(0.004)	??0.01(0.001)
					??6	?24300	???0.129(0.0315)	??0.115(0.005)	??0.009(0.001)
??7	?72900	???0.0545(0.0018)	??0.041(0.001)	??0.017(0.008)
					??8	?218700	???0.109(0.005)	??0.062(0.02)	??0.073(0.021)

* (IP)=intraperitoneal pin and syringe administration, (PJ)=PowderJect ^_The needleless injector administration uses diphtheria toxin, diphtherotoxin to carry out secondary ELISA mutually as catching in order to estimate immunoreactive specificity.In this, CRM197 is the diphtheria toxin, diphtherotoxin saltant, and highly intersection is active but CRM197 has.Therefore antiserum is attached to and is used as evaluation response on the diphtheria toxin, diphtherotoxin towards the CRM197 carrier protein.The result is reported in the following table 8, and is described among Fig. 3.Once more, this crystallization vaccine combination provides with the traditional vaccine compositions and has compared comparable result.

Table 8
						????1	??????2	????3	????4
	Dilution	Do not make overtesting	????PJ*	????IP*
					?1	????100	????0.21	????1.856	????1.8
?2	????300	????0.102	????1.846	????1.64
					?3	????900	????0.117	????1.688	????1.616
?4	???2700	????0.084	????1.38	????1.194
					?5	???8100	????0.072	????0.931	????0.797
?6	??24300	????0.077	????0.578	????0.512
					?7	??82900	????0.072	????0.3	????0.269
?8	?248700	????0.072	????0.165	????0.17

* (IP)=intraperitoneal pin and syringe administration, (PJ)=PowderJect ^_The needleless injector administration

In order to detect the reaction of PRP-specific antibody, use PRP-human serum albumin (PRP-HSA) conjugate to carry out three grades of ELISA mutually as solid (catching).Because these mouse PRP-HSA conjugate of no use immunizes, and has not been HSA and attacks in other context, therefore the appearance of anti--PRP antibody makes antibody link.The result of this ELISA is reported in the following table 9, and is described among Fig. 4.This shows that this crystallization and tradition (liquid state) Hib conjugation vaccine combination has comparable result.

Table 9
						????1	????2	????3	????4
	The serum dilution	Do not make overtesting	????PJ*	????IP*
					?1	????200	????0.184	??1.244	????1.649
?2	????400	????0.143	??0.731	????0.956
					?3	????800	????0.113	??0.455	????0.625
?4	???1600	????0.097	??0.259	????0.322
					?5	???3200	????0.075	??0.159	????0.171
?6	???6400	????0.064	??0.105	????0.094
					?7	??12800	????0.064	??0.078	????0.068

* (IP)=intraperitoneal pin and syringe administration, (PJ)=PowderJect ^_The needleless injector administration

Pass through PowderJect in order to detect ^_The antibody response of the Hib conjugation vaccine of the reduction dosage of administration or conventional needle-injector to inject carries out following research.With liquid Hib conjugation vaccine combination or crystallization Hib conjugation vaccine combination in 4 weekly intervals to twice of Swiss Webster mouse (female, 6-8 age in week) inoculation (cause and strengthen).Hib conjugation compositions to four dosage (each dosage is respectively 1 μ g, 0.2 μ g, 0.04 μ g and 0.01 μ g) is tested.With regard to the needleless injector inoculation, with each vaccine dose and the preparation of 1mg trehalose.The contrast mouse uses conventional needle and syringe through peritoneal injection.Before each inoculation and strengthen collecting blood sample after 2 weeks.Estimate the antibody of PRP and CRM197 as mentioned above by ELISA.The serum of strengthening collecting after 2 weeks is compiled, use above-mentioned elisa technique to estimate pooled serum.The results are described among Fig. 5.This shows that in 1 μ g dosage, IgG titer demonstrates by PowderJect ^_The needleless injector administration is carried out the mouse of immunity and is used conventional needle and syringe to carry out can comparing between the immune mouse.In 0.01-0.2 μ g dosage range, using PowderJect ^_Antibody horizontal in the mouse of equipment immunity demonstrates the height than the mouse of respective needle and syringe administration group.In addition, also in through the animal of immunity, measure the antibody response of CRM197.Using PowderJect ^_Demonstrate dose dependent reaction (see figure 5) in the group that equipment immunizes.Contrast mouse (those animals that immunize by conventional needle and injector to inject) is to responding with higher dosage (1 μ g and 0.2 μ g) inoculation, but than not reaction under the low dosage.These data declarations use PowderJeet ^_The transdermal immune of the particulate vaccine compositions of needleless injection device is more effective than the liquid vaccine combination administration of using conventional needle and injector to inject technology, particularly with low dosage administration antigen.

For the persistent period of the immunity that provides by crystallization Hib conjugation vaccine combination is provided, carry out following research.With the Hib conjugation crystallization vaccine combination of two dosage (cause and strengthen) 4 weeks inoculation Swiss Webster mouse (female, 6-8 age in week) at interval.With regard to PowderJect ^_The needleless injector inoculation is with 1 μ g vaccine and the preparation of 1mg trehalose excipient.In contrast, adopt conventional needle and syringe medicine-feeding technology through the peritoneal injection mouse with the liquid Hib conjugation of 5 μ g vaccine combination.Before each inoculation, strengthen 2 week back and once collected blood sample in every month afterwards.Use above-mentioned elisa technique to estimate the antibody specificity of PRP and CRM197.What ELISA estimated the results are shown among Fig. 6 A and the 6B.This shows PowderJect ^_Serum antibody level that administration 1 μ g crystallization Hib conjugation vaccine combination produces PRP and CRM197 and being on close level that 5 μ g conjugates by conventional needle and injector to inject administration cause.Antibody is strengthening reaching maximum after two weeks, and continuing did not have big reduction in 8 months.These presentation of results PowderJect ^_The transdermal administration of the particulate vaccine compositions of equipment produces the long-acting serum antibody response that can compare with the conventional needle and the syringe administration of liquid vaccine combination.

4. with the inoculation of crystallization inactivation influenza virus vaccine compositions.Use PowderJect in order to detect ^_The antibody response of the reduction influenza vaccines dosage of the liquid vaccine combination of administration crystallization vaccine combination or conventional needle or injector to inject carries out following research.In order to estimate above-mentioned crystallization inactivation influenza virus (Aichi bacterial strain) compositions of 5 various dose, set up five Balb/c mouse (female, 6-8 week age) test group.These five groups are being accepted 25 μ g, 5 μ g, 1 μ g, 0.2 μ g or 0.04 μ g inactivation influenza virus 0,4 and 10.5 weeks respectively.With regard to PowderJect ^_Transdermal administration is with the vaccine and the preparation of 1mg trehalose of each dosage.Also set up five contrast mouse groups.These contrast mouse are accepted inoculation 25 μ g, 5 μ g, 1 μ g, 0.2 μ g or 0.04 μ g inactivation influenza virus with liquid vaccine combination (by conventional needle and injector to inject through the intraperitoneal administration) respectively in 0,4 and 10.5 weeks.After inoculating for 2 weeks for the third time, the serum of 8 mouse is collected and compiled, and ELISA detects Antibody of Influenza.

After strengthening for 2 weeks, reuse the antigen titration degree that above-mentioned elisa technique detects anti-Aichi virus in the pooled serum.The result of ELISA is described among Fig. 7.This shows the PowderJect of crystal composition ^_The conventional needle of transdermal administration and fluid composition and injector to inject all are the dose dependent antibody response.But, under the vaccine of same dose, PowderJect ^_Inoculation causes higher antigen titration degree than pin and injector to inject, and this explanation is to the PowderJect of skin ^_Administration crystallization vaccine has been strengthened the vaccine performance.

The cell agglutination of testing the blood pooled serum again suppresses active (HI).The result of this HI activity rating is described in the following table 10.This shows that HI titer is dose dependent in the serum of vaccinated animal.Accept PowderJect ^_The HI titer of the administration and the animal of conventional needle and injector to inject is identical under higher vaccine dose (25 μ g, 5 μ g and 1 μ g).But, under low vaccine dose (0.2 μ g and 0.04 μ g), only accepting from PowderJect ^_Produce HI titer in the animal of the crystal composition of system.

Table 10
			HI antigen titration degree in the 12nd all serum that is compiled
Influenza vaccines (μ g)	?PowderJect _Administration	Syringe and pin injection
			????25	????40	????80
????5	????20	????20
			????1	????20	????10
????0.2	????10	????-
			????0.04	????10	????-

In order to estimate the protective that influenza virus is next stimulated, instil 1 * 10 by intranasal in the 12nd week ⁶PFU (100 * LD ₆₀) adapt to the Aichi influenza virus of mouse to stimulate vaccinated animal.More particularly, after the last immunity 10 days, be mixed with the 100mg/kg Patients Under Ketamine Anesthesia mouse of 10mg/kg xylazine by peritoneal injection.With in the 50 μ l saline 1 * 10 ⁶The influenza virus of granulating unit (PFUs) slowly is instilled in the nasal cavity mouth.These mouse suck this liquid by the light of nature.Allow these mouse recover then.Before stimulating, measure the body weight of these animals after 14 days every day with stimulation.Every day monitor animal symptom and survival condition.Alleviate 25% pre-body weight and the moribund animals of stimulating and make its euthanasia with carbon dioxide.

Write down survival condition and lose weight every day in back 14 days of stimulation.All animals all lose weight in 3-7 days after stimulation.Survivor to 14 day recovers their body weight.The results are shown in the following table 11 of protective research.The animals survived statistics that acceptance contains 25 μ g and 5 μ g inactivation viral vaccine compositionss is shown in respectively among Fig. 8 A and the 8B.This shows, under 25 μ g dosage, PowderJect ^_Fluid composition when the administration crystal composition compares through conventional needle and injector to inject administration to mortality has better protective.Find out that by the mutual relation between antibody response and the survival condition dead animal has lower antigen titration degree.Under 5 μ g dosage, and there is not protective to compare in the animal of accepting same dose by conventional needle and injector to inject, PowderJect ^_The administration crystal composition provides the part protective to mortality.Therefore, through PowderJect ^_Equipment transdermal administration crystal composition provides better protective than conventional needle and injector to inject.

Table 11
			The influenza irritant test
Influenza vaccines (μ g)	Survival number/sum
			PowderJect _Syringe and pin
	????25	????7/8			?????5/8
????5			????3/8	?????0/8
	????1	????0/8			?????0/8
????0.2			????0/8	?????0/8
	????0.04	????0/8			?????0/8

Annotate: 3 grades of vaccinated mouse accepting for 0,4 and 10 weeks stimulated through the influenza virus that 100 * LD50 adapts to mouse of the same race in the 12nd week.Data show stimulates the protective in 2 weeks of back.

5. with the inoculation of crystallization diphtheria toxin, diphtherotoxin (dT) vaccine combination.In order to detect PowderJect ^_The antibody response of the dT vaccine of the reduction dosage of administration crystal composition, and compare with those antibody responses that obtain by conventional needle and injector to inject, carry out following research.Set up two test group of Balb/c mouse (female, 6-8 week age).These animals are being accepted inoculation liquid dT vaccine combination (by pin and injector to inject administration) or crystallization vaccine combination 0 and 4 weeks (by PowderJect ^_The needleless injection device administration).Two kinds of vaccine doses are tested, promptly contained the vaccine combination of 5 μ g and 1 μ g diphtheria toxin, diphtherotoxin.With regard to PowderJect ^_Inoculation is with the vaccine and the preparation of 1mg trehalose of each dosage.With conventional needle and syringe peritoneal injection contrast mouse.After inoculating for 2 weeks at last, collect serum and compile, measure dT antibody by above-mentioned elisa technique from 8 mouse.

ELISA the results are shown in the following table 12.This shows that transdermal administration contains high 15 times than in the animal of the liquid vaccine combination of the same dose of being accepted by conventional needle and injector to inject of serum antibody response due to the crystal composition of 1 μ gdT.This illustrates that interparticle percutaneous drug delivery is effective to administration dT vaccine.Under 5 μ g dosage, pass through PowderJect ^_Equipment accept in the animal of crystal composition the serum antibody level with arrive seen in the animal of accepting fluid composition by conventional needle and injector to inject identical.

Table 12
			Measure the IgG titer of dT in the 6th all serum that is compiled by ELISA
dT(μg)	Serum IgG titer (PowderJect _)	Serum IgG titer (syringe/pin)
1	8130	570
5	188830	149560

C.3 the formation of particle adjuvant compositions and evaluation

Embodiment 4

The formation of particle adjuvant compositions

Many traditional adjunvant compositions are mixed with microgranule (powder) type according to the inventive method.Use traditional arbitrarily prilling process can easily make these and other adjuvant again.Proper excipient in the adjunvant composition comprises trehalose, sucrose, agarose, mannitol or these and/or other sugared mixture.That granulating technique can comprise is air-dry, lyophilizing, spraying and supercritical fluid processes.But unless other explanation is arranged, all used adjuvant goods all are to prepare with method for crystallising of the present invention (concrete aforesaid joint C.1 and C.2) in the test below.

The production of particle adjuvant compositions is as follows.

Obtain a large amount of aluminium hydroxide and aluminum phosphate adjuvant (" Alum adjuvant ") (produce by SuperfowBiosectora/s, obtain) from Accurate Chemical and Scientific Corp..This Alum adjuvant and trehalose and dI aqueous solution.Gained solution is mixed gently, pour in the glass Petri dish and under fume hood air-dry 2 days.In the exsiccator of crossing with nitrogen wash (Nalgene Plastic exsiccator) dry again one day.Collect this drying solid compositions by curettage, use mortar and pestle to pulverize then.The gained dried powder is weighed, the dried material total amount is obtained the amount of dry matter in each dosage divided by the quantity of need preparation dosage.The particle size distribution of the Alum adjunvant composition of preparation changes in (1-100 μ m) very on a large scale.Typically, each dosage need about 1-2mg dried material, and weighing difference is about 10% or lower.

Obtain a large amount of MPL adjuvants (the single phosphinylidyne lipoid A that purifies by Minnesota Salmonella mattress R595) (RIBI ImmunoChem Research, Inc.).With this MPL adjuvant and trehalose and dI aqueous solution.Gained solution is mixed gently, pour in the glass Petri dish and under fume hood air-dry 2 days.In the exsiccator of crossing with nitrogen wash (Nalgene Plastic exsiccator) dry again one day.Collect this drying solid compositions by curettage, use mortar and pestle to pulverize then.The gained dried powder is weighed, the dried material total amount is obtained the amount of dry matter in each dosage divided by the quantity of need preparation dosage.The particle size distribution of the MPL adjunvant composition of preparation changes in (1-100 μ m) very on a large scale.Typically, each dosage need about 1-2mg dried material, and weighing difference is about 10% or lower.

Obtain a large amount of CpG adjuvants (20 aggregate into oligonucleotide, CpG-1:ATCGACTCTCGAGCGTTCTC, SEQ ID NO.1 and CpG-2:TCCATGACGTTCCTGATGCT, SEQ ID NO.2) (GIBCO-BRL).With this CpG adjuvant and trehalose and dI aqueous solution.Gained solution is mixed gently, pour in the glass Petri dish and under fume hood air-dry 2 days.In the exsiccator of crossing with nitrogen wash (Nalgene Plastic exsiccator) dry again one day.Collect this drying solid compositions by curettage, use mortar and pestle to pulverize then.The gained dried powder is weighed, the dried material total amount is obtained the amount of dry matter in each dosage divided by the quantity of need preparation dosage.The particle size distribution of the CpG adjunvant composition of preparation changes in (1-100 μ m) very on a large scale.Typically, each dosage need about 1-2mg dried material, and weighing difference is about 10% or lower.

Obtain a large amount of PCPP adjuvants (synthetic polymer-poly-[two (carboxyl phenoxy groups) are phosphazine]) (VirusResearch Institute).With this PCPP adjuvant and trehalose and dI aqueous solution.Gained solution is mixed gently, pour in the glass Petri dish and under fume hood air-dry 2 days.In the exsiccator (Nalgene Plastic exsiccator) that a usefulness nitrogen wash is crossed dry again one day.Collect this drying solid compositions by curettage, use mortar and pestle to pulverize then.The gained dried powder is weighed, the dried material total amount is obtained the amount of dry matter in each dosage divided by the quantity of need preparation dosage.The particle size distribution of the PCPP adjunvant composition of preparation changes in (1-100 μ m) very on a large scale.Typically, each dosage need about 1-2mg dried material, and weighing difference is about 10% or lower.

Embodiment 5

Inoculate with the particle adjuvant compositions

The equipment that is used for conveying finely divided compositions (is PowderJect ^_Needleless injection device) and be used for carrying the equipment (being conventional needle and syringe) of contrast (liquid) compositions described in above-mentioned embodiment 3.Experimental animal (female Balb/c mouse) through as above handling, is also carried vaccine combination as mentioned above, aforesaid identical elisa technique is used for following research.Be used for the crystallization of following research and complete influenza virus (Aichi bacterial strain) compositions and the diphtheria toxin, diphtherotoxin compositions that contrast (liquid) vaccine combination is the inactivation described in the top embodiment 2.Described in top embodiment 3, carry out viral stimulation study (in influenza research) with the Aichi influenza bacterial strain that adapts to mouse.

1. carry out inoculation study with particles A lum adjunvant composition.With this particles A lum adjunvant composition as having the adjuvant of crystallization inactivation Inflenza vaccine composition and through PowderJect ^_The Needleless injection drug-supplying system is carried.With with 100 μ g particles A lum adjunvant compositions or do not have 5 μ g of particles A lum adjunvant composition or 1 μ g crystallization inactivated vaccines compositions inoculation mouse.Use conventional needle to inoculate control animal with the aqueous compositions of identical vaccine/adjunvant composition subcutaneous (" S/C ") with syringe.Carrying out three inoculations (in 0,4 and 10.5 weeks) afterwards, from 8 mouse, compile serum in the 12nd week, and detect Antibody of Influenza by above-mentioned elisa technique.

The result of ELISA research is reported in the following table 13.Carrying out with Alum is the percutaneous drug delivery of the influenza vaccines of adjuvant, when with accept the same dose vaccine but when not having the animal of adjuvant to compare, serum antibody response improves greatly.Through PowderJect ^_Equipment is accepted serum antibody level and the animal of adopting the conventional art inoculation with identical vaccine/adjunvant composition identical in the animal of microparticle compositions.Therefore the Alum adjuvant can be transported to skin with powdery, thereby strengthen the immunogenicity of influenza vaccines.

Table 13
				The total IgG titer of in the 12nd all serum that is compiled, measuring by ELISA
Aluminum (μ g)	Influenza vaccines (μ g)	Serum IgG titer (Powder Ject _)	Serum IgG titer (syringe/pin)
Do not have	5	6790	1505
Do not have	1	873	804
100	5	27180	19198
100	1	2539	9966

In order to estimate the next protective of influenza virus stimulation, instil 1 * 10 by intranasal in the 12nd week ⁶PFU (100 * LD ₅₀) adapt to the Aichi influenza virus of mouse to stimulate vaccinated animal.More particularly, after the last immunity 10 days, be mixed with the 100mg/kg Patients Under Ketamine Anesthesia mouse of 10mg/kg xylazine by peritoneal injection.With in the 50 μ l saline 1 * 10 ⁶The influenza virus of granulating unit (PFUs) slowly is instilled in the nasal cavity mouth.These mouse suck this liquid by the light of nature.Allow these animals recover then.Before stimulating, measure the body weight of these animals afterwards every day with stimulation.Every day monitor animal symptom and survival condition.Alleviate 25% pre-body weight and the moribund animals of stimulating and make its euthanasia with carbon dioxide.

Write down survival condition and lose weight every day in back 14 days of stimulation.These results are reported in the following table 14 and are described among Fig. 9.This shows that accepting with Alum is that the survival ratio accepted separately in the mouse of vaccine of survival ratio in the mouse of vaccine of adjuvant is much bigger.PowderJect ^_Administration is that the Inflenza vaccine composition microgranule of adjuvant prevents the (see figure 9) that loses weight better than syringe and pin injection with Alum.At first, two groups of mouse all alleviate 18% body weight, but through PowderJect ^_The animal that equipment is accepted microparticle compositions recovers body weight with the speed faster than the mouse of subcutaneous vaccination, and this explanation is better than conventional needle and syringe medication to skin transdermal delivery of particulate immunomodulator.

Table 14
				The protective that the opposing influenza stimulates
Aluminum (μ g)	Influenza vaccines (μ g)	Survival number/sum (PowderJect _)	Survival number/sum (syringe and pin)
				Do not have	????5	?????3/8	?????0/8
Do not have	????1	?????0/8	?????0/8
				??100	????5	?????8/8	?????7/8
??100	????1	?????5/7	?????6/8
				???-	Do not do the mouse of overtesting	?????????????????2/16

Annotate: data by the number of animals of survival after 14 days to being stimulated the animal sum.

Equally with adjuvant and the process PowderJect of this particles A lum adjunvant composition as crystallization diphtheria toxin, diphtherotoxin (dT) vaccine combination ^_The Needleless injection drug-supplying system is carried.With with 100 μ g particles A lum adjuvants or do not have 5 μ g of particles A lum adjuvant or 1 μ g crystallization dT vaccine combination uses PowderJect ^_Equipment is inoculated mouse by transdermal administration.Use conventional needle and the aqueous compositions subcutaneous vaccination control animal of syringe drug-supplying system with identical vaccine/adjunvant composition.Inoculate in 0 and 4 weeks.The 6th week collected serum and compiled from 8 mouse, and detected dT antibody by above-mentioned elisa technique.

The result of ELISA research is reported in the following table 15.This shows, when with accept the same dose vaccine but when not having the control animal of adjuvant to compare, through PowderJect ^_It is that serum antibody response in the animal of dT vaccine combination microgranule of adjuvant improves greatly that administration device is accepted with Alum.Seen identical of these serum antibody levels and the animal of accepting this liquid state vaccine/adjunvant composition with conventional apparatus.

Table 15
				The IgG titer of in the 6th all serum that is compiled, measuring by ELISA
Aluminum (μ g)	dT(μg)	Serum IgG titer (PowderJect _)	Serum IgG titer (syringe/pin)
Do not have	1	8130	570
100	1	58085	142120

Measure the IgG subclass titer of dT equally by ELISA, the result is reported in the following table 16.This shows, pass through PowderJect ^_The equipment transdermal administration is that the dI vaccine combination microgranule of adjuvant mainly causes the IgG1 reaction with Alum.See that with conventional needle and injector to inject identical IgG subclass distributes.This explanation Alum adjuvant has promoted the Th2 type immunoreation by this vaccine of percutaneous drug delivery.Therefore, use method of the present invention particles A lum adjuvant can be transported to skin and be used to control the immunoreation type of common administration of vaccines.

Table 16
						The IgG subclass of measuring by ELISA in the 6th all serum that is compiled distributes
Aluminum (μ g)	dT(μg)	PowderJect _		Syringe/pin
						IgG1	IgG2a	IgG1	IgG2a
		Do not have	5	14064 0	2170	11480 0	745
100	5	24907 0	810	35932 0	1145

2. the inoculation study of microgranule PCPP adjunvant composition.With adjuvant and the process PowderJect of microgranule PCPP adjunvant composition as crystallization inactivation Inflenza vaccine composition ^_The Needleless injection drug-supplying system is carried.With with 100 μ g microgranule PCPP adjunvant compositions or do not have 5 μ g of microgranule PCPP adjunvant composition or 1 μ g crystallization inactivation Inflenza vaccine composition inoculation mouse.Use conventional needle to inoculate control animal with the aqueous compositions of identical vaccine/adjunvant composition through S/C with syringe.Carrying out three inoculations (in 0,4 and 10.5 weeks) afterwards, from 8 mouse, compile serum in the 12nd week, and detect Antibody of Influenza by above-mentioned elisa technique.Equally with the naked eye estimate the toxicity sign (for example, forming granuloma) of injection position with hands.

Granuloma is the common form that gives the afterwards seen local toxic effect of many adjuvants.The subcutaneous injection liquid state is that the Inflenza vaccine composition of adjuvant causes forming granuloma at the subcutaneous tissue at injection position place with PCPP.On the contrary, use PowderJect ^_The delivery of particulate compositions detects with thick hands and naked eyes to be discovered less than granuloma.These data suggest transdermal delivery of particulate PCPP adjuvant reduces or even has avoided the toxicity relevant with PCPP usually.

In order to estimate the next protective of influenza virus stimulation, instil 1 * 10 by intranasal in the 12nd week ⁶PFU (100 * LD ₅₀) adapt to the Aichi influenza virus of mouse to stimulate vaccinated animal.Stimulate as mentioned above.Write down survival condition continuous 14 day every day and lose weight.Survival ratio is illustrated in the following table 17, and body weight is described among Figure 10.As can be seen from Table 17, compare, microgranule PCPP adjuvant is increased the survival ratio just greatly with crystallization influenza vaccines (with 5 μ g dosage) administration with the vaccine that does not have adjuvant.See identical protective with identical vaccine dose from contrast (liquid state) animal.But, as seen from Figure 10, PowderJect ^_Medicine is that the influenza vaccines microgranule of adjuvant has prevented to lose weight than the subcutaneous injection that uses conventional needle and injector system better with PCPP.Thus, two groups of mouse all alleviate about 15% body weight at first, but through PowderJect ^_The mouse of inoculation recovers body weight with the speed faster than the mouse of subcutaneous vaccination, can't see big protective in the animal of accepting 1 μ g dosage influenza vaccines (being with or without adjuvant) when carrying with microgranule or liquid form.

Equally with adjuvant and the process PowderJect of this microgranule PCPP adjunvant composition as crystallization diphtheria toxin, diphtherotoxin (dT) vaccine combination ^_The Needleless injection drug-supplying system is carried.With with 100 μ g microgranule PCPP adjuvants or do not have 5 μ g of microgranule PCPP adjuvant or 1 μ g crystallization dT vaccine combination uses PowderJect ^_Equipment is inoculated mouse by transdermal administration.Use conventional needle and the aqueous compositions subcutaneous vaccination control animal of syringe drug-supplying system with identical vaccine/adjunvant composition.Inoculate in 0 and 4 weeks.The 6th week collected serum and compiled from 8 mouse, and detected dT antibody by above-mentioned elisa technique.

The result of ELISA research is reported in the following table 18.This shows, when with accept the same dose vaccine but when not having the control animal of adjuvant to compare, through PowderJeot ^_It is that serum antibody response in the animal of dT vaccine combination microgranule of adjuvant improves greatly that administration device is accepted with PCPP.Seen identical of these serum antibody levels and the animal of accepting this liquid state vaccine/adjunvant composition with conventional apparatus.

Table 17
				The protective that the opposing influenza stimulates
?PCPP (μg)	Influenza vaccines (μ g)	Survival number/sum (PowderJect _)	Survival number/sum (syringe and pin)
				Do not have	????5	?????3/8	?????0/8
Do not have	????1	?????0/8	?????0/8
				?100	????5	?????6/8	?????7/7
?100	????1	?????2/8	?????2/8
				??-	Do not do the mouse of overtesting	??????????????2/16

Annotate: data by the number of animals of survival after 14 days to being stimulated the animal sum.

Table 18
				The IgG titer of in the 6th all serum that is compiled, measuring by ELISA
PCPP(μg)	dT(μg)	Serum IgG titer (PowderJect _)	Serum IgG titer (syringe/pin)
Do not have	1	8130	570
100	1	248550	284415

Measure the IgG subclass titer of dT equally by ELISA, the result is reported in the following table 19.This shows, pass through PowderJect ^_The equipment transdermal administration is that the dT vaccine combination microgranule of adjuvant mainly causes the IgG1 reaction with PCPP.Can see that with conventional needle and injector to inject identical IgG subclass distributes.This explanation PCPP adjuvant has promoted the Th2 type immunoreation by this vaccine of percutaneous drug delivery.Therefore, use method of the present invention microgranule PCPP adjuvant can be transported to skin and be used to control the immunoreation type of common administration of vaccines.

Table 19
						The IgG subclass of measuring by ELISA in the 6th all serum that is compiled distributes
PCPP(μg)	dT(μg)	PowderJect _		Syringe/pin
						IgG1	IgG2a	IgG1	IgG2a
		Do not have	5	140640	2170	114800	745
100	5	308160	3620	585810	3225

3. the inoculation study of microgranule CpG adjunvant composition.With adjuvant and the process PowderJect of microgranule CpG adjunvant composition as crystallization inactivation Inflenza vaccine composition ^_The Needleless injection drug-supplying system is carried.With with 100 μ g microgranule CpG adjunvant compositions or do not have 5 μ g of microgranule CpG adjunvant composition or 1 μ g crystallization inactivation Inflenza vaccine composition inoculation mouse.Use conventional needle to inoculate control animal with the aqueous compositions of identical vaccine/adjunvant composition through S/C with syringe.Carrying out three inoculations (in 0,4 and 10.5 weeks) afterwards, from 8 mouse, compile serum in the 12nd week, and detect Antibody of Influenza by above-mentioned elisa technique.

The result of ELISA research is reported in the following table 20.Through PowderJect ^_It is the administration of influenza vaccines microgranule of adjuvant that equipment carries out with CpG, when with accept the same dose vaccine but when not having the animal of adjuvant to compare, serum antibody response improves greatly.In addition, through PowderJect ^_Equipment is accepted serum antibody level and the animal of adopting the conventional art inoculation with identical vaccine/adjunvant composition identical in the animal of microparticle compositions.Therefore, the CpG adjuvant can be transported to skin with powdery, thereby strengthen the immunogenicity of influenza vaccines.The immunoenhancement result that provides by the CpG adjuvant has been provided greatly with this mode administration.

Table 20
				The total IgG titer of in the 12nd all serum that is compiled, measuring by ELISA
CpG(μg)	Influenza vaccines (μ g)	Serum IgG titer (PowderJect _)	Serum IgG titer (syringe/pin)
				Do not have	????5	????6790	????1505
Do not have	????1	????873	????804
				?10μg	????5	????6066	????874
?10μg	????1	????2862	????＜100

Measure the IgG subclass titer of influenza virus equally by ELISA, the result is reported in the following table 21.This shows PowderJect ^_The microgranule Inflenza vaccine composition that administration does not have adjuvant mainly causes the IgG1 reaction.Should can see that identical IgG subclass distributed by the liquid state vaccine combination with conventional needle with injector to inject, just much lower by the titer of this approach.These data declaration influenza vaccines itself cause Th2 type immunity.PowderJect ^_Administration is that the Inflenza vaccine composition microgranule of adjuvant mainly produces IgG2a antibody with CpG.This explanation CpG promotes the Th1 reaction.Therefore, skin is administration CpG adjuvant or is the optimum position of the vaccine of adjuvant with CpG.

Table 21
						The IgG subclass of measuring by ELISA in the 6th all serum that is compiled distributes
CpG(μg)	Influenza virus (μ g)	???PowderJect _		Syringe/pin
						?IgG1	?IgG2a	?IgG1	?IgG2a
		Do not have	????1	?367	?＜200					＜200	＜200
????10	????1					＜200	?1858	＜200	＜200
		Do not have	????5	?16340	?＜200					＜200	＜200
????10	????5					?714	?8711	＜200	?547

In order to estimate the next protective of influenza virus stimulation, instil 1 * 10 by intranasal in the 12nd week ⁶PFU (100 * LD ₅₀) adapt to the Aichi influenza virus of mouse to stimulate vaccinated animal.Stimulate as mentioned above.Write down survival and lose weight continuous 14 day every day.The survival ratio is reported in the following table 22.Weight data is described among Figure 11 A and the 11B.As can be seen from Table 22, when in order to CpG being adjuvant and through PowderJect ^_During the equipment transdermal administration, see that the survival ratio of the animal of accepting particulate vaccine compositions (under 1 μ g and 5 μ g dosage) is 100%.On the contrary, this fluid composition of subcutaneous injection can not get any protective when 1 μ g dosage.Therefore, will be the PowderJect that the influenza vaccines microgranule of adjuvant is transported to skin with CpG ^_Administration is more effective than subcutaneous injection.

Table 22
				The protective that the opposing influenza stimulates
CpG(μg ????)	Influenza vaccines (μ g)	Survival number/sum (PowderJect _)	Survival number/sum (syringe and pin)
				Do not have	????5	?????3/8	?????0/8
Do not have	????1	?????0/8	?????0/8
				???10	????5	?????7/7	?????7/8
???10	????1	?????8/8	?????3/8
				???-	Do not do the mouse of overtesting	?????????????????????2/16

Annotate: data by the number of animals of survival after 14 days to being stimulated the animal sum.

Referring now to Figure 11 A and 11B, PowderJect ^_Administration is that the influenza vaccines microgranule of adjuvant has prevented to lose weight than the subcutaneous injection that uses conventional needle and syringe administration better with CpG.Thus, see in order to CpG being that the 1 μ g of adjuvant or the mouse original weight of 5 μ g crystallization influenza vaccinations alleviate less than 10%, and the very fast recovery body weight of these mouse, on the contrary, the protective that subcutaneous injection provides is much smaller.Specifically, be that original weight alleviates near 20% in the mouse of vaccine (with 5 μ g dosage) of adjuvant with CpG in the subcutaneous injection liquid state, and the subcutaneous injection liquid state is that the vaccine (with 1 μ g dosage) of adjuvant does not provide any protective with CpG.It is about 25% that 5 days body weight of all animals to the in this matched group alleviate, just dead by the 7th day.These data suggest are when using PowderJect ^_The CpG when percutaneous drug delivery of system is more effective and potent immunomodulator.

Equally with adjuvant and the process PowderJect of this microgranule CpG adjunvant composition as crystallization diphtheria toxin, diphtherotoxin (dT) vaccine combination ^_The Needleless injection drug-supplying system is carried.With with 10 μ g microgranule CpG adjuvants or do not have 5 μ g of microgranule CpG adjuvant or 1 μ g crystallization dT vaccine combination uses PowderJect ^_Equipment is inoculated mouse by transdermal administration.Use conventional needle and the aqueous compositions subcutaneous vaccination control animal of syringe drug-supplying system with identical vaccine/adjunvant composition.Inoculate in 0 and 4 weeks.The 6th week collected serum and compiled from 8 mouse, and detected dI antibody by above-mentioned elisa technique.

The result of ELISA research is reported in the following table 23.This shows, when with accept the same dose vaccine but when not having the control animal of adjuvant to compare, through PowderJect ^_It is that serum antibody response in the animal of dT vaccine combination microgranule of adjuvant improves greatly that administration device is accepted with CpG.Through PowderJect ^_The serum antibody level that administration device is accepted in the animal of microparticle compositions is higher 10 times than the titer in the animal that inoculates identical vaccine/adjunvant composition (with liquid state) with conventional needle and syringe.Therefore, carry CpG to strengthen the immunogenicity of common administration dI vaccine combination with particulate form.

Table 23
				The IgG titer of in the 6th all serum that is compiled, measuring by ELISA
?CpG (μg)	???dT(μg)	Serum IgG titer (PowderJect _)	Serum IgG titer (syringe/pin)
				Do not have	????1	????8130	????570
?10	????1	????614470	????33680
				Do not have	????5	????188830	????149560
?10	????5	????1483450	????116660

Measure the antigenic IgG subclass of dT titer by ELISA equally, the result is reported in the following table 24.This shows PowderJect ^_The microgranule Inflenza vaccine composition that administration does not have adjuvant mainly causes the IgG1 reaction.Should can see that identical IgG subclass distributed by the liquid state vaccine combination with conventional needle with injector to inject, just much lower by the titer of this approach.These data declaration influenza vaccines itself cause Th2 type immunity.PowderJect ^_Administration is that the Inflenza vaccine composition microgranule of adjuvant mainly produces IgG2a antibody with CpG.This explanation CpG promotes.The Th1 reaction.Can see that with this vaccine combination of conventional needle and injector to inject (but with liquid state) identical IgG subclass distributes, just much lower by the titer of this approach.Therefore, skin is administration CpG adjuvant or is the optimum position of the vaccine of adjuvant with CpG.

Table 24
					The IgG subclass of measuring by ELISA in the 6th all serum that is compiled distributes
CpG(μg)	???dT(μg)	Administration	IgG subclass titer
					????IgG1	?IgG2a
			Do not have	????1			????SC	????7625	＜200
Do not have	????1	????PJ			????605	＜200
			????10	????1			????SC	??297600	?14165
????10	????1	????PJ			??41850	?2350
			Do not have	????5			????SC	??140640	?2170
Do not have	????5	????PJ			??114800	?745
			????10	????5			????SC	??41050	?14020 ????0
????10	????5	????PJ			?166130	?4000

The SC=subcutaneous injection, PJ=PowderJect ^_Equipment.Inoculate in 0 and 4 weeks.

4. the inoculation study of liquid and microgranule MPL adjunvant composition.In research at first, liquid MPL adjunvant composition is mixed with crystallization inactivation Inflenza vaccine composition (through PowderJect as adjuvant ^_The Needleless injection administration device is carried).With with the liquid MPL adjunvant composition of 50 μ g or the 5 μ g or the 1 μ g crystallization inactivation Inflenza vaccine composition inoculation mouse of this adjunvant composition.When using the MPL adjuvant, use No. 27 pin subcutaneous injections should liquid state MPL compositions, 5 minutes afterwards at same position PowderJect ^_Equipment administration crystallization vaccine.Use conventional needle the aqueous compositions of identical vaccine/adjunvant composition to be inoculated control animal through S/C with syringe.Carrying out three inoculations (in 0,4 and 10.5 weeks) afterwards, from 8 mouse, compile serum in the 12nd week, and detect Antibody of Influenza by above-mentioned elisa technique.

The result of ELISA research is reported in the following table 25.Through percutaneous drug delivery should be with MPL be the influenza vaccines of adjuvant, when with accept the same dose vaccine but when not having the animal of adjuvant to compare, serum antibody response improves greatly.Through PowderJect ^_Equipment is accepted serum antibody level and the animal of adopting the conventional art inoculation with identical vaccine/adjunvant composition identical in the animal of microparticle compositions.Therefore, the MPL adjuvant can be transported to skin with powdery, thereby strengthen the immunogenicity of influenza vaccines.

Table 25
				The total IgG titer of in the 12nd all serum that is compiled, measuring by ELISA
MPL(μg)	Influenza vaccines (μ g)	Serum IgG titer (PowderJect _)	Serum IgG titer (syringe/pin)
				Do not have	????5	????6790	????1505
Do not have	????1	????873	????804
				????50	????5	????20042	????10089
????50	????1	????1158	????2490

Annotate: inoculate in 0,4 and 10.5 weeks.MPL is to use No. 27 pins through hypodermic, uses PowderJect after 5 minutes ^_Equipment gives the vaccine powder to same position.

Measure the IgG subclass titer of influenza virus equally by ELISA, the result is reported in the following table 26.This shows PowderJect ^_The microgranule Inflenza vaccine composition that administration does not have adjuvant mainly causes the IgGl reaction.Should can see that identical IgG subclass distributed by the liquid state vaccine combination with conventional needle with injector to inject, with subcutaneous injection MPL adjuvant and PowderJect ^_Administration crystallization Inflenza vaccine composition combines and has produced IgGl and IgG2a antibody, and this explanation percutaneous drug delivery MPL causes the balance Th1/Th2 reaction of influenza vaccines.Should can see that identical IgG subclass distributed by the liquid state vaccine combination with conventional needle with injector to inject.

Table 26
						The IgG subclass of measuring by ELISA in the 6th all serum that is compiled distributes
MPL	Influenza	PowderJect _		Syringe ﹠ pin
(μg)	(μg)					IgG1	IgG2a	IgG1	IgG2a
Do not have	5	16340	<200	<200	<200
50	5	18985	1848	9548	3700

In order to estimate the next protective of influenza virus stimulation, instil 1 * 10 by intranasal in the 12nd week ⁶PFU (100 * LD ₅₀) adapt to the Aichi influenza virus of mouse to stimulate vaccinated animal.Stimulate as mentioned above.Write down survival and lose weight continuous 14 day every day.Survival ratio is illustrated in the following table 27, and weight data is described among Figure 12.As can be seen from Table 27, be that 5 μ g crystallization vaccine combinations of adjuvant (through hypodermic needle and injector to inject administration) are (through PowderJect in order to 50 μ gPML ^_The equipment administration) sees 100% survival rate in Jie Zhong the animal.6 mouse have only 4 survivals in contrast (through conventional needle and the syringe administration) group of identical vaccine and adjuvant yet accepting.Passing through PowderJect ^_Accept to see the part protective in the animal of 1 μ g vaccine combination and 50 μ gMPL with conventional needle and syringe administration.

Table 27
				The protective that the opposing influenza stimulates
??MPL ?(μg)	Influenza vaccines (μ g)	Survival number/sum (PowderJect _)	Survival number/sum (syringe and pin)
				Do not have	????5	?????3/8	?????0/8
Do not have	????1	?????0/8	?????0/8
				??50	????5	?????7/7	?????4/6
??50	????1	?????3/8	?????5/8
				??-	Do not do the mouse of overtesting	????????????????????2/16

Annotate: data are that the number of animals of survival after 14 days is to 10 ⁶The animal sum that virus stimulated of PFUs.

Referring now to Figure 12, PowderJect ^_Administration has prevented to lose weight than the identical vaccine of subcutaneous injection that uses conventional needle and syringe administration/adjuvant combination with MPL adjuvant crystallization influenza vaccines together better.Accepting 5 μ g vaccine doses (through transdermal PowderJect ^_Administration) to alleviate be 10% to weight limit and in the animal of 50 μ gMPL adjuvants, still, and the very fast recovery body weight of these animals.On the contrary, the inoculation fluid composition (S/C) the weight of animals alleviate near 20%, and their weight recovery speed slowly many.

In the research secondarily, with adjuvant and the process PowderJect of microgranule MPL compositions as crystallization diphtheria toxin, diphtherotoxin (dT) vaccine combination ^_The Needleless injection drug-supplying system is carried.With with 50 μ g microgranule MPL adjuvants or 5 μ g of this adjuvant or 1 μ g crystallization dT vaccine combination use PowderJect ^_Equipment is inoculated mouse by transdermal administration.Use conventional needle and the aqueous compositions subcutaneous vaccination control animal of syringe drug-supplying system with identical vaccine/adjunvant composition.Inoculate in 0 and 4 weeks.The 6th week collected serum and compiled from 8 mouse, and detected dT antibody by above-mentioned elisa technique.

Then transdermal administration is the dT vaccine combination microgranule of adjuvant with MPL, and serum antibody response is a little higher than to be accepted the same dose vaccine but do not have the control animal of MPL.Through transdermal PowderJect ^_Serum antibody level in the animal of administration inoculation is identical with the titer of the animal that inoculates identical vaccine/adjunvant composition by conventional needle and injector to inject.

Detect the IgG subclass titer of dI by ELISA.The result is reported in the following table 28.This shows PowderJect ^_The dI vaccine that administration does not have adjuvant mainly causes the IgG1 reaction.See that with conventional needle and injector to inject identical IgG subclass distributes.When with particulate form from PowderJect ^_During the equipment administration, should be that the dT vaccine combination of adjuvant produces IgG1 and IgG2a antibody with MPL.See that with conventional needle and injector to inject identical IgG subclass distributes.This explanation can be with the PowderJect of MPL to skin ^_Be administered for and induce equilibrated Th1/Th2 type immunity.

Table 28
					The IgG subclass of measuring by ELISA in the 6th all serum that is compiled distributes
MPL(μg)	dT(μg)	Administration	IgG subclass titer
					?IgG1	????IgG2a
			Do not have	????5			????SC	?140640	????2170
Do not have	????5	????PJ			?114800	????745
			????50	????5			????SC	?258390	????10150
????50	????5	????PJ			?353300	????2925

Mouse kind=Balb/c, SC=subcutaneous injection, PJ=PowderJect ^_Equipment.Inoculate in 0 and 4 weeks.

Therefore, the invention discloses the novel method of transdermal administration vaccine and adjunvant composition.(crystallization) pharmaceutical composition and preparation of novel processing also has been described and used its method.Although describe preferred implementation of the present invention in detail, significantly change in the spirit and scope of the present invention that are interpreted as in not deviating from the accessory claim book, to be put down in writing.

Claims (70)

1. method that improves selected antigen immune.This method comprises:

(a) antigen with effective dose gives the vertebrates main body; With

(b) be enough to improve the particle adjuvant compositions of the amount of antigen immune, wherein adjuvant transmitted into or skin or tissue by the vertebrates main body, wherein also use the transdermal administration technology to carry out described administration.

2. the process of claim 1 wherein that antigen is particulate form and uses the transdermal administration technology it to be delivered into or to pass through the skin or the tissue of vertebrates main body.

3. the process of claim 1 wherein and use needleless injector to give equipment delivery of particulate adjunvant composition.

4. the process of claim 1 wherein that antigen and adjuvant appear at separately in the compositions.

5. the process of claim 1 wherein that antigen and adjuvant appear in the same combination.

6. the process of claim 1 wherein the diverse location that antigen and adjuvant is administered to the vertebrates main body.

7. the process of claim 1 wherein the same position that antigen and adjuvant is administered to the vertebrates main body.

8. the process of claim 1 wherein and before adjunvant composition, give antigen.

9. the process of claim 1 wherein and after adjunvant composition, give antigen.

10. the process of claim 1 wherein antigen and adjunvant composition are given simultaneously.

11. the process of claim 1 wherein that antigen is virus antigen.

12. the method for claim 11, wherein virus antigen is a virus protein.

13. the method for claim 11, wherein virus antigen is a virion.

14. the process of claim 1 wherein that antigen is the subunit vaccine compositions.

15. the process of claim 1 wherein that antigen is bacterial antigens.

16. the method for claim 15, wherein bacterial antigens are bacterioprotein or polysaccharide.

17. the process of claim 1 wherein that antigen is the organism that weakens of living.

18. the method for claim 17 wherein weakens organism and is virus.

19. the method for claim 17, wherein weakening organism is antibacterial.

20. the process of claim 1 wherein that the particle adjuvant compositions provides with the crystal form of suitable transdermal administration.

21. the process of claim 1 wherein that adjuvant is the CpG oligonucleotide.

22. the method for claim 21, wherein the CpG oligonucleotide comprises sequence TCCATGACGTTCCTGATGCT (SEQ ID NO:1).

23. the method for claim 21, wherein the CpG oligonucleotide comprises sequence A TCGACTCTCGAGCGTTCTC (SEQ ID NO:2).

24. one kind causes immunoreactive method in the vertebrates main body, described method comprises that with the particulate vaccine compositions transdermal administration wherein this particulate vaccine compositions comprises in the skin of vertebrates main body or tissue or by wherein:

(a) the selected antigen of effective dose; With

(b) present in an amount at least sufficient to improve the adjuvant of antigen immune.

25. the method for claim 24 wherein uses needleless injector to give the equipment delivery of particulate vaccine compositions.

26. the method for claim 24, wherein antigen is virus antigen.

27. the method for claim 26, wherein virus antigen is a virus protein.

28. the method for claim 26, wherein virus antigen is a virion.

29. the method for claim 24, wherein vaccine combination is the subunit vaccine compositions.

30. the method for claim 24, wherein antigen is bacterial antigens.

31. the method for claim 30, wherein thin mattress antigen is bacterioprotein or polysaccharide.

32. the method for claim 24, wherein the weaken organism of antigen for living.

33. the method for claim 32 wherein weakens organism and is virus.

34. the method for claim 32, wherein weakening organism is antibacterial.

35. the method for claim 24, wherein particulate vaccine compositions provides with the crystal form of suitable transdermal administration.

36. the method for claim 24, wherein adjuvant is the CpG oligonucleotide.

37. the method for claim 36, wherein the CpG oligonucleotide comprises sequence TCCATGACGTTCCTGATGCT (SEQ ID NO:1).

38. the method for claim 36, wherein the CpG oligonucleotide comprises sequence A TCGACTCTCGAGCGTTCTC (SEQ ID NO:2).

39. suitable the use in skin that the transdermal administration technology is transported to the vertebrates main body or the tissue or the particle adjuvant compositions by wherein.

40. an adjuvant is used for transdermal administration to the skin of vertebrates main body or tissue or by the purposes in the microparticle compositions wherein in production.

41. according to the purposes of claim 40, wherein microparticle compositions comprises selected antigen, and adjuvant improves this immunogenicity of antigens.

42., wherein use the needleless injector administration device that this microparticle compositions is transported in the skin of vertebrates main body or the tissue or by wherein according to the purposes of claim 40.

43. according to the purposes of claim 40, wherein antigen is virus antigen.

44. according to the purposes of claim 43, wherein virus antigen is a virus protein.

45. according to the purposes of claim 43, wherein virus antigen is a virion.

46. according to the purposes of claim 40, wherein antigen is the subunit vaccine compositions.

47. according to the purposes of claim 40, wherein antigen is bacterial antigens.

48. according to the purposes of claim 47, wherein bacterial antigens are bacterioprotein or polysaccharide.

49. according to the purposes of claim 40, wherein antigen is the organism that weakens alive.

50., wherein weaken organism and be virus according to the purposes of claim 49.

51. according to the purposes of claim 49, wherein weakening organism is antibacterial.

52. according to the purposes of claim 40, wherein microparticle compositions provides with the crystal form of suitable transdermal administration.

53. according to the purposes of claim 40, wherein adjuvant is the CpG oligonucleotide.

54. according to the purposes of claim 53, wherein the CpG oligonucleotide comprises sequence TCCATGACGTTCCTGATGCT (SEQ ID NO:1).

55. according to the purposes of claim 53, wherein the CpG oligonucleotide comprises sequence A TCGACTCTCGAGCGTTCTC (SEQ ID NO:2).

56. a method that causes biological effect in the vertebrates main body comprises that the particle adjuvant compositions of claim 39 that will enough cause the amount of biological effect is administered in the skin of vertebrates main body or the tissue or by wherein.

57. one kind forms the crystalline drug method for compositions, described method comprises:

(a) liquid pharmaceutical is mixed so that a compositions to be provided with the sugar of suitable drugs level;

(b) said composition is being formed drying under the favourable suitable evaporation conditions to crystallization, therefore acquisition has the crystal composition of the density feature of raising; With

(c) collect this crystal composition.

58. the method for claim 57, wherein pharmaceutical composition is a vaccine combination.

Be transported in the skin of vertebrates main body or the tissue or the crystalline drug compositions by wherein 59. one kind suitable.

60. the compositions of claim 59, wherein said compositions are vaccine combination.

61. the compositions of claim 60, wherein said compositions comprise that an antigen and its amount enough improve the excipient of crystalline drug composition density.

62. the compositions of claim 61, wherein antigen is virus antigen.

63. the compositions of claim 61, wherein antigen is bacterial antigens.

64. method of handling a main body, described method comprises the crystalline drug delivery of composition of claim 59 in the skin of described main body or tissue or by wherein, wherein carries this crystal composition with the amount that enough causes prevention or therapeutic effect in main body.

65. the method for claim 64, wherein pharmaceutical composition is for comprising interested antigenic vaccine combination.

66. the method for claim 65, wherein vaccine combination is the subunit vaccine compositions.

67. the method for claim 65, wherein vaccine combination comprises virus antigen.

68. the method for claim 65, wherein a compositions comprises bacterial antigens.

69. the method for claim 64 wherein uses needleless injector that this crystal composition is transported in the main body.

70. a medicament is used for transdermal administration to the skin of vertebrates main body or tissue or by the purposes in the microparticle compositions wherein in production.

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