CN1120310A - Polymeric mucoadhesives in the delivery of immunogens at mucosal surfaces - Google Patents

Polymeric mucoadhesives in the delivery of immunogens at mucosal surfaces Download PDF

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CN1120310A
CN1120310A CN94191614A CN94191614A CN1120310A CN 1120310 A CN1120310 A CN 1120310A CN 94191614 A CN94191614 A CN 94191614A CN 94191614 A CN94191614 A CN 94191614A CN 1120310 A CN1120310 A CN 1120310A
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mucoadhesive
antigen
immunity
virus
adjuvant
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J·D·邓肯
D·P·谢弗
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SECRETECH Inc
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SECRETECH Inc
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
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    • A61K39/12—Viral antigens
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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    • A61K9/0043—Nose
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
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    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/5555—Muramyl dipeptides
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
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    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
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    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

A pharmaceutical composition for inducing an immune response against an infectious agent in an animal comprises an antigen against the infectious agent and a mucoadhesive, and optionally an adjuvant, to boost the immune response to the antigen in the animal. Preferably, the antigen is derived from an influenza virus. The pharmaceutical composition is useful in a method of immunizing an animal by oral administration of the composition to the animal.

Description

Transport immunogenic polymerization mucoadhesive at mucomembranous surface
The present invention be JIUYUE in 1993 to submit (act on behalf of case number be 05060-0003-01000) sequence number on the 13rd be the part continuation application of 08/119578 application, the latter is again that the sequence number of (proxy number is 05060-0003-00000) of submitting on March 11st, 1993 is the part continuation application of 08/029668 application.The full content of all these applications here proposes as a reference.
The former cost of oral immunity is low, and safety also can promote compliance.Now part proves, stimulates the immunocompetence tissue of small intestinal and the protective immune response that cell can stimulate other mucomembranous surface by general mucomembranous immune system.Under some occasion, this stimulation also can cause seroreaction.Yet the application of oral vaccine is confined to the minority vaccine in medical treatment now.
Representational is that oral immunity needs the immunogen of suitable high dose to cause protective immunological reaction, and some vaccine can not induce reaction when oral.In addition, attempt increasing the safety of vaccine and using synthetic and the subunit preparation through Attenuation, the antigen preparation that generates is poorer than whole holoantigen effect usually.
The various adjuvants and the experimental oral vaccine that will include muramyldipeptide and fluoride share, and improve immunoreation so that attempt through the respond of immune stimulatory cell.Another kind of adjuvant approach be the antigen bag in granule, thereby make antigen become the form that is absorbed by immune system cell easily.For example antigen is incorporated into or is incorporated in polymer particle, nanoparticle or the liposome, usually have more immunogenicity than soluble antigen, these graininess antigens are owing to drop in the mucosa, or optionally by the M cell capture of intestinal mucosa and more effective.
Immunoreation after oral immunity is former also can be owing to the immunogen of using optionally be incorporated into epithelial cell (for example influenza hemagglutinin, vibrio cholera) or immunogen is coupled to optionally (attachment proteins of intestinal bacterium for example is as shigellosis or pseudomonas disease with the bonded chemical compound of these cells; Toxoid such as cholera or DT-Pa, or pollen grain) upward improve.
But still need certain methods and compositions after taking immunogen, to strengthen immunoreation at mucomembranous surface.
The objective of the invention is after taking immunogen, to strengthen immunoreation at mucomembranous surface.One embodiment of the invention are that immunogen and polymerization mucoadhesive are associated, and induce or increased immunoreation after oral administration or other the mucosal route immunity.The mucoadhesive of transport of drug (particularly with transdermal and suck method) and the theory and the practical value of other biological adhesive are all to know in this area, but immunogen to use with the enhance immunity reaction with mucoadhesive be unknown so far.
Another embodiment of the present invention is to induce or increase oral administration or the immunoreation after other mucosal route immunity with adjuvant and immunogen and polymerization mucoadhesive.Immunogen and mucoadhesive share that the effectiveness of adjuvant also was unknown in the past when oral.
In pharmaceutical field, all know to host's subcutaneous injection immunogen and can produce acceptable immunoreation.In addition, mucosa delivery though especially oral immunity is former usually not ideal, but still can produce immunoreation under some occasion.Immune composition of the present invention and immunization method are when using immunogen for the main mucomembranous surface that contracts, can strengthen host's immunoreation, use this administering mode, the present invention can be in some cases, obtain the general immunoreation, the effect that its intensity and persistent period are reached when approaching to give host's subcutaneous injection with same antigen very much, and the reaction of the mucosal immunoreaction that obtains during than independent subcutaneous injection antigen or mucosal administration antigen is stronger.
Implementing the present invention is to use or the former immune composition of various routine immunizations for animals with containing known person, with the immunoreation of stimulation of host.Terminology used here " immunogen " is meant and is given to the material that can stimulate body fluid, mucosa or cell-mediated immunity in the body.Term " immunogen " and " antigen " are general at this.
After terminology used here " immunoreation " is meant and touches immunogen, the specificity to external antigen reactivity that is produced changes, as increasing by the immunogenic antibody of opposing in mucosa or serum, perhaps for example cytokines activity or hypertrophy are indicated by the cell-mediated immunity of tolerance.
The immunoreation that the present invention produces conventionally is the change of measuring the immunity of mucosa, for example measure the variation of saliva IgA or the variation of mensuration general immunity with ELISA, for example measure the variation of antibody horizontal with ELISA, or the variation of mensuration cellular immunity, for example measure the reaction of T proliferation of cells.Under the situation of influenza virus being carried out immunity, also can be with measuring the method that the serum hemagglutinin suppresses titre (HI).Under the situation of influenza, can to make the immunoreation that obtains in the roughly the same time behind the same material of immunoreation that mice obtains and subcutaneous injection equivalent approximately be identical on intensity in the present invention when using these analytical methods.
Oral administration or other mucosal route are carried out after the immunity of the present invention, can be used for stimulating the immunogen kind of general mucomembranous immune system to comprise (non-limiting): 1) can through mucous membrane surface and produce the antigen of immunoreactive various infectious diseases, especially comprise natural mucomembranous immunogen (for example antigen of influenza virus, intestinal bacterium such as escherichia coli, vibrio cholera button pylori), these mucomembranous immunogens can by with mucosa on part and the interaction of receptor-specific be attached on the mucomembranous epithelial cell; 2) the enteral pathogen alive of attenuation (for example spinal cord virus or rotavirus); 3) synthetic graininess antigen (for example containing antigenic microsphere and millimicro ball); 4) with the bonded anaphylactogen of mucosa (for example ragweed pollen); With 5) autoantigen and tissue antigen (for example myelin basic protein, CD4, melanoma-specific protein).
Immunogen preparation, it can be complete immunogen, the modification immunogen, synthetic immunogen or above-mentioned immunogenic subunit, can be from virus (for example influenza, HIV, rotavirus or hepatitis virus), antibacterial (for example shigellosis, bordetella pertussis or chlamydia), protokaryon parasite (for example causing the plasmodium of malaria), toxin (for example cholera toxin or endotoxin), anaphylactogen (for example ragweed pollen) or tissue marker thing (for example melanoma CD4, or myelin basic protein).
Immunogen used in the preferred embodiment of the invention is from virus.The present invention is applicable to the multiple virus that is expected with the vaccine opposing.The present invention is particularly useful for resisting common breathing or enterovirus.For purposes of illustration, be illustrated with influenza virus here.Yet the present invention is not intended to be only limited to influenza virus, also is not limited to strains of influenza viruses used in described embodiment.Other virus of available the present invention's opposing for example comprises parainfluenza virus, respiratory syncytial virus, rhinovirus, coronavirus and adenovirus.
For influenza virus, the virus of selecting to produce vaccine depends on that partly hope can obtain the strains of influenza viruses formula hypotype of prevention.In order to obtain the vaccine useful to specific strains of influenza viruses, preferably with having the raw material of the strains of influenza viruses of at least one antigenic determinant as vaccine, this Strain is identical with the Strain of available vaccine opposing.
Will be appreciated that the present invention can use influenza virus type and the hypotype of various animals, and the not homophyletic of certain hypotype system.But this influenza virus is representational to be infection animal, for example the virus of chicken, duck and pig.The present invention is particularly useful for comprising people's primate, for example available human influenza hypotype HON1, H 3N 8, H 2N 2(being very popular property Asia virus), H 3N 2(being very popular property Hong Kong virus) H1N1 (being very popular property Russia virus), or other virus subtype that causes by antigenic discontinuous variation, and the hypotype that causes by antigenic continuous variation.A kind of example that is used for certain hypotype strain of the present invention is influenza virus A/Udorn/307/72 (H3N2), and (U.S.'s National Institutes of Health, Bethesda Md) gives for doctor B.R.Murphy.This hypotype is human influenza virus's strain, but also infected mice monkey and mice.This A/Udorn/307/72 hypotype is highly suitable for zoopery, for example the mouse infection model.
Mucoadhesive is used to increase the transhipment effect of this immunogen to mucomembranous immune system with immunogen in the present invention.Existing various mucoadhesive are used for transport of drug in pharmaceuticals industry, as film former and viscosity increasing agent, application are arranged also in food industry, and are assert it generally is safe by FDA.Example comprises: sodium carboxymethyl cellulose, Carbopol, poly-close carbon agent, sodium alginate and hydroxypropyl emthylcellulose.
The present inventor be sure of the interaction for oral immunity mucoadhesive and mucin layer, can make on mucoadhesive and any immunogen that links to each other is incorporated into all intestinal mucosa link to each other the mucin.This association can increase institute's fluidization compound and the interactional effect of mucosa, be the center cavity of chemical compound from intestinal to be moved on on the continuous motionless relatively rete malpighii of mucous epithelium, have and the immunocyte of mucous membrane of small intestine and the time of immunologically competent cell interaction potentiality thereof thereby increased chemical compound by (1); (2) fluid of chemical compound volume from the chamber and material be distributed to the relatively thinner rete of the contiguous mucin of mucosa on, thereby improved the valid density of chemical compound; (3) owing to be gathered on this mucin layer, this polymerization mucin has hindered diffusion and has made immunogen avoid the influence of enteral PH variation and catabolic enzymes, make immunogen be unlikely inactivation, the interaction of this immunogen and mucoadhesive polymer can make immunogen avoid the influence of the cleaning action of phase change and bile salts.At last, the hydrophilic nmature of mucoadhesive thing causes this dehydration mucoadhesive that water is removed from its direct environment.This dehydration of mucous epithelium layer can increase immunogenic bioavailability.In addition, the association of immunogen and polymer also can make the immunogen stabilisation.Though these factors can be explained the effectiveness of mucoadhesive, the present inventor only is limited to these unintentionally and explains.
This known mucoadhesive is a polymer, it is incorporated on the polymeric electronegative mucin by one or more mechanism, these mechanism comprise hydrophobic interaction, Van der Waals force, the interaction of charged groups, polymer fusion effect and straight chain associate and use the interaction of the combination of specificity residue and receptor and part.All known mucoadhesive all are polymer, and this is known certainly in the literature, but the haplotype mucoadhesive also is possible.
Mucoadhesive itself does not generally have the immunogen effect, and many kinds is safe to the people.They are used as and intend the mucus agent now, as the surgery viscose glue, and the wound healing auxiliary agent, diarrhea and anti-emplastic, and be used for transport of drug.Mucoadhesive has been successfully used to the transdermal transfer system of medicine, comprises the nasal-cavity administration (for example diuretic and islets of langerhans poison) of peptide medicament and medicine, and transports in the controlled release and the location of oral cavity Chinese medicine (as the steroid class).Though mucoadhesive is used as depot forming adjuvant in the gastrointestinal tract immunity, and prove the potential controlled release movement system of peptide medicament after deliberation, the former mucosa transhipment that is not used to vaccine.
Can consider to include as the examples for compounds of mucoadhesive: sodium carboxymethyl cellulose, poly-(acrylic acid), tragakanta, poly-(ethylene methacrylic ether copolymerization maleic anhydride), poly-(epoxy hexane), methylcellulose, sodium alginate, hydroxypropyl emthylcellulose, karaya, methylethylcellulose, soluble starch, gelatin, pectin, poly-(vinylpyrrolidone), poly-(ethylene glycol), poly-(vinyl alcohol), poly-(ethoxy acrylic acid methyl ester .), hydroxypropyl cellulose, Carbopl, poly-close carbon agent.These mucoadhesive can be used in the compositions and methods of the invention.Preferred mucoadhesive is sodium carboxymethyl cellulose and other cellulose family, poly-close carbon agent and Carbopol.Sodium carboxymethyl cellulose is especially preferred mucoadhesive.To recognize that also the mixture of these mucoadhesive also can use.
Immune composition of the present invention also can comprise the intensity that is enough to strengthen host immune response and persistent period consumption, or strengthens the adjuvant of host's quantitative response (for example by the antibody that stimulates different immunoglobulins rather than those antibody that stimulated by immunogen).Adjuvant effectively trigger cell mediation with body fluid to antigenic immunoreation, and host system is not had general or local excitation.Adjuvant preferably has low grade fever originality.Can use known human or adjunvant composition for animals.These adjuvants can emulsion-based, is with or without mycobacteria, and perhaps adjuvant is adsorbed in aluminum salt based on antigen, especially on aluminium hydroxide or the aluminum phosphate.Oily adjuvant in these adjuvants can be based on mineral oil, animal oil or vegetable oil.The oil adjuvant is used to increase the humoral response of domestic animal to antigen vaccine, and some oily adjuvant has been tested and has been used for the people.Representational adjuvant is Freund Freund's complete adjuvant and Freund Freund.
The suitable adjuvant of exploitation recently comprises liposome, immunostimulating complex (IS-COMs) and Squalene or squalene emulsion.Also can be with the surfactant that adjuvanticity is arranged.At ISCOMs and Pluronic These adjuvants comprise saponarin sample QuilA molecule in the block copolymer, are used for preparing stable squalene emulsion.Saponarin is the surfactant that is distributed widely in the plant.
The analog of muramyldipeptide (MDP) or muramyl-tripeptide (MTP), the MDP threonine analog and the fat polysaccharide (LPS) that for example have the side effect of adjuvanticity and attenuating also are suitable for as adjuvant.The LPS proof can produce good effect to mice.The also known pyrogen that adjuvanticity and attenuating are arranged of one phosphonyl derivatives of the synthetic analogues of MDP and lipid A.Another suitable peptide be synthetic muramyl peptide MTP-PE (N-acetyl group-muramyl-L-alanyl-D-isoglutamine acyl-L-alanine-2-[1,2-two palmityls-Sn-glyceryl-3-(hydroxyl phosphorus acyloxy)] acetamide.Especially Shi Yi preparation is Syntex adjuvant formulation-1 or SAF-1, and it is with the threonyl analog of the MDP in the carrier that is made of Pluronic L-121 triblock polymer and Squalene and a spot of Tween 80 Combine Tween 80 It is the emulsifying cleaning agent.The preferred adjuvant that is used for the people is MDP and analog thereof, is with or without Squalene, a phosphonyl derivatives of saponarin and lipid A.
Other chemical compound proof is having adjuvanticity when oral with antigen is common.These adjuvants comprise Fel Bovis seu Bubali, polycation, as DEAE-4, and glucosan and poly ornithine, dodecyl phenenyl sulfate, lipid conjugates matter, streptomycin, vitamin A and other can influence the structure of mucomembranous surface or the reagent of functional completeness.
When adjuvant and immunogen and mucoadhesive make up in the present composition and the method, can be observed immunoreactive further enhancing.Moreover though without any ready-made theory explanation, this mucoadhesive can make immunogen and adjuvant be easily and pass reacting cells, thereby strengthens the effect of these materials with cooperative mode.
Can be used for taking antigenic mucoadhesive, the mucomembranous surface that is contacted includes gastrointestinal tract mucous (comprising stomach, small intestinal, large intestine, colon and rectum); Oral mucosa (comprising oral cavity and esophageal mucosa membrane injury and tonsil surface); Respiratory mucosa (comprising nose, larynx, trachea and bronchus mucosa); Genitals mucosa (comprising vagina, cervix uteri and mucous membrane of urethra) and eye mucosa.The optimization approach of taking the present composition to the host is oral, nasal cavity, rectum and tonsil swab.Especially preferred route of administration is oral, because usage is simple, and relative non-invasi.Recognize that all usages can unite use.It is oral for example beginning, auxiliary with nasal-cavity administration, otherwise good.
Recognize that immune composition of the present invention also can be used in the vaccine.This vaccine can be to contain as immunogenic antigenic therapeutant, and described antigen is from one or more pathogenic microorganisms, and giving can stimulate Active immunity behind the human or animal, and makes body avoid by the infection of these or related microorganism or lower gradient of infection.This vaccine can be live vaccine, inactivated vaccine, attenuated vaccine, subunit or mixed vaccine.
The present composition can be spiked in the various suitable movement systems.For example, antigen, mucoadhesive and adjuvant can with the medicinal fluid carrier, for example water, buffering common salt aqueous solution, or edibility animal or plant oil share.Compositions can with one or more suitable pharmaceutical excipient or cores, cellulose for example, cellulose derivative, sucrose, gelatin, starch 1500, NuTab, lactose, maltodextrin, Pulvis Talci, carboseal, magnesium stearate, alginate, Actisol, PEG400, Myvacet, glyceryl triacetate, syrup, oil, Sorbitol, mannitol and Plasdone share, and have more than to be limited to these materials of being enumerated; Also available other or other excipient or core.
Also to recognize, also can contain in the dosage form that the present composition is made in the energy and the chemical reagent of gastric acid.Suitable nertralizer comprises H 2Antagonist, sodium bicarbonate, calcium carbonate and aluminium hydroxide.
Dosage forms such as the present composition can elixir, solution, suspension, syrup, aerosol are used.Compositions also can be made into and is suitable for oral dosage forms unit, as granule agent, granule, medicine pearl, tablet, hard gelatin capsule and Perle.
Immune composition can be handled, and makes it avoid Degradation before arriving target site with protection antigen, mucoadhesive and adjuvant.A kind of embodiment is with dosage forms unit such as tablet or capsule conventional method, includes, but is not limited to pan coating; Fluidized bed coating (for example Wurster method) or top nebulization coating; Spray drying and emulsifying or microcapsule method are wrapped enteric coating.Also can randomly use inferior coating materials, hydroxypropyl emthylcellulose for example, Opadry or Dryclean.Casing is applicable to various dosage forms unit.The enteric coating agent can be various be used to protect antigen and the medicinal materials that antigen is discharged at enteral.The selection and the consumption of inferior coating materials or enteric coating agent are the ordinary skill in the art, depend in part on dosage form, for example tablet or capsule.Suitable enteric coating agent has HPMCP5s (hydroxypropyl emthylcellulose phthalic acid fat, CAP (Cellacefate), Eudragit Aguateric, Coa-teric, Surlease Shellac and wax.
The consumption that immunogen, mucoadhesive and adjuvant share should be able to make the host that infective agent is produced immunoreation.This can be by seropositive conversion, i.e. the estimation of the antibody horizontal before and after immunity is determined.If the host has antibody titer in advance to antigen, then the increase degree of this specific antibody level is depended in Mian Yi success, if do not have dependency between seropositive conversion and protective effect, then can monitor cell-mediated immunoreation.
Antigen, mucoadhesive in each dosage unit, the content of agent and adjuvant depend on required dosage and administration frequency.If antigen is that influenza virus, mucoadhesive are carboxymethyl cellulose in the compositions, adjuvant is a muramyldipeptide, and then single dosage unit can contain: the antigen amount be about 10 μ g to 150 μ g hemagglutinins (HA), be preferably about 5 μ g to 45 μ gHA; Mucoadhesive dosage is approximately 10 μ g to 1g, is preferably 1 μ g to 50 μ g; The adjuvant amount is approximately 1 μ g to 2 μ g, is preferably about 10 μ g to 200 μ g, antigen: mucoadhesive: the relative weight of adjuvant is than being approximately 5-45: 1000-50000: 0-200 this moment.Inevitable animal species and the other factors according to antigen, mucoadhesive and the adjuvant selected for use, quilt immunity of accurate compositions changes, and those skilled in the art can find optimum formula.The antigen amount that booster dose comprised will enough strengthen initial immunoreation.Each scheme that adopts all must be according to antigen and host's situation.For child and former not people's multiple dose of using preferably of contacted vaccine.An embodiment is that the influenza antigens amount that each dosage unit contains is wanted effectively to watch for animals it can not caught after touching this virus.
The definition of dosage is to improve individual immunity to react necessary immune commercial weight.For example, homology neutralizing antibody level is the sign of the individual susceptibility that the homology strains of influenza viruses is infected in the serum, and the serum blood clotting suppresses that titre is approximately 1: 32 or 1: 4 or when higher, is considered to resist the natural infection of homology virus.Therefore, the preferred dose of influenza antigens is to measure by standard method that can to make serum blood clotting titre approximately be 1: 32 or 1: 40 or higher dosage.
The raising of the also available serum hemagglutinin of immunogenic protective effect inhibitory action level or the increasing of finding after immunity of mucoantibody titre are represented; when the treatment of influenza, in immunity back 7-21 days in the serum HI titre increase by 4 times and be considered to have protectiveness.Mucoantibody titre in 7-21 days (for example in the saliva IgA or wash IgA in the snot) increases 4 times of amounts, is also thought that by some influenza is had protective effect.Therefore, the preferred dose of immune composition of the present invention can make the specific antibody level of human body bring up to such degree.Can adjust immunizing dose up to obtaining detectable antibody titer, preferably obtain NAT.
Recognize that the used immunogen of the present invention can resist the various types and the hypotype of various parasites, antibacterial or virus, and the not homophyletic of certain hypotype system.But parasite, antibacterial or virus are representational to be infection animal, for example Canis familiaris L., cat, poultry, pig, Ma Heniu, especially mammals, for example comprise that human primates, immunogen can administrations before or after giving mucoadhesive or adjuvant, but generally be immunogen mucoadhesive and adjuvant.
Immunogen of the present invention can be mixed immunogen, mucoadhesive and adjuvant for the preparation of compound simply and is not had covalent bonds, or all compositions are coupled at together.Therefore this compositions also has the advantage of preparation easily.
The following examples will illustrate in greater detail the present invention.
Embodiment 1
Vaccine antigen
Influenza virus A/Udorn/307/72 (H3N2), BK6, Egg3, clone 3A (7-25-89) is that (NIH, Bethesda MD) give doctor B.R.Murphy.This virus goes down to posterity in Embryo Gallus domesticus once, and allantoic fluid is deposited in-130 ℃ as deposit virus, and (infection titer is that every 0.2ml has 2.53 * 10 7Plaque forming unit (P.F.U).10 days Embryo Gallus domesticus (400-500) is with the deposit viral infection of 0.1ml, and this virus to be diluted in L-15 medium (2.18M sucrose, the 0.038M KH that is supplemented with SPG at 1: 1000 2PO 4, 0.072MK 2HPO 4In the 0.049M monosodium glutamate.After 48 hours, collect allantoic fluid, in 35-36 ℃ of incubation in 3700g AvMade clarificationization in centrifugal 20 minutes.Again in 1000000g AvIn supernatant, collected virus in centrifugal 45 minutes.Piller placed in 0.5ml phosphate-buffered common salt aqueous solution (PBS) in 0 ℃ spend the night, reuse PBS is diluted to 6ml acutely to be mixed, in 1300g AvCentrifugal 15 minutes to remove aggregation.This operates triplicate.After centrifugal for the third time,, centrifugal again with ultrasonicization of piller.The supernatant of collecting that contains viral suspension is added to 10-60% in the continuous saccharose gradient of PBS top, waves centrifugal 2 hours (1000000g on the rotator in nothing Av).Collect the virus band, use PBS1: 1 dilution, virus and formalin (1: 4000V/V) be inactivated in 72 hours in 37 ℃ of incubations.To the PBS dialyzed overnight, as the above-mentioned centrifugal piller that gets, resuspending makes every ml content protein 5mg in PBS or water, deposit in-80 ℃ under 4 ℃.(IL USA) measures in the protein through the ruinate virus of sodium hydroxide for Pierce, Rockford in conjunction with test with Coomassie blue.
The vaccine that preparation has mucoadhesive
The short-cut method that provides by maker prepares the gel of various mucoadhesive, the sodium carboxymethyl cellulose (CMC) that is used for this experiment available from Agualon (Wilmington, DE).The CMC gel is made 2% aqueous solution.Carbopol and poly-close carbon agent (Polycarbophil) acrylate copolymer are available from B.F.Goodrich (restraining sharp Forlan, the Ohio).By these polymer manufacture gels is that Carbopol is made 0.25% water slurry, and 0.5% water slurry is made in poly-close carbon agent.These solution pH after adding several IN NaOH approximately rises to 4 from 3.When pH reached 4, this acrylate copolymer suspension became gel.Sodium alginate (is Merck﹠amp available from Kelco; Co., Inc (San Diego, branch (A)) makes 2% aqueous solution with gel.Zilactin available from the Zila pharmaceutical factory (Phoenix, AZ).Before immunity, prepare 1: 10 Zilactin aqueous solution immediately.
Influenza virus solution (containing 50 μ g virus proteins in per 10 μ l phosphate-buffered common salt aqueous solutions) and mucoadhesive gel were with 1: 50 (vaccine: mixed gel) (each dosage contains 10 μ l vaccine solution and 490ul mucoadhesive).In all cases, mixing only needs about 1～2 minute of simple agitation, becomes homogeneous phase solution to get final product up to perusal, and mixes immediately before immunity.
The immunity of mice
BALB/C mice (8 ages in week, female Mus) derives from Charles River or Jackson laboratory, 5 one group.Give mouse stomach vaccine (in mucoadhesive) 500 μ l with irritating the stomach syringe needle.A matched group is that influenza vaccines are (in 0.1M NaHCO 3In the solution) also gastric infusion.
The general immunity of matched group is capable with being injected under free antigen (50 μ g/ mice) (in physiological salt solution) percutaneous.
The collection of sample
Blood was got from mouse tail vein in the immunity back before selected time and immunity.Centrifugalize goes out blood plasma, and is freezing.Lumbar injection carbaminoylcholine chloride (1 μ g/ mice) stimulates and secretes saliva, collects with capillary tube.Add soybean trypsin inhibitor, each 2ug of phenyl methyl sulfuryl fluoride, Hydrazoic acid,sodium salt and hyclone is stored in-80 ℃ after the clarification.
ELISA
In order to measure antigen-specific antibodies, the 96 hole polystyrenes trace that covers in the A/U-dorn influenza virus that with concentration is the purification of 4 μ g/ml drips plate, and (Va carries out ELISA on USA) for Dynatech, Alexandri-a.The terminal point titre of serum and the saliva anti-mice Ig of horseradish peroxidase-labeled or goat IgG (the Southern Biotechnolvgy As-sociates Birmingham of IgA, Al, USA) and substrate 2,2 '-azine group-two-(3-ethyl benzo thiazole phenanthroline) sulfonic acid (Sigma, St.Louis, MO USA) measures.(CA USA) measures in 414nm the color of showing for Molecular Devices, Polo Alto with the Vmax photometer.
HI measures
It is to carry out after by 1: 5 dilution proportion with PBSA with mice serum that blood clotting suppresses (HI) reaction, and the treated nonspecific inhibitor of removing (heated 30 minutes in 56 ℃; 30 fens kinds of Kaolin incubation with 15% acid treatment; With 10% chicken erythrocyte suspension incubation 30 minutes).On the trace plate of 96 holes, serum is done the twice dilution.Add viral suspension (8HA unit adds with equal-volume) in every hole, incubation is 30 minutes under the room temperature.Add 0.5% chicken erythrocyte suspension in every well, under the room temperature incubation 45-60 minute.The HI titre is represented as the inverse of the highly diluted that has suppressed erythrocytic hemagglutinative function fully.
Embodiment 2
Vaccine antigen
Influenza virus A/Udorn/307/72 (H3N2), BK6, Fgg3, clone 3A (7-25-89) is that (NIH, Bethesda MD) give doctor B.R.Murphy.This virus goes down to posterity once in the Embryo Gallus domesticus application, and allantoic fluid deposits in-130 ℃ as deposit virus that (infection titer is that every 0.2ml has 2.53 * 10 7Plaque forming unit (p.f.u).10 days Embryo Gallus domesticus (400-500) is with the deposit viral infection of 0.1ml, and this virus to be diluted in L-15 medium (2.18M sucrose, the 0.038M KH that is supplemented with SPCT at 1: 1000 2PO 4, 0.072MK 2HPO 4With the 0.049M monosodium glutamate) in., collect allantoic fluid and made clarificationization in centrifugal 20 minutes after 48 hours in 35-36 ℃ of incubation in 370gav.Piller high-volume spends the night in O ℃ in 0.5ml phosphate-buffered common salt aqueous solution (PBS), is diluted to 6ml with PBS, the violent mixing, in centrifugal 15 minutes kinds of 1300gav to remove aggregation.This operation repeats 3 times.The 3rd time centrifugal after, piller is ultrasonic, centrifugal again.The supernatant of collecting that contains viral suspension is added to the continuous saccharose gradient top of 10-60% in PBS, waved centrifugal on the rotator (1000000gav) 2 hours in nothing.Collect the virus band, use PBS1: 1 dilution, virus and formalin (1: 4000V/V) be inactivated in 72 hours in 37 ℃ of incubations.To the PBS dialyzed overnight, as the above-mentioned centrifugal piller that obtains, resuspending makes in PBS or water and contains protein 5mg among every ml, deposits in-80 ℃ under 4 ℃.(Pierce, Rock-ford IL.USA) measure in the protein through the ruinate virus of sodium hydroxide in conjunction with test with Coomassie blue.
The vaccine that preparation has mucoadhesive
The short-cut method that provides according to maker prepares the gel of various mucoadhesive.The sodium carboxymethyl cellulose (CMC) that is used for this experiment is that (Wilming-fon DE), makes 2% aqueous solution with the CMC gel to 7MF available from Agualon.Carbopol and poly-close carbon agent (Poly-carbophil) acrylate copolymer are available from B.F.Goodrich (restraining sharp Forlan, the Ohio).By these polymer manufacture gels is that Carbopol is made 0.25% water slurry, and 0.5% water slurry is made in poly-close carbon agent.These solution pH after adding several sodium hydroxide (IN) solution approximately is raised to 4 from 3.When PH reached 4, this acrylate copolymer suspension became gel.Sodium alginate (is Merck﹠amp available from Kelco; Co., Inc (San Diego, branch (A)) makes 2% aqueous solution with gel.Zilactin available from the Zila pharmaceutical factory (Phoenix, AZ).Before immunity, prepare 1: 10 Zilactin aqueous solution immediately.
Influenza virus solution (containing 50 μ g virus proteins in per 10 μ l phosphate-buffered common salt aqueous solutions) and viscose binder gel were with 1: 50 (vaccine: mixed gel) (each dosage contains 40 μ l vaccine solution and 490 μ l mucoadhesive).In all cases, mixing only needs about 1～2 minute of simple agitation, becomes homogeneous phase solution to get final product up to perusal, and mixes immediately before immunity.
The immunity of mice
BALB/C mice (8 ages in week, female Mus) derives from Charles River or Jackson laboratory, 5 one group.Give mouse stomach vaccine (in mucoadhesive) 500 μ l with irritating the stomach syringe needle.A matched group is that influenza vaccines are (in 0.1M NaHCO 3In the solution) also gastric infusion.
The general immunity of matched group with free antigen (50 μ g/ mice) in physiological salt solution) be injected under the percutaneous capable.
The collection of sample
Blood was got from mouse tail vein in the immunity back before selected time and immunity.Centrifugalize goes out blood plasma, and is freezing.Lumbar injection carbaminoylcholine chloride (1 μ g/ mice) stimulates and secretes saliva, collects with capillary tube.Add soybean trypsin inhibitor, each 2 μ g of phenyl methyl sulfuryl fluoride, Hydrazoic acid,sodium salt and hyclone are stored in-80 ℃ after the clarification.
ELISA
In order to measure antigen-specific antibodies, the 96 hole polystyrenes trace that covers in the A/U-dorn influenza virus that with concentration is the purification of 4 μ g/ml drips plate, and (Va carries out ELISA on USA) for Dynatech, Alexandri-a.The terminal point titre of serum and saliva is with the anti-mice Lg of peppery summary peroxidase labelling or goat IgG (the Sorthern BiotechnolvgyAssociates Birmingham of Ig, Al, USA) and substrate 2,2 '-azine group-two-(3-ethyl benzo thiazole phenanthroline) sulfonic acid (Sigma, St.Louis, MO USA) measures.(CA USA) measures in 414nm the color of showing for Molecular Devices, Polo Alto with Vmax luminosity square.
HI measures
It is to carry out after by 1: 5 dilution proportion with PBSA with mice serum that blood clotting suppresses (HI) reaction, and the treated nonspecific inhibitor of removing (heated 30 minutes in 56 ℃; 30 fens kinds of Kaolin incubation with 15% acid treatment; With 10% chicken erythrocyte suspension incubation 30 minutes).On the trace plate of 96 holes, serum is done the twice dilution.Add viral suspension (8HA unit adds with equal-volume) in every well, incubation is 30 minutes under the room temperature.Add 0.5% chicken erythrocyte suspension in every hole, under the room temperature incubation 45-60 minute.The HI titre is represented as the inverse of the highly diluted that has suppressed erythrocytic hemagglutinative function fully.
The result
As shown in table 1, the mice of all groups after oral virus formulation immunity in mucoadhesive, serum immune globulin, the IgA titre is all higher in hemagglutinin titre and the saliva.The mucoadhesive carboxymethyl cellulose has caused best immunoreation in the present embodiment.
Table 1
Titre HI titre in the serum
Serum Saliva
0 day 28 days 0 day 28 days 0 day 28 days
Oral contrast (is used bicarbonate, no mucoadhesive ????8,000 ????32,000 ????10 ????40 ????10 ????＜10
Subcutaneous injection contrast (in PBS, no mucoadhesive) ????8,000 ????512,000 ????10 ????10 ????＜10 ????160
Carboxymethyl cellulose/water, 2.0% (W/V) ????16,000 ????512,000/ ????1,024,000 ????＜10 ????＞80 ????＜10 ????160
Carbopol/ water, 0.25% (W/V), pH4.0 ????8,000 ????128,000 ????＜10 ????＞80 ????＜10 ????160
Poly-close carbon agent/water, 0.5% (W/V), pH4.0. ????8,000 ????256,000 ????＜10 ????＞80 ????＜10 ????40
2.0% (W/V) sodium alginate aqueous solution ????8,000 ????128,000 ????＜10 ????＞80 ????＜10 ????40
1: 10 (V/V) aqueous solution of Zilactin ????8,000 ????64,000 ????＜10 ????20 ????＜10 ????＜10
5 mice/groups, the mensuration of carrying out with collection serum data base shown with 50 μ g influenza virus A/Udorn (only with it time be matched group), or separately with or mixes before injection percutaneous under or mouthful gavage immunity with various mucoadhesive and after the immunity 28 days in saliva antibody titer (with ELISA method survey IgG) and serum in antibody titer (using the total Ig of ELISA method and hemagglutinin inhibition titer determination).
Embodiment 3
In order to determine to reach the required mucoadhesive agent concentration of optimal immune response, mice through port gavage is used in the virus formulation for preparing in carboxymethyl cellulose (the replacing model 7MF) mucoadhesive of various concentration and carries out immunity.In addition, 2 groups of mice through port gavages carboxymethyl cellulose (model is 9M31FPH or 12M31P) influenza A/Udorn virus of being used in replacement carries out making in the immunity.10 groups of mices, every group 5, female BALB/C mice, immunization method is the mucoadhesive agent formulation of using as preparation as described in the embodiment 2 that contains formalin influenza virus (A/Udorn), different is that the concentration with 0.02% (W/V) is added to the antiseptic thimerosal in the allantoic fluid when results virus, and all keeps same concentration in all subsequent steps of virus preparation.Mice by immunity after, gather serum, measure hemagglutinin and suppress titre.Though the 14th day and the 28th day serum hemagglutinin suppress titre and are positive after the immunity of subcutaneous injection free virus, but in concentration be in the presence of the mucoadhesive carboxymethyl cellulose of 0.05% to 4.0% (W/V) behind oral immunity, do not obtain the serum hemagglutinin and suppress titre.IgA and antibody horizontal fail to measure with the ELISA method in the saliva.
Embodiment 4
Content is studied by the feeding environment that changes mice the influence of immune result in the mice stomach when the mouth gavage is irritated stomach.Four groups of mices, every group of 5 female BALB/C mice, place fasting or prohibit the environment of water, oral gavage is carried out immunity with the preparation that contains 2% (W/V) carboxymethyl cellulose mucoadhesive and 50 μ g formalin influenza virus A/Udorn preparations (method for making as embodiment 3 as described in) afterwards.That is virus is made in the presence of antiseptic (0.02% thimerosal).Mice by immunity after, according to the described methods blood sampling of embodiment 2 and measure serum hemagglutinin titre.These experimental grouies (comprising can free pickuping food and the matched group of water) did not obtain the serum hemagglutinin at the 14th day and suppress titre.Go out IgA and antibody content in the saliva with ELISA method undetermined.
Embodiment 5
Embodiment 2 shows, when oral influenza virus (A/Udorn) according to embodiment 2 described scheme preparations carries out immunity, in the presence of mucoadhesive (comprising carboxymethyl cellulose), has produced male serum hemagglutinin titre.Otherwise embodiment 3 and 4 shows, influenza A/Udorn virus according to embodiment 3 preparations, behind oral immunity in the presence of the carboxymethyl cellulose, do not produce male serum hemagglutinin titre, but in subcutaneous injection free virus when immunity, but produced male serum hemagglutinin titre.Comprise use antiseptic thimerosal according to the used scheme of embodiment 3 preparation influenza virus.If omit this antiseptic, can in viral liquid storage, cause germ contamination when pressing the described preparations virus of embodiment 2.Therefore, cultivate to have determined whether germ contamination viral liquid storage.The liquid storage proof of preparation does not have germ contamination when having thimerosal to exist, and the viral liquid storage of preparation has germ contamination when not having thimerosal to exist.The pollution of carefully analyzing this virus formulation proof antibacterial is klebsiella bacillus and Xanthomonas campestris.
Influenza virus A/the Udorn/307/72 (H3N2) that contains klebsiella bacillus and Xanthomonas campestris, BK6, Egg3, clone 3A (7-25-89) deposits in American type culture collection (Rockville, Maryland).
Embodiment 6
In the presence of carboxymethyl cellulose, the germ contamination of virus liquid storage to the influence of immune result by to relatively determining by the viral liquid storage immune mouse of embodiment 3 preparations, and known not pollution, and known this liquid storage is polluted by klebsiella bacillus and Xanthomonas campestris.Mice by immunity after, blood sampling is measured the serum hemagglutinin with the ELISA method and is suppressed titre and the total immune globulin antibody of serum influenza, institute is in steps as described in the embodiment 2.
The result
As shown in table 2, to press in the influenza virus liquid storage of embodiment 2 preparation at thimerosal and known during by germ contamination with not ing, percutaneous when injection or oral administration, is measured with ELISA method or serum hemagglutinin inhibitory action down, shows that the immunoreation to the initiation mice is effective.When in the presence of mucoadhesive and bacterial pollutant, the immunoreation height that the immunoreation that oral immunity caused causes than the immunogen that does not have mucoadhesive with only containing bacterial pollutant, this shows that bacterial pollutant and mucoadhesive all are needs for causing immunoreation effectively.Otherwise, have thimerosal to have that the viral liquid storage of preparation down is known not to have germ contamination according to embodiment 3, during the subcutaneous injection immunogen, can cause immunoreation, but when oral immunity is former, even in the presence of the mucoadhesive carboxymethyl cellulose, can not cause tangible immunoreation.And to 1,2,4, after group measures resulting result and merge, summation shows immunogen of the present invention when the 5th group ELISA result compares with the ELISA detection method, between adjuvant and the mucoadhesive be have synergistic.
Table 2
The group alias Immunogen ELISA measures The serum hemagglutinin suppresses titre
0 day 28 days 0 day 28 days
In the presence of thimerosal, press the no bacterial virus liquid storage of embodiment 2 preparations
????1 Oral 50 μ g influenza virus and 500 μ l NaHCO 3Carry out immunity ????16,000 ????16,000 ????＜4 ????＜4
????2 Oral 50 μ g influenza virus and carboxymethyl cellulose carry out immunity ????16,000 ????32,000 ????＜4 ????＜4
Subcutaneous injection 50 μ g influenza virus carry out immunity ????16,000 ????128,000 ????＜4 ????32/64
Do not having in the presence of the thimerosal, by the viral liquid storage that germ contamination is arranged of embodiment 1 preparation
????4 Oral 50 μ g influenza virus and 500 μ lNaHCO 3Carry out immunity ????8,000 ????32,000 ????＜4 ????＜4
????5 Oral 50 μ g influenza virus and carboxymethyl cellulose carry out immunity ????8,000 ????128,000 ????＜4 ????64
Table 2 (continuing)
The group alias Immunogen ELISA measures The serum hemagglutinin suppresses titre
0 day 28 days 0 day 28 days
????6 Subcutaneous injection 50 μ g influenza virus carry out immunity ????16,000 ????128,000 ????＜4 ????128
Every group of 5 mices, the mensuration of carrying out with collection serum data base has shown that being used in no thimerosal exists and known (embodiment 1 scheme) that is prepared under the situation of germ contamination, or before (embodiment 2 schemes) 50 μ g influenza A/Udorn viruses that have thimerosal to exist but do not have to prepare under the situation of germ contamination (only with it time be contrast) are carried out immunity and the antibody titer (with ELISA method and the total Ig of hemagglutinin inhibition titer determination) in back 28 days serum of immunity.Virus with or not with the mucoadhesive carboxymethyl cellulose, replace model 7MF (2% aqueous solution W/V) and mix, percutaneous is injection or mouthful gavage administration down. Embodiment 7
Shown in embodiment 6, knownly can be caused immunoreation in the presence of the mucoadhesive carboxymethyl cellulose is being arranged the time by the virus formulation of germ contamination.Even but oral virus formulation is not had the mucoadhesive carboxymethyl cellulose to exist down by germ contamination, does not cause immunoreation yet.The function of antibacterial may be the adjuvant effect of embodiment 6, tests with the prescription that includes other adjuvant.In the presence of the mucoadhesive carboxymethyl cellulose, measured the effect of adjuvant lipopolysaccharide and muramyldipeptide.For this reason, with mice with 50 μ g influenza virus preparation oral immunity, said preparation known not by germ contamination, add the carboxymethyl cellulose of 500 μ l2%, add or do not add LPS or MDP as adjuvant.
The result
As shown in table 3, will be added in the carboxymethyl cellulose mucoadhesive agent formulation from klebsiella bacillus or the colibacillary LPS of Erichsen, caused the positive immunoreation that records as with serum hemagglutinin titre or saliva IgA titre.Add muramyldipeptide and also caused the immunoreation that records as with serum hemagglutinin titre or saliva IgA, but saliva IgA titre level is a little less than the titre level that obtains with LPS.
Table 3
The group alias Immunogen The serum hemagglutinin suppresses titre Saliva IgA titre
0 day 14 days 28 days 0 day 28 days
Subcutaneous immunity
????1 50 μ g influenza virus ＜4 128 256 ＜10 10
With carboxymethyl cellulose and lipopolysaccharide or muramyldipeptide oral immunity
????2 50 μ g influenza virus with 500 μ l2% (W/V) CMC ＜4 ＜4 ＜4 ＜10 ＜10
????3 50 μ g influenza virus with 500 μ l2% (W/V) CMC and 1mg LPS (klebsiella bacillus) ＜4 64 64 ＜10 ＞80
????4 50 μ g influenza virus with 500 μ l2% (W/V) 1 CMC and 1mg LPS (Erichsen escherichia coli) ＜4 128 128 ＜10 ?＞80
Table 3 (continuing)
The group alias Immunogen The serum hemagglutinin suppresses titre Saliva IgA titre
0 day 14 days 28 days 0 day 28 days
????5 50 μ g influenza virus with 500 μ l2% (W/V) CMC and 200 μ g MDP ??＜4 ???64 ???64 ???＜10 ????20
Every group of 5 mices, percutaneous down injection or mouthful gavage be mixed with or be not mixed with before the 50 μ g influenza A/Udorn viruses of mucoadhesive to 2% (W/V) aqueous solution of carboxymethyl cellulose (replacing model 7MF) (only with it time the be contrast) immunity and after the immunity 14 and 28 days in serum antibody titer (hemagglutinin inhibition titre) and before the immunity with immune back 28 days antibody titers (measuring IgA) in saliva with ELISA.

Claims (20)

1. immune composition, the antigen and the content that include the immunity amount are enough to can induce or strengthen antigenic immunoreactive mucoadhesive.
2. the compositions of claim 1, wherein antigen is influenza antigen.
3. the compositions of claim 1, wherein mucoadhesive is selected from sodium carboxymethyl cellulose and other cellulose family, poly-close carbon agent and Carbopol.
4. immune composition, include antigen of (a) immunity amount and (b) content be enough to can induce or strengthen to antigenic immunoreactive mucoadhesive and adjuvant.
5. the compositions of claim 4, wherein antigen is influenza antigen.
6. the compositions of claim 4, wherein mucoadhesive is selected from sodium carboxymethyl cellulose and other cellulose family, poly-close carbon agent and Carbopol.
7. the compositions of claim 4, wherein adjuvant is selected from muramyldipeptide, Squalene, a phosphonyl derivatives of saponarin and lipid A.
8. method that makes animal immune, wherein this method comprises the compositions of the claim 1 of the immunity amount of taking to animal.
9. the method for claim 8, wherein antigen is influenza antigen.
10. the method for claim 8, wherein mucoadhesive is selected from sodium carboxymethyl cellulose and other cellulose family, poly-close carbon agent and Carbopol.
11. the method for claim 8, the method for wherein taking this immune composition are oral, nasal cavity, rectum or the administration of tonsil swab.
12. a method that makes animal immune, wherein this method comprises the compositions of taking the claim 4 of immunity amount to animal.
13. the method for claim 12, wherein antigen is influenza antigen.
14. the method for claim 12, wherein mucoadhesive is selected from sodium carboxymethyl cellulose and other cellulose family, poly-close carbon agent and Carbopol.
15. the method for claim 12, wherein adjuvant is selected from muramyldipeptide, a phosphonyl derivatives of Squalene, saponarin and lipid A.
16. the method for claim 12, the method for wherein taking this immune composition are oral, nasal cavity, rectum or the administration of tonsil swab.
17. a method that makes animal immune, wherein this method comprises the compositions to the claim 4 of animal oral immunity amount.
18. the method for claim 17, wherein antigen is influenza antigen.
19. the method for claim 18, the antibody of taking to animal in the base are enteric coated dosage forms unit.
20. the method for claim 19, antigen, mucoadhesive and adjuvant are taken to animal simultaneously in the base.
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