CN1288384A

CN1288384A - Lipooligosaccharide-based vaccine for prevention of i(moraxella) i(branhamella) i (catarrhalis) infections in mammals

Info

Publication number: CN1288384A
Application number: CN99802142A
Authority: CN
Inventors: 古新星; J·B·罗宾斯
Original assignee: Government Of Usa Represented By Secretary Of Department Of
Current assignee: Government Of Usa Represented By Secretary Of Department Of; US Department of Health and Human Services
Priority date: 1998-01-13
Filing date: 1999-01-12
Publication date: 2001-03-21
Also published as: KR20010034124A; BR9906902A; AU2221299A; WO1999036086A1; AU2003200964A8; AU2003200964B2; IL136903A0; AU2003200964A1; JP2002509115A; CA2315746A1; EP1047447A1

Abstract

A conjugate vaccine for Moraxella (Branhamella) catarrhalis comprising isolated lipooligosaccharide from which esterified fatty acids have been removed, to produce a detoxified lipooligosaccharide (dLOS), or from which lipid A has been removed, to produce a detoxified oligosaccharide (OS), which is linked to an immunogenic carrier. The vaccine is useful for preventing otitis media and respiratory infections caused by M. catarrhalis in mammals, including humans.

Description

Be used to prevent the fat oligosaccharide vaccine of mammal moraxella catarrhalis (Branhamella) bacterium infection

Invention field

The present invention relates to be used to prevent the coupling vaccine of bacterial infection.More specifically, the present invention relates to be used to prevent the coupling vaccine of people's moraxella catarrhalis (Branhamella) bacterium infection, wherein comprise fat oligosaccharide, removed the fatty acid or the lipid A of esterification in this fat oligosaccharide, and be connected on the immunogenic carrier from antibacterial.

Background of invention

Moraxella catarrhalis (Branhamella) bacterium is a kind of pathogen, be to be positioned at the third the modal morbid substance (Bluestone that causes child's otitis media and sinusitis after streptococcus pneumoniae and the hemophilus influenza, C.D., 1986, Drugs 31 (Suppl3): 132-41; Catlin, B.W., 1990, Clin.Microbiol.Rev.3:293-320; Doern, G.V., 1986, Diagn, Microbiol.Infect.Dis.4:191-201; Endght, M.C.﹠amp; H.McKenzie, 1997, J.Med.Microbiol.46:360-71; Faden, H., et al., 1994, J.Infect.Dis.169:1312-1317).This gram-negative diplococci also can cause adult's respiratory tract infection (Boyle, F.M., et al., 1991, Med.J.Aust.154:592-596; Sarubbi, F.A., et al., 1990, Am.J.Med.88:9S-14S), those immunodeficiency or suffer from people (Enright, the M.C.﹠amp of chronic obstructive pulmonary disease (chronic obstructive pulmonary disease) particularly; H.McKenzie, 1997, J.Med.Microbiol.46:360-71).The sickness rate of the disease that morazella catarrhalis causes raise (Mcleod, D.T.et al., 1986, Br.Med.J.292:1103-1105; Fung, C.P.et al., 1992, J.Antimicrob.Chemother.30:47-55).At present, the needleless vaccine diseases induced still to morazella catarrhalis.

Although the protective antigen of morazella catarrhalis does not also clearly limit, anti-morazella catarrhalis serum antibody may brought into play important effect in the immunity of anti-morazella catarrhalis.For example, because of the natural normal adult of giving birth to or infecting the acquisition immunity that moves, its carrying rate (1～6%) is lower than child (50～78%) and old people's (＞26%), and less suffers from and infect (EjlertsenT.et al., 1994, J.Infect.29:23-31; Faden H.et al., 1994, J.Infect.Dis.169:1312-1317; Eliasson, I., 1986, Drugs 31 (Suppl3): 7-10; Vaneechoutte, M., et al., 1990., 3.Clin.Microbiol.28:2674-2680).At postnatal in the middle of 4 years, the child little by little generates the serum antibody of anti-morazella catarrhalis, this phenomenon is with the bacteremia of morazella catarrhalis initiation and relevant (the CDR WeeklyReports of reduction of otitis media sickness rate, 1992-1995, Communicable Disease Surveillance Centre, London; Goldblatt D., et al., 1990, J.Infect.Dis.162:1128-1135; Vaneechoutte, M., et al., 1990., J Clin Microbiol 28:2674-2680; Bluestone, C.D., 1986, Drugs 31 (Suppl 3): 132-141).In the acute of adult patients and convalescence serum, detected antibody (Christensen, J.J., et al., 1990, the Clin.Diagn.Lab.Immunol.3:717-721 of anti-morazella catarrhalis; Rahman, M., et al., 1997, APMIS105:213-220).Most of convalescent serums shown bactericidal activity at corresponding morazella catarrhalis (Chapman, A.J.Jr., et al., 1985, J.Infect.Dis.151:878-882).These results show that serum antibody may participate in the protective effect that anti-morazella catarrhalis infects.

Up to now, be that the research work of important pathogen concentrates on (Bhushan, R., et al., 1994, J.Bacteriol.176:6636-6643 in the description of outer membrane protein surface antigens such as (OMP) for example usually with the morazella catarrhalis; Campagnari, A.A., et al., 1994, Infect.Immun.62:4909-4914; Helminen, M.E., Et al., 1993, Infect.Immun.61:2003-2010; Helminen, M.E., et al., 1994, J.Infect.Dis.170:867-872; Murphy, T.F., etal., 1993, Mol.Microbiol.10:87-97).Two kinds of outer membrane protein have been carried out extensive studies, they are high-molecular-weight protein (UspA) and major outer membrane albumen (CD).These two kinds of albumen relatively are kept in the different strains of morazella catarrhalis, and can cause generation (Helminen, M.E., et al., 1994, the J.Infect.Dis.170:867-872 of bactericidin; Murphy, T.F., et al., 1993, Mol.Microbiol 10:87-97; Yang, Y.P., et al., 1997, FEMSImmunol.Med.Microbiol 17:187-199).In addition, the removing of pulmonary's morazella catarrhalis in the rat animal model (Helminen, M.E., et al., 1994, J.Infect.Dis.170:867-872 have been increased with anti-USPA monoclonal antibody passive immunity or with the UspA immunity; Chen, D., etal., 1996, Infect.Immun.64:1900-1905).Coding CD proteic gene cloned and check order (Murphy et al., 1993, Molec.Microbiol 10 (1): 87).

Be purified and qualitatively other outer membrane protein comprise albumen E (OMP E) (Bhushanet al., 1994, J.Bacteriol, 6636), protein B 1 (Ducey et al., 1,996 176 (21):, Abstracts, 186) and PROTEIN C OPB (Aebiet al., 1996 Gen.Mtg.Am.Soc.Microbiol., 96 (0):, Abstracts, Intersci.Conf.Antimicrobial Agents ﹠amp; Chemotherapy 36:158).Other surface antigen comprises pili (Marrs, the C.F.﹠amp that does not also find in all bacterial strains; S.Weir, 1990, Am.J.Med.88 (suppl 5A): 36S～40S) and capsular polysaccharide, whether capsular polysaccharide exists also disputable (Ahmed, K, et al., 1991, Microbiol Immunol.35:361-366).The relevant high molecular outer membrane protein of fat oligosaccharide is also identified (Klingman ﹠amp; Murphy, 1992, Abstr.Gen Mtg.Am.Soc.Microbiol.).

Fat oligosaccharide (LOS), a kind of main surface composition of morazella catarrhalis is virulence factor (Doyle, W.J., 1989, the Pediatr.Infect.Dis.J.81 (Suppl): S45-S47 of this antibacterial pathogenesis; Fomsgaard, J.S., et al., 1991, Infect.Immun.59:3346-3349).LOS is for the reason that the formation of immunoprotection plays an important role: (1) detects the serum antibody of anti-LOS in patient's body that morazella catarrhalis infects, (2) from the rehabilitation of patients phase anti--LOS IgG shows the bactericidal activity at morazella catarrhalis, and (3) LOS in human body has the reservation structure (Rahman that is based upon on its serological characteristic basis, M., et al., 1995, Eur.J.Clin.Microbiol.Infect.Dis.14:297-304; Tanaka, H., et al., 1992, J.Jpn.Assoc.Infect.Dis.66:709-715).Similarly, (for example be specific to other microorganism, b type hemophilus influenza, meningitis be Salmonella, vibrio cholera, Shigella sonnei how) serum sterilizing LPS or PS antibody give the immunity (Robbins of human body at these pathogen, J.B., et al., 1995, J.Infect.Dis.171:1387-1398; Cohen, D., et al., 1997, Lancet 349:155-159).

3 main antigenic types of LOS (A, B and C) account for 95% of morazella catarrhalis bacterial strain and (promptly are measured as 61%A in a research; 29%B; And 5%C) (Vaneechoutte, M., et al., 1990, J.Clin.Microbiol.28:182-187).Studies show that to comprise a kind of oligosaccharide that is connected on the lipid A among these LOS, do not contain the O-specific polysaccharide, form branch (Edebrink, P., etal., 1994, Carbohydr.Res.257:269-284 with a common kernel from the oligosaccharide of these 3 kinds of serotypes; Edebrink, P., et al., 1995, Carbohydr.Res.266:237-261; Edebrink, P., et al., 1996, Carbohydr.Res.295:127-146).

Usually, lipopolysaccharide (LPS) and the LOS from multiple microorganism has toxicity in vivo for mammal.Now existing several different methods can be used for the detoxification of LPS or LOS, perhaps obtains avirulent from the LPS polysaccharide or from the oligosaccharide of LOS.For example, weak acid is handled LPS or LOS and can be acted on Kdo-glycosamine key and make lipid A excise lipid A (Gu, X.X. ， ﹠amp from the LOS molecule; C.M.Tsai, 1993, Infect.Immun.61:1873-1880).Another kind method is mild alkaline treatment LOS, removes the fatty acid that ester connects but has kept fatty acid (Gupta, R.K., et al., 1992, the Infect.Immun.60:3201-3208 of the amide-connection of lipid A; Gu et al., 1996, Infect.﹠amp; Imm.64 (10): 4047).

Now attempted using several different methods to develop the vaccine of morazella catarrhalis or other microorganism (Karma et al., 1995, Int ' l.J.Ped.Otorhinolaryngol.32 (SUPPL.): S127-S134).U.S. Pat 5,607,846 and US5,556,755 disclose the morazella catarrhalis vaccine based on following ingredients: outer membrane protein E and CD, derived peptide or oligopeptide or these proteic nucleotide of encoding.U.S. Pat 5,334,379 disclose the antigen of carbohydrate containing have been connected to a kind of coupling vaccine of making on cytokine, lymphokine, hormone or the somatomedin of immunomodulating effect that has.The patent No. be 2,162,193 Canadian Patent a kind of antibacterial lactoferrin receptor protein of purification is disclosed can be as comprising that at anti-morazella catarrhalis can produce the vaccine of the pathogen of lactoferrin receptor protein.PCT application WO90/11777 discloses a kind of method that is used to obtain non-assembling pili subunit, and this pili subunit can be as the vaccine of morazella catarrhalis and other antibacterial.

Need exploitation a kind of nontoxic but have immunogenic morazella catarrhalis vaccine, be used to prevent mammal, particularly child and adult otitis media, sinusitis and similar respiratory tract infection.Although the poison-removing method from the LOS of other microorganism is known, immunogenicity is very weak usually in the body of detoxification product (being hapten).Therefore, need a kind of like this morazella catarrhalis LOS of form, it be detoxification but its immunogenicity is enough to excite the intravital immunne response of mammal, produce anti--LOS antibody, preferred IgG.

Summary of the invention

According to an aspect of the present invention, morazella catarrhalis coupling vaccine is disclosed, comprising a kind of fat oligosaccharide (LOS) and a kind of immunogenic carrier covalently bound that separates from morazella catarrhalis with it, described fat oligosaccharide by removing the LOS (dLOS) that the esterified fatty acid detoxification is generated as a kind of detoxification, perhaps is generated as a kind of oligosaccharide (OS) by removing lipid A.In the embodiment, immunogenic carrier is a protein.In another embodiment, described immunogenic carrier albumen is selected from: separate UspA from morazella catarrhalis, separate CD, tetanus toxin/toxoid from morazella catarrhalis, separate high-molecular-weight protein (HMP) from non-acquisition type (nontypeable) hemophilus influenza, pseudomonal toxin A, cholera toxin/toxoid, pertussis toxin, PT/toxoid, clostridium perfringens extracellular toxin/toxoid, hepatitis B surface antigen, hepatitis B virus core antigen, rotavirus vp 7 albumen of diphtheria toxin, diphtherotoxin/toxoid, detoxification, comprise CRM ₁₉₇(Pappenheimer et al., Immimochem.9:891-906,1972) and CRM ₃₂₀₁The CRMs (cross-linking reaction material) of (Black et al., Science 240:656-659,1988) etc. and breathe syncystial virus F and G albumen.Aspect of this vaccine, immunogenic carrier albumen is tetanus toxoid or HMP.But another embodiment is the pharmaceutical composition that comprises described vaccine conjugate on a kind of materia medica in the received vector, wherein can comprise a kind of adjuvant.Preferably, described adjuvant is the mixture of monophosphoryl lipid A and trehalose dimycolate or Alumen.In one embodiment, immunogenic carrier covalently is connected on the LOS that takes off ester by a kind of linker compounds.Preferably, this linker compounds is selected from adipic dihydrazide (adipic acid dihydrazide), episilon amino caproic acid, Mecoral dimethyl-acetal (chlorohexanol dimethyl acetal), D-glucuronolactone and p-nitrophenyl ethamine, and more preferably, described linker compounds is an adipic dihydrazide.In one embodiment, described vaccine comprises further and isolatingly from morazella catarrhalis obtains oligosaccharide (OS) by the lipid A of removing among the LOS that this oligosaccharide and immunogenic carrier are covalently bound.

According to a further aspect in the invention, the fat oligosaccharide of a kind of separation from morazella catarrhalis and the fatty acid detoxification by removing wherein ester-connection disclosed, perhaps a kind of oligosaccharide that obtains by the lipid A of removing among the LOS.In one embodiment, the described morazella catarrhalis of isolating the fat oligosaccharide purification bacterial strain that is morazella catarrhalis.

According to a further aspect in the invention; a kind of method that is used to prevent morazella catarrhalis to infect the mammal otitis media that causes is disclosed; comprising coupling vaccine to the effective immunoprotection amount of administration; comprise a kind of in this coupling vaccine by taking off the detoxification fat oligosaccharide (dLOS) that the ester processing is prepared from by morazella catarrhalis LOS; perhaps a kind of by removing the oligosaccharide that lipid A is prepared among the LOS (OS), and a kind of immunogenic carrier that is covalently attached on above-mentioned dLOS or the OS.In a preferred embodiment, described mammal is behaved.In another embodiment, described coupling vaccine is used by parenteral route.In one embodiment, described coupling vaccine is perhaps used by the combination that is deposited on intranasal mucosa or these methods by intramuscular injection, subcutaneous injection.Effective immunoprotection amount described in another embodiment is about 10 μ g～50 μ g for every dose.Inject about 10 μ g～25 μ g coupling vaccines in the time of the bimester that described method can also being included in approximately, and inoculated once in 13rd month in current inoculation back.In one embodiment, described inoculation step comprises: inoculate first dose, inoculate second dose of about 10 μ g～25 μ g coupling vaccines after then the bimester making an appointment with for first dose, the bimester making an appointment with after second dose, inoculate the 3rd dose of about 10 μ g～25 μ g coupling vaccines then, then about 12 months about 10 μ g of inoculation the 4th dosage～25 μ g coupling vaccines after the 3rd dose.

According to a further aspect in the invention, a kind of method of carrying out detoxification to from the isolating fat oligosaccharide of morazella catarrhalis (LOS) of being used for is disclosed, comprising remove the fatty acid that is connected with ester from LOS.In the embodiment, remove the fatty acid that is connected with ester with hydrazine or weak base reagent.

The present invention also comprises a kind of method of carrying out detoxification to from the LOS of morazella catarrhalis of being used for, and comprises that removing lipid A from LOS prepares OS.In one embodiment, remove lipid A by acid treatment.

According to a further aspect in the invention, a kind of method for preparing morazella catarrhalis coupling vaccine is disclosed, comprising separating from the fat oligosaccharide (LOS) of morazella catarrhalis thus in the fatty acid that is connected with ester remove the LOS (dLOS) for preparing deesterify; And it is the dLOS that obtains is covalently bound to immunogenic carrier.In one embodiment, above-mentioned removing comprises in the step with hydrazine or weak base reagent and handles LOS.In one embodiment, above-mentioned Connection Step comprises dLOS is attached on a kind of linker compounds, and this linker compounds is attached on the immunogenic carrier.Preferably, described linker compounds is adipic dihydrazide, episilon amino caproic acid, Mecoral dimethyl-acetal, D-glucuronolactone and p-nitrophenyl ethamine, and more preferably, described linker compounds is an adipic dihydrazide.In another embodiment, described vaccine combination can comprise a kind of adjuvant.

The present invention also provides a kind of coupling vaccine that is used to prevent to be infected by morazella catarrhalis the mammal otitis media that causes, comprising a kind of fat oligosaccharide (LOS) and a kind of immunogenic carrier covalently bound that separates from morazella catarrhalis with it, described fat oligosaccharide is generated as a kind of detoxification LOS (dLOS) by removing the esterified fatty acid detoxification, perhaps is generated as a kind of oligosaccharide (OS) by removing lipid A.Preferably, described immunogenic carrier is a kind of protein.Aspect of this preferred embodiment, immunogenic carrier is for separating UspA from morazella catarrhalis, separate CD, tetanus toxin/toxoid from morazella catarrhalis, separate high-molecular-weight protein (HMP) from non-acquisition type hemophilus influenza, bacillus pyocyaneus (aeruginosa) toxin A, cholera toxin/toxoid, pertussis toxin, PT/toxoid, clostridium perfringens extracellular toxin/toxoid, hepatitis B surface antigen, hepatitis B virus core antigen, rotavirus vp 7 albumen behind the diphtheria toxin, diphtherotoxin/toxoid, detoxification, comprising CRM ₁₉₇(Pappenheimeret al., Immimochem.9:891-906,1972) and CRM ₃₂₀₁The CRMs (cross reacting material) of (Black et al., Science240:656-659,1988) etc. or breathe syncystial virus F and G albumen.Preferably, described immunogenic carrier albumen is tetanus toxoid or HMP.

The accompanying drawing summary

Fig. 1 is illustrated to be sterilization titre at the rabbit anti-serum of morazella catarrhalis bacterial strain 25238, serum is taken from 2～3 battery of tests rabbit, each member all inoculates twice separately with following material in wherein should group: LOS (＂ LOS ＂), conjugate (" dLOS-TT " and ＂ dLOS-HMP ＂) perhaps add the conjugate (＂ dLOS-TT+Ribi ＂ and ＂ dLOS-HMP+Ribi ＂) of adjuvant.Based on the serum dilution that causes 50% above antibacterial to kill, the sterilization titre of each group is shown above the increase multiple of the value of preimmune serum and is expressed as geometrical mean (bar) and standard deviation (line diagram on the bar).The sterilization titre of the hyper-immuneserum that the full cell activation of morazella catarrhalis generates is 1: 1,600.

Fig. 2 is that mouse lung is removed the passive protection test figure of model when using morazella catarrhalis bacterial strain 25238 aerosol challenge.With 40 mices of anti-dLOS-TT rabbit anti-serum or preimmune serum immunity, back 18 hours then, attack by the aerosol chamber with morazella catarrhalis bacterial strain 25238 in immunity.Attack the back and put to death mice in the time of 3 and 6 hours.

FIG.3 shows is the result of the passive protection test described in Fig. 2 legend.Gathering lung and blood sample is used for analyzing.The y-axle is represented the colony-forming units (CFU) in each lung.First bar is a matched group, and second bar is vaccine group.Attacked back 3 hours, the amount of antibacterial is compared with matched group and has been reduced 50% in the vaccine group.Attacked back 6 hours, the amount of antibacterial is compared with matched group and has been reduced 61% in the vaccine group.

Detailed Description Of The Invention

The fat oligosaccharides (LOS) of moraxella catarrhalis (Branhamella) bacterium is a kind of main surface Antigen, it excites anti-can cause children's tympanitis and nasosinusitis and adult's respiratory tract infection The bactericidal properties antibody of bacterium. For simplicity, above-mentioned bacterium is not drawn referred to as catarrh hereinafter Salmonella (Moraxella catarrhalis or Mcatarrhalis). Separation is also processed catarrh not Laplace bacterium LOS has reduced approximately with limulus amoeboid cell dissolved matter experimental test virulence 20,000 times. LOS after the detoxification (dLOS) is coupled to carrier (example by a kind of connection compound As, tetanus toxoid or from non-acquisition type haemophilus influenzae purifying and the HMW that comes Albumen) upper dLOS-TT or the dLOS-HMP of forming. DLOS and TT in the conjugate that obtains And the mol ratio between the HMP was respectively 19: 1 and 31: 1. The double immunodiffusion test knot Fruit shows that the antigenicity of these two kinds of conjugates is suitable with the LOS that separates. For these two kinds of dLOS-Carrier, subcutaneous (s.c.) or intramuscular (i.m.) injection animal can excite anti-LOS immunity The rising of Lysozyme (IgG) average level. In the Mice Body, inject 3 times above-mentioned conjugate After, detect average IgG level and raise 50～100 times, in the rabbit body, inject twice after The IgG level has raise 350～700 times. In the conjugate preparation, add adjuvant, can strengthen coupling The immunogenicity of thing.

In the rabbit body, the antiserum that produces behind the immune conjugate has brought out for catarrh and has not drawn The bactericidal activity of the complement of Salmonella homology or allos bacterial strain-mediation. These results show, after the detoxification The LOS-protein conjugate can be as a kind of for immunoprophylaxis morazella catarrhalis-draw Send out the vaccine of disease.

(ATCC 25238, from US mode with the A type morazella catarrhalis of a strain purifying Culture collection center (American Type Culture Collection), Rockville, MD) As exemplary source, with standard method (Edebrink, P., et al., 1994, Carbohycir.Res. 257:269-284; Masoud, H., et al., 1994, Can.J.Chem.72:1466-1477) purifying LOS. Other known morazella catarrhalis strain wherein has many from ATCC or other guarantor Zang Chu perhaps uses the purifying clinical strains of the bacteriological method acquisition of knowing also at model of the present invention Within enclosing, as the source of LOS. In brief, morazella catarrhalis strain 25238 is existed Cultivated 8 hours on the chocolate agar, be inoculated into then 3% trypticase soya broth (TSB). 37 ℃ shaken overnight incubation. Further dilution culture is also transferred in the baffle plate flask that TSB is housed, 37 ℃ were continued shaken cultivation 24 hours. Centrifugal collecting cell, according to standard method, usefulness ethanol, Acetone and petroleum ether cell precipitation (such as Masoud, H., et al. is at 1994, Can.J.Chem. Described in the 72:1466-1477), be dried then powdered. Improve with passing through (Gu, X-X., 1995, the hot phenol-water law of standard Infect.Immun.63:41154120) (Westphal, O., et al., 1965, Methods Carbohydr.Chem.5:83-91), from cell, extracts LOS, obtain egg White or nucleic acid content is lower than 1% LOS (Smith, P.K., et al., 1985, Anal.Biochem. 150:76-85; Warburg, O.﹠ W.Christian, 1942, Biochem.Z.310:384-421). Can replace method of the present invention with the method for other known purifying LOS.

Although what the present invention described is with hydrazine the LOS from morazella catarrhalis to be carried out detoxification Method, but use any one reagent that can remove the esterified fatty acid in the lipid A or enzyme Method, mild alkaline treatment is for example namely used rare (0.1N) NaOH of pH13.2～13.6 or other Dilute alkaline aqueous solution is processed, also within the scope of the invention. Importantly, detoxification condition is necessary Enough gentle, to guarantee can not be hydrolyzed the oligosaccharides part of LOS. The hydrolysis of oligosaccharides can destroy the guarantor Protecting property epi-position. With the essentially identical condition of above-mentioned temperate condition under, process with anhydrous hydrazine, right The morazella catarrhalis LOS detoxification (Gu, X.X, et al., 1996, the Infect.Immun. that separate 64:4047-4053; Gupta, R.K., et al., 1992, Infect.Immun.60:3201-3208). Briefly, LOS is suspended in the anhydrous hydrazine and temperature in 1 ℃～100 ℃ temperature range Educate, preferred 25 ℃～75 ℃, more preferably from about 37 ℃. Mixed incubation 10 minutes～24 hours, Preferred about 2 hours～about 3 hours, cooling mixture added cold acetone and precipitates to forming then, Centrifugal collection changes sediment. Use the acetone washing precipitation, water-soluble, then super centrifugal. To surpass from The supernatant freeze-drying that obtains behind the heart, heavy molten, and carry out column chromatography, collect the carbon containing water that wash-out goes out The cut of compound, freeze-drying, called after dLOS. Weigh, the weight of dLOS is about LOS 38%.

Perhaps, can use pH to be about 2～3 rare or weakly acidic aqueous solution, weak acid is processed LOS Carry out detoxification, specifically describe and see Gu et al. (Infect.Immu～61:1873-1880,1993), The method can be removed lipid A and be obtained oligosaccharides (OS). Use then the method identical with dLOS to incite somebody to action This OS is coupled on the carrier. Although what describe among the present invention is to use acetic acid to remove catarrh Lipid A among the catarrhalis LOS, but use any one can remove the reagent of lipid A Or the method for enzyme all within the scope of the present invention. Described OS-protein conjugate is mouse and rabbit Also all have immunogenicity in the daughter, can excite the antibody of anti-LOS and carrier protein. In the mouse, conjugate (adding adjuvant)-immune serum shows for the homology moraxella catarrhalis The bactericidal activity of bacterial strain 25238. In rabbit, conjugate-immune serum shows the homology bacterium The bactericidal activity of strain 25238.

With regard to the preparation of dLOS or OS conjugate, dLOS can be directly covalently bound to carrying On the body protein, for example, use such as cross-linking reagents such as glutaraldehydes. Preferably, select various known In the method any one (for example, seen Marburg et al., 1986, J.Am Chem Soc. 108:5282 and U.S. Pat 4,882,317; US 5,153, and 312; US 5,204, and 098), make Thereby with linker compounds dLOS or OS and carrier are kept apart and to prepare dLOS or OS idol The connection thing. The existence of linker compounds promotes dLOS or OS effectively to be coupled on the carrier, optimizes The immunogenicity of conjugate. Linker compounds has length and the flexible side chain that can adjust as required, Can isolate carbohydrate and carrier components. The translation that the joint thing can strengthen coupled antigen reaches Rotational characteristic, thus increase antibody in conjunction with approach. Between difunctional site, described joint The thing side chain can comprise various architectural features, comprises heteroatom and cracking site. Although oneself is two years old Hydrazides (ADH) is a kind of preferred joint thing, but other suitable joints also comprise, for example ε-Aminocaproic acid, Mecoral dimethyl-acetal, D-glucuronolactone and p-nitrophenyl ethamine etc. Assorted bifunctional linker thing. Attempt to be used for coupling reagent of the present invention comprise HOSu NHS and Carbodiimides. Many suitable joint things and coupling reagent come for those of ordinary skills Say all be known (Dick et al., conjugate Vaccines, J.M.Cruse ﹠ R.E.Lewis, Jr., eds., Karger, New York, pp.48-114,1989).

In a preferred embodiment, at first use AH (adipic dihydrazide) (ADH) dLOS or OS are derived, ADH serves as the joint thing between the protein carrier. Letter It uses known method, and adipic dihydrazide (ADH) is connected to dLOS or OS The carboxyl of Kdo part on form AH-dLOS or H-OS derivative (Gu, XX, ﹠ C.M. Tsai, 1993, Infect.Immun.61:1873-1880). Use the excessive ADH of molal quantity, with Guarantee more effectively coupling and restriction dLOS-dLOS coupling. In the reactant mixture, ADH And the mol ratio between dLOS or the OS typically is about 10: 1～and about 250: 1, be preferably 50: 1～About 150: 1, more preferably from about 100: 1. In a preferred embodiment, each dLOS or OS Corresponding 1 ADH is present in the AH-dLOS conjugate. In a further preferred embodiment, In the final dLOS-carrier conjugates, the mol ratio between dLOS or OS and the carrier is for about 15～about 100, preferred about lower limit of 20～about 50 and preferred about upper limit of 40～about 75 it Between, preferred about 25～about 50, more preferably from about 50. Usually, by changing AH-dLOS or AH-The initial concentration of OS and carrier and reaction time, control above-mentioned mol ratio. Usually, at this In a little scopes, above-mentioned mol ratio and body are interior to positive correlation between the antibody response of this conjugate (that is, Mol ratio is more big, replys approximately strong). By column chromatography, from above-mentioned reactant mixture, be purified into DLOS after deriving or OS are contained carbohydrate and hexanoyl hydrazine (adipic simultaneously Hydrazide) eluting fraction (Kemp, A.H.﹠ M.R.A.Mo rgan, 1986,3.Immunol. Methods 94:65-72). Collect these cuts, freeze-drying, called after AH-dLOS or AH-OS.

Although the preferred embodiments of the invention are morazella catarrhalis dLOS or the OS that are connected on the protein, more preferably purification and the tetanus toxoid (TT) or the high-molecular-weight protein (HMP) that come from hemophilus influenza, but various carriers as known in the art also are suitable for preparing dLOS-of the present invention or OS-carrier conjugates.HMP is one group of high-molecular-weight protein that the surface exposes, they are that (S.J.Barenkamp is seen in the further qualification of 26S Proteasome Structure and Function for main antibody target in the people's convalescence serum that obtains from the individuality that has infected hemophilus influenza, 1992, J.Infect.Dis.165 (Suppl.1): S181-184).Carrier can strengthen the immunogenicity of oligosaccharide, and the antibody that anti-carrier produces can be used for medical aspect.Described carrier can be a water solublity or insoluble.Suitable natural or synthetic heteropolymer immunogenic carrier comprises, for example, contains the material of primary amino radical and/or secondary amino group, azido or carboxyl.

In the various immunogenic carrier albumen any one may be used to prepare dLOS-of the present invention or OS-carrier conjugates.These albumen comprise, for example toxinferous nontoxic or " toxoid " form of the excreting poison of pili, outer membrane protein and pathogen, this class, be similar to bacteriotoxic antigenicity nontoxic protein (known cross-linking reaction material or the CRMs of being called) and other protein.Preferable separate is from the outer membrane protein of gram negative bacteria.Preferred outer membrane protein comprises UspA and the CD of separation from morazella catarrhalis.Toxoid also is preferred.Trial is used for bacteriotoxin of the present invention and anatoxic limiting examples comprises, for example, pseudomonal toxin A, cholera toxin or toxoid, pertussis toxin, PT or the toxoid after tetanus toxin or toxoid, diphtheria toxin, diphtherotoxin or toxoid, the detoxification and clostridium perfringens extracellular toxin or toxoid.The present invention has also attempted with virus protein (hepatitis B surface or cAg, rotavirus vp 7 albumen and breathing syncytial virus (RSV) F and G albumen) as carrier in addition.

CRMs comprises the antigenicity CRM that is similar to diphtheria toxin, diphtherotoxin ₁₉₇The hereditism of (Pappenheimer Etal., above-mentioned) and pertussis toxin, PT operates mutant form CRM3201 (Black etal., above-mentioned).From mammiferous immunogenic carrier albumen, for example keyhole limpet hemocyanin, king crab hemocyanin (horseshoe crab hemocyanin) and plant edestin are also at this

Within the scope of invention.

In addition, also imagined the multiple coupling method that is used to prepare morazella catarrhalis dLOS-or OS-protein conjugate.For example, occur among the present invention, dLOS or OS are carried out derivatization, be connected to then on TT or the HMP with AH.Perhaps, the another kind of method that is used to prepare suitable dLOS-or OS-protein conjugate comprises the cystamine derivatization of dLOS, the derivatization of EDC-mediation, curing is coupled on the protein behind N-butanimide-3-(2-pyridine two sulfur) propionate-derivatization then.Other are well-known be used for oligosaccharide be coupled on the immunogenic carrier albumen method also within the scope of the invention, specific descriptions see that for example U.S. Patent No. 5,153,312, U.S. Patent No. 5,204,098; And European patent EP 0497525; With EP 0245045.

AH-dLOS or AH-OS be coupled to that tetanus toxoid (TT) is gone up or go up and form conjugate (Gu, X.X. ， ﹠amp from the high-molecular-weight protein (HMP) of hemophilus influenza; C.M.Tsai, 1993, Infect.Immun.61:1873-1880).In the reactant mixture, the mol ratio between AH-dLOS or AH-OS and the protein ingredient typically is about 10: 1～and about 250: 1, be preferably 50: 1～about 150: 1, more preferably from about 100: 1.For example, according to mol ratio about 100: 1 between AH-dLOS and the coupling protein, the AH-dLOS that is dissolved in the water is mixed with TT or HMP.Then, pH is transferred to 5.4 ± 0.2, and in 1～3 hour time period, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride is joined in the reactant mixture of stirring.Reactant mixture is transferred to pH7.0, centrifugal, and use column chromatography purification.Collection contains the peak of protein and carbohydrate, according to different called after dLOS-TT of the albumen that uses in the conjugate or dLOS-HMP.As object of reference, form (Dubois, M., et al., 1956, Anal.Biochem.28:250-256 with dLOS and BSA with carbohydrate and albumen that conventional method is analyzed in the conjugate; Smith, P.K, et al., 1985, Anal.Biochem.150:76-85).

Those skilled in the art can understand that the dLOS or the OS that are coupled on the carrier can prepare the multivalence mixture from single morazella catarrhalis strain or multiple bacterial strain.Perhaps, can use the single source dLOS or the OS that are used to prepare single conjugate, prepare dLOS-or OS-carrier conjugates separately, different conjugates can be mixed with out then and contain more than one type the dLOS-or the vaccine of OS-carrier conjugates.In this case, can prepare the vaccine of the morazella catarrhalis LOS that contains one or more known antigens types.

DLOS after to purification is qualitative with the SDS-PAGE of standard and silver-colored staining technique, and the LOS behind itself and the purification is compared concrete operations (Tsai, C.M.﹠amp same as above substantially; C.E.Frasch, 1982, Anal.Biochem.119:115-119).On gel, separate the five equilibrium solution of morazella catarrhalis LOS (25,50,100 and 200ng) and dLOS (20 μ g), also contain Minnesota State Salmonella LPS Ra and Re on this gel as marker.Each contains and a Mr all occurred in the swimming lane of morazella catarrhalis LOS and be about 4,000 single band (Edebrink, p.et al., 1994, Carbohydr.Res.257:269-284), but do not occur in the position of LOS can detected band for the swimming lane that contains 20 μ g dLOS.On the contrary, the swimming lane that contains 20 μ g dLOS a fuzzy band occurred at the level place of Minnesota State Salmonella LPS Ra band.These results show that the residual quantity of morazella catarrhalis in the dLOS sample is less than 0.25%.

With standard LAL (LAL) test (Hochstein, H.D., etal., 1973, Bull.Parenter.Drug Assoc.27:139-148), test separates the toxicity of LOS and dLOS.When using can be from reference material that U.S.F.D.A obtains the time, the sensitivity of lal test is 0.2EU/ml.Isolating LOS is 20,000EU/ μ g, and dLOS is 1EU/ μ g, shows that toxicity has reduced by 20,000 times.Preferably, toxicity has reduced about 500～1000 times of EU/ g or more compositions can be used to prepare vaccine.The phenomenon that this class reduces as the in vitro toxicity that determines with lal test is relevant with toxicity in vivo reduction and acceptable level.Body endogenous pyrogen method of testing with standard can easily determine toxicity in vivo (for example, in the rabbit body, using dosage is 0.1 μ g～1 μ g/kg body weight).

Use the rabbit hyper-immuneserum of the full cell of anti-morazella catarrhalis (bacterial strain 25238), with the antigenicity of two-way immunodiffusion method test dLOS, AH-dLOS and dLOS-TT and dLOS-HMP conjugate.Prepare hyper-immuneserum with standard method.Briefly, with 10 ⁹Full cell of the morazella catarrhalis of M (bacterial strain 25238) and non-complete Fu Shi adjuvant (volume ratio is 1: 1) emulsion, the subcutaneous and intramuscular double injections (subcutaneous and intramuscular injection is all carried out in per injection) to two new zealand white rabbits were spaced apart for 4 weeks twice.2 weeks of before the per injection and injection back are collected blood sample.

(PBS pH7.4) on 0.8% agarose gel in, carries out two-way immunodiffusion with standard method being dissolved in phosphate buffer.In this test, add the rabbit hyper-immuneserum of the full cell of anti-morazella catarrhalis in the centre bore, add LOS, dLOS-TT, dLOS-HMP, dLOS and HMP on every side in the hole separately.In double immunodiffusion test, hyper-immuneserum and LOS reaction produce one and significantly, easily detect the precipitation band.Similarly, hyper-immuneserum and dLOS reaction produces a wideer slightly precipitation band, shows that the dLOS after the separation has kept the antigenicity of the LOS after the separation.When comparing with LOS, hyper-immuneserum also reacts with dLOS-TT and dLOS-HMP conjugate, generates an identical precipitation band.But, hyper-immuneserum with separate after HMP between measurable reaction does not take place.

In addition, to disclosed method before this (Gu, X.X., et al., 1996, Infect.Immun64:4047-4053) do change slightly, measure antigenicity with enzyme linked immunosorbent assay (ELISA)., sealed with 3%BSA then by the ELISA flat board with the LOS bag.Then, with ELISA hole and the rabbit anteserum incubation of using after diluting, add alkali phosphatase-link coupled goat subsequently and resist-rabbit igg and IgM (Sigma).Above-mentioned institute in steps between, with each hole of the abundant repeated washing of PBS that contains polymer dispersed reagent (0.01%Tween-20).With 30 minutes, add zymolyte, then at A ₄₀₅The place carries out quantitatively reaction.With conjugate as envelope antigen and the dilution after the rabbit immune serum as binding antibody, measure the antigenicity of dLOS-carrier conjugates similarly.These two kinds of dLOS-carrier conjugates have all shown combining between comparable and rabbit hyper-immuneserum, and the antigenicity of dLOS-carrier conjugates is higher than LOS under similarity condition.

In order to measure the body endoantigen, the dLOS-carrier conjugates is expelled in mice and the rabbit body by parenteral route, measure the level of anti--LOS antibody in the animal serum subsequently with ELISA.In mice, the non-coupling mixture of dLOS and TT or HMP can not excite the generation of anti--LOS antibody.On the contrary, these two kinds of conjugates of dLOS-TT and dLOS-HMP all excite after injection for the second time and have generated low-level resisting-LOS IgG.After injecting conjugate for the third time, the level of anti--LOS IgG has raise 50～100 times.After injecting 3 times, dLOS-TT and dLOS-HMP excite the anti--LOS IgG that has generated similar level.LOS and LOS-carrier conjugates excite the anti--LOS IgG level of generation approximate separately.

The dLOS-TT and the dLOS-HMP conjugate preparation that have added adjuvant have all strengthened the intravital immunogenicity of mice significantly.That is to say that the antibody horizontal that the dLOS～carrier conjugates that has 2 dosage of adjuvant excites generation quite or be higher than the antibody horizontal of 3 dosage conjugates of independent use.Do not contain the level that obtains behind the same conjugate of adjuvant 3 times with injection and compare, inject the dLOS～carrier conjugates that has adjuvant for 3 times after, resist-LOS IgG level raise about 9～15 times.After injecting 3 conjugate-adjuvant formulations, the dLOS-TT conjugate excites the IgG water of generation to be lower than the dLOS-HMP conjugate.

The adjuvant that uses comprise monophosphoryl lipid A and trehalose dimycolate (trade name is Ribi-700, available from Ribi Immunochemical Research, Hamilton, MT).Other well-known standard adjuvants are also within the scope that the present invention attempts, for example, lipopolysaccharide, the pertussis bordetella suspension that kills, N-acetyl muramyl-L-alanyl-D-glutamic acid and known other adjuvants of those of ordinary skills of aluminium compound (being Alumen), process chemical modification.The description of other adjuvant is referring to (Ann.Rev.Biochem.4:369-388,1986 such as Warren; New Generation Vaccines, 2nd Edition, Leine, M.M.et al., Eds., Marcel Dekker, Inc., New York, 1997).Preferably use aluminium compound, especially preferably be applicable to people's adjuvant.During IgM level in analyzing mice serum, conjugate after per injection, excite be low to moderate the moderate level anti--average level of LOS antibody, and LOS after injection for the third time, excite generated high-caliber anti--LOS IgM.In the dLOS-protein conjugate, add adjuvant and improved anti--LOS IgM level of injection back generation for the second time.

When the antibody of protein ingredient (TT or HMP) is tested in the anti-dLOS-protein conjugate in similarly to mice serum, found anti--protein antibodies.With regard to anti--TT antibody, dLOS-TT excites after injection for the first time and has generated low-level IgG, and this level obviously raises after injecting for the second time or for the third time.Compare with the mice of having accepted same conjugate but not accepted adjuvant, the dLOS-TT that injection has an adjuvant can improve the IgG level in the dLOS-TT injection group.The non-coupling mixture of TT and dLOS excites the anti--TT IgG level of generation to be higher than the level that dLOS-TT excites generation.All immunogens all excite and have generated low-level resisting-TTIgM, and behind the adding adjuvant, its level raises in the injection.

To resisting in the mice serum-when the HMP antibody horizontal was tested, dLOS-HMP excited after the injection in the first time and generated low-level IgG, anti--HMP IgG level significantly raises after injecting for the second time and for the third time when similarly.The adding of adjuvant has improved the intravital IgG level of the mice of accepting the dLOS-HMP conjugate.Adding or do not adding under the situation of adjuvant, the non-coupling mixture of HMP and dLOS excites the anti--HMP IgG level of generation all to be higher than observed level in the mice of accepting dLOS-HMP (tabulating 2 as follows).All immunogens all excite and have generated low-level resisting-HMP IgM.

In addition, under rabbit skin and intramuscular injection dLOS-protein conjugate 0 and measured immunogenicity (subcutaneous and intramuscular is all carried out in per injection simultaneously) after 1 month.Two weeks and two weeks of back of injection for the second time after 0 o'clock (when promptly injecting first), injection first, the collection blood sample.Use ELISA method used when measuring mice serum, measure IgM and IgG level: LOS in the rabbit anteserum that obtains after the following immunogen of injection (every kind of immunogen is all injected 50 μ g at every turn), dLOS-TT, the dLOS-TT that contains adjuvant, dLOS-HMP, the dLOS-HMP that contains adjuvant, the non-coupling mixture of dLOS, TT and HMP, the perhaps full cell of morazella catarrhalis.The mixture of dLOS, TT and HMP or LOS originally excite behind double injection and have generated low-level resisting-LOS IgG or IgM antibody.Compare with the serum levels that injection is preceding, the dLOS-TT conjugate excites and has generated resisting-LOS IgG of remarkable rising after the first time and the injection for the second time.Level when injection dLOS-HMP conjugate excites the IgG level of generation to be lower than injection dLOS-TT conjugate.After adding adjuvant, for two kinds of conjugates, excite anti--LOS IgG level of generation all to uprise after the per injection, having behind the double injection of adjuvant does not have evident difference between these two kinds of conjugates.With regard to IgM, two kinds of conjugates all excite and have generated the anti--LOS antibody that is low to moderate medium level, and the conjugate of the same race that does not contain adjuvant with injection is compared, add adjuvant can improve usually after the per injection detected anti--level of LOS IgM antibody.

When the antibody of protein ingredient is measured in the anti-dLOS-protein conjugate in similarly to rabbit anteserum, found anti--protein antibodies.With regard to anti--TT antibody, injection back dLOS-TT excites and has generated low-level IgG for the first time, and this level significantly raises after injection for the second time.Do not add adjuvant during with injection and compare, after injection added the dLOS-TT of adjuvant, detected resisting-TT significantly raise.The non-coupling mixture of injection TT, dLOS and HMP excites anti--TT I IgG of generation to be higher than the level that dLOS-TT excites generation, adds the level that dLOS-TT behind the adjuvant excites generation but be lower than injection back for the second time.All immunogens all excite and have generated the anti--TT IgM that is low to moderate medium level.

With regard to anti--HMP antibody of finding in the rabbit anteserum, dLOS-HMP is exciting low-level IgG after the injection for the first time, and after the injection, this level significantly raises for the second time.The adding adjuvant has improved the level of anti--HMP IgG antibody in dLOS-HMP.Antibody horizontal when the mixture of HMP, dLOS and TT excites the antibody horizontal of generation to be higher than injection not contain the dLOS-HMP of adjuvant, but a little less than the dLOS-carrier conjugates that has adjuvant.All immunogens all excite and have generated the anti--HMP IgM that is low to moderate medium level.

With standard method (Gu X-X., et al., 1996, Infect.Immun.64:4047-4053), the antiserum that test prepares with mice and rabbit is at the body outer disinfecting activity of homology or allos morazella catarrhalis.In the rabbit model, do not show bactericidal activity at the strain of homology morazella catarrhalis with the serum for preparing after LOS or the non-link coupled dLOS immunity.On the contrary, when being 1: 16 (not containing adjuvant) and 1: 40 (adding adjuvant), average titer shown bactericidal activity with the serum that excites generation after the dLOS-TT immunity.With ELISA measure anti--the LOSIgG level is relevant with detected sterilization titre.

(for example, Japan the bactericidal activity of) homology or allos bacterial strain shows antiserum, and the bactericidal activity that excites the rabbit anteserum of generation to show with the dLOS-protein conjugate is higher than the mice serum for preparing with similar approach from different regions at separating.All conjugates-inductive rabbit anteserum has all shown the bactericidal activity at the strain of homology morazella catarrhalis, and typical serum has shown bactericidal activity at most non-homogeneous bacterial strain (in 10 ATCC bacterial strains and the clinical separation strain 9).On the contrary, not enough half dLOS-carrier conjugates-inductive serum has shown the bactericidal activity at homologous strain.Usually, relevant between sterilization titre and the anti-LOS IgG antibody horizontal.

Use 20 other morazella catarrhalis strains (10 wild type ATCC bacterial strains and 10 clinical separation strains), excite the bactericidal activity of the rabbit anti-serum of generation to measure having joined the dLOS-' IT behind the adjuvant.10 in 20 bacterial strains is complement or serum sensitive strain.When using remaining 10 bacterial strain, when average titer was 1: 15 (scope is 1: 2～1: 32), rabbit anti-serum showed the bactericidal activity at 4 ATCC and 5 clinical separation strains.

In the mouse model, behind 3 dLOS-carrier conjugates of injection, have 20% in the serum with dLOS-protein conjugate immune mouse preparation (20 mice in 4), and, shown low titre bactericidal activity at homology morazella catarrhalis strain (ATCC 25238) with 45% (having 9 in 20 mices) arranged in the serum for preparing behind conjugate and the adjuvant immunity.

The result who occurs in the following examples shows that after the detoxification processing, morazella catarrhalis dLOS has kept antigenic determinant but do not had immunogenicity in the body.When being coupled to dLOS on the carrier protein, this dLOS component has possessed immunogenicity.That is to say that morazella catarrhalis dLOS-carrier conjugates can be induced the remarkable IgG antibody response at mammal LOS.In the mammal, the dLOS-carrier conjugates excites to have generated and is equivalent to anti--LOS IgG antibody that LOS excites level at least.This dLOS-protein conjugate is better than mice (promptly in the intravital immunogenicity of rabbit, in the rabbit body behind 2 this conjugates of injection, the multiple that anti--LOS antibody increases usually greater than 2 times or 3 injections with in the mice body behind a kind of conjugate observed anti--the increase multiple of LOS antibody).In these two kinds of animals, the conjugate of the same race that does not contain adjuvant with injection is compared, and injects the level that the conjugate that is added with adjuvant can both improve anti--LOS antibody usually.DLOS-TT and dLOS-HMP conjugate excite the anti--LOSIgG antibody that has generated level of approximation, and antibody horizontal raises when adding adjuvant in conjugate.

Morazella catarrhalis dLOS-carrier conjugates of the present invention can be used as vaccine, induces the immunoreation of resisting mammal morazella catarrhalis, particularly prevents people's otitis media and respiratory tract disease.The method of this class of the preparation of describing among the present invention dLOS-carrier conjugates can be used for producing this class vaccine.Disclosed method also can be used to identify other dLOS-carrier conjugates (being the conjugate of dLOS and other carrier elements) among the present invention; these conjugates can be used for inducing the protective immune response of mammal at morazella catarrhalis, particularly comprise the protective immune response in child's the human body.What can understand is, the vaccine of morazella catarrhalis can comprise the dLOS-carrier conjugates, and other compositions, and for example, the inert materia medica of immunogenicity can be accepted reagent or clinical acceptable adjuvant.Those skilled in the art can also be understood that, morazella catarrhalis dLOS-carrier conjugates can be united (for example, preparation is a kind of can cause other antibacterials of children disease or the combined vaccine of virus at morazella catarrhalis and one or more) with other immunogen activity compositions at other infectants; For example, be used to prevent the trivalent vaccine of the morazella catarrhalis of bacillary otitis media, non-acquired hemophilus influenza and streptococcus pneumoniae.

With regard to inoculation, the dLOS-carrier conjugates is inoculated through parenteral route.Although various route of inoculation comprise, for example, intramuscular (i.m.), subcutaneous (s.c.), intraperitoneal (i.p.), through mucous membrane (for example, intranasal) and all attempt, preferably through mucous membrane, subcutaneous and intramuscular inoculation through the tremulous pulse inoculation.With regard to parenteral inoculation, the dLOS-carrier conjugates can adopt the form of sterilization preparation, and for example, sterile water for injection solution or oily suspension add or do not add adjuvant.According to method well-known in the art, prepare this class suspension with suitable dispersion or wetting agent and suspending agent.Described sterilization preparation can also can be accepted diluent or solvent with non-intestinal, and for example, 1,3 butylene glycol solution is mixed with aseptic injectable solution or suspension.Other suitable diluent comprise, for example, and water, Ringer's mixture and isoosmotic sodium chloride solution.In addition, aseptic expressed oi (fixed oils) conventionally also commonly used is as solvent or suspension media.For example, the expressed oi of any one brand can use, and comprises synthetic monoglyceride or diglyceride.In addition, fatty acids such as oleic acid can also be used for the preparation of ejection preparation similarly.With regard to intranasal vaccination,, with inert carrier (for example, air or Hydrocarbon) described preparation is carried out aerosolization and handle according in the various common methods any one.

DLOS-carrier conjugates in the vaccine of the present invention can be solvable or particulate form, perhaps can be incorporated in microsphere or the microcapsule, comprises liposome.In one embodiment, the dosage of the conjugate of inoculation is about 10 μ g～about 100 μ g, preferred about 20 μ g～about 50 g.In a further preferred embodiment, inoculum concentration is about 25 μ g～about 40 μ g.Precise dosage can be measured out with the known routine dose/answer method of those of ordinary skills, and dosage of inoculation is usually based on body weight, and the child is especially true.

Vaccine of the present invention can be inoculated into the mammal at arbitrary age, is suitable in young mammal, and particularly the people induces the otitis media that generation brings out at morazella catarrhalis or the active immunity of respiratory tract infection to reply.Use vaccine as the child, the dLOS-carrier conjugates was inoculated when about 2～12 monthly ages, preferred about 2～6 monthly ages.May also booster injection will be given.Typically, February, peace treaty carried out twice booster injection of about 10 μ g～about 25 μ g during March approximately for example injecting the back first.Perhaps, when injecting back 2,4 and June first, carry out booster injection.Other booster injection schemes have also been attempted in addition.

Can comprise in the vaccine combination that from the combination of the conjugate cocktail of different morazella catarrhalis strains such vaccine combination can prevent all or the relevant bacterial strain of most of medical science.According to the dLOS difference, existing 3 kinds of dissimilar morazella catarrhalis: A, B and C type, they account for 61%, 29% and 5% of clinical strains respectively.As described in example 6 above, the antiserum that produces of at bacterial strain can be with some but is not that other whole bacterial strains carry out cross reaction.Therefore, may use the chicken tail combination of different conjugates.The mixture that contains from the conjugate of the dLOS of A and Type B or OS can cover 90% of the relevant bacterial strain of all medical science, can cover 95% of the relevant bacterial strain of all medical science and contain from the dLOS of A, B and C type or the conjugate mixture of OS.

As described in following embodiment 7, the immune antiserum immune mouse with arbitrary anti-dLOS-TT carries out passive protection test, stimulates with morazella catarrhalis strain aerosol chambers 25238 then.In the vaccine group, the antibacterial CFU that observes in each lung significantly reduces.This mouse lung is removed model can simulate this antibacterial in the intravital natural transhipment of people.The advantage of this model is that it is simple, can repeat and can study a large amount of mices under with a kind of attack condition by the fine control of aerosol machinery, and do not need any surgery invasive operation when inoculated bacteria.

Although be not intended to be limited to a kind of concrete behavior or mechanism model, the bactericidin, particularly IgG of replying the generation of dLOS-carrier conjugates can ooze out the mucous membrane of nasopharynx surface.At this moment, the morazella catarrhalis inoculum of above-mentioned antibody on can the deactivation mucomembranous surface, thereby prevention or alleviate otitis media that morazella catarrhalis brings out and the symptom of respiratory tract disease.Secretory IgA, sIgA also may have been brought into play effect in the respiratory mucosa immunity, particularly when by this coupling vaccine of rhinovaccination.

Following embodiment can illustrate the preferred embodiments of the invention.

Embodiment 1: the purification of morazella catarrhalis LOS and detoxification

With demonstration source (Edebrink, P., et al., 1994, the Carbohydr.Res.257:269-284 of (A type) morazella catarrhalis strain ATCC 25238 as purification LOS; Masoud, H., et al., 1994, Can.J.Chem.72:1466-1477).At 37 ℃, 5%CO ₂Condition under, on chocolate agar, cultivated this bacterial strain 8 hours, transfer to then 250ml 3% trypticase soya broth (TSB) that is contained in the 500mL culture bottle (Difco Laboratories, Detroit, Mich.) in.This culture bottle is placed constant temperature oscillator (Model G-25, New BrunswickScientific, Co., Edison, NJ) last 37 ℃, 110rpm overnight incubation.Culture is transferred in 6 2.8 upshift plate Fernbach flasks, 1.4 liters of TSB respectively are housed in each flask.At 37 ℃, constant-temperature shaking culture is 24 hours under the 110rpm.Culture is centrifugal 10 minutes collecting cells of 15,000 * g under 4 ℃.

According to conventional method (same substantially Masoud, H., et al., 1994, the description among the Can J.Chem.72:1466-1477), cell precipitation is with 95% washing with alcohol 1 time, with washing with acetone 2 times, and with petroleum ether 2 times, is dried to powder.With the hot phenol-water law (Westphal of improved (Gu X-X., 1995, Infect.Immun 63:4115-4120) standard, O., et al., 1965, MethodsCarbohydr.Chem.5:83-91), from cell, extract LOS, obtain contained albumen or nucleic acid compositions and be lower than 1% LOS (Smith, P.K., et al., 1985, Anal.Biochem.150:76-85; Warburg, O.﹠amp; W.Christian, 1942, Biochem.Z.310:384-421).

Under the alkali condition of gentleness, handle LOS with anhydrous hydrazine, so that from lipid A, remove esterified fatty acid (Gu, X.X., et al., 1996, Infect.Immun.64:4047-4053; Gupta, R.K., Et al., 1992, Infect.Immun.60:3201-3208).Briefly, with LOS (160mg) be suspended in the 16mL anhydrous hydrazine (Sigma Chemical Co., St.Louis, MO) in, stir down 37 ℃ of incubations 3 hours.This suspension is placed cooled on ice, drip cold acetone until forming precipitation.Mixture is descended 5, centrifugal 30 minutes of 000xg at 5 ℃.With cold acetone washing precipitation 2 times, be dissolved in that final concentration is 10-20mg/mL in the pyrogen-free water, then at 5 ℃ down 150, super centrifugal 3 hours of 000xg.Lyophilizing supernatant and through ((Pharmacia LKB Biotechnology, Uppsala Sweden) carry out chromatography to 1.6 * 90cm) Sephadex G-50 on the post, with 25mM ammonium acetate eluting, and with differential refractometer (R400; Waters, Milford, Mass.) monitoring.With micro-phenol-sulfuric acid method (Dubois, M., et al., 1956, Anal.Biochem.28:250-256) carbohydrate in the eluent is analyzed.Collection contains the fraction of carbohydrate, lyophilizing, and called after dLOS, its weight is 38% of LOS.

Pass through lipid A part (that is, Gu, the X.X. ， ﹠amp in the excision LOS molecule on the cracking Kdo-glycosamine key with the acid treatment LOS of gentleness; C.M.Tsai, 1993, method described in the Infect.Immun.61:1873-1880), be coupled on the protein carrier again after, detoxification can obtain the dLOS of higher yields to morazella catarrhalis LOS in this way.

Embodiment 2:dLOS derive and with proteinic coupling

According to following method, preparation is according to adipyl trap (AH) derivant of the dLOS of method preparation among the embodiment 1, and purification.With 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) and N-hydroxyl sulfo group-butanimide (sulfo group-NHS), adipic dihydrazide (ADH) is connected on the Kdo carboxyl partly of dLOS, form AH-dLOS derivant (Pierce) (Gu, X.X. ， ﹠amp; C.M.Tsai, 1993, InfecL Immun.61:1873-1880).Briefly, with dLOS (70mg) be dissolved in 7mL 345mM ADH (about 100: 1 of the mol ratio of ADH and LOS is estimating that dLOS is 3, on the basis of 000Mr) (Edebrik, P., etal., 1994, Carbohydr.Res.257:269-284).Adding sulfo group-NHS is 8mM to concentration, and pH transfers to 4.8, and add EDC to concentration be 0.1M.Stirred reaction mixture also kept 3 hours under pH4.8.According to the method among the embodiment 1, reactant mixture is transferred to pH7.0 and on G-50, carry out column chromatography.Carbohydrate in the eluent and adipic dihydrazide (adipic hydrazide) (AH) are analyzed (Kemp, A.H.﹠amp; M.R.A.Morgan, 1986, J.Immunol.Methods 94:65-72).Collect the fraction that contains carbohydrate and AH simultaneously, lyophilizing, called after AH-dLOS.As standard, measure the composition of AH-dLOS with AH and dLOS.

According to following method AH-dLOS is coupled on the protein (TT and HMP).TT (Connaught Labs.Inc., Swiftwater, Pa.) and HMP from non-acquisition type hemophilus influenza bacterial strain 12 purification (Barenkamp, S.J., 1996, Infect.Immun.64:1246-1251).AH-dLOS is coupled to TT or last conjugate (Gu, the X.X. ， ﹠amp of forming of HMP; C.M.Tsai, 1993, Infect.Immun.61:1873-1880).Briefly, AH-dLOS (30mg) is dissolved in the 3mL water, and with 15mg TT (5.9mg/ml, or 12mg HMP (4mg/ml) mixes.Mol ratio between AH-dLOS and TT (Mr the is 150K) andHMP (Mr is 120K) is about 100: 1.PH transfers to 5.4, add EDC to concentration be about 0.05～0.1M.Stirred reaction mixture, pH kept 1～3 minute at 5.6 times.Reactant mixture is transferred to pH7.0, centrifugal, and in 0.9%NaCl, pass through Sephacryl S-300 chromatographic column (1.6 * 90cm).Collect the peak that contains albumen and carbohydrate simultaneously, called after dLOS-TT or dLOS-HMP.As standard, analyze carbohydrate and proteic composition (Dubois, M., et al., 1956, Anal.Biochem 28:250-256 in these two kinds of conjugates with dLOS and BSA; Smith, P.K., et al., 1985, Anal.Biochem.150:76-85).

According to dLOS and protein measuring amount (μ g/ml) in AH in the derivative products and dLOS or the conjugate, deutero-AH-dLOS and dLOS-protein conjugate are carried out physics analysis.According to the content of carbohydrate, the productive rate of AH-dLOS is 93%.For dLOS-TT, measure 103 μ g/ml dLOS and 266 μ g/ml protein; For dLOS-HMP, measure 220 μ g/ml dLOS and 280 μ g/ml protein.Use the molecular weight of dLOS, TT and HMP to be respectively 3,000,150,000 and 12,000,, calculate the mol ratio of conjugate with the dLOS molal quantity of every mole of protein correspondence.DLOS was respectively 19: 1 and 31: 1 with respect to the mol ratio of TT and HMP in two kinds of conjugate preparations.Measure the primary quantity of dLOS and the content in the conjugate with phenol-sulfuric acid method, calculate the productive rate of conjugate on this basis.The productive rate of dLOS-TT and dLOS-HMP is respectively 8% and 19%.The antigenicity of embodiment 3:dLOS, deutero-dLOS and dLOS-protein conjugate

With the rabbit hyper-immuneserum of the full cell of anti-morazella catarrhalis (bacterial strain 25238), by two-way immunodiffusion and enzyme-linked immunosorbent assay (ELISA) method, the antigenicity of test dLOS, AH-dLOS and conjugate.The hyper-immuneserum preparation method is same as above.

(PBS pH7.4) on 0.8% agarose gel in, carries out two-way immunodiffusion with standard method being dissolved in phosphate buffer.In this test, add the rabbit hyper-immuneserum of the full cell of anti-morazella catarrhalis in the centre bore, add following material: 1mg/mlLOS, 103 μ g/ml dLOS-TT, 220 μ g/ml dLOS-HMP (based on the amount of dLOS), 1mg/ml dLOS, 200 μ g/ml dLOS and 500 μ g/ml HMP on every side in the hole respectively.In double immunodiffusion test, hyper-immuneserum and LOS reaction produce one and significantly, easily detect the precipitation band.Similarly, hyper-immuneserum and dLOS reaction, (equal under two test concentrations) produces a wideer slightly precipitation band, shows that isolating dLOS has kept the antigenicity of isolating LOS.Hyper-immuneserum and dLOS-TT and the reaction of dLOS-HMP conjugate generate clearly precipitation band.In the two-way immunodiffusion, these two kinds of conjugates and LOS have formed essentially identical precipitation line.On the contrary, between hyper-immuneserum and the isolating HMP measurable reaction does not take place.

Carry out the ELISA test with disclosed (Gu, X.X., et al., 1996, Infect.Immun.64:4047～4053) improved method before this.With LOS bag by each hole on the standard ELISA plate (Dynatech Laboratories, Inc., Alexandria, VA) after, the 3%BSA solution sealing 1 hour with being dissolved among the PBS added rabbit anteserum (1/8,000 dilution factor) incubation 2 hours then.Add alkali phosphatase-link coupled goat subsequently and resist-rabbit igg and IgM (Sigma) incubation 1 hour.Between above-mentioned described step, with the PBS washing that contains 0.01%Tween-20.The diluent that serum and phosphatase use is the 1%BSA that is dissolved in PBS 0.01%Tween-20.After 30 minutes, add zymolyte, (EL309 is Bio-TekInstruments) at A to use microwell plate automatic reading instrument then ₄₀₅The place reads reaction.According to A ₄₀₅The ELISA reactivity at place is measured the antigenicity of dLOS-carrier conjugates similarly.With conjugate as envelope antigen (10 μ g/ml) and with the rabbit immune serum as binding antibody (1/8,000 dilution factor).

With regard to the conjugate preparation described in the embodiment 3, all shown comparability between two kinds of conjugates and the rabbit hyper-immuneserum and combined.The A of dLOS-TT ₄₀₅Value is 1.9, the A of dLOS-HMP ₄₀₅Value is 1.3.Under the similarity condition, the A of LOS (10 μ g/ml) ₄₀₅Numerical value is 1.1.

Next use animal model, promptly measure the interior immunogenicity of body of dLOS-protein conjugate with mice and rabbit.

The immunogenicity of Example 4:dLOS-protein conjugate in mice

5 all female mices in age (NIH/Swiss), every group 10～20, subcutaneous injection 5 g (based on sugar) are dissolved in the following material among the 0.2ml 0.9%NaCl: dLOS-TT, dLOS-HMP, LOS or dLOS add the mixture of TT or HMP (5 μ g protein), wherein contain or do not contain adjuvant.Contain 50 μ g monophosphoryl lipid As (MPL) and 50 μ g trehalose synthesis dicorynomycolate (STD) in the used adjuvant of per injection; it exists, and (trade name is Ribi-700 with a kind of inert carrier; available from Ribi Immunochemical Research, Hamilton, MT) in.With the interval in 3 weeks injection 3 times, the injection back is the 14th day for the first time, and second and third time injection back was given the mice blood-letting on the 7th day.

Serum is anti--and the LOS water-glass is shown ELISA unit (EU), with separating LOS from 25238 bacterial strains as envelope antigen.As reference, use the serum of anti-25238 strain whole-cells, the appointment numerical value of IgG wherein and IgM is respectively 65,000EU/ml and 800EU/ml.Measure the serum antibody of anti-TT or HMP with ELISA, wherein TT or HMP (5 μ g/mL) are as envelope antigen, and be that basic representation is an ELISA unit with reference mice serum (three injections TT or HMP produce), appointment numerical value with reference to IgG in the serum and IgM is respectively 2,000EU/ml and 10EU/ml.

For these results' statistical analysis, antibody horizontal is expressed as geometric average ELISA unit or titre (the mutual) ± standard deviation or the amplitude (n＜4) of n independent observation numerical value.Detect the measurement significance with bilateral t, the P value is considered as significantly less than 0.05.

As mark in 1 shown in the data, the non-coupling mixture of dLOS and TT or HMP can not excite and generate anti--LOS antibody.After the injection, two kinds of conjugates all excite and have generated low-level resisting-LOS IgG for the second time, but in not generation of injection back for the first time; After the injection, 50～100 times (P＜0.01) approximately raise for the third time.After 3 injections, dLOS-TT and dLOS-HMP excite the anti--LOS IgG that has generated similar level.LOS itself excites the anti--LOSIgG level of generation approximate with conjugate.

The goods of two kinds of conjugates and Ribi adjuvant have all strengthened their immunogenicity significantly: the conjugate that has 2 dosage of adjuvant excites the IgG of generation to be on close level or the IgG level when being higher than 3 dosage conjugates of independent use, anti--LOS IgG level after three injections raise about 9～15 times (P＜0.01).Be furnished with under the situation of adjuvant, after 3 injections, dLOS-TT excites the IgG level of generation to be lower than dLOS-HMP conjugate (P＜0.05).

With regard to IgM, conjugate excites and generates the anti--LOS that is low to moderate medium level after the per injection, and LOS excites the anti--LOS IgM that generates higher level after injection for the third time.The Ribi adjuvant can improve the anti--LOS IgM level in the conjugate group.The Mus source antibody response immunogen that table 1. conjugate excites at morazella catarrhalis LOS ^aBlood sample ^bGeometrical mean (± SD amplitude) ^c

ELISA unit

IgG IgMdLOS+TT 1 1 1

2 1 1(1-2)

3 3 5(1-15)dLOS+HMP 1 1 1

2 1 1(1-2)

3 1(1-2) 1dLOS-TT 1 1 1

2 5(1-19) 7(2-23)

3 52(6-447) ^** 17(3-91)dLOS-HMP 1 1 1(1-2)

2 2(1～8) ^* 6(2-19)

3 101 (15-691) ^*17 (5-63) dLOS-TF, 13 (1-11) ^*2 (1-4)+adjuvants 2 210 (48-923) ^*16 (62-396)

3 470 (266-828) 14 (8-25) dLOS-HMP, 13 (1-9) ^*9 (2-39)+adjuvants 2 101 (26-389) ^*22 (6-84)

3 1,514(299-7,658) 32(13-80)LOS 1 1 3(1-9)

2 8(2-40) ^* 2(1-4)

3 113 (20-630) ^*52 (12-230) a adds conjugate, LOS or the dLOS of Ribi adjuvant and the mixture of TT or HMP (each 5 μ g) with 5 μ g LOS, 5 μ g conjugates, 5 μ g, gives subcutaneous injection altogether three times to every group of 10～20 mices, is spaced apart for 3 weeks.B collects blood sample: 1, and two weeks of back of injection first; 2,1 week of back of injection for the second time; And 3,1 week of back of injection for the third time.C ELISA unit is based on the reference serum of antibiotic strain 25238, uses LOS from bacterial strain 25238 as envelope antigen.With ^*With ^*What the data pin of labelling was right is single immunogen, measurement result significant difference (P＜0.01).

Anti--protein antibodies in the mice.Shown in the data of listing in the table 2, dLOS-TT after injection for the first time, excite generate low-level anti--TT IgG antibody, this level significantly raise (P＜0.01) after second and third time injection.Add the IgG level that the Ribi adjuvant can improve the dLOS-TT group during injection.The mixture of TT and dLOS excites the IgG level of generation to be higher than the dLOS-TT group.All immunogens all excite and generate low-level resisting-TT IgM.

Also listed in the table 2 for anti--result that HMP antibody obtains.DLOS-HMP excites after injection for the first time and generates low-level IgG, this level significantly raise (P＜0.01) after second and third time injection.The Ribi adjuvant has improved the IgG level of dLOS-HMP group.The mixture of HMP and dLOS excites the IgG level of generation to be higher than dLOS-HMP.All immunogens all excite and have generated low-level resisting-HMP IgM.The Mus source antibody response immunogen a blood sample that table 2. conjugate excites at protein (TT or HMP) ^bGeometrical mean (± SD amplitude)

ELISA unit

IgG IgM

Anti--TTdLOS-TT 11 (1-2) 1

2 34(21-54) 1

3 90 (35-237) 2 (1-3) dLOS-TT 1 14 (6-35) 3+ adjuvants, 2 303 (191-481) 11 (7-18)

3 2,430 27(13-56)dLOS+TT 1 11(7-18) 1

2 303(191-481) 1(1-2)

3 729 2(1-3)

Anti--HMPdLOS-HMP 111

2 2(1-8) 1

3 11 (3-43) 1dLOS-HMP, 14 (2-9) 2 (1-6)+adjuvant 2 52 (30-92) 7 (3-17)

3 810(389-1,685) 11(7-18)dLOS+HMP 1 2(1-6) 2(1-3)

2 377(149-952) 2(1-3)

31,403 (645-3,051) 10 (7-14) a adds the conjugate of Ribi adjuvant or the mixture of dLOS and TT or HMP (each 5 μ g) with 5 μ g LOS, 5 μ g conjugates, 5 μ g, gives subcutaneous injection three times to every group of 10～20 Mus, is spaced apart for 3 weeks.B collects blood sample: 1, and two weeks of back of injection first; 2,1 week of back of injection for the second time; And 3,1 week of back of injection for the third time.

Embodiment 5:dLOS-protein conjugate is in the intravital immunogenicity of rabbit

By the mixture (each component is 50 μ g) that the amount of 50 μ g/ injection is injected subcutaneous and 2 following material: LOS of intramuscular, contained or do not contain the dLOS-TT conjugate of adjuvant, the dLOS-HMP conjugate, the dLOS that contain or do not contain adjuvant adds TT or HMP, double injection was spaced apart for 4 weeks to rabbit (2～3 every group).Do not contain the injected material of adjuvant for all, immunogen is dissolved among the 1mL 0.9%NaCl.For the injected material that contains adjuvant, immunogen is dissolved among the 1mL 0.9%NaCl, wherein contain 250 μ g monophosphoryl lipid As and 250 μ g trehalose dimycolates (the Ribi-700 adjuvant, Ribi Immunochemical Research, Hamilton, MT).In 2 weeks after the per injection, from ear vein, gather the blood sample of 10～20ml with conventional method.

Shown in the result that table 3 is listed, behind double injection, the mixture of LOS, TT and HMP or LOS excite and have generated low-level resisting-LOS IgG or IgM antibody.The dLOS-TT conjugate excites the anti--LOSIgG (higher 37 times and 700 times than preimmune serum) that has generated remarkable elevated levels after first and second injections.The IgG level that dLOS-HMP shows is lower than dLOS-TT (higher 6 times and 347 times than preimmune serum respectively).The Ribi adjuvant has improved the anti--LOS IgG level (higher 40 times and 2000 times than preimmune serum respectively) after two kinds of conjugate per injections.After 2 injections, there is not significant difference between the replying of this two kinds of conjugates.

With regard to IgM, two kinds of conjugates all excite and have generated the anti--LOS antibody that is low to moderate medium level, are added with all to excite after the conjugate per injection of Ribi adjuvant to have generated the anti--LOS antibody that is low to moderate medium level.Rabbit source antibody response immunogen a blood sample b geometrical mean (amplitude) the ELISA unit that table 3. conjugate excites at morazella catarrhalis LOS

IgG IgMLOS 0 6(3-10) 6(3-10)

1 10(3-30) 52(30-90)

2 52(30-90) 90dLOS-TT 0 5(3-10) 14(10-30)

1 187(90-270) 90

23,505 (2,430-7,290) 187 (90-187) dLOS-TT 0 10 5 (3-10)+adjuvants 1 810 (270-2,430), 389 (270-810)

2 21,870 270(90-810)dLOS-HMP 0 7(3-10) 10

43(30-90) 30(10-90)

22,430 90 (30-270) dLOS-HMP, 0 10 (3-30) 7 (3-10)+adjuvants 1 389 (30-2,430), 90 (30-270)

2 21,870 270(90-810)dLOS+TT+HMP?0 10(3-30) 17(10-30)

1 10(3-30) 30

2 52 (30-90) 30 full cells 06 (3-10) 10

1 270 156(90-270)

2 65,610 49 (3-810) a is adding the conjugate of Ribi adjuvant or the mixture of dLOS and TT or HMP (each 50 μ g) with 50 μ g LOS, 50 μ g conjugates, 50 μ g, carry out subcutaneous and intramuscular injection is immune after 0 o'clock and one month to 2～3 rabbits in every group.The hyper-immuneserum of anti-morazella catarrhalis is described in previous section of the present invention.B collects blood sample: 0, before injecting immune is former; 1, injected back 14 days for the first time; And 2, injected back 14 days for the second time.

Anti--protein antibodies of rabbit.Shown in the data of relevant anti--TT antibody of listing in the table 4, dLOS-TT excites after 2 injections and has generated remarkable high-caliber IgG (higher 389 times than preimmune serum level), and the Ribi adjuvant makes dLOS-TT excite the IgG level of generation to improve 4 times after 2 injections.The mixture of dLOS and TT excites the anti--TT IgG level of generation to be higher than the dLOS-TT conjugate, particularly after injection for the first time.All immunogens all excite and generate low-level resisting-TT IgM.

Also listed the data of anti--HMP antibody in the table 4, dLOS-HMP excites the IgG (higher 81 times than preimmune serum level) that generates significant level after 2 injections, adds the Ribi adjuvant and makes dLOS-HMP excite the IgG level of generation to improve 4 times after 2 injections.HMP and dLOS mixture excite the IgG level of generation to be higher than dLOS-HMP, particularly after injection for the first time.All immunogens all excite and have generated low-level resisting-HMP IgM.Rabbit source antibody response immunogen a blood sample b geometrical mean (amplitude) the ELISA unit that table 4. conjugate excites at morazella catarrhalis LOS

IgG IgM

Anti--TTdLOS-TT 033

1 7(3-10) 10

21,168 (810-2,430) 14 (10-30) dLOS-TT 05 (3-10) 3+ adjuvants, 1 14 (10-30) 14 (10-30)

2 5,055(2,430-21,870) 10(3-30)dLOS+TT+HMP 0 10 10

1 270 90

2 2,430 30

Anti--HMPdLOS-HMP 0 10 (3-30) 7 (3-10)

1 14(10-30) 10

2 810 10dLOS-HMP 07 (3-10), 10 (3-30)+adjuvants 1 130 (30-270) 14 (10-30)

2 3,505(2,430-7,290) 30dLOS+TT+HMP 0 10 10

1 156(90-270) 30

22,430 52 (30-90) a is adding the conjugate of Ribi adjuvant or the mixture of dLOS and TT or HMP (each 50 μ g) with 50 μ gLOS, 50 μ g conjugates, 50 μ g, carry out subcutaneous and intramuscular injection is immune after 0 o'clock and one month to 2～3 rabbits in every group.The serum of exempting from of anti-morazella catarrhalis is described in previous section of the present invention.B collects blood sample: 0, before injecting immune is former; 1, injected back 14 days for the first time; And 2, injected back 14 days for the second time.Embodiment 6: the bactericidal activity of the animal serum of anti-morazella catarrhalis strain

Among this embodiment, tested among the embodiment 4 and 5 bactericidal activity of the animal serum of preparation at following bacterial strain: (ATCC 25238 to be used to separate the bacterial strain of the same race of LOS; " homology strain ") and other wild strains (" allos strain ") of morazella catarrhalis.(the ATCC preserving number is 8176,8193,23246 to 11 morazella catarrhalis wild-type strains, 25238,25239,25240,43617,43618,43627,43628 and 49143) (available from US mode culture collection center, Rockville, MD), and 10 Japanese clinical separation strains that are separated in otitis media or the respiratory tract infection patient body (called after M1～M10) (Goro Mogi is so kind as to give, and Oita Medical University Japan) tests.One of ordinary skill in the art will appreciate that, can from clinical sample, isolate the other bacterial strain of morazella catarrhalis and similarly test with conventional biology techniques.

For bactericidal activity test, with before the rabbit immunity and immunity back (2 injection backs) serum 56 ℃ of incubations 30 minutes, thereby deactivation complement component wherein.Use the bactericidal assay of complement-mediated then, the test inactivated serum is at the bactericidal activity of morazella catarrhalis, test method with basic identical (Gu, X.X. are described before this; Et al., 1996, Infect.Immun.64:40474053), difference is to use guinea pig serum (dilution in 1: 1, every hole 20 μ l) as complement source (Sigma, St.Louis, MO) and before the agar plate bed board with reactant mixture 37 ℃ of following incubations 30 minutes.Cause the highest serum dilution factor of 50% above kill ratio to be expressed as mutual sterilization titre.

In the mouse model, after injecting 3 dLOS-TT or dLOS-HMP, 20% conjugate immune serum (having 4 in 20 mices), and 45% conjugate (adding adjuvant) immune serum (having 9 in 20 mices) have shown the low titre bactericidal activity at homologous strain.

Fig. 1 shows to scheme to show, in the rabbit model, do not show bactericidal activity from the serum of the animal of LOS (or dLOS) immunity at homology morazella catarrhalis strain (ATCC 25238), on the contrary, it is the bactericidal activity of 1: 16 (not adding adjuvant) and 1: 40 (adding adjuvant) that the dLOS-TT immune serum has but shown average titer, and the dLOS-HMP-immune serum shows the bactericidal activity that average titer is 1: 10 (not adding adjuvant) and 1: 40 (an adding adjuvant).(r=0.60, P=0.02), but the IgM level is not associated to have dependency relation between LOS IgGELISA level and the sterilization titre.

Use other 10 wild types (ATCC bacterial strain) and 10 Japanese clinical separation strains, excite the bactericidal activity of the rabbit anti-serum of generation further to measure the dLOS-TT that has prepared the Ribi adjuvant.In 20 bacterial strains 10 be the complement sensitivity (bacterial strain 23246,43617, M9) or serum sensitivity (bacterial strain 43627,43628,49143, M4, M7, M8, M10) bacterial strain.When using remaining 10 bacterial strain, when average titer was 1: 15 (scope is 1: 2～1: 32), rabbit anti-serum showed the bactericidal activity at 4 ATCC and 5 Japanese clinical separation strains.There is 1 bacterial strain (ATCC 25240) to be negative in this bactericidal assay.The present invention is used for the cross matching of exhibit bactericidal activity and shows that the conjugate that can be prepared from the LOS by different strains of relative minority makes a kind of vaccine (seeing the 21st page, the bottom) that protection can be provided for all morazella catarrhalis toxic strains.Embodiment 7: mouse lung is removed the passive protection test of model

With 40 mices of anti-dLOS-TT rabbit anti-serum or preimmune serum immunity, back 18 hours then in immunity, with 10 ⁸Cfu morazella catarrhalis bacterial strain 25238 is attacked by the aerosol chamber.After attack, kill mice (Fig. 2) 3 and 6 hours the time.Gathering lung tissue and blood sample is used for analyzing.The results are shown in Figure 3.In attack back 3 hours, the amount of bacteria in the vaccine group was compared with matched group and has been reduced 50%.In attack back 6 hours, vaccine group was compared with matched group and has been reduced 61%.At each time point, all there is notable difference between matched group and the vaccine group.Go back the antagonist level in addition and analyze, antibody horizontal is relevant with antibacterial cfu conversely.These results show that dLOS-TT conjugate inductive antibody strengthened the pulmonary of morazella catarrhalis in the mice and removed.Embodiment 8: with morazella catarrhalis dLOS-protein conjugate immunity people

At first, select adult's test its safety and immunogenicity for the OS-carrier conjugates for preparing among dLOS-carrier conjugates for preparing in embodiment 1 and 2 or the embodiment 9-11.Screening endogenous antibody (for example owing to the childhood infection morazella catarrhalis generates) individuality that level is low is relatively selected to compare relative low-level adult with total population and is used for research.(25 μ g～50 μ g, view volume is heavily decided) carry out intramuscular injection to these individualities with the dLOS-TT conjugate that is dissolved in pharmacological-acceptable carrier, dLOS-HMP conjugate, OS-TT conjugate or OS-HMP conjugate.For the adult, injecting to excite usually for 1 time to generate is enough to show the antibody response in 3 days～2 weeks.With the basic identical method of in embodiment 4～6, describing, measure the immunogenicity and the bactericidal activity of the serum that obtains.For the first time the injection back was carried out injection second time in about 1～6 month, after this 1 week measured serum to resist-level of morazella catarrhalis antibody.The same dosage of the individual injection of contrast be dissolved in corresponding protein composition (separately) in the above-mentioned conjugate in the pharmacological-acceptable carrier of the same race, and be grown up according to same infusion protocol immunity.

For the individuality of accepting dLOS-carrier conjugates or OS-carrier conjugates, the serum antibody level after the immunity shows that these conjugates have immunogenicity in the body and do not produce unacceptable side-effects.Bactericidal activity is with relevant from these individual serum antibodys.With regard to these are individual, preferably carry out repeatedly immunity to reach best immunne response.On the contrary, the bactericidal activity of the anti--morazella catarrhalis of serum before the serum that obtains from the contrast individuality of accepting the contrast injection does not show any immunogenicity and is higher than its injection.That is to say, compare that the antiserum of accepting the adult of dLOS-TT conjugate, dLOS-HMP conjugate, OS-TT conjugate or OS-HMP conjugate shows obviously high immunogenicity or anti--morazella catarrhalis bactericidal activity with matched group.In addition, the occurrence frequency of these individual middle ear infections of monitoring during this period of time, is used dLOS-TT in the several years, dLOS-HMP, and middle ear infection takes place in none example of adult that OS-TT or the immunity of OS-HMP conjugate are crossed.

On the antiserum result's that the adult obtains basis, expand vaccine test to child, the vaccine of intramuscular injection predose between 2～4 monthly ages (the amount view volume is heavy and decide, be generally 10～40 μ g), carry out twice booster shot 2～April after initial immunity.One group of child accepts 3 booster shot when being received in initial immunity back 2,4 and June.Do not accept suitable child of age of vaccine in contrast.Monitoring immunity back child's serum levels, and monitoring according to the method described above, the result shows to have immunogenicity and bactericidal activity simultaneously.From be inoculated into for the first time about 4 years old during this period of time in, these children are carried out otitis media and sinusitis is measured.Otitis media and sinusitis take place in child hardly that accept inoculation, even during this period of time monitor otitis media and/or sinusitis, its symptom is compared also lighter with matched group.Embodiment 9: acid hydrolysis morazella catarrhalis 25238 preparation oligosaccharide (OS)

Handle LOS with weak acid, and the lipid A from Kdo-glycosamine key excision LOS molecule (be Gu, X.X. ， ﹠amp; C.M.Tsai, 1993, the method described in the Infect.Immun.61:1873-1880), to morazella catarrhalis LOS detoxification.Briefly, with 421mg LOS be dissolved in the distilled water to concentration be 10mg/ml, 100 ℃ of hydrolysis 2～3 hours in 1% acetic acid then.150,000xg removed lipid A part and hydrolysis LOS not in centrifugal 3 hours.The supernatant lyophilizing is dissolved in the water of small size, is divided into two, and every part all (is carried out chromatography at the Sephadex G-25 post of crossing with 25mM ammonium acetate balance on 1.6 * 90cm).Analyze sugared content, the main OS fraction of merging in the eluent, lyophilizing removes freshen three times.By weight, the productive rate of OS from initial LOS is 47%.

Embodiment 10: with ADH OS is carried out derivation

According to before this description (Gu et al., 1993, above-mentioned), by with EDC and sulfo group-NHS by the condensation reaction of carbodiimides mediation, ADH is coupled on the OS.With Bio-Gel P-4 post (1.6 * 90cm; Bio-Rad, Hercules, CA) purification reaction mixture.Analyze sugar and AH content in the eluent.Merge the peak that contains sugar and AH simultaneously, lyophilizing removes salinity 3 times.The ratio of OS and ADH is about 1.The productive rate of sugar is about 74% in the AH-OS derivant.Embodiment 11:AH-OS and proteinic coupling

Under the condition of pH5.2 ± 0.2, carry out coupling reaction with 0.1M EDC.With regard to the OS-TT reaction, 30mg AH-OS is dissolved in the 3ml distilled water, and mixes with 15mg TT (5.90mg/ml).With regard to the OS-HMP reaction, 20mg AH-OS is dissolved in the 2ml water, and mixes with 8.4mg HMP (4.2mg/ml).Mol ratio between AH-OS and TT or the HMP is 150 or 141 to 1 (based on molecular weight: OS are 2,000, and TT is 150,000, and HMP is 120,000).Identical among purification step and the embodiment 2 to the description of dLOS-protein conjugate.These OS-protein conjugates have and the identical purposes of above-mentioned dLOS-protein conjugate, i.e. the vaccine that infects as people's moraxella catarrhalis (Branhamella) bacterium.The composition of OS-TT and OS-HMP conjugate, productive rate and antigenicity see Table 5.Composition, productive rate and the antigenicity of table 5.OS conjugate

Amt (ug/ml) mol ratio ^aProductive rate ^bA ₄₀₅ ^cConjugate OS albumen OS/ albumen (%) hyper-immuneserum OS-TT1 23 157 11 2.4 1.1OS-TT2 43 152 21 6.1 0.6OS-HMP1 47 182 16 8.8 2.0OS-HMP2 32 112 17 4.5 1.4a. schedules of proportion are shown the OS molal quantity of every mole of albumen correspondence, dLOS, TT, HMP molecular weight are respectively 2,000,150,000 and 120,000.B. measure based on the primary quantity of OS and the OS content in the conjugate with micro-phenol-sulfuric acid method.C. use conjugate as envelope antigen (10 g/ml) and rabbit hyper-immuneserum during as binding antibody (1/8,000 dilution factor), the antigenicity of conjugate is expressed as A ₄₀₅The ELISA of place response value, under similarity condition, the A that LOS (10ug/ml) shows ₄₀₅Numerical value is 1.1.

The OS-protein conjugate all excites the antibody response that has generated anti-LOS in mice and rabbit.Protein conjugate also all excites the antibody that has generated anti-TT and HMP in mice and rabbit.Excite the Mus source antibody response at morazella catarrhalis bacterial strain 25238 LOS of generation to be shown in table by conjugate.Female mice (NIH/Swiss), every group 10, subcutaneous injection 5 g (calculating by carbohydrate) are dissolved in the following material among the 0.2ml 0.9%NaCl: OS-TT, OS-HMP, LOS or OS add the mixture of TT or HMP (5 μ g protein), and they contain or do not contain adjuvant.The adjuvant that uses is identical with the dLOS antibody response.Inject 3 times, be spaced apart for 3 weeks, the injection back is the 14th day for the first time, and second and third time injection back was given the mice blood-letting on the 7th day.

Serum is anti--and the LOS water-glass is shown ELISA unit (EU), with separating LOS from 25238 bacterial strains as envelope antigen.As reference, use the hyper-immuneserum of anti-25238 strain whole-cells, the appointment numerical value of IgG wherein and IgM is respectively 65,000EU/ml and 800EU/ml.According to the method that the dLOS conjugate is described, measure the serum antibody of anti-TT or HMP with ELISA.For these results' statistical analysis, antibody horizontal is expressed as geometric average ELISA unit or the titre ± standard deviation or the amplitude (n＜4) of n (mutual) independent observation numerical value.Detect the measurement significance with bilateral t, P is considered as significantly less than 0.05.

Shown in data in the table 6, the non-coupling mixture of OS, TT and HMP does not excite and generates anti--LOS antibody.After the injection, two kinds of conjugates all excite and have generated low-level resisting-LOS IgG, but are not exciting after the injection for the first time for the second time; After the injection, slightly raise (P＜0.01) for the third time.After 3 injections, OS-TT and OS-HMP excite the anti--LOSIgG that has generated similar level.After injecting 2 times, LOS itself excites the anti--LOS IgG level of generation approximate with conjugate; But after injection for the third time, LOS itself excites the level of generation to be significantly higher than conjugate.The goods that two kinds of conjugates add behind the adjuvant have all strengthened their immunogenicity significantly.Table 6. conjugate excites the Mus source antibody response at morazella catarrhalis bacterial strain 25238 LOS of generation

GM (± SD amplitude) ELISA unit ^bImmunogen ^aFrequency injection IgG IgMOS-TT1 111

2 6(1-30) 2(1-3)

34 (1-35) 5 (1-46) OS-TT1,12 (1-3) 3 (1-9)+adjuvant 24 (1-22) 3 (1-9)

3 7(148) 50(12-209)OS-TT2 1 1 2(1-6)

2 1 4(1-14)

38 (1-70) 7 (2-28) OS-IT2,11 (1-2) 2 (1-5)+adjuvant 2 34 (5-249) 19 (6-62)

3 113(13-957) 126(39-409)OS-HMP1 1 1 1

2 2(1-4) 2(1-5)

35 (1-72) 4 (1-40) OS-HMP1,11 (1-2) 2 (1-9)+adjuvant 223 (1-9)

3 90(5-1,585) 81(26-257)OS-HMP2 1 1 1

2 1(1-2) 4(1-15)

33 (1-13) 7 (2-25) OS-HMP2,117 (2-25)+adjuvants 26 (1-26) 34 (12-92)

3 38(8-176) 195(46-828)OS+TT+HMP 1 1 1

2 1 1

3 1(1-2) 3(2-5)LOS 1 1 3(1-9)

2 8(2-40) 2(1-4)

3 113 (20-630) 52 (12-230) a mices, every group 10,3 times 5 following materials of g of subcutaneous injection: the mixture (each 5 μ g) of conjugate, LOS or OS, TT and the HMP of conjugate, adding Ribi adjuvant was spaced apart for 3 weeks.In 2 weeks of back of injection for the first time, 1 week of second and third time injection back is gathered the mice blood sample.B ELISA unit based on antibiotic strain 25238 with reference to serum, use LOS from bacterial strain 25238 as envelope antigen.Preimmune serum contains 1 (1-2) U IgG and 1U IgM.

Provided the Mus source antibody response that the OS-TT conjugate excites generation in the table 7 at TT.The IgG antibody response is better than the IgM antibody response.Adjuvant has strengthened the immunne response of OS-TT2 significantly, but not as obvious to the amplitude of OS-TT 1 increase.Table 7.OS-TT conjugate excites the Mus source antibody response at TT of generation

GM (± SD amplitude) ELISA unit ^bImmunogen ^aFrequency injection IgG IgMOS-TT1 12 (1-3) 1

2 22(7-72) 1

3 90 (34-237) 1OS-IT1,14 (1-9) 4 (2-6)+adjuvant 2 126 (69-229) 5 (3-9)

3 90(64-128) 22(9-51)OS-TT2 1 95(33-269) 2(1-3)

2 52(9-296) 2(1-4)

3 140 (12-1,614) 5 (2-11) OS-TT2,1 113 (66～191) 3 (1-5)+adjuvant 2 271 (81-908) 19 (9-41)

3 1,756(454-6,769) 90(48-169)OS+TT+HMP 1 14(8-25) 1(1-2)

2 470(257-851) 2(1-6)

3 908 (332-2,487) 14 (5-40) a see Table 6 footnote ab ELISA units based on anti-TT with reference to serum, as envelope antigen, preimmune serum shows the IgG or the IgM of＜1 unit with TT.

Provided the Mus source antibody response that the OS-HMP conjugate excites generation in the table 8 at HMP.With regard to the TT antibody response, the IgG antibody horizontal obviously raises, and the IgM level is still very low.Add adjuvant and strengthened the IgG antibody response widely, exceed much than the TT antibody response of listing in the table 7.Table 8.OS-HMP conjugate excites the Mus source antibody response at HMP of generation

GM (+SD amplitude) ELISA unit ^bImmunogen ^aFrequency injection IgG IgMOS-HMP1 131 (1-2)

2 65(40-105) 14(5-37)

3 183 (83-398) 8 (3-21) OS-HMP1,19 (2-34) 2 (1-3)+adjuvants 2 585 (330-1,442), 5 (3-8)

3 1,506(507-4,464) 6(3-12)OS-HMP2 1 9(4-19) 3(2-5)

2 81(39-168) 4(2-12)

3 175 (34-902) 2 (1-6) OS-HMP2,1 81 (45-146) 6 (3-14)+adjuvant 21,131 (642-1,997) 17 (8-38)

3 4,228(2,600-7,454) 38(12-120)OS+TT+HMP 1 2(1-4) 1

2 470(257-851) 3

31,573 (752-3,281) 34 (11-104) a see Table 6, footnote ab ELISA unit based on anti-HMP with reference to serum, as envelope antigen, preimmune serum shows the IgG or the IgM of 1～3 unit with HMP.

Provided the rabbit source antibody response that conjugate excites generation in the table 9, provided the rabbit source antibody response that conjugate excites generation in the table 10 at TT or HMP at morazella catarrhalis LOS.Table 9. conjugate excites the rabbit source antibody response at morazella catarrhalis LOS of generation

GM (± SD amplitude) ELISA unit ^bImmunogen ^aFrequency injection IgG IgMOS-TT2 0 22 (3-90) 7 (3-30)

1 140(30-810) 52(30-90)

2 7,290(2,430-21,870) 68(30-90)OS-TT2+Ribi 0 4(3-10) 10(3-30)

1 729(270-2,430) 156(90-270)

2 12,627(7,290-21,870)?90OS-HMP2 0 17(10-30) 7(3-30)

1 467(90-2,430) 10(3-30)

2 7,290(2,430-21,870) 90(30-270)OS-HMP2+Ribi 0 9(3-30) 7(3-30)

1 810 30(10-90)

2 12,627(7,290-21,870)?118(90-270)OS+TT+HMP 0 5(3-10) 17(10-30)

1 5(3-10) 5(3-10)

2 38(3-90) 17(10-30)LOS 0 6(3-10) 6(3-10)

1 10(3-30) 52(30-90)

2 52 (30-90) 90a rabbit, every group 2～3, the immune 2 times 50 following materials of μ g of subcutaneous and intramuscular injection: the mixture (each 50 μ g) of conjugate, LOS or OS, TT and the HMP of conjugate, adding Ribi adjuvant is spaced apart for 1～February.Reach back 14 days blood sample collections of injection before the per injection.B sees Table 6, footnote b.Table 10. conjugate excites the Mus source antibody response at protein (TT or HMP) of generation

GM (± SD amplitude) ELISA unit ^bImmunogen ^aFrequency injection IgG IgM is anti--the analysis OS-TT2 0 10 10 of TT

1 270 17(10-30)

2 4,209(2,430-7,290) 118(90-270)OS-TT2+Ribi 0 17(10-30) 10

1 2,430 90

2 21,870 355(90-810)OS+TT+HMP 0 30 17(10-30)

1 468(270-810) 155(90-270)

The analysis OS-HMP2 0 52 (30-90) 17 (10-30) of 22,430 90 anti--HMP

1 118(90-270) 30

2 2,430 52(30-90)OS-HMP2+Ribi?0 30 17(10-30)

1 468(270-810) 30

2 7,290 30OS+TT+HMP 0 52(30-90) 17(10-30)

1 270 30

22,430 52 (30-90) a sees Table 9 footnote ab and sees Table 7 and 8 footnote b

After giving 3 OS-TT of injected in mice or OS-HMP conjugate, there is not a bactericidal activity that shows at homology morazella catarrhalis bacterial strain 25238 in OS protein conjugate-immune serum, and 40% in conjugate (adding adjuvant)-immune blood (20 mice in 8) has shown the bactericidal activity at homology morazella catarrhalis bacterial strain 25238, and scope is 1: 8～1: 64.To 2 OS-TT of rabbit injection or after adding the OS-HMP conjugate of adjuvant, 37.5% conjugate-immune serum (8 rabbit in 3) shows the bactericidal activity at homology morazella catarrhalis bacterial strain 25238, and scope is 1: 2～1: 8.

Though the present invention is described in detail particular,, those skilled in the art can understand that these technical schemes just illustrate and are not construed as limiting, true scope of the present invention is limited by following claim.

Claims

1. morazella catarrhalis coupling vaccine, comprising a kind of fat oligosaccharide (LOS) and a kind of immunogenic carrier covalently bound that separates from morazella catarrhalis with it, described fat oligosaccharide is handled the LOS (dLOS) that is generated as a kind of detoxification by removing esterified fatty acid, perhaps is generated as a kind of oligosaccharide (OS) by removing lipid A.

2. the vaccine of claim 1, wherein said carrier is a protein.

3. the vaccine of claim 2, wherein said immunogenic carrier albumen is selected from: separate UspA from morazella catarrhalis, separate CD, tetanus toxin/toxoid from morazella catarrhalis, separate high-molecular-weight protein (HMP) from non-acquisition type hemophilus influenza, pseudomonal toxin A, cholera toxin/toxoid, pertussis toxin, PT/toxoid, clostridium perfringens extracellular toxin/toxoid, hepatitis B surface antigen, hepatitis B virus core antigen, rotavirus VP 7 albumen, CRM, CRM behind the diphtheria toxin, diphtherotoxin/toxoid, detoxification ₁₉₇, CRM ₃₂₀₁And breathing syncystial virus F and G albumen.

4. the vaccine of claim 3, wherein said immunogenic carrier albumen is tetanus toxoid or HMP.

5. Pharmaceutical composition is comprising the described vaccine conjugate of claim 1 that can accept on materia medica in the carrier.

6. the Pharmaceutical composition of claim 5 wherein further comprises a kind of adjuvant.

7. the Pharmaceutical composition of claim 6, wherein said adjuvant is the mixture for monophosphoryl lipid A and trehalose dimycolate or Alumen.

8. the coupling vaccine of claim 1, wherein said immunogenic carrier is covalently bound by a kind of linker compounds and dLOS or OS.

9. the coupling vaccine of claim 8, wherein said joint thing is selected from adipic dihydrazide, episilon amino caproic acid, Mecoral dimethyl-acetal, D-glucuronolactone and p-nitrophenyl ethamine.

10. the coupling vaccine of claim 8, wherein said linker compounds is an adipic dihydrazide.

11. separate from morazella catarrhalis and by removing the fat oligosaccharide of esterified fatty acid detoxification wherein.

12. the fat oligosaccharide of claim 11, the wherein said morazella catarrhalis that is used to separate described fat oligosaccharide is the purification bacterial strain of morazella catarrhalis.

13. separate from morazella catarrhalis and by removing the fat oligosaccharide of lipid A detoxification wherein.

14. one kind is used for preventing mammal to be infected the method for the otitis media that causes by morazella catarrhalis; comprising coupling vaccine from effective immunoprotection amount to mammal that inoculate; comprise in this coupling vaccine and a kind ofly deriving and the detoxification fat oligosaccharide (dLOS) or the oligosaccharide (OS) that come by separating from the fat oligosaccharide of morazella catarrhalis, and a kind of immunogenic carrier that is covalently attached on the above-mentioned dLOS.

15. the method for claim 14, wherein said mammal is behaved.

16. the method for claim 14, wherein said coupling vaccine is inoculated through the parenteral route approach.

17. the method for claim 16, the route of inoculation of wherein said coupling vaccine are the combination of intramuscular injection, subcutaneous injection or intranasal mucosa deposition or these methods.

18. the method for claim 14, wherein said effective immunoprotection amount are every dose of about 10 μ g～50 μ g.

19. the method for claim 14 wherein further comprises with about 10 μ g～about 25 μ g conjugate booster injection.

20. the method for claim 14, wherein said inoculation step comprises the about 1 μ g of inoculation first dosage～50 μ g coupling vaccines, inoculate the about 10 μ g of second dosage～25 μ g coupling vaccines after then bimester that first dosage is about, the bimester making an appointment with behind second dosage, inject the about 10 μ g of the 3rd dosage～25 μ g coupling vaccines then, then about 12 months about 10 μ g of injection the 4th dosage～25 μ g coupling vaccines behind the 3rd dosage.

21. one kind to separating the method for carrying out detoxification from the fat oligosaccharide of morazella catarrhalis, comprising the esterified fatty acid of removing among the isolating LOS.

22. the method for claim 21, removing by handling LOS with hydrazine or weak base reagent of wherein said esterified fatty acid realizes.

23. a method for preparing morazella catarrhalis coupling vaccine, comprising:

Remove the LOS (dLOS) after the esterified fatty acid of separation in the fat oligosaccharide (LOS) of morazella catarrhalis obtains detoxification; And

DLOS is covalently bound to immunogenic carrier.

24. the method for claim 23, wherein said removing comprises in the step with hydrazine or weak base reagent and handles described LOS.

25. the method in the claim 23, wherein said Connection Step comprise dLOS is connected on a kind of linker compounds, and this linker compounds is connected on the immunogenic carrier.

26. the method for claim 25, wherein said linker compounds are adipic dihydrazide, episilon amino caproic acid, Mecoral dimethyl-acetal, D-glucuronolactone or to nitro-ethyl amine.

27. the method for claim 25, wherein said linker compounds is an adipic dihydrazide.

28. the method for claim 23 wherein further comprises among the dLOS on being connected to immunogenic carrier adding adjuvant.

29. a method for preparing morazella catarrhalis coupling vaccine, comprising:

Remove the lipid A of separation in the fat oligosaccharide (LOS) of morazella catarrhalis and obtain oligosaccharide (OS); And

OS is covalently bound to immunogenic carrier.

30. the method for claim 29, the wherein said step of removing comprises and uses acid treatment LOS.

31. the method for claim 29, wherein said Connection Step comprise OS is connected on a kind of linker compounds, and this linker compounds is connected on the immunogenic carrier.

32. the method in the claim 31, wherein said linker compounds are adipic dihydrazide, episilon amino caproic acid, Mecoral dimethyl-acetal, D-glucuronolactone or to nitro-ethyl amine.

33. the method for claim 32, wherein said linker compounds is an adipic dihydrazide.

34. the method for claim 29 wherein further comprises among the OS on being connected to immunogenic carrier adding adjuvant.

35. coupling vaccine, comprising a kind of fat oligosaccharide (LOS) and a kind of immunogenic carrier covalently bound that separates from morazella catarrhalis with it, described fat oligosaccharide is generated as a kind of LOS (dLOS) of detoxification or is generated as a kind of oligosaccharide (OS) by removing lipid A by removing the esterified fatty acid processing, and this vaccine can be used for preventing the otitis media of mammal morazella catarrhalis infection-induced.

36. the vaccine of claim 35, wherein said immunogenic carrier is a protein.

37. the vaccine of claim 36, wherein said immunogenic carrier albumen is selected from: separate UspA from morazella catarrhalis, separate CD, tetanus toxin/toxoid from morazella catarrhalis, separate high-molecular-weight protein (HMP) from non-acquisition type hemophilus influenza, pseudomonal toxin A, cholera toxin/toxoid, pertussis toxin, PT/toxoid, clostridium perfringens extracellular toxin/toxoid, hepatitis B surface antigen, hepatitis B virus core antigen, rotavirus VP 7 albumen, CRM, CRM behind the diphtheria toxin, diphtherotoxin/toxoid, detoxification ₁₉₇, CRM ₃₂₀₁And breathing syncystial virus F and G albumen.

38. the vaccine of claim 37, wherein said immunogenic carrier albumen is tetanus toxoid or HMP.