CN1399539A - Adjuvant comprising polyxyethylene alkyl ether or ester and at least one nonionic surfactant - Google Patents

Abstract

The present invention relates to a novel adjuvant system comprising a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant. Preferably said additional non-ionic surfactant is an Octoxynol (the TRITON<TM> series). The present invention provides said novel adjuvants, vaccines comprising them, and methods of their manufacture and their formulation into vaccines. The use of the adjuvants or vaccines of the present invention in the prophylaxis or therapy of disease is also provided.

Description

The adjuvant that contains polyoxyethylene alkyl ether or polyxyethylated ester and at least a non-ionic surface active agent

The present invention relates to a kind of new adjuvant system, comprise and at least a other non-ionic surface active agent and the polyoxyethylene alkyl ether or the polyxyethylated ester surfactant of usefulness.Preferred described other non-ionic surface active agent is a hot benzene polysaccharide (Octoxynol).The invention provides described new adjuvant, comprise they vaccine, and preparation method thereof and it is made into the method for vaccine.Adjuvant of the present invention or the vaccine purposes in prevention or treatment disease also is provided.A kind of method that strengthens the host immune response that uses adjuvant of the present invention and vaccine also is provided.This adjuvant is effective especially as mucosal adjuvants, but also effective to whole body.

Except having avoided because " fearing pin " and injection pain and the patient is produced the influence of related side effects; the mucosal vaccination vaccine is attractive; because shown for animal; mucosal administration antigen more high efficiency impels mucomembranous surface to produce protective response, and mucomembranous surface is the admission passage of many pathogen.In addition, shown the mucosal vaccination vaccine, for example intranasal vaccination not only can be at nasal mucosa, and mucosal sites causes mucosal immunity a long way off, as genitals mucosa (Mestecky, 1987, " clinical immunology magazine " (Journal of ClinicalImmunology), 7,265-276; McGhee and Kiyono, " infectiousness medicament and disease " (Infectious Agents and Disease), 1993,2,55-73).Although many researchs have been carried out in this field, the safety and the effective mucosal adjuvants that are fit to the people is used still have to be determined.The invention provides a solution to this problem.

The medical application of some non-ionic surface active agent has been described.For example, having described intranasal gives polyoxyethylene ether and polyoxyethylene ester (Hirai etc. 1981, " international journal of Practical Pharmacy " (International Journal of Pharmaceutics), 9,165-172 to strengthen the nasal absorption insulin; Hirai etc. 1981, " international journal of Practical Pharmacy " (International Journal ofPharmaceutics), and 9,173-184).

(JP 05 201877 as the description of the composition of fat liquor or acrylate copolymer adjuvant for existing polyoxyethylene alkyl ether; US 3,919, and 411).

Other non-ionic surface active agent has been used to vaccine mixture.For example, comprise polyoxyethylene castor oil or sad/certain herbaceous plants with big flowers acid glyceride, with the bacterin preparation of polyethenoxy sorbitan monoesters and antigenic mixture, after the mucosa topical administration, can cause systemic immune response (WO94/17827).This patent application discloses non-ionic surface active agent TWEEN20^TM(polyethenoxy sorbitan monoesters) and Imwitor742^TM(sad/the certain herbaceous plants with big flowers acid glyceride) associating, or TWEEN20^TMUnite use with polyoxyethylene castor oil and after the intranasal immunity inoculation, can strengthen systemic immune response.At document (Gizurarson etc. 1996 " vaccine research " (Vaccine Research), 5,69-75; Aggerbeck etc. 1997, " vaccine " (Vaccine), 15,307-316; Tebbey etc., Viral Immunol 1999; 12 (1): described this preparaton gives antigenic immunoreation potentiation at intranasal details 41-5).

Non-ionic surface active agent has formed nonionic surfactant vesicle (known NISV, US 5,679,355) in one way.The preparaton of this non-ionic surface active agent usually in the presence of cholesterol, forms two lipoid layer vesicle, and it is remaining antigen in internal layer aqueous phase or two lipoid layer itself.

(US 5 for International Patent Application WO 96/36352,653,987) described a kind of liquid pharmaceutical formulation, it comprises at least two kinds of absorption enhancers and water, be mainly the transmission of oral insulin, the amount of wherein each absorption enhancer is that the concentration with the 1-10%w/w of total preparaton exists.

The preparaton whole body that surfactant usually is used for the oil emulsion adjuvant gives, and plays the stabilize oil drop.For example, Sorbitan ethoxylate (TWEEN^TM) and fatty acid esters of sorbitan (SPAN^TM) be used for the stabilize water latex oil (EP 0 399 843 B, WO95/17210).

The applicant provides surprising discovery here, and polyoxyethylene alkyl ether or polyxyethylated ester with at least a other non-ionic surface active agent and usefulness, play a role as effective vaccine adjuvant together.Advantageously, but the said composition whole body give, but when vaccine combination adopts mucosal administration, also can effectively induce systemic immune response.The vaccine-induced immunoreation of mucosal administration of the present invention may be the same high with observed immunoreation behind the traditional vaccine systemic injection at least.

The invention provides safe and potent adjuvants, it is easy to produce, and comprises at least a polyoxyethylene alkyl ether or polyxyethylated ester and at least a other non-ionic surface active agent.The surfactant that uses among the present invention can be the suspension that aqueous solution maybe can form grain structure, as vesicle or micelle (micelle).Preferred this surfactant is aqueous solution or micellar form.

Can be allocated into the polyoxyethylene ether of vaccine of the present invention and adjuvant or the molecule that ester comprises general formula (I):

HO (CH₂CH₂O)_n-A-R wherein, n is 1-50, A be key or-C (O)-, R is C_1-50Alkyl or phenyl C_1-50Alkyl.

Thereby a scheme of the present invention comprises a kind of vaccine mixture, and this preparaton comprises the polyoxyethylene alkyl ether of general formula (I), and wherein n is 1 to 50, preferred 4-24, more preferably 6-12, most preferably 9; Composition R is C_1-50, preferred C₄-C₂₀Alkyl, more preferably C₁₂Alkyl.Polyoxyethylene ether concentration should be in the scope of 0.1-20%, preferred 0.1-10%, the more preferably scope of 0.1-1%.Suitable polyoxyethylene ether is selected from down group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-stearoyl ether, polyoxyethylene-8-stearoyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether.

More preferably, described polyoxyethylene alkyl ether is polyoxyethylene-9-lauryl ether (laureth 9).The replacement term of polyoxyethylene lauryl ether or title are open in the CAS registration.The CAS number of registration of polyoxyethylene-9-lauryl ether is: 9002-92-0.Polyoxyethylene ether, as polyoxyethylene lauryl ether " Merck index " (the 12nd edition: clauses and subclauses 7717, Merck﹠amp; Co.Inc., Whitehouse Station, New Jersey, the U.S.; ISBN 0911910-12-3) open in, described therapeutic use comprises: local anesthesia; Antipruritic; With the sclerosing agent activity.As a class, this class polyoxyethylene ether or polyoxyethylene ester are non-ionic surface active agents.Oxirane and dodecanol reaction can form Laureth 9, and 9 ethylene oxide unit(s)s are on average arranged.In one embodiment, use the surfactant mixtures of such general formula (I), meant the average of the ethylene oxide unit(s) that exists in all surface activating agent in the n=mixture in the article of the present invention.

The length of polyoxyethylene part and the ratio of alkyl chain length (are n: the ratio of alkyl chain length), influence the dissolubility of this class cleaning agent (detergent) in aqueous medium in the surfactant.Thereby adjuvant of the present invention can be that solution maybe can form micrograined texture, as capsule or vesicle.As solution, adjuvant safety of the present invention for example adopts and is easy to sterilization by one deck 0.22 μ m film, and it is simple to give (administer), and can be by the plain mode manufacturing, and need not GMP relevant and QC permission (issue) with the formation of homogeneous micrograined texture.Some polyoxyethylene ether as laureth 9, can form no vesicle solution.Yet, polyoxyethylene-8 palmityl ether (C₁₈E₈) can form vesicle.Thereby, expect that especially the vesicle of polyoxyethylene-8 palmityl ether and at least a other non-ionic surface active agent unite use, to form adjuvant of the present invention.

Preferably, the polyoxyethylene alkyl ether composition that exists in the adjuvant compound of the present invention has hemolytic activity.But the hemolytic activity external test of polyoxyethylene alkyl ether with reference to following method, is represented with the maximum concentration that does not cause haemoclastic cleaning agent (detergent):

1. the Cavia porcellus fresh blood washs 3 times in desk centrifuge with phosphate buffered saline (PBS) (PBS).Again make suspension to initial volume, further dilute 10 times with PBS.

2. 50 these blood suspension of μ l being joined 800 μ l contains among the PBS of the cleaning agent of diluting 2 times.

3.8 after hour, naked eyes or by measuring the optical density assessment haemolysis of supernatant.Red supernatant occurs, show in the 570nm absorbing light to have haemolysis.

4. the result represents with the concentration that hemolytic first time of detergent dilution no longer takes place.

In the inherent experiment transmutability of this biological method, the surfactant of polyoxyethylene alkyl ether of the present invention or general formula (I), preferably has hemolytic activity, between about 0.5-0.0001%, more preferably between the 0.05-0.0001%, even more preferably between the 0.005-0.0001%, and most preferably between the 0.003-0.0004%.Ideally, described polyoxyethylene ether or polyoxyethylene ester should similar to the hemolytic activity of polyoxyethylene-9 lauryl ether or polyoxyethylene-8 stearoyl ether (promptly in 10 times of differences).

For at least a other non-ionic surface active agent that adds in polyoxyethylene alkyl ether or the polyxyethylated ester, can be any cleaning agent with suitable surface activity ability.Suitable cleaning agent is described among Ed:Attwood and the Florence (1983, Chapman and Hall) at " surfactant system ".

Preferred nonionic surfactants is not to fall into those of general formula (I), as hot benzene polysaccharide (octoxynol) and Sorbitan ethoxylate.Especially preferred hot benzene polysaccharide comprises Triton X-45, uncle's octylphenoxy polyethoxy ethanol (Triton X-100), Triton X-102, Triton X-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57, Triton N-101, Triton N-128.Preferred especially Triton X-100.Hot benzene polysaccharide series comprises uncle's octylphenoxy polyethoxy ethanol (TRITONX100^TM) the clauses and subclauses 6858 of " Merck index " (1162 pages, the 12nd edition, Merck﹠amp; Co.Inc., Whitehouse Station, New Jersey, the U.S.; ISBN 0911910-12-3) describes in.

Other preferred nonionic surfactants is a Sorbitan ethoxylate, and these esters comprise polyethenoxy sorbitan monooleate (TWEEN80^TM), the clauses and subclauses 7742 of " Merck index " (1308 pages, the 12nd edition, Merck﹠amp; Co.Inc., Whitehouse Station, New Jersey, the U.S.; ISBN 0911910-12-3) in description is arranged.Hot benzene polysaccharide and Sorbitan ethoxylate all can be bought from Sigma Inc..Preferred Sorbitan ethoxylate is polyethenoxy sorbitan monooleate (TWEEN80^TM).

The most preferred adjuvant of the present invention comprises polyoxyethylene alkyl ether and hot benzene polysaccharide, as uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM).Optional described mixture can further comprise Sorbitan ethoxylate, as polyethenoxy sorbitan monooleate (TWEEN80^TM).Most preferably, described polyoxyethylene alkyl ether is that polyoxyethylene-9-lauryl ether and described hot benzene polysaccharide are uncle's octylphenoxy polyethoxy ethanol (TRITONX100^TM).In these preparatons, can add the ion-type cleaning agent, as the derivant of bile salts or cholic acid.

Thereby adjuvant formulations can comprise polyoxyethylene alkyl ether or polyxyethylated ester (general formula I), and hot benzene polysaccharide randomly comprises Sorbitan ethoxylate and randomly comprises bile salts or chlolic acid derivatives.The preferred version of this preparaton comprises polyoxyethylene-9 lauryl ether, uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM), polyethenoxy sorbitan monooleate and NaTDC.

Polyoxyethylene alkyl ether or ester, as polyoxyethylene-9 lauryl ether, the typical concentration in adjuvant of the present invention will be in the scope of 0.001-20%, preferred 0.001-10% and more preferably 0.001-1% and most preferably 0.001-0.8% or about 0.5% (w/v).The other non-ionic surface active agent that adds wherein is not polyoxyethylene ether or polyoxyethylene ester.Each other non-ionic surface active agent typically will the concentration with 0.001-20% exist in final vaccine mixture, and more preferably 0.01-10% is most preferably up to about 2% (w/v).If there are two kinds of described other non-ionic surface active agents, preferably they exist up to about 2% concentration with every kind in final preparaton, typically exist up to about 0.6% concentration with every kind.If have 3 kinds or how other non-ionic surface active agent, they exist up to about 1% concentration with every kind usually, and typically every kind of trace is to up to about 0.2% or 0.1%.Any mixture of surfactant can be present in according in the vaccine mixture of the present invention.Non-ionic surface active agent, those surfactants as discussed above preferred concentration in final vaccine combination is as follows: hot-or the ninth of the ten Heavenly Stems-phenoxy group polyoxy ethanol (octyl-or nonylphenoxy polyoxyethanols), as Triton X-100^TMOr other cleaning agent in the Triton series: from 0.001%-20%, preferred 0.001%-10%, more preferably 0.001-1%, most preferably 0.005-0.1% (w/v); With if there is Sorbitan ethoxylate, as Tween 80^TM: 0.01-1%, more preferably from about 0.1% (w/v).

Total concentration of detergent in vaccine of the present invention or the adjuvant formulations, comprise polyoxyethylene ether or polyoxyethylene ester and one or more other non-ionic surface active agents, typical scope is 0.001-40%, preferably between 0.001 to 20%, more preferably between 0.001 to 10%, still more preferably between 0.001 to 1%, most preferably between 0.001 to 0.7% (w/v).

Give bacterin preparation of the present invention by mucosal route,, can be used for protecting or treat easily ill or ill mammal as oral cavity/cheek/intestinal/vagina/rectum or nasal mucosa approach.This administering mode can be droplet, spraying or dry powder form.The vaccine mixture of atomizing or aerosolization also constitutes a part of the present invention.The suppository of the intestinal preparation of oral administration such as anti-capsule for treating gastropathy (gastroresistant capsule) and granule, rectum or vagina administration also constitutes a part of the present invention.The present invention also can be used for strengthening the immunogenicity of antigens that is applied to skin (percutaneous or subcutaneous transmission).In addition, adjuvant of the present invention can give through parenteral route, for example intramuscular or subcutaneous administration.When being used for intranasal vaccination, the character of preferred vaccine of the present invention is hemolytic.

According to route of administration, can use multiple doser.For example, the sprayer unit that intranasal administration uses is as the Accuspray that can use the merchant to sell^TM(Becton Dickinson).

The sprayer unit of preferred intranasal administration is the device that its performance does not rely on the user applied pressure.Known these devices are devices that pressure limit is arranged.Have only when reaching pressure limit, liquid just flows out from nozzle.These devices are easier to form the regular spraying of droplet liquid measure.The device that pressure limit is arranged that the present invention is suitable for is that prior art is known, for example states in WO91/13281 and EP311 863B.This device can buy from Pfeiffer GmbH.

Droplet (water is cooked liquid and the measured) scope that preferred intranasal device produces is at 1 to 200 μ m, preferred 10 to 120 μ m.Less than 10 μ m the danger that is inhaled into is arranged, therefore the droplet less than 10 μ m preferably is no more than about 5%.The above droplet of 120 μ m can not spread well as less droplet, and the droplet that therefore surpasses 120 μ m preferably is no more than about 5%.

Dose double transmission (bi-dose delivery) is the further preferred feature that is used for the intranasal transmitting device of vaccine of the present invention.The dose double device contains two sub-doses (subdose) of a vaccine dose, and a sub-doses gives a nostril.

The present invention further provides a test kit (kit), comprised the intranasal administration device that contains vaccine mixture of the present invention described herein.

For some vaccine mixture, can comprise other vaccine composition.Same adjuvant formulations of the present invention also can comprise the derivant of bile acid and cholic acid.Preferred chlolic acid derivatives is its salt, is more preferably its sodium salt.The example of bile acid and derivant thereof comprises cholic acid itself; the glycerol of deoxycholic acid, tauroursodeoxycholic acid (salt), chenodeoxy cholic acid, lithocholic acid, ursodesoxycholic acid, Hyodeoxycholic Acid and aforementioned bile acid-, cattle sulphur-, aminopropyl-1-third sulphur-, aminopropyl-2-hydroxyl-1-third sulphur-derivant; or N, two (3D gluconic acid aminopropyl) the deoxidation cholamine of N-.Particularly preferred example is the NaTDC (NaDOC) that can be present in the final bacterin preparation.

Preferably, when adjuvant formulations of the present invention is the form of suspension of aqueous solution form or still (non-vesicular), has superiority.This preparaton is easy to repeat make, also be easy to sterilization (by have 450 or the film end-filtration in 220nm aperture) and be easy to Sprayable through nasal mucosa medicine administration, the complicated physical arrangement of adjuvant is not degraded simultaneously.Polyoxyethylene-9 lauryl ether and TRITON-X 100^TMCoupling is made into aqueous solution (also can have little micelle).

A scheme of the present invention provides a kind of bringing out (inducing) or has strengthened the method for host immune response, comprises antigen is mixed with adjuvant of the present invention, and use described mixture to the host.Preferably, described host's route of administration is by mucomembranous surface, more preferably passes through nasal mucosa.When giving mixture by nasal mucosa, preferred mixture gives with Sprayable.Preferably bring out in the immunoreactive method at one, give vaccine of the present invention by intranasal and induce systemic immune response.The method of preferred enhance immunity reaction can be used the initial dose or the booster dose of vaccine, and preferably this vaccine comprises influenza antigens or antigen preparation (antigenic preparation).In these methods, the preferred adjuvant preparaton of nasal mucosa medicine administration is that polyoxyethylene alkyl ether and hot benzene polysaccharide are united use, for example preferred polyoxyethylene-9 lauryl ether and uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM) associating, (monooleate for example, TWEEN 80 additionally to comprise Sorbitan ethoxylate in the optional described adjuvant associating agent^TM) and/or bile salts or chlolic acid derivatives, as NaTDC.

Predict compositions of the present invention and can be used for preparing the antigenic vaccine that contains multiple extensive source.For example, antigen can comprise people, antibacterial or viral nucleic acid, the deutero-antigen of pathogen or antigen preparation, the deutero-antigen of tumor or antigen preparation, comprises albumen or peptide and chimeric fusion protein that GnRH and IgE peptide, reorganization produce.

Preferred vaccine mixture of the present invention contains a kind of immunoreactive antigen or antigen composition that can cause anti-human pathogen (humanpathogen), this antigen or antigen composition are derived from HIV-1, (as tat, nef, gp120 or gp160), the herpes virus hominis, as the gD or derivatives thereof or immediately early protein as ICP27 from HSV1 or HSV2, cytomegalovirus ((especially human cytomegalic inclusion disease virus) (as gB or derivatives thereof), rotavirus (comprising active attenuated virus), Epstein-Barr virus (as the gp350 or derivatives thereof), varicella zoster virus (Varicella Zoster Virus) is (as gpI, II and IE63), or from hepatitis virus such as hepatitis B virus (for example hbs antigen or derivatives thereof), hepatitis A virus, hepatitis C virus and hepatitis E virus, or from other viral pathogen, as paramyxovirus: respiratory syncytial virus (as F and G albumen or derivatives thereof), parainfluenza virus, Measles virus, mumps virus, human papillomavirus (HPV6 for example, 11,16,18, ..), Flavivirus is (as yellow fever virus, dengue virus, tick borne encephalitis virus, Japanese encephalitis virus) or be grown in influenza virus in ovum or the mdck cell (active complete (whole) or inactivation of viruses, division influenza virus (split influenza virus), or Vero cell or complete (whole) influenza virus particles (whole flu virosomes) are (as R.Gluck, " vaccine " (Vaccine), 1992,10, describe among the 915-920) or purification or its recombiant protein, as HA, NP, NA, or M albumen, or their mixture (combination)), or, comprise Diplococcus gonorrhoeae and Neisseria meningitidis (for example capsule polysaccharide and conjugate thereof (conjugate) from bacterial pathogens such as neisseria, transferrin binding protein, lactoferrin binding protein, PilC, adhesin); Streptococcus pyogenes (for example M albumen or its fragment, C5A protease, lipoteichoic acid), streptococcus agalactiae, Streptococcus mutans; Haemophilus ducreyi; Moraxella comprises moraxella catarrhalis, also comprises known mucositis branhamella (for example high and low-molecular-weight adhesin and hyaluronidase); Bordetella comprises Bordetella pertussis (for example pertactin, pertussis toxin, PT or derivatives thereof, filamentous hemagglutinin, adenyl cyclase, pili), Bordetella parapertussis and bordetella bronchiseptica; Mycobacterium comprises mycobacterium tuberculosis (for example ESAT6, Antigen 85A ,-B or-C antigen), Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, mycobacterium paratuberculosis, mycobacterium smegmatis; Legionnella comprises legionella pneumophilia; Escherichia comprises and produces enterotoxigenic Escherichia coli (for example colonizing factor, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorrhagic Escherichia coli, enteropathogenic E.Coli (for example shiga toxin sample toxin or derivatives thereof); Vibrio comprises vibrio cholera (for example cholera toxin or derivatives thereof); Shigella comprises bacillus ceylonensis A, shigella dysenteriae, shigella flexneri; Yersinia comprises yersinia enterocolitica (for example Yop albumen), Yersinia pestis, artificial tuberculosis yersinia genus; Campylobacter comprises campylobacter jejuni (for example toxin, adhesin and hyaluronidase) and large intestine Campylobacter; Salmonella comprises salmonella typhi, salmonella paratyphi, Salmonella choleraesuls, Salmonella enteritidis; Growth comprises the monocyte hyperplasia Listeria; Helicobacterium comprises helicobacter pylori (for example urease, catalase, VacA); Rhodopseudomonas comprises Pseudomonas aeruginosa; Staphylococcus comprises staphylococcus aureus, staphylococcus epidermidis; Enterococcus comprises enterococcus faecalis, enterococcus faecalis; Fusobacterium comprises clostridium tetani (for example tetanus toxin and derivant thereof), bacillus botulinus (for example creotoxin and derivant thereof), clostridium difficile (for example clostridial toxin A or B and derivant thereof); Bacillus comprises Bacillus anthracis (for example Botulinum toxin (botulinum toxin) and its derivant); Corynebacterium comprises diphtheria corynebacterium (for example diphtheria toxin, diphtherotoxin and its derivant); Borrelia comprises B. burgdorferi (OspA for example, OspC, DbpA, DbpB), loud, high-pitched sound borrelia burgdorferi (OspA for example, OspC, DbpA, DbpB), Ah's borrelia burgdorferi (OspA for example, OspC, DbpA, DbpB), B.andersonii (OspA for example, OspC, DbpA, DbpB), He Shi Ticks burgdorferi; The ehrlichiosis body belongs to, and comprises the pathogen of horse ehrlichiosis body and people's granular cell ehrlichiosis disease; Rickettsiae comprises rickettsia rickettsii; Chlamydiaceae comprises sand holes chlamydia (for example MOMP, heparin is conjugated protein), Chlamydia pneumoniae (for example MOMP, hepatic binding protein (HBP)), chlamydia psittaci; Leptospira comprises leptospira interrogans; Treponema comprises Treponoma palladium (for example rare outer membrane protein, treponema denticola, swine dysentery treponema; Or from parasite, for example Plasmodium comprises Plasmodium falciparum; Toxoplasma, comprise the Mus toxoplasma (SAG2 for example, SAG3, Tg34); Entamoeba comprises Entamoeba histolytica; Babesia comprises the vole babesia; Trypanosoma comprises schizotrypanum cruzi; Giardia comprises Giardia lamblia; Leishmania comprises large-scale Leishmania; Pneumocystis comprises Pneumocystis carinii; Trichomonas comprises trichomonal vaginitis; Schistosoma comprises Schistosoma mansoni; Or from yeast, for example Candida comprises Candida albicans; Cryptococcus comprises Cryptococcus histolyticus (C.neoformans).

The preferred bacterium vaccine comprises from Streptococcus, comprises streptococcus pneumoniae (for example capsule polysaccharide and its conjugate, PsaA, PspA, streptolysin, choline binding protein) and pneumolysin proteantigen (Biochem Biophys Acta, 1989,67,1007; Rubins etc., " microorganism pathology " (Microbial Pathogenesis), 25,337-342) and its sudden change detoxifcation derivant (WO 90/06951; WO 99/03884).Other preferred bacterium vaccine comprises from Haemophilus spp, comprise Type B type sepecies Haemophilus influenzae (for example PRP and couplings thereof), non-molding (nontypeable) Haemophilus influenzae, for example (US 5 for the antigen of OMP26, high molecular adhesin (adhesins), P5, P6, D albumen and lipoprotein D and actin and actin derived peptide, 843,464) or its multicopy variant or fusion rotein.Other preferred bacterium vaccine comprises from moraxella catarrhalis (comprise its outer membrane vesicles, and OMP106 (WO 97/41731)) with from the antigen of Type B Neisseria meningitidis (comprise its outer membrane vesicles, and NspA (WO96/29412)).

The hbs antigen derivant is to know in the prior art, and comprise other at European patent application EP-A-414 374; Those PreSl that propose among EP-A-0304 578 and the EP 198-474, PreS2 S antigen.In a preferred version, vaccine mixture of the present invention comprises HIV-1 antigen, gp120, especially when they are expressed in Chinese hamster ovary celI.In further scheme, vaccine mixture of the present invention comprises the gD2t of definition as mentioned.

In a preferred version of the present invention, vaccine contains the adjuvant of claim, comprise antigen from the human papillomavirus (HPV) of the reason that is considered to cause the genitals tumor, (HPV6 or HPV 11 and other), with HPV virus be the reason that forms cervical cancer (HPV16, HPV 18 and other).

Particularly preferred genitals tumor prevention or treatment vaccine form comprise L1 microgranule or capsomere, and the preferred fusion protein form, and it comprises that one or more are selected from the antigen of HPV6 and HPV11 albumen E6, E7, L1 and L2.

Most preferred fusion rotein form is: disclosed D (1/3)-E7 albumen among disclosed L2E7 and the GB9717953.5 (PCT/EP98/05285) among the WO96/26277.

Preferably be used to prevent or treat the vaccine of HPV cervical infection or HPV cervical cancer, its component can comprise HPV16 or 18 antigens.For example, L1 or L2 antigen monomer or L1 or L2 antigen occur simultaneously as a kind of viral sample microgranule (VLP), or L1 albumen occurs separately in VLP or capsomere structure separately.This antigen, viral sample microgranule and capsomere itself are known.For example referring to WO94/00152, WO94/20137, WO94/05792 and WO93/02184.

Other early protein, for example preferred E7, E2 or E5 can be comprised separately or as fusion rotein; Special preferred version comprises the VLP (WO96/11272) that includes the L1E7 fusion rotein.

Particularly preferred HPV 16 antigens comprise the early protein E6 that merges with the D protein carrier or E7 forming protein D-E6 or the E7 fusion rotein from HPV16, or their mixture (combination); Or the mixture of E6 or E7 and L2 (WO 96/26277).

Perhaps, HPV 16 or 18 early protein E6 and E7 can be present in the individual molecule, and optimization protein D-E6/E7 merges.This vaccine can randomly comprise from the E6 of HPV 18 or E7 albumen the two one or both of the form of optimization protein D-E6 or protein D-E7 fusion rotein or protein D E6/E7 fusion rotein is all arranged.

Vaccine of the present invention also can comprise the antigen from other HPV strain, preferably from HPV6, and 11,31,33 or the antigen of 45 strains.

Vaccine of the present invention further comprises from the parasite antigen that can cause malaria.For example, preferred antigens comprises RTS from Plasmodium falciparum, S and TRAP.RTS is a kind of hybridization albumen, comprises proteic all C-end portion basically of ring-type zygoblast (CS) of Plasmodium falciparum, and it is connected with surface (S) antigen of hepatitis B virus by four aminoacid of the PreS2 part of hbs antigen.Its entire infrastructure is disclosed in International Patent Application PCT/EP92/02591, publication number WO93/10152, the claim priority is UK Patent Application No.9124390.7.When it is expressed in yeast, produce hdl particle RTS, when the S antigen coordinate expression of it and HBV, produce known hybrid fine particles RTS, S.TRAP antigen has description in international patent application No.PCT/GB89/00895, its publication number is WO90/01496.A preferred version of the present invention is a malaria vaccine, and wherein antigen preparation comprises RTS, the antigenic mixture of S and TRAP.May be Plasmodium falciparum MSP1 as other plasmodium antigens of each stage malaria vaccine candidate composition, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfs16, Pfs48/45, the analog of Pfs230 and Plasmodium thereof.

This preparaton also can contain a kind of tumor-resistant antigen, and can be used for immunization therapy ruling by law treatment cancer.For example, the discovery adjuvant formulations can be share with tumor rejection antigen, as the rejection antigen of prostate, chest, colorectum, lung, pancreas, kidney or melanoma cancer.Example antigen comprises and is used for the treatment of melanomatous MAGE 1 and MAGE 3 or other MAGE antigen, PRAME, BAGE or GAGE (Robbins and Kawakami, 1996, " the up-to-date opinion in the immunology " (Current Opinions in Immunology) 8, the 628-636 page or leaf; People such as Van denEynde, the international magazine of laboratory research " clinical and " (International Journalof Clinical﹠amp; Laboratory Research) (is published in 1997); People such as Correale (1997), " national cancer association magazine " (Journal of the National CancerInstitute) 89,293 pages.In fact, in the tumor type of wide scope, can both express these antigens, for example melanoma, pulmonary carcinoma, sarcoma and bladder cancer.Other tumor specific antigen is suitable for together using with adjuvant of the present invention, includes but not limited to prostate specific antigen (PSA) or Her-2/neu, KSA (GA733), MUC-1 and carcinoembryonic antigen (CEA).According to a scheme of the present invention, a kind of vaccine is provided, comprise a kind of according to adjunvant composition of the present invention and a kind of tumor rejection antigen.

Described antigen in addition can also be the self peptide hormone, and as total length gonadotropin releasing hormone (GnRH, WO 95/20600), it is that length is 10 amino acid whose small peptides, can be used for treating many cancers, or is applied to the immunity excision.

Can precognition compositions of the present invention can be used for preparing the antigenic vaccine that contains from Borrelia.For example, antigen can comprise albumen or peptide and the chimeric fusion protein that nucleic acid, the deutero-antigen of pathogen or antigen preparation, reorganization produce.This antigen is OspA especially.OspA can be the fat form (Lipo-OspA) or the non-fat derivant of the complete maturation protein (full matureprotein) of host cell (escherichia coli (E.Coli)) definition.This non-fat derivant comprises non-fat NS1-OspA fusion rotein, and it has preceding 81 the N terminal amino acids and the complete OspA albumen of the non-structural protein (NS1) of influenza virus; And another kind of, MDP-OspA is the non-fat form of OspA, has 3 other N terminal amino acids.

Vaccine of the present invention can be used for prevention hypersensitive or treatment.This vaccine can comprise allergen specificity antigen (for example Der p1) and anaphylactogen heterogenetic antigen (for example from the peptide of people IgE, including but not limited to stanworth decapeptide (EP 0 477 231 B1)).

The most preferred vaccine of the present invention and bring out immunoreactive method comprises influenza antigen.The inactivating influenza virus preparation can be obtained by conventional embryonated egg method, maybe can obtain for method (new generation method) by any new biography of viral growth that makes with tissue culture.Be used to make the suitable cell substrate of viral growth to comprise, such as Madin-Darby canine kidney(cell line) (MDCK), as MDCK or from the clone's of MDCK cell, MDCK like cell, monkey-kidney cells, as the AGMK cell, comprise any cell type that is suitable for producing the influenza virus that is used to prepare the vaccine purpose of African green monkey kidney cell or other.Suitable cell substrate comprises people's cell, as the MRC-5 cell.Suitable cell substrate is not limited to cell line; For example primary cell also can be included such as chick embryo fibroblast.Any obtaining in the processing method that the influenza antigen preparation can be sold by a large amount of merchants is as the division influenza antigens processing method of describing among the patent DD 300 833.Can buy the Fluarix that the division influenza that obtains comprises that SmithKline Beecham sells^TM, same Fluarix and adjuvant of the present invention are united use and are constituted the preferred vaccine of the present invention.

The preferred a kind of multivalence influenza vaccines of influenza vaccines according to the present invention comprise two or more influenza strains.More preferably it is a kind of trivalent vaccine that comprises 3 strains.The conventional flow influenza vaccine generally includes three kinds of strains of influenza, two A strains and a B strain.Yet, the present invention do not repel for example may be useful to popularity univalent vaccine.A kind of unit price pandemic influenza vaccine will contain the influenza antigens from single A strain probably.

Therefore, a kind of preferred vaccine mixture comprises ovum or tissue culture's influenza antigens, preferably divides influenza antigens, and a kind of polyoxyethylene alkyl ether and at least a other non-ionic surface active agent can optionally comprise the derivant of bile salts or cholic acid.The preferred version of this vaccine comprises division influenza antigen, polyoxyethylene-9 lauryl ether and TRITON-X 100^TMRandomly, this most preferred vaccine can further comprise a kind of Sorbitan ethoxylate, as TWEEN80^TMAnd/or NaTDC.

Protein content in every kind of vaccine dose is chosen to be can the induce protective immunity reaction and do not produce the dosage of tangible adverse side effect in typical vaccine.How this dosage can and produce (presented) and change along with the specific immunogens that uses.In general, expect that each dosage comprises 1-1000 μ g albumen, preferred 1-500 μ g, preferred 1-100 μ g, most preferably 1-50 μ g.The optimal dose of a specific vaccine can be determined by research on standard, comprises and observes the immunoreation that the experimenter matches.After the first vaccination, the experimenter can accept one and several abundant isolated booster immunization inoculation.

A preferred version of the present invention is can work in coordination with the adjuvant effect that strengthens polyoxyethylene alkyl ether by other non-ionic surface active agent.In this, the immunoreation degree that produces from the blended adjuvant formulations of coupling is higher than the immunoreation sum that is produced separately when each composition uses separately, promptly can be observed synergism.Another situation, when the polyoxyethylene ether and the other non-ionic surface active agent of low dosage produces tangible immunoreation, and even when being used alone or each composition all can not produce obviously or can detected immunoreation the time, also can be observed synergism.

A scheme of the present invention is adjuvant and vaccine mixture, and it comprises polyoxyethylene alkyl ether or polyxyethylated ester and at least a other non-ionic surface active agent, and wherein the antigen that exists in the vaccine does not wrap and is loaded within the non-ionic surface active agent vesicle.

But vaccine of the present invention also by oral route gives.In this case, pharmaceutically acceptable excipient also can comprise alkaline buffer or enteric bag quilt or microgranule.Vaccine of the present invention also can give by vaginal approach.In this case, pharmaceutically acceptable excipient also can comprise emulsifying agent, polymer such as the CARBOPOL of vaginal cream and suppository^_And other known stabilizing agent.Vaccine of the present invention also can give by the rectum approach.In this case, pharmaceutically acceptable excipient also can comprise wax and the polymer that is used to form rectal suppository well known in the prior art.

Preparaton of the present invention can be used for prevention and therapeutic purposes.Therefore, the invention provides a kind of method for the treatment of mammalian infections disease or cancer or irritated or autoimmune disease, or treat the method for the susceptibility of these diseases.Further scheme of the present invention provides a kind of adjuvant mixture (adjuvant combination) and the vaccine that is used for medicine described herein.The preparation of vaccine is usually at " new trend of vaccine and progress " (New Trends and Developments inVaccines), volumes such as Voller, and University Park publishes, Baltimore, the Maryland State, the U.S. states in 1978.

A scheme of the present invention relates to non-ionic surface active agent for example polyoxyethylene alkyl ether or the polyxyethylated ester and the application of hot benzene polysaccharide in the preparation adjuvant formulations of general formula (I).The present invention also relates to the polyoxyethylene alkyl ether or the application in the preparation vaccine mixture of polyxyethylated ester, hot benzene polysaccharide and antigen of general formula (I).The described adjuvant and the vaccine of method preparation can optionally further comprise Sorbitan ethoxylate as described.In all these schemes of the present invention, preferred polyoxyethylene alkyl ether is polyoxyethylene-9 lauryl ether, and preferred hot benzene polysaccharide is uncle's octylphenoxy polyethoxy ethanol (Triton X-100^TM).

In selectable relevant programme of the present invention, adjuvant of the present invention can further be united use with other adjuvant, comprises choiera toxin and B subunit thereof, the heat-labile enterotoxin LT of escherichia coli and B subunit LTB and their detoxifcation type such as mLT; Monophosphoryl lipid A (Monophosphoryl Lipid A) and its non-toxic derivative 3-O-deacylated tRNA monophosphoryl lipid A (3D-MPL, at British patent GB 2,220, description is arranged in 211), immunologic competence saponin fragment, as from the Quil A of the bark of South America Quillaja Saponaria Molina tree and derivant thereof (QS21 for example, US patent 5,057,540), and oligonucleotide (oligonucleotide) adjuvant system CpG (as describing among the WO 96/02555), especially 5 ' TCG TCG TTT TGT CGTTTT GTC GTT3 ' (SEQ ID NO.1).

In the technical program, preferred especially polyoxyethylene alkyl ether (as polyoxyethylene-9 lauryl ether), other non-ionic surface active agent are (as uncle's octylphenoxy polyethoxy ethanol (Triton X-100^TM) and the adjuvant mixture of 3-O-deacylated tRNA monophosphoryl lipid A (3D-MPL).This preferred version can further comprise Sorbitan ethoxylate such as TWEEN80 optionally^TM, and/or bile salts or chlolic acid derivatives such as NaTDC.Preferably include this adjuvant formulations and influenza antigens especially, especially divide the vaccine of influenza antigens.

The following example is used for explaining but does not limit the present invention.

Embodiment

Embodiment 1, be used for detecting the method that serum (sera) antibody (Ab) reactsELISA (enzyme disimmunity determining adsorption) measures monkey influenza-specific serum Ig Abs

Dilute β-propanoic acid lactone (BPL) inactivating influenza virus (by SSD GmBH producer, Dresden, Germany provides) 1 μ g/ml HA with PBS, wrap by MaxisorpNunc immunity dull and stereotyped (immunoplate) in 50 μ l/ holes (μ l/well), spends the night under 4 ℃.Room in the flat board select use saturated buffer: PBS to contain 1%BSA, 0.1% polyoxyethylene sorbitan monolaurate (TWEEN 20) is filled out envelope (1 hour, 37 ℃).Then, successive 2 times of diluents of the reference serum that adds as standard curve are (in saturated buffer, 50 μ l/ holes) (serum with mid point titer (titer) is represented with ELISA unit/ml, it is capable to be put into A) and serum sample (since 1/100 dilution factor and to be put into B capable to H), cultivated 1 hour 30 minutes for 37 ℃.Then with lavation buffer solution (PBS, 0.1% polyoxyethylene sorbitan monolaurate (TWEEN 20)) washing (* 3) flat board.Then, at saturated buffer biotinylated goat Anti-Human Ig (goat anti-human Ig) (Amersham) is diluted to 1/3000,37 ℃ of cultivation (50 μ l/ hole) 1 hour 30 minutes.Wash 3 times, add streptavidin-horseradish peroxidase conjugate (Amersham) subsequently, dull and stereotyped 5 times and with indication buffer (revelation buffer) (the OPDA 0.4mg/ml (Sigma) and the H in 50 μ l/ holes of washing₂O₂0.03% in 50mM pH 4.5 citrate buffers) at room temperature cultivated 20 minutes.Add 50 μ l/ hole H₂SO₄2N stops indication.With Biorad 3550 immune readers (immunoreader) 492 and 630nm obtain the optical density (OD) reading.4 parameter mathematical method calculating antibody titer with SoftMaxPro software.

The hemagglutination of monkey influenza-specific serum Abs suppresses (HAI) activity

In order to eliminate the hemagglutinative non-specific inhibitory action in the primate serum, these (25 μ l) 37 ℃ of overnight incubation, the every ml VCN of described mixed solution (BoerhingerMannheim) contains 400 receptor destroying enzyme units with 100 μ l chlorination (chlorure) calcium/Calcium pyroborate/sodium borate mixed solutions.After adding 2.5% sodium citrate solution, 75 μ l, heated serum 30 minutes at 56 ℃.Add 50 μ l PBS solution serum is diluted to 1/10 at last^ThThen, the serum of on 96 hole Greiner flat boards, handling with 25 μ l PBS (since 1/10, continuous 2 times of dilutions) dilution, 25 μ l.Added BPL deactivation intact virus (25 μ l/ hole) with 30 minutes with 4 hemagglutinating-unit concentration (promptly than the last low 4 times dilution factor that excites red cell agglutination) in room temperature (RT) with under stirring.Follow under RT with 1 hour adding chicken red blood cell (25 μ l/ hole).The last dull and stereotyped reading again that spends the night of under 4 ℃, preserving.HAI titer is equivalent to suppress the inverse of the hemagglutinative final serum dilution of virus induction.

ELISA measures mice tetanus toxoid (TT) specific serum IgG

The 50 μ l/ holes that are used in the 1 μ g/ml antigen (TT that Behring provides) of (B to H at flat board is capable) dilution among the PBS are wrapped by Maxisorp Nunc immunity dull and stereotyped, or the goat that is used in 5 μ g/ml purification among the PBS (A is capable) anti--the 50 μ l bag of mice (goat anti-mouse) Ig (Boerhinger) spent the night under 4 ℃ by dull and stereotyped.Room in the flat board is selected and is used saturated buffer: PBS (containing 1%BSA, 0.1% polyoxyethylene sorbitan monolaurate (TWEEN 20) and 4% standard calf serum (NBS)) to fill out envelope (1 hour, 37 ℃).Then, adding as continuous 2 times of diluents of IgG homotype (isotype) mixture of a standard curve (in saturated buffer, 50 μ l/ holes) (use mouse monoclonal antibody IgG1, the IgG2a of Sigma and the mixture of IgG2b, begin and to be put into A capable from 200ng/ml) and serum sample (since 1/100 dilution factor and to be put into B capable to H), cultivated 1 hour 30 minutes at 37 ℃.Then with lavation buffer solution (PBS, 0.1% polyoxyethylene sorbitan monolaurate (TWEEN 20)) washing (* 3) flat board.Then, the biotinylated goat that is diluted in saturated buffer in 1/5000 resisted-mice IgG (Amersham), 37 ℃ of cultivations (50 μ l/ hole) 1 hour 30 minutes.Wash 3 times, add streptavidin-horseradish peroxidase conjugate (Amersham) subsequently, dull and stereotyped 5 times and with indication buffer (OPDA 0.4 mg/ml (Sigma) and the H in 50 μ l/ holes of washing₂O₂0.03% in 50mM pH 4.5 citrate buffers) at room temperature cultivated 20 minutes.Add 50 μ l/ hole H₂SO₄2N stops indication.With Biorad 3550 immune readers 492 and the 630nm place obtain optical density readings.4 parameter mathematical method calculating antibody titer with SoftMaxPro software.

Embodiment 2, and common immunogenic influence to primed Rhesus Macacus intranasal influenza vaccine is used in uniting of laureth9 and TWEEN80 and TritonX100

Be contained in A/ Beijing/262/95 and B/ Harbin/7/94 influenza virus, the 25 μ gHA of the every strain β-propanoic acid lactone deactivation among the 100 μ l PBS for each nostril perfusion of Rhesus Macacus with sprayer unit (anesthesia down).After 28 days, give monkey (4 or 5/group) intranasal (anesthesia down) booster dose with 200 μ l solution (each nostril 100 μ l carries with sprayer unit), solution is at A: in polyoxyethylene-9-lauryl ether 0.5% (L9); Or at B: contain 30 μ g HA/BPL-deactivation A/ Beijing/262/95 and B/ Harbin/7/94 strains of influenza viruses among polyoxyethylene-9-lauryl ether 0.5%+TWEEN80 (0.11%)+triton-X-100 (0.074%); Or pass through C: intramuscular injection 15 μ gHA/ influenza vaccines strains, vaccine strain contains the strain identical with A and B.Virus antigen is grown in the ovum of the seed bank that is provided by manufacturer (SSD GmBH, Dresden, Germany).As HAI and IgAb reaction in the mensuration serum as described in the embodiment 1.The result improves the shared percentage of animal of 4 times of Ab after with booster immunization and recently represents.

Experience with 0.5% polyoxyethylene-9-lauryl ether showed that this preparaton was to inducing the influenza systemic immune response effective in the past.Yet, as shown in table 1, can significantly improve adjuvant complementary (adjuvanticity) level by adding other non-ionic surface active agent.When replenishing polyoxyethylene-9-lauryl ether with TWEEN80 and triton-X-100, this preparaton can strengthen the general IgAb that has set up in advance effectively as classical parenteral route influenza vaccines and react like this.

Also measure hemagglutination and suppress (HAI) reaction (table 2), again, best intranasal preparaton remains the polyoxyethylene-9-lauryl ether that has replenished TWEEN80 and triton-X-100.This preparaton is identical with the immunogenicity of traditional parenteral route vaccine.

Table 1, monkey serum Ig reaction

IgELISA antibody	Ab raises 4 times of seroconversion (%) extremely:
			Group	A/ Beijing/262/95	B/ Harbin/7/94
????A	????0	????0
			????B	????100	????100
????C	????75	????75

Table 2, monkey serum HAI titer

HAI antibody	Ab raises 4 times of seroconversion (%) extremely:
			Group	A/ Beijing/262/95	B/ Harbin/7/94
????A	????0	????0
			????B	????20	????0
????C	????25	????0

Embodiment 3: relatively add the intranasal that TWEEN80 and triton-X-100 be mixed with laureth9 and divide influenza vaccines and the traditional parenteral route vaccine (Fluarix that has gone through to use^TM) to healthy adult experimenter's immunogenicity.

Assess the intranasal preparaton that adds the ovum source division influenza antigens of TWEEN80 and triton-X-100 (A) preparation with laureth 9, and and Fluarix^TM/ α-Rix^_(B) compare.The preparaton that contains three kinds of deactivation division virus antigens is recommended the strain preparation by 1998/1999 season WHO.The device that is used to grant vaccine is the Accuspray from Becton Dickinson^TMThe nasal injection device, this device is similar with the conventional syringe operation principle, but has a special needle point that contains spirality channel, and when exerting pressure to piston, it can produce spraying.Each nostril is sprayed into 100 μ l preparatons.

The composition of preparaton

Intranasal preparaton (A) contains following deactivation division virion:

1.30 μ g HA A/ Beijing/262/95 (HlNl)

2.30 μ g HA A/ Sydney/5/97 (H3N2)

3.30 μ g HA B/ Harbin/7/94 and phosphate buffered saline pH7.4 ± 0.1,0.1%TWEEN80,0.015%Triton-X-100,0.0045% NaTDC and be lower than the thiomersalate of 35 μ g/ml.

The volume of a preparation is 200 μ l (each nostril 100 μ l sub-doses).

Assist in the formulation H into adjuvant laureth 9 so that final concentration reaches 0.5% (w/v).

Comparison Fluarix^TM/ α-Rix^_(B) be that the merchant of SmithKlineBeecham Biologicals sells inactivated trivalent division influenza vaccines, intramuscular administration, dosage is 500 μ l.

Immunogenicity research

Estimate the immunogenicities of intranasal division influenza vaccines with opening, contrast and randomised study, this vaccine is formulated by the laureth 9 that has replenished TWEEN80 and triton-X-100, and with traditional parenteral route vaccine (be Fluarix^TM) compare.20 healthy adult experimenters (age 18-40 year) accept the Fluarix of a dosage^TM, 10 experimenters accept the intranasal influenza vaccine an of dosage (two sub-doses, the sub-doses in each nostril).

About safety and reactionogenicity, two kinds of vaccines can both be stood by fine in the hope of producing part and General Symptoms in continuous 8 days.There is not the report serious side effects relevant with vaccination.

The immunogenicity determining of vaccine is: suppress (HI) titer by assessment serum hemagglutination and determine that seroconversion rate is (for each vaccine strain; its definition is to compare with the 0th day; serum HI titer was increased to 4 times the shared percentage ratio of vaccine at least in the 21st day); transform factor (for each vaccine strain; its definition is to compare with the 0th day; the 21st day serum HI geometric mean titer (GMT) is increased to multiple) and the serum protective rate (its definition is; the shared percentage ratio of vaccine of (for each vaccine strain) serum HI titer 〉=40 after the immunity inoculation is accepted its indication as protection).In addition, measure mucosa IgA antibody response with enzyme-linked immunosorbent assay (ELISA).

The Fluarix of a dosage of inoculation^TMOr behind the intranasal preparaton 21 days, HI seropositivity, seroconversion and serum protective rate can see Table 3.

Table 3: inoculate 21 days HI seropositivities, seroconversion and serum protective rates behind the dosage:

Strain	Group	Time	??N	Seropositivity n %	Serum protection n %	Seroconversion n %
							A/ Beijing	The intranasal vaccine is added with Laureth 9	It is 0 day 21	??20 ??20	??5????25.0 ?19????95.0	??1????5.0 ?10????50.0	?15????75.0
??FluarixTM	It is 0 day 21	??19 ??19	??4????21.1 ?19????100.0	??3????15.8 ??18???94.7	?19????100.0
						A/ Sydney		The intranasal vaccine is added with Laureth-9	It is 0 day 21	??20 ??20	?16????80.0 ?20????100.0	??4????20.0 ?19????95.0	?15????75.0
??FluarixTM	It is 0 day 21	??19 ??19	?14????73.7 ?19????100.0	??1????5.3 ?18????94.7	?16????84.2
							B/ Harbin	The intranasal vaccine is added with Laureth-9	It is 0 day 21	??20 ??20	?18????90.0 ?20????100.0	?11????55.0 ?19????95.0	?12????60.0
??FluarixTM	It is 0 day 21	??19 ??19	?17????89.5 ?19????100.0	?11????57.9 ?19????100.0	?15????78.9

Seropositivity (n, %): the experimenter's quantity and the percentage ratio of titer 〉=10

The serum protection (n, %): the experimenter's quantity and the percentage ratio of titer 〉=40

(n, %): titer was increased at least 4 times experimenter's quantity and percentage ratio in from 0 to 21 day in seroconversion

Table 4 as seen, between 21 days and 0 day (1 dosage), specificity/total mucosa IgA antibody ratio is increased to the shared percentage ratio of experimenter of 2 times or 4 times.

Table 4: between 21 days and 0 day (1 dosage), specificity/total IgA ratio is increased to 2Doubly or 4 times the shared percentage ratio of experimenter

Strain	Group	??N	Be increased to 2 times (%)	Be increased to 4 times (%)
					A/ Beijing	????Laureth-9 ????FluarixTM	??20 ??19	????50.0 ????52.6	????20.0 ????26.3
A/ Sydney	????Laureth-9 ????FluarixTM	??20 ??19	????55.0 ????47.4	????25.0 ????5.3
					B/ Harbin	????Laureth-9 ????FluarixTM	??20 ??19	????15.0 ????26.3	????10.0 ????5.3

Sum up

Immunogenicity result in the tabulation shows intranasal preparaton and the traditional parenteral route vaccine (Fluarix that gives a dosage above^TM) back 21 days, the seropositivity of generation, seroconversion and serum protect level similar.After the dosage, the intranasal preparaton is usually than traditional parenteral route vaccine (Fluarix^TM) the mucosa IgA reaction that produces is better.Embodiment 4, and laureth 9 and Triton X100 share the immunogenic influence to primed mice intranasal tetanus toxoid vaccine

In the present embodiment, after we have estimated to low dosage and add Triton X100 in inferior to the Laureth-9 of optimal dose, intranasal is strengthened the effect of tetanus toxoid (TT)-specific serum antibody.The merchant that female balb/c mice intramuscular gives 20% (2 * 50 μ l) human dosage sells DTPa vaccine (Diptheria, tetanus, acellular pertussis vaccine: INFANRIX^TMSmithKline Beecham, Belgium).After one month, with the A:PBS that contains 5 μ gTT; B:0.5% polyoxyethylene-9 lauryl ether; C:0.1% polyoxyethylene-9 lauryl ether; D:0.1% polyoxyethylene-9 lauryl ethers+0.02%Triton X100 gives mice intranasal booster dose (under the anesthesia, each nostril 5 μ l), or uses E: by intramuscular booster injection DTPa vaccine (2 * 50 μ l).Strengthen two weeks of back, the TT-specific IgG of serum analysis.

As shown in Figure 1, opposite with 0.5% dosage, low dosage Laureth-9 (0.1%) can not effectively strengthen the reinforcement reaction to TT.Yet, by replenishing the adjuvanticity that Triton X100 (p＜0.0001) can improve this preparaton greatly.The antibody response that causes is sold the vaccine-induced reacting phase of DTPa seemingly with the merchant.

Claims (36)

1. adjunvant composition, it comprises the polyoxyethylene alkyl ether or the polyxyethylated ester of (a) general formula (I):

HO (CH₂CH₂O)_n-A-R wherein, n is 1-50, A be key or-C (O)-, R is C_1-50Alkyl or phenyl C_1-50Alkyl; (b) at least a other non-ionic surface active agent.

2. adjunvant composition according to claim 1, wherein said other non-ionic surface active agent is hot benzene polysaccharide.

3. adjunvant composition according to claim 2, wherein said hot benzene polysaccharide is uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM).

4. one kind according to each adjunvant composition of claim 1 to 3, also comprises Sorbitan ethoxylate or cholic acid or derivatives thereof in addition or has both comprised in addition also that Sorbitan ethoxylate also comprised the cholic acid or derivatives thereof.

5. one kind according to each adjunvant composition of claim 1 to 4, it is characterized in that the polyoxyethylene alkyl ether of general formula (I) or polyxyethylated ester are hemolytic.

6. the adjunvant composition according to claim 5 is characterized in that measuring the hemolytic activity degree of polyoxyethylene alkyl ether or polyxyethylated ester in the scope of 0.05-0.0001% with guinea pig blood cell hemolytic experiment.

7. adjunvant composition according to claim 5 or 6 is wherein measured the difference of hemolytic activity of the hemolytic activity of the polyoxyethylene alkyl ether of general formula (I) or polyxyethylated ester and polyoxyethylene-9 lauryl ether or polyoxyethylene-8-stearoyl ether within 10 times with guinea pig blood cell hemolytic experiment.

8. one kind according to each adjunvant composition of claim 1 to 7, comprises the polyoxyethylene alkyl ether or the polyxyethylated ester of general formula (I), and wherein n is 4-24.

9. adjunvant composition according to Claim 8, wherein n is 9.

10. one kind according to each adjunvant composition of claim 1 to 7, comprises the polyoxyethylene alkyl ether or the polyxyethylated ester of general formula (I), and wherein R is C_8-20Alkyl or phenyl C_8-20Alkyl.

11. the adjunvant composition according to claim 10, wherein R is C₁₂Alkyl.

12. one kind according to each adjunvant composition of claim 1 to 11, comprises the polyoxyethylene alkyl ether or the polyxyethylated ester of general formula (I), wherein A is a key, thereby forms ether.

13. one kind according to each adjunvant composition of claim 1 to 12, comprises the polyoxyethylene alkyl ether or the polyxyethylated ester of general formula (I), wherein A be-C (O)-, thereby form ester.

14. one kind according to each adjunvant composition of claim 1 to 13, the polyoxyethylene ether or the polyoxyethylene ester of its formula of (I) are selected from: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-Lauryl Ester, polyoxyethylene-9-stearoyl ether, polyoxyethylene-8-stearoyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, polyoxyethylene-23-lauryl ether.

15. an adjuvant mixture comprises polyoxyethylene-9-lauryl ether and uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM).

16. one kind according to each adjunvant composition of claim 1 to 15, wherein the total concentration of detergent of Cun Zaiing is 0.001-10%.

17. the adjunvant composition according to claim 16, wherein total concentration of detergent is 0.001-1%.

18. the adjunvant composition according to claim 17, wherein total concentration of detergent is 0.001-0.7%.

19. an adjuvant mixture comprises the adjuvant of uniting in the claim 1 to 17 of use each with at least a other immunostimulant.

20. the adjuvant mixture according to claim 19, wherein at least a other immunostimulant is selected from: LT, CT, 3D-MPL, CpG and QS21.

21. the adjunvant composition according to claim 20, wherein the CpG adjuvant is: TCCATG ACG TTC CTG ACG TT (SEQ ID NO.1).

22. one kind comprises polyoxyethylene-9 lauryl ether, uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM) and the adjuvant mixture of 3D-MPL.

23. one kind comprises each the vaccine of adjuvant of claim 1 to 22, it also comprises antigen.

24. the vaccine according to claim 23, wherein said antigen is selected from: human immunodeficiency virus, varicella zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalic inclusion disease virus, dengue virus, first type, B-mode, third type or hepatitis E, respiratory syncytial virus, human papillomavirus, influenza virus, Hib, meningitis virus; Salmonella, neisseria, Borrelia, chlamydiaceae, Bordetella, Streptococcus, Mycoplasma, Mycobacterium, Haemophilus spp, Plasmodium or toxoplasma, stanworth decapeptide; Or tumor associated antigen (TMA), MAGE, BAGE, GAGE, MUC-1, Her-2 neu, LnRH, CEA, PSA, KSA or PRAME.

25. the vaccine according to claim 24, wherein said antigen are antigen or the antigen preparations from influenza virus.

26. a vaccine combination comprises polyoxyethylene-9 lauryl ether, uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM) and influenza antigen.

27. one kind according to each vaccine of claim 23 to 26, wherein vaccine is aerosol or Sprayable.

28. one kind according to each vaccine of claim 23 to 27, is used for medicine.

29. each adjunvant composition of claim 1 to 22 is applied to application in the medicine on patient's mucomembranous surface or the skin in preparation.

30. polyoxyethylene-9 lauryl ether and uncle's octylphenoxy polyethoxy ethanol (TRITON X100^TM) mixture be applied to application in the vaccine on patient's mucomembranous surface in preparation.

31. a sprayer unit, a kind of more specifically dose double sprayer unit wherein is equipped with each vaccine of claim 23 to 27.

32. the vaccine combination of each qualification of claim 23 to 27 is used for the treatment of application in the vaccine of virus, antibacterial, parasitic infection, allergy or cancer in preparation.

33. treat mammal pathogenic infection or cancer or irritated or for one kind, comprise each the vaccine combination of claim 23 to 27 of granting a kind of safe and effective amount to mammal to the method for the susceptibility of these diseases.

34. treat mammal pathogenic infection or cancer or irritated or for one kind, comprise each the vaccine combination of claim 23 to 27 of granting a kind of safe and effective amount to the mammal through mucous membrane to the method for the susceptibility of these diseases.

35. treat mammal pathogenic infection or cancer or irritated or for one kind, comprise each the vaccine combination of claim 23 to 27 of granting a kind of safe and effective amount to mammal through intranasal to the method for the susceptibility of these diseases.

36. one kind prepares each the method for vaccine combination of claim 23 to 27, comprises each adjunvant composition of (a) claim 1 to 22, (b) pharmaceutically acceptable excipient and (c) a kind of antigen or antigen composition mix.

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