CN101208101B - Immunogenic composition - Google Patents

Abstract

The present application discloses an immunogenic composition comprising a Hib saccharide conjugate and at least two further bacterial saccharide conjugates wherein the Hib conjugate is present in a lower saccharide dose than the mean saccharide dose of all the at least two further bacterial saccharide conjugates.

Description

Immunogenic composition

The present invention relates to immunogenic composition and vaccine, it comprises Hib glycoconjugate and at least a extra antibacterial glycoconjugate, itself and other antigen combinations that comprise whole cell pertussis and/or hbs antigen, relate to the method for this type of immunogenic composition of preparation and vaccine, and use purposes and the method for described immunogenic composition and vaccine immunity.

Shown that bacterial polysaccharides is the effective immunogen for vaccine, especially when being conjugated to carrier protein.Can obtain anti-b type hemophilus influenza (Hibtiter

Wyeth-Lederle), pneumococal polysaccharide (Prevnar -Wyeth-Lederle) and meningococcal polysacharide (Meningitec -Wyeth-Lederle and Menactra

-Sanofi) commercial conjugate vaccines.

The immunogenic composition and the vaccine that comprise Hib conjugate, Neisseria meningitidis glycoconjugate, whole cell pertussis and hbs antigen have also been described.For example, WO02/00249 discloses 7 valency immunogenic compositions, and it comprises diphtheria toxoid, tetanus toxoid, whole cell pertussis, hbs antigen, Hib conjugate, MenA conjugate and MenC conjugate.The clinical test results of presenting uses every kind of antibacterial glycoconjugate of 5 μ g sugar dosage.

The present invention relates to provide combination-vaccine, it comprises Hib conjugate, extra antibacterial glycoconjugate (for example, the Neisseria meningitidis glycoconjugate) and other antigens, comprises in whole cell pertussis and the hbs antigen one or both.The sugared dosage permission anti-Hib of generation that uses and extra antibacterial sugar and the good immunne response of anti-pertussis component and/or hbs antigen component.

The inventor it has surprisingly been found that in the combination-vaccine that comprises whole cell pertussis and/or hbs antigen, sugared dosage by reducing Hib and/or extra antibacterial conjugate is to being lower than every dose of 5 μ g, maintenance is to the immunogenicity of the good immunne response of glycoconjugate and whole cell pertussis and hbs antigen be enhanced (for example, so that as the GMC of the Pw that measures by ELISA and/or hepatitis B be higher than the level that the immunogenic composition immunity with the every kind of conjugate that contains 5 μ g sugar dosage realizes afterwards).

Therefore, a first aspect of the present invention provides immunogenic composition, it comprises Hib glycoconjugate, at least a extra antibacterial glycoconjugate, and is selected from another antigen of whole cell pertussis and hbs antigen, and wherein the sugared dosage of Hib glycoconjugate is less than every dose of 5 μ g.

Describe in detail

Immunogenic composition of the present invention comprises the Hib glycoconjugate, and/or at least or just 1,2,3 or 4 kind of antibacterial glycoconjugate, for example, the Neisseria meningitidis glycoconjugate, with another antigen that is selected from whole cell pertussis and hbs antigen, wherein the sugared dosage of Hib glycoconjugate is less than 5 μ g or less than 4 μ g, perhaps at 1-4 μ g or 1-3 μ g, or between 2-4 μ g or the 2-3 μ g, perhaps 2.5 μ g approximately or just, randomly, at least or just 1,2,3 or 4 kind of extra antibacterial glycoconjugate (for example, the Neisseria meningitidis glycoconjugate) each sugared dosage of whole or its is less than 10 μ g, 9 μ g, 8 μ g, 7 μ g, 6 μ g, 5 μ g or, 4 μ g, or 1-10 μ g, 1-8 μ g, 1-6 μ g, 1-5 μ g, 1-4 μ g, 1-3 μ g, or 2.5 μ g between 2-4 μ g or the 2-3 μ g or approximately or just.

Term " sugar " comprises polysaccharide or oligosaccharide.Polysaccharide is graded by sized to a certain degree by Micro Fluid from the antibacterial separation or from the antibacterial separation and by known method (seeing for example EP497524 and EP497525) is also optional.Polysaccharide can be graded by sized in order to reduce the viscosity in the polysaccharide sample and/or improve the filtration rate of conjugation product.Oligosaccharide has recurring unit's (usually 5-30 recurring unit) of low number and the polysaccharide through being hydrolyzed normally.

With the immunogenic composition that is applied to the people or the measurement amount " sugared dosage " of vaccine.

Hib sugar is the oligosaccharide of PRP (PRP) capsular polysaccharide or b type hemophilus influenza.

" at least a extra bacterial polysaccharides conjugate " refers to be connected to the conjugate of any antibacterial saccharide of carrier protein.The antibacterial saccharide randomly is from the capsular polysaccharide of Neisseria meningitidis bacterial strain, streptococcus pneumoniae (S.pneumoniae) bacterial strain, salmonella typhi (S.typhi) bacterial strain or any antibacterial saccharide as herein described.

" at least a Neisseria meningitidis glycoconjugate " refers to Neisseria meningitidis serogroups A capsular saccharides (MenA), Neisseria meningitidis serogroup C capsular saccharides (MenC), Neisseria meningitidis W135 capsular saccharides (MenW), Neisseria meningitidis sero-group Y capsular saccharides (MenY), Neisseria meningitidis serogroup B capsular saccharides (MenB), serogroup C and Y capsular saccharides (MenCY), serogroup C and A capsular saccharides (MenAC), serogroup C and W135 capsular saccharides (MenCW), serogroups A and Y capsular saccharides (MenAY), serogroups A and W135 capsular saccharides (MenAW), sero-group W135 and Y capsular saccharides (MenWY), serogroups A, C and W135 capsular saccharides (MenACW), serogroups A, C and Y capsular saccharides (MenACY), serogroups A, W135 and Y capsular saccharides (MenAWY), serogroup C, W135 and Y capsular saccharides (MenCWY) or serogroups A, C, W135 and Y capsular saccharides (MenACWY), serogroup B and C capsular saccharides (MenBC), serogroup B, C and Y capsular saccharides (MenBCY), serogroup B, C and A capsular saccharides (MenABC), serogroup B, C and W capsular saccharides (MenBCW), serogroups A, B and Y capsular saccharides (MenABY), serogroups A, B and W capsular saccharides (MenABW), serogroup B, W135 and Y capsular saccharides (MenBWY), serogroups A, B, C and W135 capsular saccharides (MenABCW), serogroups A, B, C and Y capsular saccharides (MenABCY); Serogroups A, B, W135 and Y capsular saccharides (MenABWY), serogroup B, C, W135 and Y capsular saccharides (MenBCWY); Or serogroups A, B, C, W135 and Y capsular saccharides (MenABCWY).

For example, adding listed above or do not add the Hib glycoconjugate the Neisseria meningitidis glycoconjugate arbitrary combination can with less than 5 μ g less than 4 μ g or 1-4 μ g or 1-3 μ g or 2-4 μ g or 2-3 μ g or approximately or just the sugared dosage of 2.5 μ g exist.

For the purposes of the present invention, " approximately " or " approximately " is defined as about in the of 10% of given numeral.

In one embodiment, the sugared dosage of Hib can be identical, larger with the sugared dosage of Neisseria meningitidis glycoconjugate or still less.The sugared dosage of Hib glycoconjugate can for for example at least a extra antibacterial (for example, Neisseria meningitidis) glycoconjugate on average or low sugar dosage 100% or less than 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.The sugared dosage of Hib sugar for for example at least a extra antibacterial (for example, Neisseria meningitidis) glycoconjugate on average or low sugar dosage 20% to 60%, 30% to 60%, 40% to 60% or approximately or just 50%.

In one embodiment of the invention, each dosage of described at least a extra antibacterial (for example, Neisseria meningitidis) sugar or its is identical or approximately identical.

The example of immunogenic composition of the present invention by form below or comprise following compositions:

Hib conjugate and MenA conjugate and MenC conjugate, randomly, sugared dose ratio is 2: 2,1: 2: 1,1: 4: 2,1: 6: 3,1: 3: 3,1: 4: 4,1: 5: 5,1: 6: 6 (w/w).Randomly, the sugared dosage of MenA is greater than the sugared dosage of MenC.

Hib conjugate and MenC conjugate and MenY conjugate, randomly, sugared dose ratio is 1: 2: 2,1: 2: 1,1: 4: 2,1: 4: 1,1: 8: 4,1: 6: 3,1: 3: 3,1: 4: 4,1: 5: 5,1: 6: 6 (w/w).Randomly, the sugared dosage of MenC is greater than the sugared dosage of MenY.

Hib conjugate and MenC conjugate and MenW conjugate, randomly, sugared dose ratio is 1: 2: 2,1: 2: 1,1: 4: 2,1: 4: 1,1: 8: 4,1: 6: 3,1: 3: 3,1: 4: 4,1: 5: 5,1: 6: 6 (w/w).Randomly, the sugared dosage of MenC is greater than the sugared dosage of MenW.

Hib conjugate and MenA conjugate and MenW conjugate, randomly, sugared dose ratio is 1: 2: 2,1: 2: 1,1: 4: 2,1: 4: 1,1: 8: 4,1: 6: 3,1: 3: 3,1: 4: 4,1: 5: 5,1: 6: 6 (w/w).Randomly, the sugared dosage of MenA is greater than the sugared dosage of MenW.

Hib conjugate and MenA conjugate and MenY conjugate, randomly, sugared dose ratio is 1: 2: 2,1: 2: 1,1: 4: 2,1: 4: 1,1: 8: 4,1: 6: 3,1: 3: 3,1: 4: 4,1: 5: 5,1: 6: 6 (w/w).Randomly, the sugared dosage of MenA is greater than the sugared dosage of MenY.

Hib conjugate and MenW conjugate and MenY conjugate, randomly, sugared dose ratio is 1: 2: 2,1: 2: 1,1: 1: 2,1: 4: 2,1: 2: 4,1: 4: 1,1: 1: 4,1: 3: 6,1: 1: 3,1: 6: 3,1: 3: 3,1: 4: 4,1: 5: 5,1: 6: 6 (w/w).Randomly, the sugared dosage of MenY is greater than the sugared dosage of MenW.

" other antigen " comprises one or both in whole cell pertussis (Pw) and the hbs antigen (HepB).In one embodiment, described other antigens also comprise diphtheria toxoid (DT), tetanus toxoid (TT), acellular pertussis (Pa) and/or poliovirus (IPV).In one embodiment, described other antigens comprise DT, TT, pertussis antigen (Pa or Pw) and HepB or consisting of.In one embodiment, described other antigens comprise or DT, TT, pertussis antigen (Pa or Pw), HepB and IPV its by forming.

Be included in other non-toxic mutants of non-toxic mutant that Hib in the immunogenic composition of the present invention and/or Neisseria meningitidis sugar is conjugated to carrier protein such as tetanus toxoid, tetanus toxoid fragment C, tetanus toxin, diphtheria toxoid, CRM197, diphtheria toxin, diphtherotoxin [such as CRM176, CRM197, CRM228, CRM45 (Uchida et al J.Biol.Chem.218; 3838-3844,1973); CRM9, CRM45, CRM102, CRM103 and CRM107, and Nicholls and Youle be at genetically engineered toxin (Genetically Engineered Toxins), Ed:Frankel, Maecel Dekker Inc, other sudden changes of describing in 1992; Disappearance or sudden change and other sudden change of disclosed Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly among US 4709017 or the US 4950740; Sudden change or other sudden changes of disclosed at least one or a plurality of residue Lys516, Lys526, Phe530 and/or Lys534 among US5917017 or the US6455673; Perhaps disclosed fragment among the US5843711], pneumococcal pneumolysin, OMPC (meningococcus outer membrane protein-usually from the Neisseria meningitidis serogroup B extract-EP0372501), synthetic peptide (EP0378881, EP0427347), heat shock protein (WO93/17712, WO94/03208), pertussis albumen (WO98/58668, EP0471177), cytokine, lymphokine, somatomedin or hormone (WO 91/01146), comprise artificial proteins from the people CD4+T cell epitope of the antigen in multiple pathogens source (people (2001) the Eur J Immunol 31 such as Falugi; 3816-3824), such as N19 albumen (people (2004) InfectImmun72 such as Baraldoi; 4884-7) streptococcus pneumoniae surface protein PspA (WO02/091998) pneumolysin (people (1995) the Infect Immun63 such as Kuo; 2706-13), ferrum picked-up albumen (WO01/72337), difficulty are distinguished toxin A or B (WO 00/61761) or the protein D (EP594610 and WO00/56360) of clostridium (C.difficile).

In one embodiment, immunogenic composition of the present invention is chosen wantonly at Hib conjugate and the identical carrier protein of every kind of Neisseria meningitidis glycoconjugate (all glycoconjugates that exist in the optional immunogenic composition) middle use in Hib conjugate and at least a Neisseria meningitidis conjugate.

In one embodiment, described immunogenic composition randomly comprises Hib glycoconjugate and Men polysaccharide conjugates, Hib glycoconjugate and MenC glycoconjugate, Hib glycoconjugate and MenW glycoconjugate, Hib glycoconjugate and MenY glycoconjugate, Hib glycoconjugate and Men and MenC glycoconjugate, Hib glycoconjugate and Men and MenW glycoconjugate, Hib glycoconjugate and Men and MenY glycoconjugate, Hib glycoconjugate and MenC and MenW glycoconjugate, Hib glycoconjugate and MenC and MenY glycoconjugate, Hib glycoconjugate and MenW and MenY glycoconjugate, Hib glycoconjugate and MenA, MenC and MenW glycoconjugate, Hib glycoconjugate and MenA, MenC and MenY glycoconjugate, Hib glycoconjugate and MenA, MenW and MenY glycoconjugate, Hib glycoconjugate and MenC, MenW and MenY glycoconjugate or Hib glycoconjugate and MenA, MenC, MenW and MenY glycoconjugate.

In one embodiment, single carrier protein can carry more than one carbohydrate antigen (WO04/083251).For example, single carrier protein can be conjugated to Hib and MenA, Hib and MenC, Hib and MenW, Hib and MenY, MenA and MenC, MenA and MenW, MenA and MenY, MenC and MenW, MenC and MenY or MenW and MenY.

In one embodiment, immunogenic composition of the present invention comprises the Hib sugar that is conjugated to carrier protein, and described carrier protein is selected from fragment C and the protein D of TT, DT, CRM197, TT.

Wherein for Hib, described carrier protein is TT or its fragment and at least a Neisseria meningitidis sugar, and the accumulated dose of carrier is 2.5-25 μ g, 3-20 μ g, 4-15 μ g, 5-12.5 μ g, 15-20 μ g, 16-19 μ g or 17-18 μ g.

In one embodiment, immunogenic composition of the present invention comprises at least 1,2,3 or the 4 kind of Neisseria meningitidis antibacterial sugar that is conjugated to carrier protein, and described carrier protein is selected from fragment C and the protein D of TT, DT, CRM197, TT.

Immunogenic composition of the present invention randomly comprises the Hib glycoconjugate, and the ratio of its Hib and carrier protein is 1: 5 to 5: 1; 1: 2 to 2: 1; 1: 1 to 1: 4; 1: 2 to 1: 3.5; Or approximately or just 1: 2.5 or 1: 3 (w/w).

Immunogenic composition of the present invention is optional (for example to comprise at least a meningococcus sugar, MenA and/or MenC and/or MenW and/or MenY) conjugate, the ratio of its Men sugar and carrier protein is 1: 5 to 5: 1,1: 2 to 5: 1,1: 0.5 to 1: 2.5 or 1: 1.25 to 1: 2.5 (w/w).

Use the conjugate of sterilizing to measure the ratio of sugared in the conjugate and carrier protein (w/w).Use the Lowry algoscopy (such as the people such as Lowry (1951) J.Biol.Chem.193, the people such as 265-275 or Peterson, Analytical Biochemistry100,201-220 (1979)) sugar is used for MenA with the ratio ((w/w)) of carrier protein (w/w) and with ICP-OES (plasma-optical emission spectroscopy of inductive) in the mensuration conjugate, the DMAP algoscopy is used for MenC and resorcinol algoscopy and is used for MenW and MenY people (1988) Anal.Biochem.175,525-530 such as () Monsigny to measure the amount of sugar.

In an embodiment of immunogenic composition of the present invention, Hib sugar is conjugated to carrier protein by joint such as bifunctional linker.Joint randomly is different bifunctional or with bifunctional, for example has an active amino and active carboxylic acid's group, 2 active aminos or 2 active carboxylic acid's groups.Joint has for example 4 to 20,4 to 12,5 to 10 carbon atoms.Possible joint is ADH.Other joints comprise B-propionamido (WO 00/10599), nitrobenzophenone-ethamine (people (1979) Med.Microbiol.Immunol.165 such as Gever; 171-288), haloalkyl halogen (US4057685) glycosidic bond (US4673574, US4808700) and 6-aminocaprolc acid (US4459286).

The glycoconjugate that exists in immunogenic composition of the present invention can be by any known coupling technology preparation.For example, can be by thioether bond with sugared coupling.Conjugation methods can depend on cyano group-4-dimethylaminopyridine with Tetrafluoroboric acid 1-

Activation sugar is to form cyanate.Thereby the sugar of activation can be directly or is coupled to amino on the carrier protein by interval base (joint).Randomly, with hexamethylene diamine or ADH cyanate coupling and use are related to the assorted chemistry sugar that amino is derivative that connects that thioether bond forms and be conjugated to carrier protein, perhaps use carbodiimide (for example, EDAC or EDC) chemistry that it is conjugated to carrier protein.This type of conjugate is described in application WO 93/15760 Uniformed Services University that PCT announces and WO 95/08348 and WO 96/29094.

Utilization carbimide, hydrazides, active ester, norbornane, Nitrodracylic acid, N-hydroxy-succinamide, S-NHS, EDC, TSTU that other are suitable.Many descriptions in WO 98/42721.Put together and can relate to the carbonyl joint, it can react by free hydroxyl group and CDI of sugar (the people J.Biol.Chem.1979 such as Bethell, 254; 2572-4, the people J.Chromatogr.1981.218 such as Hearn; 509-18), then form by forming amino-formate bond with proteins react.This can relate to different end is reduced into primary hydroxyl, randomly protects/goes and protect primary hydroxyl, makes primary hydroxyl and CDI reaction form the CDI carbamate intermediate and the amino on CDI carbamate intermediate and the protein is reacted.

By also preparing conjugate such as the direct-reduction amination method of describing among US4365170 (Jennings) and the US 4673574 (Anderson).Additive method is described in EP-0-161-188, EP-208375 and EP-0-477508.

Relate on the other hand by carbodiimides condensation (the people Infect.Immunity such as Chu C., 1,983 245 256), for example use EDAC, to be coupled to the sugar of the derivative Bromine cyanide. (or CDAP) of adipic acid hydrazides (ADH) activation carrier protein people Infect.Immunity such as (, 1,983,245 256) Chu C..

In one embodiment, the hydroxyl on the sugar directly or indirectly is connected to (passing through joint) amino or carboxyl on the protein.When having joint, randomly for example use CDAP to put together, the hydroxyl on the sugar is connected to amino on the joint.Another amino on the joint (for example ADH) can be conjugated to the hydroxy-acid group on the protein, for example, and by using the carbodiimides chemistry, for example, by using EDAC.In one embodiment, before joint is conjugated to carrier protein, at first Hib or at least a Neisseria meningitidis sugar are conjugated to joint.

In one embodiment, when Hib sugar existed, it was used CNBr or CDAP, and the combination of the perhaps combination of CDAP and carbodiimides chemistry (such as EDAC), or CNBr and carbodiimides chemistry (such as EDAC) is conjugated to carrier protein.Randomly, use CNBr and carbodiimides chemistry (optional EDAC) to put together Hib.For example, connect sugar and joint with CNBr, then use carbodiimides chemistry jointing and protein carrier.

In one embodiment, at least a at least a Neisseria meningitidis sugar directly is conjugated to carrier protein.Randomly, MenW and/or MenY and/or MenC and/or MenA sugar directly are conjugated to carrier protein.For example, with MenW; MenY; MenC; MenA; MenW and MenY; MenW and MenC; MenY and MenC; Or MenW, MenY and MenC are directly connected to carrier protein.Randomly, by CDAP at least a at least a Neisseria meningitidis sugar directly puted together.For example, with MenW; MenY; MenC; MenW and MenY; MenW and MenC; MenY and MenC; Or MenW, MenY and MenC are directly connected to carrier protein (seeing WO 95/08348 and WO96/29094) by CDAP.

Randomly, the ratio of MenW and/or Y sugar and carrier protein is 1: 0.5 to 1: 2 (w/w) or MenC sugar with the ratio of carrier protein is 1: 0.5 and 1: 2 or 1: 1.25 and 1: 1.5 or 1: 0.5 and 1: 1 (w/w), especially randomly uses CDAP that these sugar are directly connected to protein.

In one embodiment, by joint, such as bifunctional linker at least a Neisseria meningitidis sugar is conjugated to carrier protein.Joint randomly is different bifunctional or with bifunctional, for example has an active amino and active carboxylic acid's group, 2 active aminos or 2 active carboxylic acid's groups.Joint has for example 4 to 20,4 to 12 and 5 to 10 carbon atoms.Possible joint is ADH.

In one embodiment, by joint with MenA; MenC; Or MenA and MenC are conjugated to carrier protein.

In one embodiment, use CDAP and EDAC, by joint Neisseria meningitidis sugar is conjugated to carrier protein.For example, use CDAP described above and EDAC, by joint (for example, having two amino at its end, such as ADH) with MenA; MenC; Or MenA and MenC are conjugated to protein.For example, with CDAP sugar is conjugated to joint and with EDAC joint is conjugated to protein.Randomly, for MenA; MenC; Or MenA and MenC, be 1: 0.5 to 1: 6 by causing sugar and the ratio of carrier protein puting together of joint; 1: 1 to 1: 5 or 1: 2 to 1: 4.

In one embodiment of the invention, immunogenic composition comprises the Neisseria meningitidis capsular polysaccharide from least 1,2,3 among serogroups A, C, W and the Y or 4 kind that is conjugated to carrier protein, wherein at least 1,2,3 or 4 kind or every kind of N.Meningitidis PsC be that natural polysaccharide or the coefficient that is up to x1.5, x2, x3, x4, x5, x6, x7, x8, x9, x10 or x20 are graded by sized.For example, at least 1,2,3 or the mean size of 4 kind or every kind N.Meningitidis PsC be approximately 50kDa, 60kDa, 75kDa, 100kDa, 110kDa, 120kDa or 130kDa.

" natural polysaccharide " refers to also not accept purpose is the polysaccharide of processing that reduces the size of this polysaccharide.

" coefficient that is up to x2 is graded by sized " refers to this polysaccharide is carried out a kind of processing, and the purpose of this processing is the big or small size that still keeps above half of natural polysaccharide size that reduces polysaccharide.But X3, x4 etc. are explained in an identical manner, that is, polysaccharide is processed, and its objective is the size that reduces polysaccharide keep to surpass 1/3,1/4 etc. size of natural polysaccharide size.

In one aspect of the invention, immunogenic composition comprises at least 1,2,3 or 4 kind the Neisseria meningitidis capsular polysaccharide from serogroups A, C, W and Y that is conjugated to carrier protein, wherein at least 1,2,3 or 4 kind or every kind of N.Meningitidis PsC be natural polysaccharide.

In one aspect of the invention, immunogenic composition comprises at least 1,2,3 or 4 kind the Neisseria meningitidis capsular polysaccharide from serogroups A, C, W and Y that is conjugated to carrier protein, wherein at least 1,2,3 or 4 kind or the every kind of N.Meningitidis PsC coefficient that is up to x1.5, x2, x3, x4, x5, x6, x7, x8, x9 or x10 be graded by sized.

In one embodiment, at least 1,2,3 or the mean size of 4 kind or every kind N.Meningitidis PsC as measuring by MALLS, for 50KDa to 1500kDa, 50kDa to 500kDa, 50kDa to 300KDa, 101kDa to 1500kDa, 101kDa to 500kDa, 101kDa is to 300kDa.

In one embodiment, when MenA sugar existed, it had such as 50-500kDa, 50-100kDa, 100-500kDa, 55-90KDa, 60-70kDa or the 70-80kDa that measures by MALLS or the molecular weight of 60-80kDa.

In one embodiment, when MenC sugar existed, it had the molecular weight such as 100-200kDa, 50-100kDa, 100-150kDa, 101-130kDa, 150-210kDa or the 180-210kDa that measures by MALLS.

In one embodiment, when MenY sugar existed, it had the molecular weight such as 60-190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa or 110-140kDa, 50-100kDa, 100-140kDa, 140-170kDa or the 150-160kDa that measures by MALLS.

In one embodiment, when MenW sugar existed, it had the molecular weight such as 60-190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa, 110-140kDa, 50-100kDa or the 120-140kDa that measures by MALLS.

The molecular weight of sugar referred to before puting together and the molecular weight of the polysaccharide measured by MALLS.

In one embodiment, Neisseria meningitidis sugar be natural polysaccharide or in normal extraction thing process the natural polysaccharide that reduces of size.

In one embodiment, Neisseria meningitidis sugar is passed through mechanical breaking, as being graded by sized by Micro Fluid or supersound process.Micro Fluid and supersound process have advantages of the size of the natural polysaccharide that reduction is larger and are enough to provide filterable conjugate.

In one embodiment, the polydispersity of sugar is 1-1.5,1-1.3,1-1.2,1-1.1 or 1-1.05 and after being conjugated to carrier protein, the polydispersity of conjugate is 1.0-2.5,1.0-2.0,1.0-1.5,1.0-1.2,1.5-2.5,1.7-2.2 or 1.5-2.0.All polydispersity are all by being the MALLS measurement.

MALLS for meningococcus sugar analyzes, and can be used in combination two pillars (TSKG6000 and 5000PWxl TOSOH Bioscience) and wash desaccharide with water.Use light scattering detector (for example, the Wyatt Dawn DSP of equipment 10mW argon laser is under 488nm) and interferometry refractometer (for example, the Wyatt OtilabDSP of equipment P100 cell and red filter is under 498nm) to detect saccharide.

In one embodiment, MenA sugar when existing, it is by at least in part O-acetylation so that at least 50%, 60%, 70%, 80%, 90%, 95% or 98% recurring unit at least one position by the O-acetylation.The O-acetylation for example is present at least O-position.

In one embodiment; MenC sugar when existing; it is by at least in part O-acetylation so that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% (2 → 9)-NeuNAc recurring unit that connects at least one or two positions by the O-acetylation.The O-acetylation for example is present in O-7 and/or O-8 position.

In one embodiment, MenW sugar when existing, it is by at least in part O-acetylation so that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% recurring unit at least one or two positions by the O-acetylation.The O-acetylation for example is present in O-7 and/or O-9 position.

In one embodiment, MenY sugar when existing, it is by at least in part O-acetylation so that at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% recurring unit at least one or two positions by the O-acetylation.The O-acetylation is present in 7 and/or 9.

The acetylizad percentage ratio of O-refers to contain the percentage ratio of the acetylizad recurring unit of O-.This can measure in sugar before puting together and/or after puting together.

Another aspect of the present invention is the vaccine that comprises immunogenic composition of the present invention and pharmaceutically acceptable excipient.

Randomly, immunogenic composition or vaccine contain enough enhancings to the adjuvant of immunogenic immunne response amount.Suitable adjuvant comprises, but be not limited to, aluminum salt (aluminum phosphate or aluminium hydroxide), Squalene mixture (SAF-1), muramyl peptide, saponin derivative, mycobacteria cell wall preparation, monophosphoryl lipid A, derivatives of mycophenolic acid, non-ionic block copolymer surfactant, QuilA, b subunit of cholera toxin, polyphosphazene and derivant, and immunostimulating complex (ISCOMs), those that describe such as the people such as Takahashi (1990) Nature344:873-875.

For HibMen combination discussed above, advantageously Hib and Men glycoconjugate are not adsorbed on Alum adjuvant or any adjuvant.

In one embodiment, immunogenic composition is without adjuvant.For the present invention, " without adjuvant " refers to self not do is that the adjuvant component of antigenicity component is not present in the immunogenic composition.

In one embodiment of the invention, HepB is adsorbed to aluminum phosphate (WO93/24148).

In one embodiment, immunogenic composition comprises by joint and is conjugated to the Hib sugar of tetanus toxoid and directly or by joint is conjugated to the MenC sugar of tetanus toxoid and directly or be conjugated to the MenA sugar of tetanus toxoid by joint.

In one embodiment, immunogenic composition of the present invention is buffered in or is adjusted to pH7.0 to 8.0, pH7.2 to 7.6 or pH7.4 approximately or just.

Randomly lyophilizing immunogenic composition of the present invention or vaccine in the presence of stabilizing agent such as polyhydric alcohol such as sucrose or trehalose.

For all immunogenic compositions or vaccine, must determine immunogenic immunology effective dose by experience.Factor to be considered comprise immunogenicity, immunogen whether will with adjuvant or carrier protein or other protein be compound or covalently bound, route of administration and immunizing dose number to be administered.This type of factor is that the known and immunologist in vaccine field can make this type of without undo experimentation fully and determines.

Activating agent can be present in different concentration in pharmaceutical composition of the present invention or the vaccine.Usually, the Cmin of material is the necessary amount of desired use that realizes it, although Cmax is to remain in the solution or evenly to be suspended in maximum in the initial mixture.For example, the minimum of therapeutic agent will be randomly for providing the amount of single therapy effective dose.For bioactive substance, the essential amount of biological activity when Cmin is reconstruct, and Cmax is the concentration in the time of can not keeping unit for uniform suspension.For single dose unit, described amount is the amount that single therapy is used.Usually, expect that every dose will comprise 1-100 μ g proteantigen, randomly 5-50 μ g or 5-25 μ g.Bacterin preparation of the present invention can for the protection of or treatment to infecting responsive mammal (for example, human patients), can use described vaccine by whole body or mucosal route.Human patients randomly for baby (below 12 months), begin to learn the child (12-24,12-16 or 12-14 month) that walks, child (2-10,3-8 or 3-5 year) and teenager (12-25,14-21 or 15-19 year) or be grown up (12,15,18 or more than 21 years old).These are used and can comprise by intramuscular, intraperitoneal, Intradermal or subcutaneous route injection; Perhaps arrive oral cavity/esophagus, respiratory tract, genitourinary tract by mucosal administration.Preferred intranasal administration vaccine is with treatment pneumonia or otitis media (carry as can effectively preventing the nasal mucosa of Diplococcus pneumoniae, thereby the infection that alleviates its earliest stages).Although vaccine of the present invention can be used as single dose and uses, but its component also can (for example be used together or in the different time jointly, be present in the vaccine such as fructose, so these at the same time separate administration or behind the bacterioprotein vaccine administration, use to coordinate best mutual immunne response 1-2 week).Except the single administration approach, can also use 2 kinds of different route of administration.For example, virus antigen can be used by ID (Intradermal), and bacterioprotein can IM (intramuscular) or IN (intranasal) use.If there is saccharide, they can IM (or ID) be used and virus protein can be used by IN (or ID) so.In addition, vaccine of the present invention can be used as amount of initiator by IM, and IN uses as booster dose.

Bacterin preparation generally is described in vaccine design (" The subunit and adjuvantapproach " (Powell M.F. and Newman M.J. work) (1995) Plenum Press NewYork).Fullerton, United States Patent (USP) 4,235,877 have described the parcel in liposome.

Another aspect of the present invention is for the vaccine kit that accompanies or order is used; it comprises two kinds of multivalent immunogenic compositionss, is used for giving the host protection of the disease that anti-bordetella pertussis (Bordetellapertussis), Clostridium tetani (Clostridium tetani), corynebacterium diphtheriae (Corynebacterium diphtheriae), hemophilus influenza (Haemophilusinfluenzae) and Neisseria meningitidis (Neisseria meningitidis) cause.For example, this test kit randomly comprises first container and second container, and the first container comprises one or more:

Tetanus toxoid (TT),

Diphtheria toxoid (DT), and

The whole cell pertussis component and

Randomly also comprise hbs antigen,

Second container comprises

The Hib glycoconjugate, and

At least 1,2,3 or 4 kind of Neisseria meningitidis glycoconjugate,

Wherein the sugared dosage of Hib conjugate is less than 5 μ g or 4 μ g, or 1-4 μ g or 1-3 μ g, or 2-4 μ g or 2-3 μ g or 2.5 μ g approximately or just, randomly at least or just 1,2,3 or each sugared dosage of 4 kind of extra antibacterial glycoconjugate (for example, Neisseria meningitidis glycoconjugate) or its less than 2.5 μ g between 10 μ g, 9 μ g, 8 μ g, 7 μ g, 6 μ g, 5 μ g or 4 μ g or 1-10 μ g, 1-8 μ g, 1-6 μ g, 1-5 μ g, 1-4 μ g, 1-3 μ g or 2-4 μ g or the 2-3 μ g or approximately or just.

The example of Hib conjugate and at least a extra antibacterial sugar (for example, Neisseria meningitidis sugar) conjugate such as above-mentioned.Any character of above-mentioned conjugate may reside in the vaccine kit.

Randomly, vaccine kit of the present invention comprises the third component.For example, test kit of the present invention randomly comprises first container and second container and the 3rd container, and first container comprises one or more:

Tetanus toxoid (TT),

Diphtheria toxoid (DT), and

The whole cell pertussis component, and

Choose wantonly and comprise hbs antigen;

Second container comprises:

Carrier protein and from one or more conjugates of the capsular saccharides of streptococcus pneumoniae [wherein capsular saccharides optional from be selected from 1,2,3,4,5, the streptococcus pneumoniae serology of 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F]

And the 3rd container comprises:

The Hib glycoconjugate, and

At least a Neisseria meningitidis glycoconjugate,

Wherein the sugared dosage of Hib conjugate is less than 5 μ g or 4 μ g, or 1-4 μ g or 1-3 μ g, or 2-4 μ g or 2-3 μ g or 2.5 μ g approximately or just, and randomly at least or just 1,2,3 or each sugared dosage of 4 kind of extra antibacterial glycoconjugate (for example, Neisseria meningitidis glycoconjugate) or its less than 2.5 μ g between 10 μ g, 9 μ g, 8 μ g, 7 μ g, 6 μ g, 5 μ g or 4 μ g or 1-10 μ g, 1-8 μ g, 1-6 μ g, 1-5 μ g, 1-4 μ g, 1-3 μ g or 2-4 μ g or the 2-3 μ g or approximately or just.

Comprise meningococcal conjugate, such as the immunogenic composition of HibMenC, HibMenAC, HibMenAW, HibMenAY, HibMenCW, HibMenCY, HibMenWY, MenAC, MenAW, MenAY, MenCW, MenCY, MenWY or MenACWY, comprise to the test kit of above-mentioned those similar components randomly comprising antigen from measles and/or parotitis and/or rubella and/or chickenpox.For example, the meningococcus immunogenic composition contains the antigen from measles, parotitis and rubella or measles,mumps,rubella and chickenpox.In one embodiment, these virus antigens randomly are present in the container identical with meningococcus and/or Hib glycoconjugate.In one embodiment, with these virus antigen lyophilizing.

Another aspect of the present invention is the method for preparation immunogenic composition of the present invention, it with Hib glycoconjugate and at least a extra antibacterial (for example comprises, Neisseria meningitidis) glycoconjugate mixes and the step of formation compositions with another antigen that is selected from whole cell pertussis and hbs antigen, wherein the sugared dosage of Hib conjugate is less than 5 μ g or 4 μ g, or 1-4 μ g or 1-3 μ g, or 2.5 μ g between 2-4 μ g or the 2-3 μ g or approximately or just, and randomly, at least or just 1,2,3 or each sugared dosage of 4 kind of extra antibacterial (such as Neisseria meningitidis) glycoconjugate or its less than 10 μ g, 9 μ g, 8 μ g, 7 μ g, 6 μ g, 5 μ g or 4 μ g, or 1-10 μ g, 1-8 μ g, 1-6 μ g, 1-5 μ g, 1-4 μ g, 1-3 μ g, or 2.5 μ g between 2-4 μ g or the 2-3 μ g or approximately or just.

The present invention is immune human host with influenza haemophilus and the method that infects of Neisseria meningitidis randomly on the other hand, and it comprises immunogenic composition of the present invention or vaccine or the test kit of the host being used immunoprotection dosage.

Another aspect of the present invention is immunogenic composition of the present invention, the disease that it is used for the treatment of or prevents to be caused by hemophilus influenza and/or Neisseria meningitidis.

The purposes of the medicine of another aspect of the present invention disease that to be immunogenic composition of the present invention or vaccine or test kit caused by hemophilus influenza and/or Neisseria meningitidis for the production for the treatment of or prevention.

As used herein term " comprise " and " comprising " by the inventor be intended to term " by ... form " and " consisting of " can randomly substitute every-way.

All lists of references or the patent application of quoting in the patent specification all are incorporated herein by reference.

In subsidiary embodiment, illustrate the present invention.Except other detailed description, use as well known to those skilled in the art and conventional standard technique to carry out the following examples.These embodiment are illustrative, and do not limit the present invention.

Embodiment

The preparation of embodiment 1-polysaccharide conjugates

By the people such as Chu (Infection and Immunity 1983,40 (1); 245-256) the coupling chemical method of exploitation is carried out the covalent bond of hemophilus influenza (Hib) PRP polysaccharide and TT.By adding CNBr and activating Hib PRP polysaccharide in 6 minutes at the pH10.5 incubation.PH is reduced to pH8.75 and adds adipic dihydrazide (ADH) and continued incubation 90 minutes.Use 1-ethyl-3-(3-dimethyl-amino propyl group) carbodiimides (EDAC) PRP of activation to be coupled to the tetanus toxoid of purification by the carbodiimides condensation.Add EDAC to the PRP of the activation final ratio with the PRP that reaches 0.6mg EDAC/mg and activate.The tetanus toxoid of regulating pH to 5.0 and adding purification is to the PRP that reaches the 2mgTT/mg activation.Gained solution was placed 3 days under gentle agitation.Behind 0.45 μ m membrane filtration, purification conjugate on Sephacryl S500HR (Pharmacia, the Sweden) post of using 0.2M NaCl balance.

Use (measuring more than 150kDa by MALLS) natural polysaccharide or produce PSC by gentle Micro Fluid.Use natural polysaccharide or as the polysaccharide of the above gentle Micro Fluid of 60kDa measured by the method for embodiment 2 produce PSA.Use the polysaccharide that is graded by sized generation MenW and PSY (seeing embodiment 2) such as the approximately 100-200kDa that measures by MALLS.Use homogenizer EmulsiflexC-50 device to be graded by sized by Micro Fluid.Then filter polysaccharide by 0.2 μ m filter.

Activating and coupling described in WO96/29094 and WO00/56360.In brief, the polysaccharide of 10-20mg/ml concentration among the 2M NaCl pH5.5-6.0 and CDAP solution (with the 100mg/ml of the fresh preparation of acetonitrile/WFI50/50) being mixed to final CDAP/ polysaccharide ratio is 0.75/1 or 1.5/1.1.5 after minute, with sodium hydroxide rising pH to pH10.0.After 3 minutes, add tetanus toxoid take reach sugar/protein ratio for MenW as 1.5/1, be 1.2/1 for MenY, be 1.5/1 or be 1.5/1 for MenC for MenA.Reaction continues 1 to 2 hour.

After coupling step, add glycine to the final ratio of glycine/PS (w/w) and be 7.5/1 and regulate pH to pH9.0.Mixture was placed 30 minutes.Use 10 μ m Kleenpak filters clarification conjugate, then be loaded on the Sephacryl S400HR post, use 150mMNaCl, the eluent of 10mM or 5mM Tris pH7.5.Filter clinical lot number at Opticap 4 sterile films.The average polysaccharide of the conjugate that obtains: protein ratio is 1: 1-1: 5 (w/w).

In order by the interval base MenA capsular polysaccharide to be conjugated to tetanus toxoid, make in the following method.Carry out the covalent bond of polysaccharide and interval base (ADH) by the coupling chemical method, by the method, under controlled condition by cyaniding (cyanylating) reagent Tetrafluoroboric acid 1-cyano group-4-dimethylaminopyridine

(CDAP) activated polysaccharide.Make the interval base by its diazanyl and the PS reaction of cyaniding, between interval base and polysaccharide, form stable isourea.

Obtaining the CDAP/MenA ratio with the 100mg/ml CDAP solution-treated 10mg/ml MenA solution in the acetonitrile/water (50/50 (v/v)) of fresh preparation is 0.75 (w/w).1.5 after minute, rising pH is to pH10.0.After 3 minutes, it is 8.9 that adding ADH obtains the ADH/MenA ratio.Reduce the pH to 8.75 of solution and reacted 2 hours.

Before conjugation reaction, with the TT fusion of purification with PSA _AHSolution dilution and reach the PSA of 10mg/ml _AHThe TT concentration of concentration and 10mg/ml.

To PSA _AHAdd EDAC in the solution in order to reach 0.9mg EDAC/mg PSA _AHFinal ratio.Regulate pH to 5.0.The tetanus toxoid that adds purification with peristaltic pump (in 60 minutes) reaches 2mgTT/mg PSA _AHGained solution was under agitation placed 60 minutes at+25 ℃, obtained 120 minutes final coupling time.Use 10 μ m filters clarification conjugate and carry out purification with Sephacryl S400HR post.

Embodiment 2-uses the MALLS determining molecular weight

Detector is connected to the HPLC size-exclusion column, from its elution samples.On the one hand, the laser light scattering detector is measured the light intensity of macromole solution scattering at 16 angle places, and on the other hand, online interferometry refractometer of placing allows the amount of the sample of mensuration eluting.Can measure macromolecular size and shape the solution from these intensity.

Mean molecule quantity (M by weight _w) weight that is defined as all species multiply by their molecular weight separately and and divided by all species weight sums.

A) weight-mean molecule quantity :-Mw-

$M_w=\frac{\mathrm{\Σ}{W}_i,M_i}{\mathrm{\Σ}{W}_i}=\frac{m_2}{m_1}$

B) number average molecular weight :-Mn-

$M_n=\frac{\mathrm{\Σ}{N}_i,M_i}{\mathrm{\Σ}{N}_i}=\frac{m_1}{m_0}$

C) root-mean-square radius :-Rw-and R ²W is square radius by defining below:

(m _i-be the quality of scattering center i ,-r _i-be the distance between scattering center i and the macromole center of gravity).

D) polydispersity is defined as ratio-Mw/Mn-.

By being loaded on two the HPLC posts (TSKG6000 and 5000PWxlTOSOH Bioscience) that are used in combination, analyze meningococcal polysacharide by MALLS.The water elution that 25 μ l polysaccharide is loaded on the pillar and filters with 0.75ml.Use light scattering detector (the Wyatt Dawn DSP of equipment 10mW argon laser is at 488nm) and interferometry refractometer (the Wyatt Otilab DSP of equipment P100 measuring cell and red filter is under 498nm) to detect polysaccharide.

In Astra4.72 software, use Polydispersity and the recovery that a time fitting of a polynomial calculates all samples by the debye method.

The II of the HibMenAC-TT conjugate vaccine that embodiment 3 mixes with DTPw-HepB Clinical trial phase

Research design: use 5 groups of disclosed, randomized (1: 1: 1: 1) single center research of carrying out.Vaccination regimen below 5 groups are accepted when 6,10 and 14 age in week respectively:

.Tritanrix ^TM-HepB/Hib-MenAC2.5/2.5/2.5: be called later on 2.5/2.5/2.5

.Tritanrix ^TM-HepB/Hib-MenAC2.5/5/5: be called later on 2.5/5/5

.Tritanrix ^TM-HepB/Hib-MenAC5/5/5: be called later on 5/5/5

.Tritanrix ^TM-HepB+Hiberix ^TM: be called later on Hiberix

.Tritanrix ^TM-HepB/Hiberix ^TM+ Meningitec ^TM: be called later on Meningitec

1 month (behind the dosage-3) blood sample collection behind the vaccine dose when the vaccine dose first time (Pre) and for the third time.

Tritanrix ^TMIt is the DTPw vaccine of GlaxoSmithKline Biologicals S.A listing.

In each group of 5 groups, use 105 experimenters, in this research, altogether use 525 experimenters.

Table 1

^*2.5/2.5/2.5 vaccine is the dose dilution liquid of Hib-MenAC5/5/5 vaccine that contains the GSK Biologicals of every kind of PRP-TT of 2.5 μ g, MenA-TT and MenC-TT.

The Hib-MenAC bacterin preparation is mixed immediately with Tritanirix-HepB.The diph/tet of the combination of GSKBiologicals-whole cell pertussis bacillus-hepatitis B (DTPw-HB) vaccine (Tritanrix-HepB) contains and is no less than 30 ius (IU) diphtheria toxoid, be no less than the 60IU tetanus toxoid, be no less than bordetella pertussis that 4IU kills and the hbs antigen of 10 μ g restructuring.

Reference treatment, dosage and method of application, lot number:

Vaccination regimen/position: when 6,10 and 14 age in week, in left vastus, accept the Tritanrix.-HepB vaccine and in right vastus, accept Hiberix for one group ^TMAnother group is accepted Tritanrix in left vastus when 6,10 and 14 week ^TM-HepB/Hiberix ^TMVaccine and in right vastus, accept Meningitec ^TMVaccine.

Vaccine/compositions/dosage/lot number: the Tritanrix of use ^TM-HepB vaccine is as described below.

The b type hemophilus influenza conjugate vaccine of potion (0.5ml) GSK Biologicals: Hiberix ^TMContain the PRP that 10 μ g are conjugated to tetanus toxoid.In the Hiberix group, mixed being incorporated in the Meningitec group of itself and sterile diluent mixed it with Tritanrix-HepB.

The MENINGITEC of potion (0.5ml) Wyeth Lederle ^TMVaccine contains: 10 μ g are conjugated to capsular polysaccharide and the aluminum salt of the meningococcus group C of 15 μ g corynebacterium diphtheriae CRM197.

Result's-Hib, MenA and MenC are produced immunne response

Table 2a resists-PRP (μ g/ml)

Table 2b SBA-MenC

Table 2c SBA MenA

Table 2d resists-PSC (μ g/ml)

Table 2e resists-PSA (μ g/ml)

Table 2f resists-BPT (EL.U/ml)

Table 2g resists-HBs (mIU/ml)

Conclusion

Table 2f and 2g show, when HibMenAC mixes in vaccine with DTPwHepB, 2.5/2.5/2.5 dose ratio 2.5/5/5 dosage of Hib/MenA/MenC produces higher anti-DT-Pa and the immunne response of hbs antigen, and 2.5/5/5 dosage produces higher anti-DT-Pa and the immunne response of hbs antigen than 5/5/5 dosage again.Table 2a-e shows that the HibMenA of low dosage and MenC still realize the good immunne response to Hib, MenA and MenC, and the patient of 99-100% realizes being higher than the immunne response of selected threshold value.

Embodiment 4 HibMenAC clinical trial-usefulness HibMenAC conjugate causes

Carry out disclosed randomised study of II phase with assessment Tritanrix ^TMThe immunological memory that the preliminary seeded process of-HepB/HibMenAC vaccine is induced, and be evaluated at and use Tritanrix ^TMThe age that-HepB/Hib-MenAC causes be among 15 to 18 months the experimenter with the Hib-MenAC conjugate vaccines of GSK Biologicals or the Hib of GSK Biologicals _2.5The Tritanrix of the GSKBiologicals that vaccine mixes ^TMThe immunogenicity of the booster dose of-HepB vaccine and reactionogenicity.When 6,10 and 14 week, accept preliminary vaccination regimen for 5 groups, as listing in the table 3.

Table 3

(before the PS-October) and common polysaccharide are strengthened rear 1 month (behind the PS-November) when common polysaccharide (PS) is strengthened, from organizing 1,3,5,7 and 9 blood sample collections.

Attention: provided the immunogenicity result who obtains in 5 groups accepting common polysaccharide reinforcement (that is, group 1,3,5,7 and 9).

Experimenter's number: plan: 450 (every group of 45 experimenters)

Participate in: in accepting the group 1,3,5,7 and 9 that common polysaccharide strengthens, totally 193 experimenters (in the group 1 42, in the group 3 39, in the group 5 37, in the group 7 36, in the group 9 39) participate in.Finish: inapplicable

Immunogenicity: the crowd who altogether participates in=193 experimenters

Attention: in this research, the crowd of the crowd's who altogether participates in=total inoculation.

Diagnosis and the standard that adds: the age is 10 months sex experimenter, it has finished three doses of preliminary seeded process describing in embodiment 1, there is not obvious health problem, the booster shot for diphtheria, tetanus, pertussis, hepatitis B, meningococcus serogroups A or C and/or Hib disease before since the research of preliminary study adds access, also not accepted.Before research enters, obtain written informed consent from father and mother/guardian of experimenter.

Research vaccine, dosage, method of application, lot number: all vaccines that use in this research are all by GSK Biologicals research and production.

Vaccination regimen/position: Polysaccharide A and polysaccharide C vaccine that the experimenter in the group 1,3,5,7 and 9 accepts to make up, the Mencevax of 1/5 dosage ^TMAC and the common PRP of 10 μ g during as 10 monthly ages respectively in the intramuscular injection of left and right front outside thigh.

The persistent period for the treatment of: the persistent period of whole research is every experimenter approximately 6 to 9 months, the booster shot of using when it is included in 15 to 18 monthly ages.In the time of 11st month, carry out intermediate analysis (after common polysaccharide is strengthened using during at 10 months 1 month).

Evaluation criteria: before common polysaccharide is strengthened using and after using 1 month, the evaluation criteria of group 1,3,5,7 and 9 was as follows:

-SBA-MenA antibody titer 〉=1: 8

-SBA-MenC antibody titer 〉=1: 8

-anti-PSA antibody concentration 〉=0.3 μ g/ml

-anti--PSC antibody concentration 〉=0.3 μ g/ml

-anti--PRP antibody concentration 〉=0.15 μ g/ml.

Statistical method: this intermediate analysis is based on the crowd of total participation.All analyses all are pure descriptive and do not have calculating to the statistical inference of any terminal point.Only analyze accepting 5 groups (organizing 1,3,5,7 and 9) that common polysaccharide strengthens 10 months the time.Although these 5 groups are subgroups of main group in the preliminary study, distribute according to main seminar to provide the result.

Analysis of Immunogenicity: provided in this embodiment the result who obtains at three time points, be in the main inoculation study for the third time vaccine dose (embodiment 1), before polysaccharide is strengthened using when 10 monthly ages (that is) in order to the persistency of immunne response after assessing preliminary inoculation with use polysaccharide (during 11 monthly ages) immunological memory of inducing in order to assess preliminary inoculation of strengthening rear 1 month.At each time point: show to have the geometric average antibody concentration of 95% confidence interval (CIs) or (GMCs or GMTs)-MenC, the SBA-MenA that tire, anti--PSC, anti--PSA and anti--PRP for serum kills bacteria-measuring method (GMCs or GMTs) tabulation.For calculating, every kind of antibody has just seropositivity or the serum protection of 95%CI.Use reverse summation curve (RCCs) research polysaccharide to strengthen and polysaccharide is strengthened rear one month antibody concentration or tired for every kind of antigen and serology.

The result

Demographic result: the crowd's of all participation mean age was 43.2 weeks, and standard deviation was 6.5 weeks.Male and women's ratio are 1.3 (110/83).All experimenters belong to East Asia or Southeast Asia ethnic group.

Immunogenicity result: the crowd's of full entry immunogenicity result lists in table 4.

Table 4a

Table 4b

Conclusion

In the SBA algoscopy, the HibMenAC 2.5/5/5 conjugate vaccine preparation that contains low amount Hib tends to bacterin preparation than all three kinds of conjugates that contain equivalent and produces better immunological memory for MenA and MenC.This can relatively finding out from the POST-PS reading.Therefore, in initiation, use 2.5/5/5 preparation to cause more excellent immunological memory to be replied.

Observe PIII (M3) data, for Hib (22.5v17) and MenC (by SBA, 76v48 or 56 and 5339v3342 or 3863), observe higher reading with 2.5/5/5 preparation.

Embodiment 5a: 2,4 and 6 months babies in use and Infanrix penta and Prevenar The clinical trial of the HibMenCY that gives simultaneously

Research design: use 5 parallel group II phase of carrying out, disclosed (the part double blinding ^*), the multicenter study of randomized (1: 1: 1: 1: 1), contrast, when 2,4 and 6 monthly ages, accept following together vaccine and tentatively inoculate as 3 dosage for described parallel group:

~ group Hib-MenCY2.5/5/5:Hib-MenCY (2.5/5/5)+Infanrix Penta+Prevenar

~ group Hib-MenCY5/10/10:Hib-MenCY (5/10/10)+Infanrix

Penta+Prevenar

~ group Hib-MenCY5/5/5:Hib-MenCY (5/5/5)+Infanrix

Penta+Prevenar

~ group Menjugate:Menjugate + Act HIB

+ Infanrix

Penta**

~ group ActHIB:ActHIB

+ Infanrix

Penta+Prevenar

^*Hib-MenCY (2.5/5/5) and Hib-MenCY (5/10/10) are applied in the mode of double blinding.Hib-MenCY (5/5/5) preparation can not be used by double blinding, because it is by (half solution abandons with 1.0ml diluent reconstruct Hib-MenCY (10/10/10) preparation, and use remaining 0.5ml), and Hib-MenCY (2.5/5/5) and Hib-MenCY (5/10/10) preparation are applied after with the reconstruct of 0.5ml diluent.

^*To when finishing, strengthening research 792014/002 provide the streptococcus pneumoniae conjugate vaccines that two doses of licenses are provided according to prescription information to the experimenter from this group.

At before the preliminary seeded process and after finishing one month, obtain blood sample (4.0ml) (research month 0 and research month 5) from all experimenters.

Project is for to carry out with 400 experimenters, every group of 80 experimenters in 5 groups.Finish (group Hib-MenCY 2.5/5/5:80 group Hib-MenCY5/10/10:81 with totally 398 experimenters under study for action; Group Hib-MenCY 5/5/5:78; Group Menjugate:81; Group ActHIB:78).

Vaccination regimen/position: as described below when about 2,4 and 6 monthly ages, with three dosage of interval intramuscular injection in two months:

Table 5: the vaccine of using and position

Table 6: candidate vaccine preparation and lot number

^*By preparing Hib-MenCY5/5/5 with 1.0ml diluent dissolving Hib-MenCY10/10/10 preparation; Use 0.5ml and abandon remaining 0.5ml.

Evaluation criteria:

Immunogenicity: the dosage first time (0th month) approximately 1 month (5th month) surveyingpin tiring/concentration to the antibody of every kind of vaccine antigen in all experimenters behind the dosage before and for the third time.(measure cutoff value: the dilution factor of 1: 8 and 1: 128) measure for the bacterial antibodies that kill of Neisseria meningitidis serogroup C and Y (SBA-MenC and SBA-MenY) and tire by killing the antibacterial test, measure anti-Neisseria meningitidis serogroup C and Y (anti--PSC and anti--PSY by ELISA, measure cutoff value 〉=0.3 μ g/ml and 〉=2 μ g/ml), Hib polysaccharide PRP is (anti--PRP, measure cutoff value 〉=0.15 μ g/ml and 〉=1.0 μ g/ml), three kinds of pertussis antigens are (anti--PT, anti--FHA, anti--PRN, measure the antibody of cutoff value 〉=5EL.U/ml), the resistance of hepatitis B surface antigen is (anti--HBs, the mensuration cutoff value 〉=10mIU/mL), diphtheria and tetanus toxoid (anti--diphtheria and anti--tetanus are measured cutoff value 0.1IU/ml); Anti--1,2 and 3 type polioviruses (measuring cutoff value 1: 8); 7 kinds of streptococcus pneumoniae sero-groups resist-4, and are anti--6B, anti--9V, resist-14, anti--18C, resist-19F, the antibody of anti--23F (measuring cutoff value 0.05 μ g/ml).

The elementary vaccine of pertussis antigen replied be defined as among the former experimenter who can't detect antibody behind dosage for the third time as seropositivity (detectable antibody) or antibody concentration before in initial seropositive experimenter, keeping at least inoculation.

Safety (evaluation criteria): follow up a case by regular visits to 8 days (the 0th to 7 day) after using every kind of vaccine dose, by father and mother/guardian of experimenter in the part (pain, rubescent, swelling) of diary card report request and general (sleepy, heating, irritability and anorexia) symptom; With adverse events (SAE) serious during whole research.

Statistical method:

Immunogenicity

To have the geometric average antibody concentration of 95% confidence interval (CI) or tire (GMC/Ts) is listed for every kind of antigen.By getting log10 concentration or the end of meansigma methods of the conversion of tiring is that 10 antilogarithm is (anti--as log10) to carry out the calculating of GMC/Ts.Be lower than the antibody concentration of algoscopy cutoff value or tire and provide half the arbitrary value of cutoff value that calculates purpose for GMC/T.Calculating has the antibody concentration of the algoscopy cutoff value that is higher than appointment/tire or has the experimenter's that the vaccine of 95%CI just replys percentage ratio.Use oppositely accumulation Antibody curve Effect of Anti bulk concentration/tire for every kind of antigen after the inoculation.Antibody concentration distribution tabulation with 7 kinds of PNEUMOVAX-23.

For the difference of every kind of antibody to compare with matched group between the exploring mode assessment Hib-MenCY group, just except SBA-MenY and the anti--PSY, just between (1) matched group (negative) and the Hib-MenCY group, be higher than the cutoff value of appointment or have the difference of the experimenter's that the vaccine of standardized asymptomatic 95%CI replys percentage ratio, (2) matched group with respect to the GMC of Hib-MenCY group or GMT than (95%CI).For SBA-MenC and anti--PSC, matched group is Menjugate; For every other antigen, matched group is group ActHIB.Carry out identical comparison to assess the difference between every pair of Hib-MenCY preparation for anti--PRP, SBA-MenC, anti--PSC, SBA-MenY, anti--PSY and anti--tetanus antibody.

Serum protection/Xue Qingyangxingshuai ﹠amp; GMC/Ts (immunogenic ATP group)

Table 7a resists-PRP (μ g/ml)

Table 7b SBA-MenC (1/Dil)

Table 7c resists-PSC (μ g/ml)

Table 7d SBA-MenY (1/Dil)

Table 7e resists-PSY (μ g/ml)

Conclusion

According to immunogenicity and SBA result, 2.5/5/5 and 5/10/10 preparation causes higher the tiring of anti-Hib, MenC and MenY.Therefore, comprise in the conjugate vaccines of combination that the Hib conjugate than low dosage obtains more excellent result.

Jointly use Hib-MenCY and Infanrix penta and Prevenar ^TMObtain gratifying result.

Embodiment 5b uses HibMenCY and Prevenar jointly ^TMFor the impact of replying on pneumococal polysaccharide

The research of embodiment 3 be on the other hand research for Prevenar ^TMThe level of the antibody that 7 kinds of pneumococal polysaccharides that exist in the vaccine produce is so that assessment is used HibMenCY jointly on the impact of the antibody titer that produces for pneumococal polysaccharide.

Be displayed in Table 87 kinds of streptococcus pneumoniae sero-groups have been had 〉=experimenter's of the antibody of 0.05 μ g/ml and 〉=0.2 μ g/ml GMC and percentage ratio.Except the 6B sero-group, the seroprevalence of 7vPn component is that 95.5-100% (antibody concentration 〉=0.05 μ g/ml) is to 93.9-100% (antibody concentration 〉=0.2 μ g/ml) between each group.For the 6B sero-group, seroprevalence is 88.4-98.6% (antibody concentration 〉=0.05 μ g/ml) and 81.2-91.4% (antibody concentration 〉=0.2 μ g/ml) (ActHIB group: 92.3% 〉=0.05 μ g/ml between each group; 86.2% 〉=0.2 μ g/ml).

Table 8a anti--4

Table 8b resists-6B

Table 8c resists-9V

Table 8d anti--14

Table 8e resists-18C

Table 8f resists-19F

Table 8g resists-23F

Conclusion

Jointly use the HibMenCY of all three kinds of dosage forms and Prevnar and cause immunne response for the satisfaction of 7 kinds of streptococcus pneumoniae sero-groups.Sero-group 6B is the immunogen of replying that is difficult to cause for it.For 6B, use the lower Hib dosage particles of HibMenC to realize that higher experimenter's GMC and percentage ratio have reached two threshold levels.Therefore, use the Hib conjugate vaccines than low dosage to cause replying preferably of anti-6B antigen jointly to use with streptococcus pneumoniae capsular polyaccharide conjugates.

Embodiment 6-according to 2,3 and 4 months schemes and Infanrix penta jointly use Hib The II clinical trial phase of MenCY

Research design: to 5 groups of three dosage tentative programmes use accepting following vaccine carry out the II phase disclosed (the part double blinding ^*) multicenter study of randomization contrast:

Group Hib-MenCY 2.5/5/5:Hib-MenCY (2.5/5/5)+Infanrix ^TMPenta

Group Hib-MenCY5/10/10:Hib-MenCY (5/10/10)+Infanrix ^TMPenta

Group Hib-MenCY5/5/5:Hib-MenCY (5/5/5)+Infanrix ^TMPenta

Group Hib-MenC:Hib-MenC (5/5)+Infanrix ^TMPenta

Group Menjugate:Menjugate ^TM**+ Infanrix ^TMHexa (contrast).

^*With Hib-MenCY2.5/5/5, Hib-MenCY 5/10/10 and Hib-MenC use in the double blinding mode and Hib-MenCY5/5/5 group and Menjugate to organize be disclosed.

^*Menjugate ^TMIt is the vaccine that is applied to all experimenters in the group.

Inoculation when+/-2,3,4 months ages (study the 0th month, January and February), and before preliminary inoculation and inoculate rear one month (studying the 0th month and March) and collecting blood sample (3.5ml) from all experimenters.

Research vaccine, dosage, method of application, lot number: approximately 2,3 and during 4 months ages with 1 month interval intramuscular injection Three doses, as described below:

Table 8: the vaccine of using (research and contrast), group and scheme/position and dosage

Immunogenicity: every kind of vaccine antigen is measured antibody titer/concentration:

In all experimenters for SBA-MenC and SBA-MenY, anti--PSC and anti--PSY, anti--PRP, anti--T, anti--FHA, anti--PRN and anti--PT, before first time dosage (the 0th month) and for the third time approximately 1 month (3rd month) behind the dosage.Use the serum of anti-Neisseria meningitidis serogroup C and Y (SBA-MenC and SBA-MenY cutoff value: 1: 8 and 1: 128) to kill bacterial activity; (anti--PSC IgG and anti--PSYIgG), the ELISA algoscopy has cutoff value 〉=0.3 μ g/ml and 〉=2 μ g/ml for anti-Neisseria meningitidis serogroup C and Y polysaccharide; Gather-ribosyl-ribitol-phosphoric acid (anti--PRP IgG) 〉=0.15 μ g/ml and 〉=1.0 μ g/ml for the Hib polysaccharide; For anti--FHA, anti--PRN, anti--PT, be 5EL.U/ml; 〉=0.1 IU/ml is anti--tetanus toxoid (anti--TT).For anti--D, anti--HBs and anti--poliomyelitis 1,2 and 3, in all experimenters behind dosage for the third time only one month (3rd month).Use the cutoff value of ELISA algoscopy: (resist-D) be 0.1IU/ml for diphtheria; (anti--as HBs) to be 〉=10mIU/ml for anti-hepatitis B virus; With the microneutralization test cutoff value: be 1: 8 for poliomyelitis 1,2 and 3 types (poliomyelitis 1,2 and 3).

Statistical method:

For SBA-MenC, anti--PSC, SBA-MenY, anti--PSY, anti--PRP, anti--tetanus, anti--PT, anti--FHA and anti--PRN before inoculation and one month afterwards; for anti--diphtheria, anti--HBs, anti--poliomyelitis 1, anti--poliomyelitis 2 and anti--poliomyelitis 3 rear one month in inoculation, calculate every group and have the serum protection/seroprevalence of 95% confidence interval (95%CI) and geometric average concentration/(GMCs/GMTs) tires.In inoculation rear one month, also calculate the vaccine that has a 95%CI for anti--PT, anti--PRN and anti--FHA and reply (antibody in initial seronegative experimenter, occurs or in initial seropositive experimenter, keep at least antibody concentration).The reverse cumulative curve of every kind of antibody (Reverse cumulativecurve) when giving 3rd month.For every kind of antibody, assess and Menjugate in the mode of extrapolation ^TMMatched group is compared, the difference between Hib-MenCY and Hib-MenC group, and just except SBA-MenY and the anti--PSY, just (1) is for Menjugate ^TMBe higher than the cutoff value of appointment between group (negative) and Hib-MenCY and the Hib-MenC group or have difference between experimenter's percentage ratio that the vaccine of standardized asymptomatic 95%CI replys, (2) Menjugate ^TMGroup is with respect to GMC or the GMT ratio of Hib-MenCY and Hib-MenC group (95%CI with them).Carry out identical comparison with assessment for anti--PRP, SBA-MenC, anti--PSC, SBA-MenY, anti--PSY and anti--TT antibody, the difference between the every pair of Hib-MenCY preparation.

According to the type of symptom, their intensity and with the relation of inoculation, calculate the overall incidence (reporting experimenter's percent of whole body, part and any symptom that causes and their 95%CI in rear 8 days in inoculation) of the symptom of part and whole body by batch total.Calculate the incidence rate of unreported symptom by batch total.For the 3rd level symptom, provide≤48 hours outbreak, medical observation, continue event, with the relation of inoculation.Serious adverse events has also been described fully.

Serum protection/seroprevalence and GMC/Ts (immunogenic ATP group)

Table 9a resists-PRP (μ g/ml)

Table 9b SBA-MenC (tiring)

Table 9c resists-PSC (μ g/ml)

Table 9d SBA-MenY (tiring)

Table 9e resists-PSY (μ g/ml)

Table 9e resists-tetanus (IU/ml)

Group Hib-MenCY 2.5/5/5:Hib-MenCY (2.5/5/5)+Infanrix ^TMPenta

Group Hib-MenCY 5/10/10:Hib-MenCY (5/10/10)+Infanrix ^TMPenta

Group Hib-MenCY 5/5/5:Hib-MenCY (5/5/5)+Infanrix ^TMPenta

Group Hib-MenC:Hib-Men (5/5)+Infanrix ^TMHexa

Group Menjugate:Menjugate ^TM+ Infanrix ^TMPenta

N=has experimenter's number of available result.%=has experimenter's percent of concentration in the particular range/tire.GMC/T: the geometric average concentration/95%CI=95% confidence interval of tiring; The LL=lower limit; The UL=upper limit.

Conclusion

Use has two kinds of preparations of the Hib that reduces dosage, is more excellent for the immunne response of Hib and MenC.For MenY, use 2.5/5/5 is compared the SBA that sees raising with 5/10/10 preparation with 5/5/5 preparation and is replied.

Claims (25)

1. immunogenic composition, another antigen that it comprises Hib glycoconjugate, at least a extra antibacterial glycoconjugate and is selected from the group that is comprised of whole cell pertussis and hbs antigen, wherein the ratio of Hib and carrier protein is 1: 1 to 1: 4 (w/w) in the Hib glycoconjugate, described antibacterial glycoconjugate comprises Neisseria meningitidis serogroup C capsular saccharides (MenC), wherein the sugared dosage of Hib glycoconjugate is 1-3 μ g, and the sugared dosage of described at least a extra antibacterial glycoconjugate is less than 5 μ g.

2. the immunogenic composition of claim 1, it comprises Neisseria meningitidis serogroups A capsular saccharides (MenA).

3. the immunogenic composition of claim 1, it comprises Neisseria meningitidis sero-group Y capsular saccharides (MenY).

4. the immunogenic composition of claim 1, it comprises Neisseria meningitidis sero-group W135 capsular saccharides (MenW).

5. the immunogenic composition of claim 1, it comprises DT, TT, Pw and HepB.

6. the immunogenic composition of claim 1, it comprises Neisseria meningitidis serogroup B adventitia infusion agent.

7. the immunogenic composition of claim 1, it comprises the pneumoniae capsular from the bacterial strain that is selected from sero-group 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F.

8. the immunogenic composition of claim 1, it comprises the salmonella typhi capsular saccharides.

9. the immunogenic composition of claim 1, wherein identical carrier protein be used to Hib conjugate and described at least a Neisseria meningitidis glycoconjugate or its each.

10. the immunogenic composition of claim 1, wherein Hib sugar is conjugated to the carrier protein of the fragment C, protein D, OMPC and the pneumolysin that are selected from TT, DT, CRM197, TT.

11. the immunogenic composition of claim 1, wherein at least a Neisseria meningitidis sugar or its each be conjugated to the carrier protein of the fragment C, protein D, OMPC and the pneumolysin that are selected from TT, DT, CRM197, TT.

12. the immunogenic composition of claim 1, wherein the ratio of Hib and carrier protein is 1: 2 to 1: 3.5 or approximately 1: 3 (w/w) in the Hib glycoconjugate.

13. the immunogenic composition of claim 2, wherein when MenA sugar exists, it has the molecular weight that is higher than 50kDa, 75kDa, 100kDa or the mean size between 50-100kDa, 55-90KDa or the 60-80kDa.

14. the immunogenic composition of claim 1, wherein when MenC sugar exists, it has the molecular weight that is higher than 50kDa, 75kDa, 100kDa or the mean size between 100-200kDa, 100-150kDa or the 150-200kDa.

15. the immunogenic composition of claim 1, wherein when MenC exists, it is by at least in part O-acetylation so that at least 30% recurring unit at least one position by the O-acetylation.

16. the immunogenic composition of claim 2, wherein when MenA exists, it is by at least in part O-acetylation so that at least 50% recurring unit at least one position by the O-acetylation.

17. the immunogenic composition of claim 1, wherein hib glycoconjugate and at least a Neisseria meningitidis glycoconjugate are not absorbed on the aluminum salt.

18. the immunogenic composition of claim 1, it is without adjuvant.

19. vaccine, it comprises among the claim 1-18 each immunogenic composition and pharmaceutically acceptable excipient.

20. be used for together or the vaccine kit of order administration; it comprises two kinds of multivalent immunogenic compositionss; said composition is used for giving the protection that resists the disease that is caused by bordetella pertussis (Bordetella pertussis), Clostridium tetani (Clostridium tetani), corynebacterium diphtheriae (Corynebacterium diphtheriae) and hemophilus influenza (Haemophilus influenzae) the host; described test kit comprises first container and second container, and first container comprises:

Tetanus toxoid (TT), and

Diphtheria toxoid (DT), and

Whole cell pertussis component, and/or hbs antigen;

Second container comprises the immunogenic composition that contains each immunogenic composition among the claim 1-18.

21. the vaccine kit of claim 20, wherein said first container also comprises the hbs antigen that is adsorbed onto on the aluminum phosphate.

22. the vaccine kit of claim 20, wherein said first or second container also comprise the poliovirus (IPV) of inactivation.

23. the method for the preparation of each immunogenic composition among the claim 1-18, it comprises the step of Hib glycoconjugate and at least a extra antibacterial glycoconjugate and another antigen of being selected from whole cell pertussis and hbs antigen being mixed to form compositions, wherein the ratio of Hib and carrier protein is 1: 1 to 1: 4 (w/w) in the Hib glycoconjugate, and wherein the sugared dosage of Hib glycoconjugate is that each sugared dosage of 1-3 μ g and at least a extra antibacterial glycoconjugate or its is less than 5 μ g.

24. each immunogenic composition, vaccine or test kit are used for the treatment of or prevent purposes in the meningitic medicine in production among the claim 1-18.

25. among the claim 1-18 each immunogenic composition, vaccine or test kit production be used for the treatment of or the medicine of the disease preventing to be caused by hemophilus influenza in purposes.