CN101450209B - Preparation method of transdermal immune influenza multivalent vaccine - Google Patents
Preparation method of transdermal immune influenza multivalent vaccine Download PDFInfo
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Abstract
The invention provides a transdermal immunity flu polyvalent vaccine and a preparing method. The transdermal immunity flu polyvalent vaccine comprises transdermal immunity adjuvant, flu polyvalent vaccine antigen, permeation agent and medical dressing. The flu polyvalent inactivation or attenuated live vaccine is differ from prior vaccine immunity approach and adjuvant. A result through permeation purpose immunity to Balb/c mouse, ferret, monkey and human body improves that the transdermal immunity flu polyvalent vaccine can generate IgA and IgG antibody with high valency, namely, can induce immune system and mucosal immune simultaneously, also can be used for immunoprophylaxis to popular flu and highly pathogenic avian influenza.
Description
Technical field
The present invention relates to a kind of vaccine, particularly a kind of influenza multivalent vaccine of transdermal immune.
Background technology
Influenza be by influenza virus (influenza virus) cause a kind of be master's disease with the infringement respiratory system, be widely current in countries in the world, be one of important infectious disease of harm humans health.Therefore, WHO announces will strictly monitor influenza.3 global flu outbreaks were once arranged in 20th century; Only be very popular for the first time (1918-1919) just cause more than 2,000 ten thousand people dead; The total number of persons more dead than the World War I is also many, and dead great majority are between twenty and fifty, causes huge social and economic loss.In recent years; The eruption and prevalence that seasonal influenza is gone round and begun again, the incident of H5N1 subtype highly pathogenic avian influenza virus infected person, and the appearance of H7N7, H9N2 subtype influenza virus; And the fact of having crossed over the kind transmission of infection; Beaten alarm bell to the mankind once more, breaking out of influenza no longer is the problem that has or do not have, but early with the problem in evening.According to national authority department, the morbidity of the annual influenza of China is up to more than 1,000,000,000 person-times, more than the tens billion of units of economic loss.In the last few years, the ecological environment heavy damage of China, bigger variation has taken place in the generation of influenza and law of development, and frequency of disease development rises, and has caused serious social influence and great economic loss.
At present, still do not have the efficacious therapy means for influenza, and vaccine virus immunization is effective means, and brings into play the effect of wanting emphatically.Inoculate influenza vaccines for the healthy adult crowd; The people of 60-80% can play the effect (when virus stain of propagating and vaccine adopt strain identical) of prevention morbidity; The more important thing is that the generation of can obviously reduce influenza for high-risk group's vaccination reduces admission rate and mortality rate.WHO provides the Strain of preparation influenza vaccines for various countries; The widely used both at home and abroad vaccinum influenzae inactivatum of producing based on Embryo Gallus domesticus the seventies in last century that remains; Be main mainly with injection; And inactivated whole virus vaccines can not be used for child's below 12 years old weak, the component vaccine weak effect of inoculation, split vaccine antigenicity; All be not suitable for the prevention of the flu outbreak in a short time that sudden flu outbreak and bio-terrorism/biological warfare agent cause, particularly existing immune programme for children is not good enough to old people and protection of the child effect, is badly in need of solving that needleless is painless, easy fast, no cross infection, problem safely and efficiently.Therefore, the research of carrying out transdermal immune influenza multivalent vaccine preparation and method thereof becomes urgent and very important problem in science.
Since inactivated influenza virus whole virus vaccine, split vaccine in 1940 were come out one after another, the case fatality rate of elderly population and the incidence rate of infection had all obviously descended.At present; The influenza inactivated virus vaccine of having got permission to go on the market mainly comprises influenza trivalent inactivated whole virus vaccines, trivalent deactivation split vaccine and the subunit vaccine to H1N1, H3N2 and Type B; These vaccines are mainly through injecting pathway inoculation, the anti-HA/IgG production of antibodies in blood serum induced, and can not induce mucosa-immune sIgA production of antibodies; Yet the intersecting protective reaction to special-shaped influenza virus depends mainly on inductive IgA antibody cross reaction property in the natural infection process.And in June, 2003; The influenza trivalent attenuated live vaccine (Flumist) of drugs approved by FDA listing; Through intranasal inoculation, can induce body to produce and comprise the more comprehensive immunological effect of cellular immunization, humoral immunization and mucosa local immunity, but mainly be applicable to the healthy population in 2-49 year; And there is safety hidden danger in the morbidity high-risk group old people and the child of influenza, big limitations its development and application.Therefore; Along with developing rapidly of modern immunology theory and technology and related discipline, a kind of to the influenza multivalent vaccine that stimulates body to produce to have mucosa IgA immunne response and systemic immunity effect and a kind of new the immunity inoculation approach--transdermal immune arises at the historic moment.
Up to earlier 1900s, people think that still skin is the barrier of microorganism, chemical substance and medicine impermeable, and once accidental discovery can be absorbed after dimethyl sulfoxide and the contact skin and get into the body circulation, just makes above-mentioned viewpoint be able to change.Nineteen twenty-four, Rein proposes the topmost resistance that the skin outermost stratum corneum layer is the drug transdermal infiltration, and Blank has done further confirmation to this hypothesis.People such as Scheuplein pointed out that transdermal penetration was the passive diffusion process that receives the horny layer restriction afterwards.Michaels etc. have measured the apparent diffusion coefficient of some model drugs in horny layer, though point out skin good barrier characteristics are arranged, and some drugs still has bigger permeability.The basis of transdermal drug delivery system research and development after the nineteen seventies has been established in these researchs, and has therefore produced first commodity through the FDA authentication, and promptly dosage is 3d/1 time Scopolamine Patch, is used to treat motion sickness.FDA has passed through 35 kinds of transdermal patch products in two nearest more than ten years, and nearly 17 kinds of effective ingredient are used for the patch product, for theoretical basis has been established in the research of transdermal immune vaccine.
From the health perspectives of humans and animals, a kind of safe and effective approach of development remains in the delivery of vaccine needs top-priority problem.Because vaccine may cause vaccinate's dermapostasis and skin trauma pain through syringe or needle inoculation; Simultaneously might make that also the operator is injured, and serious infectious diseases sickness rate such as AIDS, hepatitis are high relevant with the injecting immune cross infection.In addition, the delivery of new generation vaccine technology mucosa-immune comprises collunarium and oral immunity, and, safety simpler, easy than traditional parenteral route approach have successfully shown a kind of new immunity inoculation approach on humans and animals.But oral immunity is prone to cause antigen to receive enzyme and sour destruction at digestive tract, and the collunarium immunity is prone to cause most of antigen to flow into esophagus, and by the part of nasal mucosa absorption seldom, how the controlled quentity controlled variable effect relationship becomes collunarium, oral immunity problem demanding prompt solution.In recent years; A kind of new immunity inoculation approach-transdermal immune (Transcutaneous immunization; TCI) arise at the historic moment, this approach has been avoided the shortcoming of above approach, the potential complication that reduces injecting immune; The Digestion and the unmanageable influence of dose-effect relationship of gastric acid have been avoided, for the delivery of vaccine provides new technique.
The bacterin preparation majority in past is injected in the skin with pin.With the pin vaccinate many shortcomings are arranged, with pain, need aseptic syringe needle and syringe like injection, need well-trained medical worker to accomplish vaccination, injection can cause discomfort and can bring some potential complication with the pin skin puncture.Transdermal immune does not re-use syringe, but through cutaneous inoculation, has avoided above-mentioned shortcoming, thereby is a much progress of vaccine delivery.
In addition, transdermal immune is superior to injecting pathway inoculation, because in the former case, uses the large-area multidigit point immunity inoculation of targeting skin surface, may targeting in more immunocyte.The effective dose antigen that is enough to induce immune response not only can be at single skin part, but also can carry out dermal delivery covering intact skin zone, a plurality of draining lymph nodes district.With at single position, compare through Intradermal, the subcutaneous or a small amount of antigen of intramuscular injection, inoculate and can produce more extensively and strong impulse near this position of a large amount of different lymph nodes at each position that spreads all over whole body immune system.
Transdermal immune, i.e. percutaneous immunoprophylaxis, through at skin surface external application antigen and adjuvant, inducible system immunity and mucosa-immune generation antibody.In this method, antigen is applied to complete skin surface, after passing through horny layer, is settled down Langerhans cell picked-up and processing in skin, is transported to draining lymph node then to start immunne response.Adjuvant commonly used mainly comprises CT and the colibacillary LT of vibrio cholera; When using patch (patch) with dysentery bacterium and Salmonella antigen, escherichia coli CS6 antigen and LT or CT transdermal immune; In different animal model and recent human body volunteer, can excite antibody response (IgG and IgA); Can excite intensive cellular immunization and mucosal immune response again, and not find any untoward reaction.The IOMAI Co. that is positioned at Maryland, USA is the creator of transdermal vaccine administration, proves that not only the transdermal vaccine can provide strong immunoreation, proves safe.The research worker of IOMAI is verified, when passing through skin patch transdermal administration with various dose, can induce the immunoreation that has same order with oral immunity safely, and these discoveries are significant for effective needleless vaccination strategy.The mechanism of transdermal immune also is not fully aware of at present, but existing research shows, originates from the BMDC of bone marrow----Langerhans cell LCs and therewith settles down at intraepidermal M cell and in the immunne response process, brought into play important function.LCs mainly contains two kinds of functions: immune surveillance and antigen presentation function, after by antigenic activation, move out of skin, and get into draining lymph node, induce the antigenic specificity reaction of T, B cell.Using the antigenic while; Transdermal immune has also added immunostimulant (bacteriotoxin or activation signal), can provide to activate the sophisticated necessary signals of DCs, high level expression costimulatory molecules, EF; Make it to become powerful antigen presenting cell, associating antigen causes primary immune response.Behind confirmer and the animal via transdermal immune, these toxin can not cause the appearance of side effect, thereby have guaranteed the safety of inoculation at present, and transdermal immune avoided the first pass effect of liver, have improved bioavailability; The pain of having avoided injection to cause; Prolong action time, reduced the medication number of times; Medication voluntarily, and drug withdrawal at any time, easy to use, the user compliance is good.Be specially adapted to child, old man and assemble the immunity inoculation of colony greatly.
The dosage form that the transdermal immune approach is commonly used is patch (patch), and said preparation both can cause local response through the skin mode immunity of applying ointment or plaster, and can cause general reaction again.In addition, also comprise ointment, plaster, varnish and aerosol etc.Preparation capable of permeating skin is because low price and the good compliance of patient are widely used, and use is obvious increase trend, especially child and the old people prefers using preparation capable of permeating skin.The application of estrone and testosterone preparation capable of permeating skin is exactly good example.To the women, available 7 days Climara/E preparation capable of permeating skin (estradiol transdermal system) has the side effect minimum, without the advantage of frequent change medication, thereby has obtained liking of women patient very soon after the listing.To elderly men, the testosterone preparation capable of permeating skin volume that will occur in several years is little, easy to use, and price also may be lower.The transdermal technology that also has several renewals now is also in research and development.The Pharmetrix of the U.S. and NexMed company are developing permeation enhancer, improve lipotropy and the penetrating power of hydrophilic medicament in skin.Other company uses electroporation technology makes medicine pass through skin more easily; Using the electron ion infiltration method like Noven company and Elan company; Make different drug (comprising polypeptide) infiltrate skin more easily, Noven company also has the preparation capable of permeating skin of a methylphenidate to be in II phase clinical stage.Promote that at present the method for antigen skin permeation is varied; Except that penetrating agent; Effective chemical method and physical method that some use on medicine, the short technology of oozing such as iontophoresis, electroporation and ultrasonic, laser and heat release that comprise are come in the middle of also just being introduced in vaccine.Penetrating agent generally is divided into two types: one type only increases antigen in cuticular diffusion, for example, and acetone, ethanol etc.Another kind ofly then not only increase antigenic diffusion, can also promote antigen from horny layer to living tissue distribution, for example, dimethyl sulfoxide (DMSO), oleic acid, certain herbaceous plants with big flowers ylmethyl sulfoxide (DCMS) and some surfactants etc.
In transdermal immune, adjuvant and antigen common application just can be induced intensive body fluid, cell and mucosal immune response, therefore seek safe and effective transdermal immune adjuvant and also have important practical significance.In a word, exploitation influenza transdermal vaccine replaces traditional vaccinate to have great application prospect because influenza transdermal vaccine has simply, safety, effectively, be applicable to all groups and the characteristics of can the oneself inoculating.
Summary of the invention
The object of the invention is to provide transdermal immune influenza multivalent vaccine and preparation method thereof.
Transdermal immune influenza multivalent vaccine of the present invention comprises transdermal immune adjuvant, influenza multivalent vaccine antigen, penetrating agent.
Influenza multivalent vaccine of the present invention has comprised influenza trivalent (H1N1, H3N2, B), tetravalence (H1N1, H3N2, B, H5N1) or the multivalence of combination in any (hypotypes such as H1N1, H3N2, B, H5N1, H7N7, H9N2) vaccine antigen on this basis; Also can be to have comprised the H1-H16 hypotype, the influenza multivalent vaccine antigen that all combinations of N1-N9 hypotype obtain; Influenza multivalent vaccine antigen is killed vaccine antigen or attenuated live vaccine antigen; Influenza vaccines antigen is common employed amount in the vaccine, and killed vaccine antigen (totivirus, lytic virus, virion, virus-like particle) HA content is 1.5 μ g-100 μ g/0.2ml/ person-portions, and the antigenic amount of attenuated live vaccine is 1 * 10
6EID
50-50 * 10
6EID
50/ 0.2ml/ person-portion or 0.1 * 10
6.5-7.5TCID
50-10 * 10
6.5-7.5TCID
50/ 0.2ml/ person-portion.
The penetrating agent that relates among the present invention is dimethyl sulfoxide (DMSO), oleic acid, imiquimod, ethanol, propylene glycol, acetone or certain herbaceous plants with big flowers ylmethyl sulfoxide (DCMS) and 2-pyrrolidinone derivative, preferred oleic acid or imiquimod; The mixed volume ratio of antigen and penetrating agent is 5-20: 1, preferred 10: 1 (V/V).
The transdermal adjuvant that relates among the present invention is antibacterial ADP-extracellular toxin (bacterialADP-ribosylating exotoxin; HARE) member; Comprise cholera toxin (choleratoxin; CT), (Escherichia coli heat-labileenterotoxin LT) and their mutant, comprises other types adjuvants such as CpG to the escherichia coli heat-labile toxin in addition; Transdermal adjuvant and antigenic mixed weight ratio are 1: 1-1: 10 (w/w), preferred 1: 1,1: 2,1: 9,1: 5,1: 7.
Relating to influenza multivalent vaccine antigen among the present invention comprises: tetravalence (H1, H3, H5, B), pentavalent (H1, H3, H5, H7, B) be (H1, H3, H5, H9, B) also combination such as (H1, H3, H5, H7, H9) equivalent also; Sexavalence (H1, H3, H5, H7, H9, B) is (H1, H2, H3, H5, H7, H9) also combination such as (H1, H2, H3, H4, H5, H6) equivalent also; Octavalence (H1, H2, H3, H4, H5, H6, H7, H9) is (H1, H2, H3, H4, H5, H6, H7, B) also combination such as (H1, H2, H3, H4, H5, H6, H9, B) equivalent also; Ten valencys (H1, H2, H3, H4, H5, H6, H7, H9, H10, B) also can be combinations such as (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10) equivalent; Also combination such as (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, B) equivalent of ten bivalence (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12); Also combination such as (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, B) equivalent of ten tetravalences (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14); Ten sexavalences (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16) also can be combinations such as (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, B) equivalent; Also can be to have comprised the H1-H16 type, the influenza multivalent vaccine antigen that all combinations of N1-N9 type obtain.Above-mentioned influenza multivalent vaccine antigen all can adopt the method preparation of CN20061007909.5 patent application.
The preparation of the above-mentioned a kind of flu polyvalent inactivation vaccine patch of the present invention: use Embryo Gallus domesticus to cultivate and traditional isolation and purification method; The antigenic preparation of influenza vaccines is infected the Embryo Gallus domesticus acquisition according to the method for routine, and also available mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) is cultivated acquisition; The vaccine antigen that obtains is at first through the formalin deactivation, and degerming then, ultrafiltration and concentration, column chromatography or density gradient centrifugation are carried out separation and purification; Vaccine antigen after concentrated and purified mixes with transdermal immune adjuvant such as LT, CT or CpG etc., penetrating agent such as oleic acid or imiquimod etc., adds conventional medical dressing and processes transdermal vaccine patch.
The method of passing through Embryo Gallus domesticus production flu polyvalent inactivation vaccine that the invention described above provided may further comprise the steps:
1) Embryo Gallus domesticus is cultivated virus in a large number, collects virus and carries out the formalin deactivation, then through degerming, ultrafiltration and concentration, density gradient centrifugation or other chromatography method separation and purification.
2) influenza vaccines antigen and transdermal immune adjuvant such as LT, CT or CpG etc., penetrating agent such as oleic acid or imiquimod after concentrated and purified add conventional medical dressing to process transdermal vaccine patch;
The method of passing through mammalian cell (like Vero cell, mdck cell, diploid cell etc.) production flu polyvalent inactivation vaccine that the invention described above provided may further comprise the steps:
1) use serum-free medium to cultivate a large amount of viruses of cultivating of mammalian cell (like Vero cell, mdck cell, diploid cell etc.); Collect virus and carry out the formalin deactivation, then through degerming, ultrafiltration and concentration, density gradient centrifugation or other chromatography method separation and purification;
2) the influenza vaccines antigen after concentrated and purified and transdermal immune adjuvant such as LT, CT or CpG etc., penetrating agent such as oleic acid or imiquimod mix, and add conventional medical dressing and process transdermal vaccine patch.
The present invention also provides a kind of preparation of Gripovax patch: through the reverse genetics method; A, Type B polyvalent vaccine antigen serve as the viral skeleton of rescue with cold adaptation attenuated influenza virus strain A/Ann Arbor/6/60 and B/Ann Arbor/1/66 respectively; Integrate the surface antigen gene of epidemic diseases strain then; Utilize influenza virus plasmid rescue systems produce polyvalent vaccine antigen; Cultivate degerming then, ultrafiltration and concentration, SDGC or other chromatography method separation and purification through infecting Embryo Gallus domesticus or mammalian cell (like Vero cell, mdck cell, 2BS cell etc.); Polyvalent vaccine antigen after concentrated and purified mixes with transdermal immune adjuvant such as LT, CT or CpG etc., penetrating agent such as oleic acid or imiquimod etc., adds conventional medical dressing and processes transdermal vaccine patch.
The above-mentioned method of passing through Embryo Gallus domesticus production flu polyvalent attenuated live vaccine that provides of the present invention may further comprise the steps:
1) utilize the reverse genetics method, 8,3 or 4 recombiant plasmid cotransfection mammalian cells (like cell or co-cultured cells such as COS/MDCK, 293T/MDCK such as COS 1, COS7, Vero, MDCK, 293T) produce polyvalent vaccine antigen;
2) Embryo Gallus domesticus is cultivated virus in a large number, collects virus through degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification;
3) the influenza multivalent vaccine antigen after concentrated and purified and transdermal immune adjuvant such as LT, CT or CpG etc., penetrating agent such as oleic acid or imiquimod mix, and add conventional medical dressing and process transdermal vaccine patch.
The present invention is above-mentioned provide pass through mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) the antigenic method of production flu polyvalent attenuated live polyvalent vaccine, may further comprise the steps:
1) utilize the reverse genetics method, 8,3 or 4 recombiant plasmid cotransfection mammalian cells (like cell or co-cultured cells such as COS/MDCK, 293T/MDCK such as COS 1, COS7, Vero, MDCK, 293T) produce polyvalent vaccine antigen;
2) use serum-free medium to cultivate mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) and cultivate polyvalent vaccine antigen in a large number, collect virus through degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification;
3) the influenza multivalent vaccine antigen after concentrated and purified mixes with transdermal immune adjuvant such as LT, CT or CpG etc., penetrating agent such as oleic acid or imiquimod etc., adds conventional medical dressing and processes transdermal vaccine patch.
The influenza virus trivalent that the present invention comprises, tetravalence and polyvalent vaccine antigen can be the antigenic any antigen of attenuation or deactivation polyvalent vaccine, and wherein deactivation polyvalent vaccine antigen can be wherein any such as inactivated whole virus vaccines, split vaccine, virion or virus-like particle.Vaccine form of the present invention can adopt liquid, Powdered, creaminess and emulsive penetrating agent type, preferred powder powder.Vaccine composition can also contain just like a series of additives such as stabilizing agent, wetting agent and antiseptic.
Transdermal immune of the present invention is applied to skin inducing antigen-specific immunne response with antigen and adjuvant; It is a kind of immunization strategy of novelty; On humans and animals, have the double effects that to induce intensive mucosa IgG and sIgA production of antibodies and general immunity to reply, and the mucosa attack of contratoxin and virulent strain virus there is immune protective effect preferably.In addition, transdermal immune has the double effects of inducible system immunity and mucosa-immune.The transdermal immune inoculation also has following characteristics: (1) is safe in utilization, easy, and everybody can inoculate by own percutaneous, is suitable for flu outbreak or family oriented; (2) keep constant blood drug level or pharmacodynamics effect, strengthened therapeutic effect, reduced side effect, avoided the pain and the cross infection of injection; (3) one Seedling multiple-effect both can be prevented common influenza, can prevent the human and bird fluenza again; (4) double effects both can produce systemic immunity, can produce mucosa-immune again; (5) side reaction is low, both can be used for growing up, and applicable again child is with old; (6) preparation is simple, with low cost, and technological process is simple, can reduce production costs greatly.
The present invention is a kind of influenza multivalent vaccine of transdermal immune; The main component of this vaccine is to utilize influenza trivalent, tetravalence and multivalence (hypotypes such as H1N1, H3N2, B, H5N1, H7N7, H9N2) the attenuated live virus antigen or the inactivating influenza virus antigen of the preparation of reverse genetics reprovision cold adaptation Strain technology; (both can prolong the targeting property of the antigen time of staying with a kind of transdermal immune adjuvant respectively to Skin Cell; But the angtigen presentation function of enhancement antigen presenting cells again), a kind of penetrating agent (both can increase the antigen diffusion; Can promote antigen to antigen presenting cell, to assemble again from horny layer) the preparation patch, can effectively prevent the infection of the common influenza virus of people, highly pathogenic human and bird fluenza virus.This vaccine of experiment proof induce mucosa sIgA and systemic IgG antibody mediated immunity reply aspect effect remarkable, can safety, effectively the common influenza of prevention people and the highly pathogenic human and bird fluenza.
The present invention be advantageous in that; The influenza tetravalence or polyvalent inactivation or the attenuated live vaccine that have adjuvant transdermal immunes such as LT, CT or CpG; In mammal, can induce effective mucosa-immune and systemic immunity simultaneously; And make body produce height tire sIgA and IgG, IgA, the more present applied vaccine component of its effect---be all ideals of simple influenza inactivated virus particle, the inactivation of viruses that adds adjuvant and live virus.This vaccine is through experimental verification, can improve the immunogenicity of vaccine through using a kind of transdermal immune adjuvant such as LT, CT or CpG etc. effectively through transdermal immune, and without any side effects, and showing has good safety.
Flu polyvalent inactivation of the present invention or attenuated live vaccine were with vaccine immunity approach and adjuvant were all different in the past.Through transdermal route immunity Balb/c mice, the result proves, by the 0th, 14 day twice transdermal immune program, can produce IgA and IgG antibody that height is tired, can induce mucosa-immune and systemic immunity simultaneously.Can well protect mice to avoid the attack of influenza virus, and produce certain intersecting protective, this is that the inactivated influenza vaccine institute of common injecting immune is unrivaled.Through transdermal route immunity ferret and monkey, no matter be flu polyvalent inactivation vaccine or attenuated live vaccine, all can produce higher H I and tire, further specifying the transdermal immune approach is one of alternative desirable approach of influenza multivalent vaccine.
Description of drawings
Two kinds of approach immunity of Fig. 1 influenza virus tetravalent inactivated vaccine Balb/c mice serum IgG/IgA titre relatively
The specific embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method; Percentage composition among the following embodiment if no special instructions, is the mass and size percentage composition.
Embodiment 1:
The Embryo Gallus domesticus of influenza virus is cultivated, and adopts traditional cultural method.A type strains of influenza viruses comprises that (hypotypes such as H1, H3, H5) and Type B strains of influenza viruses are provided by Chinese CDC and Hua Lan biotechnology limited company.
Influenza virus is cultivated and to take to inoculate 9-11 age in days SPF chick embryo allantoic cavity, cultivates 48-72h (it is slightly different to look the different incubation times of virus subtype) for 33-35 ℃, 4 ℃ of refrigerator overnight Embryo Gallus domesticus of freezing to death, and the results chick embryo allantoic liquid obtains a large amount of viruses.
Mammalian cell (like Vero cell, mdck cell, diploid cell etc.) is cultivated influenza virus, and A type strains of influenza viruses comprises that (hypotypes such as H1, H3, H5) and Type B strains of influenza viruses are provided by Chinese CDC and Hua Lan biotechnology limited company.
Adopt micro-carrier system (like Cytodex 1,3 etc.) in the 100mlWAVE reaction vessel, to cultivate; Carry out the transition to gradually in the biomass generator of 10L and cultivate; And adding 3g/L microcarrier can obtain the good growth of Vero cell; Infect 36-48 hour after can obtain TCID50 and reach 10 with influenza infection Vero cell this moment
6-10
7The virus quantity of/ml.
The antigenic preparation of influenza tetravalent inactivated vaccine:
A type strains of influenza viruses H1, H3, H5 and Type B strains of influenza viruses are provided by Chinese CDC and Hua Lan biotechnology limited company.Adopt traditional chick embryo allantoic cavity to cultivate influenza virus method or microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) and cultivate influenza virus.
1. inoculate 9-11 age in days chick embryo allantoic cavity and cultivate H1, H3, H5 and Type B influenza virus in a large number, collect virus, degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization through the formalin deactivation.
2. produce H1, H3, H5 and Type B influenza virus by mammalian cell (like Vero cell, mdck cell, 2BS cell etc.); Use serum-free medium, the method for employing has: microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) are cultivated influenza virus.Collect virus through the formalin deactivation, degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization, preparation influenza tetravalent inactivated vaccine antigen.
The antigenic preparation of influenza tetravalence attenuated live vaccine:
A type strains of influenza viruses H1, H3, H5 and Type B strains of influenza viruses are provided by Chinese CDC and Hua Lan biotechnology limited company.Adopt traditional chick embryo allantoic cavity to cultivate influenza virus method or microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) and cultivate influenza virus.
1. utilize existing reverse genetics method; A type multivalent virus is viral skeleton with cold adaptation attenuated influenza virus strain A/Ann Arbor/6/60; The Type B multivalent virus serves as the viral skeleton of rescue with B/Ann Arbor/1/66; Integrate popular then A, the HA of Type B strains of influenza viruses, NA surface antigen gene respectively, be about to 4 kinds of multivalent virus of 8,3 or 4 recombiant plasmid cotransfection mammalian cells (like cell or co-cultured cells such as COS/MDCK, 293T/MDCK such as COS1, COS7, Vero, MDCK, 293T) generation that H1N1, H3N2, H5N1 and Type B influenza virus gene fragment make up.
2. 4 kinds of multivalent virus of a large amount of cultivations of Embryo Gallus domesticus are collected virus through degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization.
3. produce 4 kinds of multivalent virus by mammalian cell (like Vero cell, mdck cell, 2BS cell etc.): use serum-free medium, the method for employing has: microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) are cultivated influenza virus.Collect virus through degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization, preparation influenza tetravalence attenuated live vaccine antigen.
The preparation of flu polyvalent inactivation vaccine antigen:
Hypotypes such as A type strains of influenza viruses H1, H3, H5, H7, H9 and Type B strains of influenza viruses are provided by Chinese CDC and Hua Lan biotechnology limited company.Adopt traditional chick embryo allantoic cavity to cultivate influenza virus method or microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) and cultivate influenza virus.
1. inoculate 9-11 age in days chick embryo allantoic cavity and cultivate hypotype and Type B influenza virus such as H1, H3, H5, H7, H9 in a large number, collect virus, degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization through the formalin deactivation.
2. produce H1, H3, H5 and Type B influenza virus by mammalian cell (like Vero cell, mdck cell, 2BS cell etc.); Use serum-free medium, the method for employing has: microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) are cultivated influenza virus.Collect virus through the formalin deactivation, degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization, preparation flu polyvalent inactivation vaccine antigen.
The antigenic preparation of flu polyvalent attenuated live vaccine:
Hypotypes such as A type strains of influenza viruses H1, H3, H5, H7, H9 and Type B strains of influenza viruses are provided by Chinese CDC and Hua Lan biotechnology limited company.Adopt traditional chick embryo allantoic cavity to cultivate influenza virus method or microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) and cultivate influenza virus.
1. utilize existing reverse genetics method; A type multivalent virus is viral skeleton with cold adaptation attenuated influenza virus strain A/Ann Arbor/6/60; The Type B multivalent virus serves as the viral skeleton of rescue with B/Ann Arbor/1/66; Integrate popular then A, the HA of Type B strains of influenza viruses, NA surface antigen gene respectively, be about to 8,3 or 4 recombiant plasmid cotransfection mammalian cells (like cell or co-cultured cells such as COS/MDCK, 293T/MDCK such as COS1, COS7, Vero, MDCK, 293T) that hypotype such as H1, H3, H5, H7, H9 and Type B influenza virus gene fragment make up and produce multiple multivalent virus.
2. the multiple multivalent virus of a large amount of cultivations of Embryo Gallus domesticus is collected virus through degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization.
3. produce multiple multivalent virus by mammalian cell (like Vero cell, mdck cell, 2BS cell etc.): use serum-free medium, the method for employing has: microcarrier suspension culture and taming mammalian cell (like Vero cell, mdck cell, 2BS cell etc.) are cultivated influenza virus.Collect virus through degerming, ultrafiltration and concentration, SDGC or column chromatographic isolation and purification, dialysis and filtration sterilization, preparation flu polyvalent attenuated live vaccine antigen.
The preparation of flu polyvalent inactivation transdermal vaccine patch
1. with A, Type B and human and bird fluenza viral infection mammalian cell (like Vero cell, mdck cell, 2BS cell etc.), cultivate virus, or from the allantoic fluid that infects Embryo Gallus domesticus, obtain virus according to the traditional method that Embryo Gallus domesticus is cultivated with serum-free medium.
2. produce inactivation of viruses by cell culture: in the cell conditioned medium liquid that infects, add formalin; Its final concentration is 0.025%; Hatched 24 hours in 32 ℃; Adopt 0-50% sucrose continuous gradient to carry out band centrifugation then, obtain finished product through DNA removal, dialysis and filtration sterilization, in-70 ℃ of storages.Finished product meets standards through check:
3. adjuvant mixes with antigenic: with transdermal immune adjuvant LT, CT or CpG etc. and the polyvalent vaccine antigen mixed according to 1: 1,1: 2,1: 9,1: 5 or 1: 7 (w/w), use the 10000MW bag filter, PBS dialysis 8-10 days promptly is prepared into vaccine; Vaccine and penetrating agent dimethyl sulfoxide, oleic acid or imiquimod or certain herbaceous plants with big flowers ylmethyl sulfoxide are processed patch by 10: 1 (V/V) and conventional medical dressing mixed.
The preparation of flu polyvalent attenuated live vaccine transdermal patch:
1. utilize the reverse genetics method, 8,3 or 4 recombiant plasmid cotransfection mammalian cells (like cell or co-cultured cells such as COS/MDCK, 293T/MDCK such as COS 1, COS7, Vero, MDCK, 293T) produce the multivalence influenza virus.
2. Embryo Gallus domesticus is cultivated virus in a large number, collects virus and adopts 0-50% sucrose continuous gradient to carry out band centrifugation, obtains finished product through dialysis and filtration sterilization, in-70 ℃ of storages.
3. produce virus by mammalian cell (like Vero cell, mdck cell, 2BS cell etc.): cultivate virus with serum-free medium; Adopt 0-50% sucrose continuous gradient to carry out band centrifugation; Obtain finished product through DNA removal, dialysis and filtration sterilization, in-70 ℃ of storages.
4. adjuvant mixes with antigenic: with transdermal immune adjuvant LT, CT or CpG etc. and the vaccine mixed according to 1: 1 (w/w) or 1: 2,1: 9,1: 5,1: 7, use the 10000MW bag filter, PBS dialysis 8-10 days promptly is prepared into vaccine; Vaccine and penetrating agent dimethyl sulfoxide, oleic acid or imiquimod or certain herbaceous plants with big flowers ylmethyl sulfoxide are processed patch by 10: 1 (V/V) and medical dressing mixed.
Embodiment 2: effect observation in the mice body of flu polyvalent inactivation/attenuated live vaccine
1, Immunization programme scheme, (transdermal, injecting immune is contrast) influenza virus vaccine immune mouse of use different dosage form
1. with the 40# shears 6-8 Balb/c mice in age in week is shaved hair.Shave mao process and should skin not caused any damage.Shave mao scope and carry on the back the bottom from the width of chest middle part to being close to strength.Let mice rest 24h.Mice has been made labelling before this so that recognize, and got blood in advance to obtain preimmune serum.
2. immune mouse as follows then.With 20mg/ml xylazine solution 0.03-0.06ml, 100mg/ml ketamine 0.5ml anesthetized mice; The anesthetis of this dosage makes the about 1h of mice transfixion.
3. be added to immune solution on the QUMAO skin of back of mice by following method: the hollow out XPS of 1.2cm * 1.6cm is placed on mouse back gently; Sterile gauze fractional wettability skin (available immune solution) with saline soaked; With dropper immune solution is added in the hollowed-out board place then, forms 2cm
2Immune solution patch.Perhaps shaving the immune solution that fixed volume is coated at mao position.Carefully make dropper not scratch of point or scratch mark.
4. make immune solution (about 100 μ l to 200 μ l) leave standstill 60 to 180min at mouse back.Behind the 60min, catch neck and the afterbody of mice gently, washed for 10 seconds with the tap water of a large amount of tepors.With a sterile gauze mice is clapped gently driedly then, washed again 10 seconds; Bat is put back to mice in the cage after doing once more.Mice seems anesthesia, immunity inoculation, washing process or ectotoxic toxicity are not shown any discomfort reaction.After the immunity inoculation, do not see skin irritation or redness, the mice survival condition is good.
2,, use PBS to be diluted to its HA concentration antigen simultaneously and be>=15 μ g/ml, the amount of attenuated live vaccine>=5 * 10 according to example 1 prepared adjuvant/mixed antigen body
6EID
50, as no adjuvant matched group (Tab1).For preparation is applicable to the dosage form of mice transdermal immune, can solution be done suitably dilution with PBS.
21 groups of Balb/c mices are accepted the transdermal vaccine immunity of different dosage form respectively, and every mice 100 μ l contain the antigenic solution of adjuvant.Respectively at carrying out immunity on the the 0th, 14 day, and measured serum in the 28th day, the IgG in saliva and the lung-douching fluid, IgA and HAI tire.
The immunization protocol of table 1 transdermal vaccine immunity Balb/c mice
3, serum, the mensuration that the IgG in saliva and the lung-douching fluid, IgA and HAI tire
1. immune back 28 days, get saliva, lungs and the serum specimen of animal, measure the antibody titer of every part of BIAO and BEN.
2. inject the mice oral cavity with 0.5mlPBS, collect saliva and measure its IgA content.
3. the preparation of lung tissue cracking BIAO and BEN: put to death mice, open throat, take out lungs and, grind lung tissue and centrifugal homogenate de-cellular system, collect supernatant and be stored in-20 ℃, be used for the IgA titration with the PBS cleaning.
4. IgG and IgA titration commodity in use first type or Influenza B virus ELISA detection kit (Genzyme Virotech).Hatch for 37 ℃ and add sheep anti-mouse igg or IgA antibody (Pharmigen) and substrate solution and colour developing liquid detection specificity IgG and IgA antibody titer after 2 hours.
5. HAI titration: get mouse blood, tire according to Palmer et al designation method mensuration first type and Influenza B virus HAI.
6. data show is under the situation of no adjuvant; The whole virus particles of deactivation can not excitating organism IgA and IgG immunne response; On the contrary; If in the whole virus particles of live virus or deactivation, add adjuvants such as LT, CT or CpG, but excitating organism produces the higher IgA that tires, IgG and HAI (Tab2).
The mensuration result that IgA tires and serum IgG and HAI tire in saliva, the lung-douching fluid behind the table 2 variety classes influenza vaccines strain transdermal immune mice
NC representes: New Caledonia/20/99, and WS representes: Wisconsin/67/2005, XJ representes XiJiang/424/2004, and ML representes Malaysia/2506/2004, and PL representes lung-douching fluid, and n.d representes not measure
3, be the comparison of the different immunization routes of influenza tetravalent inactivated vaccine of adjuvant with LT, vaccine is carried out transdermal immune respectively, two kinds of different immunization routes of intramuscular injection are inoculated.The result shows, though intramuscular injection has produced the higher IgG that tires, does not detect tangible IgA antibody.But transdermal immune has produced the high IgA that tires.IgA antibody plays a significant role in the infectious disease to preventing respiratory, explains that influenza multivalent vaccine transdermal immune approach is one of alternative desirable approach (Fig. 1).
Embodiment 3: effect observation in the ferret body of flu polyvalent inactivation/attenuated live vaccine
1, Immunization programme, the influenza virus vaccine immunity ferret of using different dosage form
1. according to the method for example 1 prepared adjuvant/mixed antigen body, preparation is applicable to the dosage form of ferret transdermal immune, can solution be made suitably dilution LT with PBS.
2. male and the female ferret in age in 6-10 week, choosing body weight is this experiment that is used for of 1-2kg, all these animals are seronegativity before in experiment.Back 14 days of immunity, ferret is got hematometry HI through jugular vein and tires.
3. 6 groups of ferrets are accepted the transdermal vaccine immunity of different dosage form respectively, and every 1ml contains the antigenic solution of adjuvant such as LT, CT or CpG etc., and patch is used for hard of hearing or preaxial, carries out immunity respectively according to the group of table 3, and measures HI in the 14th day and tire.
2, serum HI titration: the ferret jugular vein is got blood, carries out HI according to OIE standard appointed method and measures.
3, experimental result shows, no matter deactivation of influenza tetravalence or attenuated live vaccine are that Embryo Gallus domesticus is cultivated and obtained, and still the virus antigen that obtains of Vero cell culture adds adjuvant such as LT, CT, CpG etc. behind the immune ferret of transdermal route, all can produce higher H I and tire.Further specifying the transdermal immune approach is one of the alternative desirable approach of influenza multivalent vaccine (table 3).
HI titration result behind the table 3 vaccine immunity ferret
C:Geometri mean titer geometric mean titer
Embodiment 4: effect observation in the monkey body of transdermal flu polyvalent inactivation/attenuated live vaccine
1, Immunization programme, the influenza virus vaccine immunity monkey of using different dosage form
1. according to the method for example 1 prepared adjuvant/mixed antigen body, preparation is applicable to the dosage form of monkey transdermal immune, can solution be made suitably adjuvants such as dilution LT, CT, CpG with PBS.
2. choose healthy monkey and be used for this experiment, all these animals were seronegativity before experiment.Immunity 14 days afterwards, monkey is got hematometry HI through jugular vein and tires.
3. 6 groups of monkeys are accepted the transdermal vaccine immunity of different dosage form respectively, and every 1ml contains the antigenic solution of adjuvant such as LT, CT or CpG etc., and patch is used for hard of hearing or preaxial, carries out immunity respectively according to the group of table 4, and measures HI in the 14th day and tire.
2, serum HI titration: the monkey jugular vein is got blood, carries out HI according to OIE standard appointed method and measures.
3, experimental result shows, no matter deactivation of influenza tetravalence or attenuated live vaccine are that Embryo Gallus domesticus is cultivated and obtained, and still the virus antigen that obtains of Vero cell culture adds adjuvant such as LT, CT, CpG etc. behind the immune ferret of transdermal route, all can produce higher H I and tire.Further specifying the transdermal immune approach is one of the alternative desirable approach of influenza multivalent vaccine (table 4).
HI titration result behind the table 4 vaccine immunity monkey
C:Geometri mean titer geometric mean titer
Embodiment 5: effect observation in the human body body of influenza tetravalence attenuated live vaccine
1, in the healthy population volunteer, evaluation and the administration of comparison transdermal route add AL (OH)
3The vaccinum influenzae inactivatum of adjuvant and influenza tetravalence attenuated live vaccine and immune effect, patch is used for hard of hearing or preaxial.
2, that open, in check, the vaccine of having estimated at random reacts at masculinity and femininity experimenter's part and systemic immunity.
3, all experimenters carry out following detection:
0 day, 21 days: ELISA detected mucosa IgA (local immunity reaction), detects each influenza representative strains in the vaccine respectively.
0 day, 21 days: detect serum HA-HI antibody titer, detect each influenza representative strains respectively.
4, the following project of Data Source:
(1) the geometric mean GMTs (95% virtual value) of all time point serum HI antibody.
Seroconversion rate during (2) to HI:21 days.
Transformation ratio during (3) to HI:21 days.
Protective rate during (4) to HI:21 days.
(5) only to IgA: increase 2-4 percentage rate doubly from 0-21 days experimenter IgA antibody titers.
5, through 8 days follow-up period to part and General Symptoms, two kinds of vaccines all tolerate safety and reactivity.Show about the no serious illeffects of inoculation.
6, the detection of vaccine immunogenicity suppresses titre through blood clotting and decides seroconversion rate (percentage rate of vaccine is to increase by 4 times at least in 0 day to limit according to 21 days HI titres; Each vaccine strain); Transformation ratio (be according to 21 days than 0 day the HI geometric mean titer (GMTs) the multiplication value limit each vaccine strain), the serum protective rate (generally acknowledge and show protection HI titre >=40 and limit by inoculation back; Each vaccine strain), mucosa IgA antibody response detects with ELISA in addition.
7, inoculation one dosage transdermal vaccinum influenzae inactivatum or tetravalence attenuated live vaccine HI seropositivity after 21 days, seroconversion rate and serum protective rate are seen table 5.
Seropositivity (n, %): numbering and experimenter's titre >=10
The serum protective rate (n, %): numbering and experimenter's titre >=40
(n, %): numbering and experimenter are 4 times propagation from 0 day to 21 days titre to seroconversion rate at least
8, the result shows: dosage inoculation is after 21 days, and transdermal immune influenza multivalence attenuated live vaccine produces similar seropositivity, seroconversion rate and serum protective rate with inactivated vaccine.
Claims (6)
1. a transdermal immune influenza multivalent vaccine is characterized in that this transdermal immune influenza multivalent vaccine comprises transdermal immune adjuvant, influenza multivalent vaccine antigen and penetrating agent; Influenza multivalent vaccine has comprised influenza trivalent or tetravalence; Influenza multivalent vaccine is inactivated vaccine or attenuated live vaccine; Killed vaccine antigen content is 1.5 μ g-100 μ g/0.2ml/ person-portions, and the antigenic amount of attenuated live vaccine is 5 * 10
6ETD
50-50 * 10
6EID
50/ 0.2ml/ person-portion or 0.1 * 10
6.5-7.5TCID
50-10 * 10
6.5-7.5TCID
50/ 0.2ml/ person-portion;
Penetrating agent is dimethyl sulfoxide, oleic acid, imiquimod, ethanol, acetone and 2-pyrrolidinone derivative or certain herbaceous plants with big flowers ylmethyl sulfoxide; The mixed volume ratio of antigen and penetrating agent is 10: 1; The transdermal immune adjuvant is the ectotoxic member of antibacterial ADP-, comprises cholera toxin, escherichia coli heat-labile toxin and their mutant, comprises other types adjuvants such as CpG in addition; Transdermal immune adjuvant and antigenic mixed weight ratio are 1: 2,1: 9,1: 5 or 1: 7.
2. transdermal immune influenza multivalent vaccine as claimed in claim 1 is characterized in that in this transdermal immune influenza multivalent vaccine that inactivated vaccine wherein is any in inactivated whole virus vaccines, split vaccine, virion or the virus-like particle.
3. transdermal immune influenza multivalent vaccine as claimed in claim 1 is characterized in that this transdermal immune influenza multivalent vaccine adopts the transdermal dosage form of liquid, powder, butterfat or emulsifying or paster.
4. transdermal immune influenza multivalent vaccine as claimed in claim 2 is characterized in that this transdermal immune influenza multivalent vaccine adopts the transdermal dosage form of liquid, powder, butterfat or emulsifying or paster.
5. like the method for preparing of claim 1,2,3 or 4 described transdermal immune influenza multivalent vaccines; It is characterized in that this method is: through the reverse genetics method; A, Type B polyvalent vaccine antigen serve as the viral skeleton of rescue with cold adaptation attenuated influenza virus strain A/Ann Arbor/6/60 and B/Ann Arbor/1/66 respectively; Integrate the surface antigen gene of epidemic diseases strain; Utilize influenza virus plasmid rescue systems produce polyvalent vaccine antigen, cultivate degerming then, ultrafiltration and concentration, SDGC or other chromatography method separation and purification through infecting Embryo Gallus domesticus, Vero cell, mdck cell or diploid cell; Polyvalent vaccine antigen and transdermal immune adjuvant, penetrating agent after concentrated and purified are mixed and made into the vaccine patch.
6. the method for preparing of transdermal immune influenza multivalent vaccine as claimed in claim 5 is characterized in that the transdermal immune adjuvant in this method is LT, CT or CpG; Penetrating agent is oleic acid or imiquimod.
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