CN101208102B - Immunogenic composition - Google Patents
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- CN101208102B CN101208102B CN2006800231423A CN200680023142A CN101208102B CN 101208102 B CN101208102 B CN 101208102B CN 2006800231423 A CN2006800231423 A CN 2006800231423A CN 200680023142 A CN200680023142 A CN 200680023142A CN 101208102 B CN101208102 B CN 101208102B
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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
The present invention relates to comprise the immune composition of Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together with carrier protein.The invention still further relates to vaccine and the vaccine kit that comprises Neisseria meningitidis (N.meningitidis) polysaccharide conjugates, the method for preparing immunogenic composition and vaccine and vaccine of the present invention and the application of immunogenic composition in treatment.The present invention also relates to adopt the immunity of Neisseria meningitidis (N.meningitidis) polysaccharide conjugates to come the method and the application of Neisseria meningitidis (N.meningitidis) polysaccharide conjugates in the preparation medicine of anti-Neisseria infection.
Neisseria meningitidis (Neisseria meningitidis) is a Gram-negative human disease bacterium, and it can cause antibacterial and give birth to meningitis.Based on the capsular polysaccharide of biology, 12 kinds of sero-group Neisseria meningitidiss (N.meningitidis) (A, B, C, H, I, K, L, 29E, W135, X, Y and Z) have been identified.Serogroups A (MenA) is the most common epiphytotics pathogenesis in Sub-Saharan Africa.Serogroup B and C are the main cases that exists in the developing country, and other case is then caused by W135 and Y.
The immunogenic composition that comprises Neisseria meningitidis (N.meningitidis) sugar of puting together with carrier protein is known in the art.For example, WO 02/58737 discloses to comprise and has puted together the vaccine of Neisseria meningitidis (N.meningitidis) capsular polysaccharide of serogroups A, C, W135 and Y from purification with carrier protein.Yet Neisseria meningitidis (N.meningitidis) capsular polysaccharide that this application instruction is extracted should come depolymerization through heating in hydrogenperoxide steam generator before puting together.
WO 03/07985 discloses the conjugate vaccines that comprises Neisseria meningitidis (N.meningitidis) sugar that is selected from serogroups A, C, W135 and Y.Extract the meningococcal capsular polysaccharide, hydrolysis then is used for puting together with carrier protein to select the capsular polysaccharide oligosaccharide of deriving.
WO 04/103400 also discloses the multivalent meningococcal derivatized polysaccharide-protein conjugate of Neisseria meningitidis (N.meningitidis) capsular polysaccharide that comprises serogroups A, C, W135 and Y.This uses instruction, does not use natural bigger capsular polysaccharide, preferably uses less meningococcal polysacharide.Document prompting depolymerizes to mean size less than 100,000 dalton with capsular polysaccharide with the mild oxidation condition part, and is preferred 12,000-25,000 dalton.
Still need develop the improved conjugate vaccine of anti-Neisseria meningitidis (Neisseria meningitidis).The present invention relates to provide a kind of meningococcal polysacharide conjugate vaccine, wherein existing teach literature of polysaccharide size is bigger.The focus of prior art is, uses oligosaccharide to be easy to conjugate production.The inventor finds to use natural or the polysaccharide conjugates of grade by sized slightly, and will realize one or more following advantages: 1) conjugate has high immunogenicity and can filter; 2) but enhance immunity memory () of embodiment 3; 3) can change polysaccharide and proteic ratio in the conjugate, polysaccharide and proteic ratio (w/w) increase (this can reduce the carrier depression effect) to some extent in the conjugate thus; 4) immunogen conjugates (for example MenA conjugate) tends to hydrolysis, can stablize through in puting together, using big polysaccharide.Use big polysaccharide, can produce more crosslinkedly with puting together carrier, thus, free sugar will be dissociated from conjugate is less.The conjugate vaccines of putting down in writing in the prior art tends to before puting together, the polysaccharide depolymerization puted together to improve it.The present invention relates to a kind of Different Strategies, and be surprised to find, the meningococcus conjugate vaccines that keeps the large-size polysaccharide can provide good immunne response to meningococcal disease.
Therefore; According to an aspect of the present invention; A kind of immunogenic composition of puting together with carrier protein that comprises from Neisseria meningitidis (N.meningitidis) capsular polysaccharide of at least a, two kinds, three kinds or four kinds serogroups A, C, W and Y is provided, and wherein the mean size of each Neisseria meningitidis (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa.
According to a further aspect of the invention, the invention provides the vaccine that comprises immunogenic composition of the present invention and pharmaceutically acceptable carrier.
According to a further aspect of the invention; The invention provides and be used to follow or the vaccine kit of order administration; It comprises that two kinds of multivalent immunogenic compositionss are to protect host's opposing by Bordetella pertussis (Bordetella pertussis); Clostridium tetani (Clostridium tetani); Diphtheria corynebacterium (Corynebacteriumdiphtheriae); Disease due to hemophilus influenza (Haemophilus influenzae) and the Neisseria meningitidis (Neisseria meningitidis); Wherein said test kit comprises first container and second container
First container comprises:
Tetanus toxoid (TT),
Diphtheria toxoid (DT) and
Full cell or acellular pertussis composition,
Second container comprises:
Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds among serogroups A, C, W and the Y, with carrier protein, wherein the mean size of these or each Neisseria meningitidis (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa.
According to a further aspect in the invention; The invention provides the method that is used to prepare immunogenic composition of the present invention or vaccine; It comprises Neisseria meningitidis (N.meningitidis) capsular polysaccharide with at least a, two kinds, three kinds or four kinds among the serogroups A, C, W and the Y that put together with carrier protein; Randomly; With the step of pharmaceutically acceptable mixed with excipients, wherein the mean size of these or each Neisseria meningitidis (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa.
The present invention on the other hand; The invention provides a kind of immune human host with the method for opposing by the disease due to the Neisseria meningitidis (Neisseria meningitidis), this method comprises immunogenic composition of the present invention or the vaccine that gives host immune protection dosage.
The present invention the invention provides and is used for treatment or prevention by the immunogenic composition of the present invention of the disease due to the Neisseria meningitidis (Neisseria meningitidis) on the other hand.
The present invention the invention provides immunogenic composition of the present invention or vaccine and is being used for preparation treatment or the prevention application by the medicine of the disease due to the Neisseria meningitidis (Neisseria meningitidis) on the other hand.
Accompanying drawing is described
Fig. 1-A-bar diagram has shown the GMC figure of anti-MenYELISA.ENYTT012 is the Men Y-TT conjugate from natural MenY polysaccharide preparation.ENYTT014 takes turns the MenY-TT conjugate that miniflow circulates, Micro Fluid prepares from the MenY polysaccharide for experience 40.ENYTT015bis takes turns the Men Y-TT conjugate that miniflow circulates, Micro Fluid prepares from Men Y polysaccharide for experience 20.
The B-bar diagram has shown the GMT figure that anti-MenY SBA analyzes.ENYTT012 is the Men Y-TT conjugate from natural MenY polysaccharide preparation.ENYTT014 takes turns the Men Y-TT conjugate that miniflow circulates, Micro Fluid prepares from Men Y polysaccharide for experience 40.ENYTT015bis takes turns the Men Y-TT conjugate that miniflow circulates, Micro Fluid prepares from Men Y polysaccharide for experience 20.
Detailed Description Of The Invention
Immunogenic composition of the present invention comprise from least a, two kinds, three kinds or four kinds among serogroups A, C, W and the Y, with Neisseria meningitidis (N.meningitidis) capsular polysaccharide that carrier protein is puted together, the mean size (weight average molecular weight of wherein at least a, two kinds, three kinds or four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide; Mw) be 50kDa, 60kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa.
Of the present invention one independently aspect in; Immunogenic composition comprise from least a, two kinds, three kinds or four kinds among serogroups A, C, W and the Y, with Neisseria meningitidis (N.meningitidis) capsular polysaccharide that carrier protein is puted together, wherein at least a, two kinds, three kinds or four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide are natural polysaccharide or by the coefficient adjustment size with the x1.5, x2, x3, x4, x5, x6, x7, x8, x9 or the x10 that are at most the natural polysaccharide weight average molecular weight.
With regard to the present invention, " natural polysaccharide " is meant that undressed polysaccharide, the purpose of wherein said processing are to reduce the polysaccharide size.Polysaccharide has the minimizing of slight size in conventional purifying process.Such polysaccharide is still natural polysaccharide.As long as polysaccharide is through the technology of adjustment size, this polysaccharide just is not considered to natural polysaccharide.
With regard to the present invention, " with the coefficient adjustment of about x2 size " is meant polysaccharide through handling, and tends to reduce the polysaccharide size but still keeps the half the size that surpasses the natural polysaccharide size.X3, x4 or the like explain in an identical manner, promptly polysaccharide are handled, and purpose is to reduce the size of polysaccharide, but still keep 1/3,1/4 or the like above size of natural polysaccharide size respectively.
In another aspect of this invention; Immunogenic composition comprise from least a, two kinds, three kinds or four kinds among serogroups A, C, W and the Y, with Neisseria meningitidis (N.meningitidis) capsular polysaccharide that carrier protein is puted together, wherein at least a, two kinds, three kinds or four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide are natural polysaccharide.
In another aspect of this invention; Immunogenic composition comprises Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds among serogroups A, C, W and the Y, with carrier protein; Wherein at least a, two kinds, three kinds or four kinds, perhaps each Neisseria meningitidis (N.meningitidis) glycocalix is with the coefficient adjustment of about x1.5, x2, x3, x4, x5, x6, x7, x8, x9 or x10 size.
Immunogenic composition of the present invention randomly comprises following conjugate: Neisseria meningitidis (N.meningitidis) serogroup C capsular polysaccharide (MenC); Serogroups A capsular polysaccharide (MenA); Sero-group W135 capsular polysaccharide (MenW); Sero-group Y capsular polysaccharide (MenY); Serogroup C and Y capsular polysaccharide (MenCY); Serogroup C and A capsular polysaccharide (MenAC); Serogroup C and W capsular polysaccharide (MenCW); Serogroups A and Y capsular polysaccharide (MenAY); Serogroups A and W capsular polysaccharide (MenAW); Sero-group W and Y capsular polysaccharide (MenWY); Serogroups A; C and W capsular polysaccharide MenACW); Serogroups A; C and Y capsular polysaccharide (MenACY); Serogroups A, W135 and Y capsular polysaccharide (MenAWY), serogroup C, W135 and Y capsular polysaccharide (MenCWY); Perhaps serogroups A, C, W135 and Y capsular polysaccharide (MenACWY).This promptly is " a kind of, two kinds, three kinds or four kinds " or the definition of " at least a " serogroups A, C, W and Y or each Neisseria meningitidis (N.meningitidis) polysaccharide that this paper mentions.
In one embodiment, at least a, two kinds, three kinds or four kinds of measuring according to the MALLS method or the mean size (or molecular weight) of each Neisseria meningitidis (N.meningitidis) polysaccharide are 50KDa-1500kDa, 50kDa-500kDa, 50kDa-300 KDa, 101kDa-1500kDa, 101kDa-500kDa or 101kDa-300kDa.
In one embodiment, if the MenA polysaccharide exists, having the molecular weight of measuring according to MALLS is 50-500kDa, 50-100kDa, 100-500kDa, 55-90KDa, 60-70kDa, 70-80kDa or 60-80kDa.
In one embodiment, if the MenC polysaccharide exists, having the molecular weight of measuring according to MALLS is 100-200kDa, 50-100kDa, 100-150kDa, 101-130kDa, 150-210kDa or 180-210kDa.
In one embodiment; If the MenY polysaccharide exists, having the molecular weight of measuring according to MALLS is 60-190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa, 110-140kDa, 50-100kDa, 100-140kDa, 140-170kDa or 150-160kDa.
In one embodiment, if the MenW polysaccharide exists, having the molecular weight of measuring according to MALLS is 60-190kDa, 70-180kDa, 80-170kDa, 90-160kDa, 100-150kDa, 110-140kDa, 50-100kDa or 120-140kDa.
The polysaccharide weight average molecular weight of measuring before the molecular weight of this paper polysaccharide or mean molecule quantity are meant and put together (Mw), it is measured according to the MALLS method.
The MALLS technology is the known technology of this area, carries out with embodiment 2 described records usually.With regard to the MALLS analysis of meningococcus sugar, two kinds of pillars of use capable of being combined (TSKG6000 and 5000PWxI TOSOH Bioscience), and with water elution sugar.These sugar are detected with light dispersion dispersion (for example, the WyattDawnDSP of equipment 10mW488nm argon laser) and interferometer refractometer (for example, equipment P100 photoelectric cell and the filtering Wyatt Otilab of 498nm HONGGUANG DSP).
In one embodiment, Neisseria meningitidis (N.menmgitidis) polysaccharide is a natural polysaccharide, the natural polysaccharide that has perhaps reduced for size in conventional extraction step.
In individual embodiment, through mechanical lysis, the size of Micro Fluid or ultrasonic adjustment Neisseria meningitidis (N.meningitidis) polysaccharide for example.Micro Fluid and ultrasonic has the big natural polysaccharide size of abundant reduction, thereby the advantage that can filter conjugate is provided.The adjustment size is carried out with the coefficient that is no more than x20, x10, x8, x6, x5, x4, x3, x2 or x1.5.
In one embodiment, immunogenic composition comprises Neisseria meningitidis (N.meningitidis) conjugate, and it is by natural polysaccharide with by made with the mixture of the big or small polysaccharide of the coefficient adjustment that is no more than x20.For example, MenC and/or MenA polysaccharide are natural polysaccharides.For example, MenY and/or MenW glycocalix are to be no more than the coefficient adjustment size of x20, x10, x8, x6, x5, x4, x3, x2 or x1.5.For example, immunogenic composition comprises the conjugate of being processed by MenY and/or MenW and/or MenC and/or MenA, and wherein their are by being no more than the coefficient adjustment size of x20, x10, x8, x6, x5, x4, x3, x2 or x1.5, and/or Micro Fluid.For example, immunogenic composition comprises the conjugate by natural MenA and/or MenC and/or MenW and/or MenY preparation.For example, immunogenic composition comprises the conjugate by natural MenC preparation.For example, immunogenic composition comprises the conjugate by natural MenC and MenA preparation, and wherein their are by being no more than the coefficient adjustment size of x20, x10, x8, x6, x5, x4, x3, x2 or x1.5, and/or by Micro Fluid.For example, immunogenic composition comprises the conjugate by natural MenC and MenY preparation, and wherein their are by being no more than the coefficient adjustment size of x20, x10, x8, x6, x5, x4, x3, x2 or x1.5, and/or by Micro Fluid.
In one embodiment; The polydispersity of polysaccharide be 1-1.5,1-1.3,1-1.2,1-1.1 or 1-1.05 and also put together with carrier protein after, 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 measured and are carried out with the MALLS technology.
Randomly, compare with the size of isolating polysaccharide from antibacterial, the size of polysaccharide can be at the most by 1.5,2,4,6,8,10,12,14,16,18 or 20 times of adjustment.
In one embodiment, immunogenic composition of the present invention further comprises the antigen from Neisseria meningitidis (N.meningitidis) serogroup B.Randomly, this antigen is Neisseria meningitidis (N.meningitidis) serogroup B capsular polysaccharide (MenB) or the polysaccharide or the oligosaccharide of deriving of being adjusted size.Randomly, this antigen is the outer membrane vesicles prepared product of Neisseria meningitidis (N.meningitidis) serogroup B of record among EP301992, WO 01/09350, WO 04/14417, WO 04/14418 and the WO 04/14419.
In one embodiment, immunogenic composition of the present invention further comprises hemophilus influenza (H.influenzae) b (Hib) capsular saccharides of puting together with carrier protein.
Being included in Neisseria meningitidis (N.meningitidis) polysaccharide in the pharmaceutical composition of the present invention (with Hib capsular saccharides randomly) can put together with following carrier protein, for example other non-virulent mutant of tetanus toxoid, tetanus toxoid fragment C, the non-virulent mutant of tetanus toxin, diphtheria toxoid, CRM197, diphtheria toxin, diphtherotoxin [for example CRM176, CRM197, CRM228, CRM45 (people J.Biol.Chem.218 such as Uchida; 3838-3844,1973); CRM9, CRM45, CRM02, CRM103 and CRM107 and other mutant, they are at Nicholls and Youle in Genetically EngineeredToxins, Ed:Frankel, Maecel Dekker Inc, on the books in 1992; Glu-148 to Asp, the disappearance of Gln or Ser or sudden change, and/or disappearance or the sudden change of Ala 158 to Gly, and other mutant, they are disclosed among US4709017 or the US 4950740; Lys516, Lys526, at least one of Phe530 and/or Lys534 or the sudden change of a plurality of residue and other mutant, they are disclosed among US5917017 or the US6455673; Perhaps disclosed fragment among the US5843711], (meningococcus outer membrane protein-extract usually is from Neisseria meningitidis (N.meningitidis) serogroup B-EP0372501), synthetic peptide (EP0378881, EP0427347), heat shock protein (WO93/17712, WO94/03208), pertussis albumen (WO98/58668, EP0471177), cytokine, lymphokine, somatomedin or hormone (WO91/01146), such as N19 albumen (Baraldoi et al (2004) Infect lmmun72 for pneumolysin, OMPC; 4884-7) streptococcus pneumoniae surface protein PspA (WO02/091998) pneumolysin (Kuo et al (1995) Infect lmmun63; 2706-13) contain Different Kinds of Pathogens antigenic multivalence people CD4+T cell epitope artificial protein (Falugietal (2001) the Eur J Immunol 31 that derives; 3816-3824), ferrum absorbs toxin A or the B (WO 00/61761) or the protein D (EP594610 and WO 00/56360) of albumen (WO01/72337), difficult clostridium perfringens enterotoxin (C.difficile).
In one embodiment, immunogenic composition of the present invention is used at least two kinds, three kinds, four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide with same carrier protein (independently).In one embodiment, when Hib existed, Hib can put together with at least a, two kinds, three kinds, four kinds or the identical carrier protein of each Neisseria meningitidis (N.meningitidis) polysaccharide.For example, 1,2,3 or 4 kind of Neisseria meningitidis (N.meningitidis) polysaccharide can put together with tetanus toxoid respectively, to prepare 1,2,3 or 4 kind of conjugate.
In one embodiment, single carrier protein can be loaded with the CA (WO04/083251) more than.For example, single carrier protein can with MenA and MenC; MenA and MenW; MenA and MenY; MenC and MenW; MenC and MenY; Men W and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY; MenA, MenC, MenW and MenY; Hib and MenA; Hib and MenC; Hib and MenW; Perhaps Hib and MenY put together.
In one embodiment; Immunogenic composition of the present invention comprises Neisseria meningitidis (N.meningitidis) polysaccharide of puting together with carrier protein, and wherein said carrier protein is selected from the group of being made up of the fragment C of TT, DT, CRM197, TT and protein D.
In one embodiment, immunogenic composition of the present invention comprises the Hib sugar of puting together with carrier protein, and wherein said carrier protein is selected from the group of being made up of the fragment C of TT, DT, CRM197, TT and protein D.
Randomly, immunogenic composition of the present invention comprises at least a meningococcus sugar (MenA for example; MenC; MenW; MenY; MenA and MenC; MenA and MenW; MenA and MenY; MenC and Men W; Men C and MenY; MenW and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MwnY; MenC, MenW and MenY; Perhaps MenA, MenC, MenW and MenY) conjugate, wherein said conjugate Men sugar is 1 with the ratio of carrier protein: 5-5: 1,1: 2-5: 1,1: 0.5-1: 2.5 or 1: 1.25-1: 2.5 (w/w).
Randomly, immunogenic composition of the present invention comprises the Hib glycoconjugate, and the Hib that it has and the ratio of carrier protein are 1: 5-5: 1; 1: 2-2: 1; 1: 1-1: 4; 1: 2-1: 3.5; Perhaps be approximately or just in time 1: 2.5 or 1: 3 (w/w)." approximately " is meant in 10% scope of said ratio.
Sugar can adopt the sterilization conjugate to measure with the ratio (w/w) of carrier protein in the conjugate.Protein content is with Lowry assay determination (for example people (1951) J.Biol.Chem.193 such as Lowry, people Analytical Biochemistry 100 such as 265-275 or Peterson, 201-220 (1979)); The polysaccharide amount; MenA is measured with ICP-OES (inducing coupling plasma-optical emission spectroscopy); MenC is used the DMAP assay determination, to MenW and MenY then use the Resorcinol assay determination (people (1988) Anal.Biochem.175 such as Monsigny, 525-530).
In one embodiment, through connexon, for example difunctional connexon is puted together the immunogen conjugates and the carrier protein of Neisseria meningitidis of the present invention (N.meningitidis) polysaccharide and/or Hib sugar.Randomly, connexon is an isodigeranyl function or with difunctional connexon, for example its have 1 reaction amino with 1 reaction carboxyl, 2 reactions are amino or 2 react carboxyls.Connexon for example has 4-20,4-12, a 5-10 carbon atom.A kind of feasible connexon is ADH.Other connexon comprises B-propionamido-(WO00/10599), nitrobenzophenone-ethamine (Gever et al (1979) Med.Microbiol.Immunol.165; 171-288), alkyl halide halogenide (US4057685), glycosidic bond (US4673574, US4808700), hexane diamidogen and 6-aminocaprolc acid (US4459286).
The polysaccharide conjugates that in immunogenic composition of the present invention, exists, available any known coupling technology preparation.Conjugation methods can be dependent on 1-cyanic acid-4-dimethylamino naphthyridine
tetrafluoroborate (CDAP) sugared activation, forms cyanate and carries out.Thus, activatory sugar can be directly or via spacerarm (connexon) group, is connected to carrier protein amino.For example; Spacerarm can be cystamine or cysteamine; To give sulfide polysaccharide, wherein said polysaccharide can be via being connected with carrier with maleimide activated carrier albumen (for example using GMBS) or the back thioester bond that is obtained of halogen acetyl carrier protein (for example using iodoacetamide or N-butanimide bromacetate) reaction.Randomly, cyanate (randomly, with the preparation of CDAP chemical method) can be connected with hexamethylene diamine or ADH, and thus, the sugar of amino derivatization can adopt carbodiimides chemistry (for example EDAC or EDC) and carrier protein to put together.Such conjugate is documented among the disclosed application of PCT WO93/15760 (Uniformed Services University), WO95/08348 and the WO96/29094.
Other appropriate technology can use carbon imines, hydrazides, Acibenzolar, norbornane, Nitrodracylic acid, N-hydroxy-succinamide, S-NHS, EDC, TSTU.Manyly all be documented among the WO98/42721.Put together and can relate to the carbonyl connexon, its by free hydroxyl group of sugar earlier with CDI reaction (people J.Biol.Chem.1979 such as Bethell, 254; 2572-4, Hearn et al J.Chromatogr.1981.218; 509-18) again with albumino reaction, form amino-formate bond and form.This process can relate to terminal anomeric carbon and be reduced into primary hydroxyl, randomly, relates to the protection/protective reaction of the primary hydroxyl of primary hydroxyl and CDI, to form the CDI carbamate intermediate, then, with the CDI carbamate intermediate, is connected with proteic amino.
Conjugate also can be by the direct reductive amination method preparation of record among US 4365170 (Jennings) and the US 4673574 (Anderson).Other method is documented among EP-0-161-188, EP-208375 and the EP-0-477508.
Further method relates to through carbodiimide polymerization (Chu C.et al Infect.Immunity, 1,983,245 256), for example uses EDAC, and the activatory sugar by AH (ADH) derivatization of Bromine cyanide. (or CDAP) is connected with protein carrier.
In one embodiment, the hydroxyl on the sugar (hydroxyl of optional activation, the for example activatory hydroxyl of cyanate) directly or indirectly (through connexon) be connected with proteic amino or carboxyl.When having connexon, sugared hydroxyl randomly is connected with the amino of connexon, for example puts together method with CDAP and connects.Connexon for example ADH unnecessary amino can with the carboxyl coupling on the albumen, for example connect, as using EDAC with the carbodiimides chemical method.In one embodiment, Hib or Neisseria meningitidis (N.meningitidis) capsular polysaccharide before connexon and carrier protein are puted together, is puted together with connexon earlier.
In one embodiment, Hib sugar if exist, is used CNBr, or CDAP, the perhaps combination of CDAP and carbodiimides chemistry (for example EDAC), and perhaps itself and carrier protein are puted together in the combination of CNBr and carbodiimides chemicals (for example EDAC).Randomly, with CNBr and carbodiimides chemistry (optional EDAC), Hib is puted together.For example, CNBr is used to connect sugar and connexon, then the carbodiimides chemicals is used for connexon is connected to protein carrier.
In one embodiment, with at least a Neisseria meningitidis (N.meningitidis) capsular polysaccharide directly and carrier protein put together; Randomly, make MenW and/or MenY and/or MenC sugar directly and carrier protein put together.For example, make MenW; MenY; MenC; MenW and MenY; MenW and MenC; MenY and MenC; Perhaps MenW, MenY and MenC directly are connected with carrier protein.Randomly, through CDAP at least a Neisseria meningitidis (N.memngitidis) capsular polysaccharide is directly puted together.For example, make MenW through CDAP; MenY; MenC; MenW and MenY; MenW and MenC; MenY and MenC; Perhaps MenW, MenY and MenC directly are connected (referring to WO95/08348 and WO96/29094) with carrier protein.In one embodiment, all Neisseria meningitidiss (N.meningitidis) capsular polysaccharide and tetanus toxoid are puted together.
Randomly; Men W and/or Y sugar are 1 with the ratio of carrier protein: 0.5-1: 2 (w/w); And/or the ratio of MenC sugar and carrier protein is 1: 0.5-1: 4 or 1: 1.25-1: 1.5 or 1: 0.5-1: 1.5 (w/w), and particularly when randomly using CDAP that these sugar directly are connected with albumen.
In one embodiment, at least a Neisseria meningitidis (N.meningitidis) capsular polysaccharide, through connexon, for example difunctional connexon is puted together with carrier protein.Randomly, connexon is an isodigeranyl function or with difunctional connexon, and for example it has 1 reactive amino and 1 reactive carboxyl, 2 reactive amino or 2 reactive carboxyls.For example, connexon has 4-20,4-12, a 5-10 carbon atom.Possible connexon is ADH.
In one embodiment, MenA; MenC; Perhaps MenA and MenC put together through connexon and carrier protein (for example tetanus toxoid).
In one embodiment, with CDAP and EDAC, at least a Neisseria meningitidis (N.meningitidis) polysaccharide and carrier protein are puted together through connexon.For example, use aforesaid CDAP and EDAC, through connexon (the for example terminal connexon that contains two diazanyls is like ADH) with MenA; MenC; Perhaps MenA and MenC and albumen are puted together.For example, use CDAP that sugar and connexon are puted together, and EDAC is used for connexon and albumen are puted together.To MenA; MenC; Perhaps MenA and MenC, randomly, by puting together of connexon, the ratio that can produce polysaccharide and carrier protein is 1: 0.5-1: 6; 1: 1-1: 5 or 1: 2-1: 4.
In one embodiment, the MenA capsular polysaccharide, if exist, it is at least in part by the O-acetylation, make at least 50%, 60%, 70%, 80%, 90%, 95% or 98% repetitive at least one site by the O-acetylation.For example, the O-acetylation appears at the O-3 site of at least 50%, 60%, 70%, 80%, 90%, 95% or 98% repetitive at least.
In one embodiment; The MenC capsular polysaccharide; If exist; It is at least in part by the O-acetylation, make NeuNAc repetitive that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% (α 2-＞9) connect at least one or two site by the O-acetylation.For example, the O-acetylation appears at the O-7 and/or the O-8 site of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% repetitive.
In one embodiment; The MenW capsular polysaccharide; If exist, its at least part by the O-acetylation, make at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% repetitive at least one or two site by the O-acetylation.For example, the O-acetylation appears at the O-7 and/or the O-9 site of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% repetitive.
In one embodiment; The MenY capsular polysaccharide; If exist, it is at least in part by the O-acetylation, thus at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% repetitive at least one or two site by the O-acetylation.The O-acetylation appears at 7 and/or 9 site of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% repetitive.
O-acetylation percentage ratio is meant the percent that contains the acetylizad repetitive of O-.This can put together preceding and/or put together after polysaccharide in measure.
In further embodiment; Immunogenic composition of the present invention comprises Hib glycoconjugate and at least two kinds of Neisseria meningitidiss (N.meningitidis) polysaccharide conjugates, and wherein the sugared dosage of Hib conjugate is lower than the average sugared dosage of at least two kinds of Neisseria meningitidiss (N.meningitidis) polysaccharide conjugates.Randomly, the sugared dosage of Hib conjugate is all lower than every kind of sugared dosage of at least two kinds of Neisseria meningitidiss (N.meningitidis) polysaccharide conjugates.For example, the dosage of Hib conjugate is than the average sugared dosage of at least two kinds of Neisseria meningitidiss (N.meningitidis) polysaccharide conjugates or low sugar dosage low at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%.
Term " sugar " comprises polysaccharide or oligosaccharide.Separation of polysaccharides is from antibacterial or separate from antibacterial, and with known method (referring to for example EP497524 and EP497525), and randomly, use the Micro Fluid method, its size is adjusted to a certain degree.Polysaccharide is carried out the size adjustment, can reduce the viscosity of polysaccharide sample and/or the filterability of the product that raising is puted together.Oligosaccharide typically is characterised in that it is the Polysaccharides (typically, 5-30 repetitive) with a small amount of repetitive.
Mean dose is through owning the dosage addition of " further " polysaccharide, and is definite divided by the quantity of " further " polysaccharide again." further " polysaccharide is meant the polysaccharide of all except that Hib in immunogenic composition, and it comprises Neisseria meningitidis (N.meningitidis) capsular polysaccharide." dosage " is in the quantity of administration of human para-immunity originality compositions or vaccine.
Hib sugar is that hemophilus influenza (Haemophilus influenzae) b type gathers ribose phosphoric acid (PRP) capsular polysaccharide or its deutero-oligosaccharide.
" at least two kinds of further antibacterial glycoconjugates " is meant that at least two kinds except that the Hib conjugate add the antibacterial glycoconjugate.At least two kinds of further antibacterial glycoconjugates comprise Neisseria meningitidis (N.meningitidis) capsular polysaccharide conjugates.
Immunogen conjugates of the present invention can comprise from one or more Neisseria meningitidiss (Neisseriameningitidis); Streptococcus pneumoniae (streptococcus pneumoniae); A group streptococcus (Group AStreptococci); B group streptococcus (Group B Streptococci); Salmonella typhi (S.typhi); The further glycoconjugate of staphylococcus aureus (Staphylococcus aureus) or staphylococcus epidermidis (Staphylococcus epidermidis).In one embodiment, immunogenic composition comprises, from the capsular polysaccharide or the oligosaccharide of the Neisseria meningitidis (Neisseria meningitidis) of one or more serogroups A, C, W135 and Y.Further embodiment comprises capsular polysaccharide or oligosaccharide from streptococcus pneumoniae (streptococcus pneumoniae).Pneumococcal capsular polysaccharide or oligosaccharide antigen randomly are selected from serotype 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 (randomly, being selected from serotype 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F).Further embodiment comprises 5 types of staphylococcus aureus (Staphylococcusaureus), 8 types or 336 type capsular polysaccharide or oligosaccharide.Further embodiment comprises the I type of staphylococcus epidermidis (Staphylococcus epidermidis), II type or III type capsular polysaccharide.Further embodiment comprises the Vi sugar (polysaccharide or oligosaccharide) from salmonella typhi (S.typhi).Further embodiment comprises Ia type, Ic type, II type, III type or the V-type capsular polysaccharide or the oligosaccharide of B group streptococcus.Further embodiment comprises the capsular polysaccharide or the oligosaccharide of A group streptococcus, randomly, can further comprise at least a M albumen and randomly comprise polytype M albumen.
In one embodiment, immunogenic composition of the present invention comprises each Neisseria meningitidis (N.meningitidis) capsular polysaccharide, and its dosage is 0.1-20 μ g; 1-10 μ g; 2-10 μ g, 2.5-5 μ g perhaps is about or is 5 μ g exactly; Perhaps be about or be 2.5 μ g exactly.
In one embodiment; Immunogenic composition of the present invention for example comprises the Hib glycoconjugate, and its sugared dosage is 0.1-9 μ g, 1-5 μ g or 2-3 μ g; Perhaps be about or be 2.5 μ g exactly; And it comprises each Neisseria meningitidis (N.meningitidis) polysaccharide conjugates, and its sugared dosage is 2-20 μ g, 3-10 μ g or 4-7 μ g, perhaps is about or is 5 μ g exactly.
With regard to the present invention, " pact " or " approximately " is meant in 10% scope that is greater than or less than given numeral.
In one embodiment; Immunogenic composition of the present invention contains the sugared dosage of Hib glycoconjugate, for example this dosage be lower than at least two kinds, three kinds, four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide conjugates average sugared dosage 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.For example, the sugared dosage of Hib sugar for the 20%-60% of at least two kinds, three kinds, four kinds or the average sugared dosage of each Neisseria meningitidis (N.meningitidis) polysaccharide conjugates, 30%-60%, 40%-60% or approximately or be 50% exactly.
In one embodiment; Immunogenic composition of the present invention contains the sugared dosage of Hib glycoconjugate, for example be lower than at least two kinds, three kinds, four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide conjugates low sugar dosage 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.For example, the sugared dosage of Hib sugar for the 20%-60% of the low sugar dosage of at least two kinds, three kinds, four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide conjugates, 30%-60%, 40%-60% or approximately or be 50% exactly.
In embodiment of the present invention, each sugared dosage of at least two kinds, three kinds, four kinds or each Neisseria meningitidis (N.meningitidis) polysaccharide conjugates randomly is identical or approximately identical.
The instance of immunogenic composition of the present invention for what be made up of following component, perhaps comprises the compositions of following component:
Hib conjugate and MenA conjugate and MenC conjugate, randomly, its sugared dosage ratio is 1: 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, MenA sugar dosage is greater than MenC sugar dosage.
Hib conjugate and MenC conjugate and MenY conjugate, randomly, its sugared dosage 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, MenC sugar dosage is greater than MenY sugar dosage.
Hib conjugate and MenC conjugate and MenW conjugate, randomly, its sugared dosage 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, MenC sugar dosage is greater than MenW sugar dosage.
Hib conjugate and MenA conjugate and MenW conjugate, randomly, its sugared dosage 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, MenA sugar dosage is greater than MenW sugar dosage.
Hib conjugate and MenA conjugate and MenY conjugate, randomly, its sugared dosage 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, MenA sugar dosage is greater than MenY sugar dosage.
Hib conjugate and MenW conjugate and MenY conjugate; Randomly, its sugared dosage 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, MenY sugar dosage is greater than MenW sugar dosage.
MenA, MenC, MenW and MenY, its sugared dosage ratio is 1: 1: 1: 1 or 2: 1: 1: 1 or 1: 2: 1: 1 or 2: 2: 1: 1 or 1: 3: 1: 1 or 1: 4: 1: 1 (w/w).
Further aspect of the present invention is the vaccine that comprises immunogenic composition of the present invention and pharmaceutically acceptable excipient.
In one embodiment, immunogenic composition of the present invention is buffered to or is adjusted to pH7.0-8.0, pH7.2-7.6 or is pH7.4 approximately or exactly.
Immunogenic composition of the present invention or vaccine randomly under there is situation in stabilizing agent, for example exist under the situation at many alcohol matters such as sucrose or trehalose, are frozen drying.
Randomly, immunogenic composition of the present invention or vaccine comprise a certain amount of adjuvant that is enough to the former immunne response of enhance immunity.Suitable adjuvant includes but not limited to, aluminum salt (aluminum phosphate or aluminium hydroxide), zamene mixture (SAF-1), muramyl peptide, saponin derivative, mycobacterium cell wall preparation, monophosphoryl lipid A, chorismic acid derivant, non-ionic block copolymer surfactant, Quil A, b subunit of cholera toxin, polyphosphazene acid derivative and the immunostimulation conjugate of for example putting down in writing among people (1 990) the Nature 344:873-875 such as Takahashi (ISCOMs).
With regard to the Neisseria meningitidis (N.meningitidis) or HibMen compositions of above-mentioned discussion, need not perhaps not use any adjuvant by any aluminum salt adjuvant, be preferred.
With regard to all immunogenic compositions or vaccine, immunogenic immune effective dose needs rule of thumb to confirm.The factor of considering comprises whether immunogenicity, immunogen put together with adjuvant or carrier protein or other carrier or covalently bound, route of administration and the immunizing dose that gives.These factors are that the vaccine field is known, and the immunological technique personnel need not too much experiment, can skillfully make such decision.
Active agent can multiple concentration exist in pharmaceutical composition of the present invention or the vaccine.Typically, the Cmin of material is the necessary amounts that obtains required application, and Cmax then is suspended in the maximum in the original mixture for remaining in solution or homogeneous.For example, the minimum of therapeutic agent randomly provides with the single therapy effective dose.As far as bioactive substance, Cmin is the bioactive necessary amounts of reconstruct, the amount that Cmax then can not be kept for the homogeneous suspension again.Under single dose unit's situation, this amount is used required amount for single therapy.Usually, can expect that each dosage comprises the proteantigen of 1-100 μ g, randomly comprise 5-50 μ g or 5-25 μ g.The instance of antibacterial sugar dosage is 10-20 μ g, 5-10 μ g, 2.5-5 μ g or 1-2.5 μ g.The preferred amounts of material changes with material, and those skilled in the art confirm easily.
Via system or the said vaccine of mucosal route administration, vaccine production thing of the present invention can be used for protection or treats susceptible mammal (for example human patients).Human patients randomly is baby's (December below), the child that learns to walk (12-24,12-16 or 12-14 month), child (2-10,3-8 or 3-5 year), teenager (12-25,14-21 or 15-19 year) or adult's (surpassing for any ages of 12,15,18 or 21).These administrations comprise intramuscular injection, peritoneal injection, intradermal injection or subcutaneous injection; Perhaps via mucosa delivery to oral cavity/esophagus, respiratory tract, urogenital tract.It is preferred (because can more effectively prevent nasopharynx synovial membrane Diplococcus pneumoniae, can alleviate early infection thus) for treatment pneumonia or otitis media that intranasal gives vaccine.Though but vaccine single dose of the present invention gives; But its component also can be simultaneously or timesharing give jointly (for example to be present under the situation in the vaccine at sugar; These components can give respectively simultaneously or after giving the bacterioprotein vaccine, give in 1-2 week, to work in coordination with immunne response each other best).Except single route of administration, can use two kinds of different route of administration.For example, virus antigen is given with ID (Intradermal), bacterioprotein is given with IM (intramuscular) or IN (intranasal).Under the situation that polysaccharide exists, can it be given with IM (or D), bacterioprotein then gives with IN (or ID).In addition, also can vaccine of the present invention be given as amount of initiator with IM, then give as booster dose with IN.
Vaccine production is on the books in Vaccine Design (" The subunit and adjuvant approach " (eds Powell M.F.&Newman M.J.) (1995) Plenum Press New York) usually.Liposome embedded technology is at Fullerton, and is on the books among the US Patent 4,235,877.
Of the present inventionly advance on the one hand for following or the vaccine kit of order administration; Wherein said test kit comprises that two kinds of multivalent immunogenic compositionss avoid by Bordetella pertussis (Bordetellapertussis), clostridium tetani (Clostridium tetani), diphtheria corynebacterium (Corynebacterium diphtheriae) and Neisseria meningitidis (Neisseria meningitidis) and randomly with the protection host, the disease due to the hemophilus influenza (Haemophilus influenzae).For example, test kit randomly comprises first container and second container, and first container comprises one or more following materials:
Tetanus toxoid (TT),
Diphtheria toxoid (DT) and
Full cell or acellular pertussis composition,
Second container comprises:
Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds in A, C, W and the Y sero-group, with carrier protein; Wherein the mean size of each all Neisseria meningitidiss (N.meningitidis) polysaccharide be 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or 130kDa with; Randomly, its be frozen exsiccant;
Perhaps
The Hib glycoconjugate and
Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds in A, C, W and the Y sero-group, with carrier protein; Wherein the mean size of each all Neisseria meningitidiss (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa; Randomly, its be frozen exsiccant.
The formulation examples of Hib conjugate and Neisseria meningitidis (N.meningitidis) polysaccharide conjugates, such as preceding text record.
Of the present inventionly advance on the one hand for following or the vaccine kit of order administration, wherein said test kit comprises that two kinds of multivalent immunogenic compositionss avoid by streptococcus pneumoniae (streptococcuspneumoniae) and Neisseria meningitidis (Neisseria meningitidis) and the disease due to the hemophilus influenza (Haemophilus influenzae) randomly with the protection host.For example, test kit randomly comprises first container and second container, and first container comprises:
1,2,3,4,5 one or more carrier proteins and streptococcus pneumoniae (streptococcus pneumoniae) capsular polysaccharide conjugates [wherein capsular polysaccharide randomly is selected from the streptococcus pneumoniae sero-group by the following group of forming:, 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F].
Second container comprises:
Neisseria meningitidis (N. meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds in A, C, W and the Y sero-group, with carrier protein; Wherein the mean size of each all Neisseria meningitidiss (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or 130kDa; Randomly, its be frozen exsiccant;
Perhaps
The Hib glycoconjugate and
Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds in A, C, W and the Y sero-group, with carrier protein; Wherein the mean size of each all Neisseria meningitidiss (N.meningitidis) polysaccharide be 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or 130kDa with; Randomly, its be frozen exsiccant.
The instance of Hib conjugate and Neisseria meningitidis (N.meningitidis) polysaccharide conjugates, such as preceding text record.
Typically; Streptococcus pneumoniae in the vaccine kit of the present invention (streptococcus pneumoniae) vaccine; Comprise that CA (randomly; Put together), 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 wherein polysaccharide is from least four kinds of streptococcus pneumoniae sero-groups that are selected from by the following group of forming:.Randomly, four kinds of sero-groups comprise 6B, 14,19F and 23F.Randomly, at least 7 kinds of sero-groups are included in the compositions, and for example they are from sero-group 4,6B, 9V, 14,18C, 19F and 23F.Randomly, be included in the compositions for example at least 10,11,12,13 or 14 kind of sero-group more than 7 kinds of sero-groups.For example, in one embodiment, compositions comprises 11 kinds of capsular polysaccharides (randomly, puting together) from sero-group 1,3,4,5,6B, 7F, 9V, 14,18C, 19F and 23F.In one embodiment of the invention; At least comprise 13 kinds of polysaccharide antigens (randomly; Put together), although more polysaccharide antigen, for example 23 valency antigens are (for example; Sero-group 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F), also be included in the scope of the invention.
Streptococcus pneumoniae sugar can be puted together with any known carrier albumen respectively, for example aforesaid CRM197, tetanus toxoid, diphtheria toxoid, protein D or any other carrier protein.
Randomly, vaccine kit of the present invention comprises the 3rd component.For example, test kit randomly comprises first container and second container and the 3rd container, and container comprises one or more following materials:
Tetanus toxoid (TT),
Diphtheria toxoid (DT) and
Full cell or acellular pertussis composition,
Second container comprises:
1,2,3,4,5 the conjugate of one or more carrier proteins and streptococcus pneumoniae (streptococcus pneumoniae) capsular polysaccharide [wherein capsular polysaccharide randomly is selected from the streptococcus pneumoniae sero-group by the following group of forming:, 6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F].
The 3rd container comprises:
Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds in A, C, W and the Y sero-group, with carrier protein; Wherein the mean size of each all Neisseria meningitidiss (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa; Randomly, its be frozen exsiccant;
Perhaps
The Hib glycoconjugate and
Neisseria meningitidis (N.meningitidis) capsular polysaccharide of puting together from least a, two kinds, three kinds or four kinds in A, C, W and the Y sero-group, with carrier protein; Wherein the mean size of each all Neisseria meningitidiss (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa; Randomly, its be frozen exsiccant.
Contain the for example immunogenic composition of HibMenC, HibMenAC, HibMenAW, HibMenAY, HibMenCW, HibMenCY, HibMenWY, MenAC, MenAW, MenAY, MenCW, MenCY, MenWY or MenACWY of meningococcal conjugate; Comprise test kit with the similar compositions of above-mentioned composition; Randomly, all can comprise antigen from measles and/or parotitis and/or rubella and/or chickenpox.For example, the meningitis immunogenic composition comprises measles, parotitis and rubella antigen, perhaps comprises measles,mumps,rubella and vzv antigen.In one embodiment, these virus antigens randomly exist in the same container of meningitis and/or Hib glycoconjugate.In one embodiment, these virus antigens have been frozen drying.
It is of the present invention that to advance on the one hand be the method for preparing immunogenic composition of the present invention; It comprises with from A, C, W and the Y sero-group at least a, two or three, Neisseria meningitidis (N.meningitidis) capsular polysaccharide puted together with carrier protein; With the blended step of antibacterial glycoconjugate, wherein the mean size of each all Neisseria meningitidiss (N.meningitidis) polysaccharide is 50kDa, 75kDa, 100kDa, 110kDa, 120kDa or more than the 130kDa.
Vaccine production is on the books in Vaccine Design (" The subunit and adjuvant approach " (edsPowell M.F.&Newman M.J.) (1995) Plenum Press NewYork) usually.Liposome embedded technology is at Fullerton, and is on the books among the US Patent 4,235,877.
It is of the present invention that to advance be immune human host with opposing by Neisseria meningitidis (N.meningitidis) and randomly on the one hand; The method of the disease due to hemophilus influenza (Haemophilus influenzae) infects, this method comprise immunogenic composition of the present invention or vaccine or the test kit that gives host immune protection dosage.
Of the present invention advance be on the one hand be used for treatment or prevention by Neisseria meningitidis (N.meningitidis) with randomly, the immunogenic composition of the present invention of the disease due to hemophilus influenza (Haemophilus influenzae) infection.
It is of the present invention that to advance on the one hand be immunogenic composition of the present invention or vaccine or test kit; Preparation be used for treatment or prevention by Neisseria meningitidis (N.meningitidis) with randomly, the application in hemophilus influenza (Haemophilus influenzae) the infection associated diseases medicine.
Term among this paper " comprises " that " comprising " mean the inventor, randomly respectively in every instance, can with term " by ... form " mutual alternative.
All lists of references or patent application that patent specification is quoted all are merged in as a reference at this.
Will be in the present invention of the following example illustrated.The following example all uses the standard technique that well known to a person skilled in the art with conventional to carry out, only if detailed description is arranged in addition.Embodiment is indicative, and it can't limit the present invention.
Embodiment
The preparation of embodiment 1-polysaccharide conjugates
Hemophilus influenza (Haemophilus influenzae) is the covalent bond of PRP polysaccharide and TT (Hib), according to people such as Chu (Infection and Immunity 1983,40 (1); 245-256) the coupling chemistry of development carries out.Through adding CNBr, pH 10.5 incubations 6 minutes are with the activation of HibPRP polysaccharide.PH is reduced to pH8.75, adds fatty acid hydrazide (ADH), continued incubation again 90 minutes.With activatory PRP with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimides (EDAC) via the carbodiimides concentration method, with the tetanus toxoid coupling of purification.EDAC is added to activatory PRP, until reaching whole ratio 0.6mgEDAC/mg activation PRP.Adjustment pH to 5.0, the tetanus toxoid that adds purification is to 2mg TT/mg activation PRP.Gained solution was placed three days gentle agitation.Behind 0.45 μ m membrane filtration, with conjugate in that (Pharmacia Sweden) goes up purification with the equilibrated sephacryl S500HR of 0.2M NaCl pillar.
With natural polysaccharide (MALLS measures more than the 150kDa) preparation MenC-TT conjugate.The polysaccharide of surveying the slight Micro Fluid more than the 60kDa with natural polysaccharide or embodiment 2MALLS method prepares the MenA-TT conjugate.The big or small size of measuring with MALLS of about 100-200kDa prepares (referring to embodiment 2) MenW and MenY-TT conjugate through the polysaccharide of adjusting.Use Emulsiflex C-50 homogenizer device to adjust size through Micro Fluid.Then, polysaccharide is passed 0.2 μ m membrane filtration.
Activation and coupling are undertaken by the method for WO96/29094 and WO 00/56360 record.In brief, the polysaccharide of the 10-20mg/ml concentration among the 2MNaCl pH5.5-6.0 and CDAP solution (100mg/ml, at second cyanogen/WFI, prepared fresh in 50/50 solvent) are mixed, reach 0.75/1 or 1.5/1 until the final ratio of CDAP/ polysaccharide.1.5 after minute, with sodium hydroxide rising pH to pH10.0.After three minutes, adding tetanus toxoid is 1.5/1 (as far as MenW), 1.2/1 (as far as MenY), 1.5/1 (as far as MenA), 1.5/1 (as far as MenC) until albumen/polysaccharide ratio.Reaction is proceeded 1-2 hour.
After coupling step, the adding glycine is until final ratio glycine/PS (w/w) 7.5/1 and adjust pH to pH9.0.Mixture was left standstill 30 minutes.With 10 μ m Kleenpak filters conjugate is clarified, used 150mM NaCl then, 10mM or 5mM Tris pH7.5 elution buffer are uploaded to Sephacryl S400HR pillar with it.With clinical packing conjugate with Opticap 4 membrane filtrations.The average polysaccharide of gained conjugate: the albumen ratio is 1: 1-1: 5 (w/w).
For MenA capsular polysaccharide and tetanus toxoid are puted together through spacerarm, can use following method.Polysaccharide and spacerarm (ADH) covalent bond; Can carry out with the coupling chemistry at cyanidization agent 1-cyanic acid-4-dimethylamino naphthyridine
tetrafluoroborate (CDAP) activated polysaccharide under the condition of control.With the PS reaction of spacerarm through diazanyl and cyanic acidization, the stable isourea of formation is connected between spacerarm and polysaccharide thus.
With 100mg/ml CDAP second cyanogen/water (50/50 (the v/v)) solution-treated of 10mg/ml MenA solution, be 0.75 (w/w) to obtain the CDAP/MenA ratio with prepared fresh.After 1.5 minutes, pH is increased to pH10.0.After three minutes, add ADH, obtaining the ADH/MenA ratio is 8.9.PH value of solution is reduced to 8.75, reacted 2 hours.
Before conjugation reaction, with the TT solution and the PSA of purification
AHSolution dilution is to the PSA of 10mg/ml
AHThe TT concentration of concentration and 10mg/ml.
EDAC is added to PSA
AHIn the solution, until obtaining final ratio 0.9mg EDAC/mgPSA
AHPH is adjusted to 5.0.With the tetanus toxoid (in 60 minutes) of peristaltic pump adding purification, until obtaining 2mg TT/mg PSA
AHWith gained solution at+25 ℃ at stirring condition held 60min, until the final coupling time that reaches 120min.Conjugate is clarified reuse SephacrylS400HR pillar purification with 10 μ m filter membranes.
Embodiment 2-uses the MALLS determining molecular weight
HPLC size exclusion column coupling with detector and elution samples.On the one hand, the laser light scattering detector is used under 16 angles measuring the scattered light intensity of macromole solution, on the other hand, the interferometer refractometer of online placement allows to measure the quantity of elution samples.From these intensity, can measure macromolecular size and shape in the solution.
Weight average molecular weight (Mw) is meant, the weight of all species multiply by they separately the summation of molecular weight again divided by the weight summation of all materials.
A) weight average molecular weight :-Mw-
Mw＝∑W
i.M
i/∑W
i＝m
2/m
1
B) number average molecular weight :-Mn-
Mn＝∑N
i.M
i/∑N
i＝m
i/m
0
C) root-mean-square radius :-Rw-, wherein R
2W is a square radius, is defined as:
R
2W or (r
2) w=∑ m
i.r
i 2/ ∑ m
i
(m
i-be the quality of the i of distribution center ,-r
i-be the distance between i of distribution center and the macromole center of gravity)
D) polydispersity is defined as-ratio of Mw/Mn-.
Through the HPLC pillar (TSKG6000 and 5000PWxI) that is uploaded to two couplings, analyze meningococcal polysacharide through MALLS.25 μ l polysaccharide are uploaded to pillar, with 0.75ml filtered water eluting.Detect polysaccharide with light dispersion dispersion (the Wyatt Dawn DSP of equipment 10mW488nm argon laser) and interferometer refractometer (equipment P100 photoelectric cell and the filtering Wyatt Otilab of 498nm HONGGUANG DSP).
The molecular weight polydispersity and the response rate of all samples are calculated with the 1 order polynomial match of Astra4.72 software through the debye method.
Embodiment 3-adopts the clinical experiment relative immunity of the Men C-TT conjugate of Meningitec or large-size
Carry out the open comparative study of II phase, so that the meningococcus serogroup C conjugate vaccines (MenC) of GSK Biologicals company and hemophilus influenza (Haemophilusinfluenzae) the b-meningococcus serogroup C conjugate vaccine (Hib-MenC) or the Meningitec
of GSK Biologicals company are compared.Every dosage Meningitec
contains the 10 μ g meningococcus serogroup C oligosaccharide of puting together with 15 μ g CRM197, and it is prepared by Wyeth company.GSK Men C conjugate contains the natural polysaccharide of the about 200kDa that puts together with tetanus toxoid (TT).
Research comprises five groups, and every group plan contains 100 objects, is divided into following two parallel classes:
In this research, all two class objects are in the time of 12-15 month (the research moon is 0) all, accepts the Mencevax of 1/5 dosage
TMACWY and the Infanrix that follows dosage
TMHexa.From all objects collect two kinds of blood samples (research month be 0 with research month be 1).Form by four groups that carry out just exempting to study for the 1st type, they are 3,4 and with following vaccine they were caused in 5 months:
● group K: MenC (10 μ g)-tetanus toxoid (TT) conjugate and the Infanrix that is not adsorbed to aluminum salt (not adding adjuvant)
TMHexa (MenC10-TT+Infanrix
TMHexa)
● not interpolation adjuvant TT conjugate and the Infanrix of group L:Hib (10 μ g)-MenC (10 μ g)
TMPenta (Hib10-MenC10-TT+Infanrix
TMPenta)
● group M:Hib (5 μ g)-MenC (not interpolation adjuvant TT conjugate and Infanrix of 5 μ g
TMPenta (Hib5-MenC5-TT+Infanrix
TMPenta)
● group N:Meningitec
TMAnd Infanrix
TMHexa (Meningitec
TM+ Infanrix
TMHexa)
Two Hib-MenC-TT vaccine group (group L and M) excite in the research at definite candidate vaccine preparation, keep double blinding.
The 2nd type (group O) by not inoculating meningitis serogroup C vaccine (not carrying out immunity) in the past but the of the right age object of accepting the conventional department of pediatrics vaccine of German immune standing committee regulation form.
Evaluation criterion
Immunogenicity: in the blood sample of all objects that before immunity, approximately obtain January with the immunity back; Through sterilization experiment measure the bactericidal properties antibody (SBA-MenC) of meningococcemia C titre (cutoff: 1: 8 dilution factor), through ELISA measure antimeningococcic serum crowd C antibody (analyze cutoff: 0.3 μ g/ml), anti-Hib polysaccharide PRP antibody (analyze cutoff: 0.15 μ g/ml) and tetanus toxoid antibody (analyze cutoff: 0.1IU/ml).
Statistical method:
Demography: average monthly age (intermediate value, scope and standard deviation [SD]), race and the sex of measuring ATP immunity crowd and panimmunity crowd are formed.
Immunogenicity:
Based on ATP immunogenicity crowd (being used to analyze immunological memory and booster response) or ATP safety crowd (being used to analyze persistency), carry out two kinds of immunogenicity analyses.These comprise:
Measurement is to the immunne response of MenC with to Hib and tetanic booster response (before giving the conventional polysaccharide vaccine of 1/5 dosage with one month after):
● in 95% confidence interval (95%CI), confirm geometric mean titer and concentration (GMTs and GMCs).
● in 95%CI, confirm to have the individual percent (seropositivity/serum protective rate) of the antibody titer/concentration more than the cutoff of suggestion
● use the titre/concentration of reverse cumulative curve research immunity back antibody
● to the difference between the seropositivity/serum protective rate between initiation group (group K, L, M and N) and the not initiation group (group O), the asymptotic 95%CI of the basis of calculation
● in 95%CI, measure the geometrical mean that the SBA-MenC titre surpasses the individual ratio of anti--PSC concentration
● with the ANOVA model to immunity after the GMT/C ratio carry out 95%CI and measure; Antagonism PRP and tetanus; Between group K, L, M and matched group N, measure,, between each initiation group (group K, L, M and N) and not initiation group (group O), measure SBA-MenC and anti--PSC.
The result
SBA-MenC titre and anti--PSC AC behind table 1 booster immunization
| Antibody | Group | N | ?GMT/C | ?95％CILL | ?95％CIUL |
| SBA-MenC | K-MenC-TT L-HibMenC M-HibMenC N-Meningitec O-contrast | 7179818591 | ?3508.9?2530.1?5385.4?1552.6?9.3 | ?2580.1?1831.7?4425.0?1044.4?6.3 | ?4772.2?3494.7?6554.2?2307.9?13.6 |
| Anti--PSC | K-MenC-TT L-HibMenC M-HibMenC N-Meningitec O-contrast | 7071767894 | ?28.10?30.01?34.58?16.59?3.05 | ?22.59?24.09?29.10?12.98?2.36 | ?34.95?37.38?41.09?21.21?3.93 |
Group K: with the object of MenC10-TT+Infanrix.hexa initiation; Group L: with the object of Hib10-MenC10-TT+Infanrix.penta initiation; Group M: with the object of Hib5-MenC5-TT+Infanrix.penta initiation; Group N: with the object of Meningitec.+Infanrix.hexa initiation; Group O: contrast object (object that does not promptly cause) with the MenC conjugate vaccines; N: the number of objects that can obtain the result
Compare with Menngitec oligosaccharide conjugate vaccines, (group K, L and M) causes with big MenC polysaccharide conjugate vaccines, can produce the SBA titre of antibody titer and the Geng Gao of higher anti-MenC.
The geometric average ratio of table 2:SBA MenC titre/anti--PSC concentration
| Group | Time | ?N | ?GMR | ?LL | ?UL |
| K | After the preceding immunity of immunity | ?70?66 | ?49.470?126.138 | ?34.939?101.419 | ?70.044?156.882 |
| L | After the preceding immunity of immunity | ?76?70 | ?36.528?90.200 | ?25.849?70.153 | ?51.621?115.975 |
| M | After the preceding immunity of immunity | ?77?74 | ?51.298?164.950 | ?36.478?139.304 | ?72.139?195.318 |
| N | After the preceding immunity of immunity | ?84?76 | ?22.571?90.168 | ?16.521?67.757 | ?30.837?119.991 |
| O | After the preceding immunity of immunity | ?3?87 | ?91.634?2.708 | ?0.651?1.767 | ?12889?8?4.149 |
In all four groups of initiation groups (group K, L, M and N), GMR shows the maturation and the functionalization that there are antibody to booster immunization, significantly increasing before the booster immunization.Group M (causing with Hib5-MenC5-TT) (uses Meningitec than group N
TMInitiation) it is high that GMR wants.
Table 3: the persistency during 12-15 monthly age before strengthening vaccine just
| Terminal point | Group | ?N | ?％ | Group | ?N | ?％ | Difference | Value % |
| SBAMenC≥1∶8 | ?K?L?M | ?79?84?85 | ?88.6?93.3?87.1 | ?N?N?N | ?91?91?91 | ?80.2?80.2?80.2 | ?N-K?N-L?N-M | -8.4-3.1-6.8 |
| SBAMenC≥1∶128 | ?K?L?M | ?79?84?85 | ?65.8?56.0?64.7 | ?N?N?N | ?91?91?91 | ?51.6?51.6?51.6 | ?N-K?N-L?N-M | -14.2-4.3-13.1 |
| Anti--PSC >=0.3 μ g/ml | ?K?L?M | ?79?84?88 | ?100.0?100.0?98.9 | ?N?N?N | ?91?91?91 | ?100.0?100.0?100.0 | ?N-K?N-L?N-M | 0.00.01.1 |
| Anti--PSC >=2 μ g/ml | ?K?L?M | ?79?84?88 | ?72.2?64.3?64.3 | ?N?N?N | ?91?91?91 | ?81.3?81.3?81.3 | ?N-K?N-L?N-M | 9.217.08.6 |
| Anti--PRP >=0.15 μ g/ml | ?K?L?M | ?81?86?90 | ?88.9?96.5?98.9 | ?N?N?N | ?91?91?91 | ?85.7?85.7?85.7 | ?N-K?N-L?N-M | -3.2-10.8-13.2 |
| Anti--PRP >=1 μ g/ml | ?K?L?M | ?81?86?90 | ?33.3?55.8?74.4 | ?N?N?N | ?91?91?91 | ?28.6?28.6?28.6 | ?N-K?N-L?N-M | -4.8-27.2-45.9 |
| Tetanus >=0.1IU/ml | ?K?L?M | ?81?86?90 | ?100.0?100.0?100.0 | ?N?N?N | ?91?91?91 | ?96.7?96.7?96.7 | ?N-K?N-L?N-M | -3.3-3.3-3.3 |
Group K: with the object of MenC10-TT+Infanrix.hexa initiation; Group L: with the object of Hib10-MenC10-TT+Infanrix.penta initiation; Group M: with the object of Hib5-MenC5-TT+Infanrix.penta initiation; Group N: with the object of Meningtec.+Infanrix.hexa initiation; N: the number of objects that can obtain the result.
Compare with causing, to cause (group K, L and M), can obtain higher anti-MenC SBA titre with big MenC with MenC-oligosaccharide conjugate Menmgitec.
Immunological memory (ATP immunogenicity crowd)
Give the common Polysaccharide A CWY vaccine of 1/5 dosage; In all four groups of initiation groups, all produced very high SBA-MenC titre; The object of the 98.7%-100% that wherein causes with the candidate vaccine system and the object of 97.5%-100% demonstrate titre >=1: 8 and >=1 respectively: 128.Using Meningitec
TMIn the initiation group of system, tend to low scale object and show titre>=1: 128 (91.8%).Comparatively speaking, 17.6% not initiation object shows SBA-MenC titre >=1: 8 and >=1: 128.
Embodiment 4 and the blended HibMenAC-TT conjugate vaccines of DTPw-HeDB II phase clinical experiment
Research design: at random (1: 1: 1: 1: 1) of the opening of five groups single center research.Make these five groups when 6,10 and 14 ages in week, accept following immunization protocol respectively.
● Tritanrix.-HepB/Hib-MenAC 2.5 μ g/2.5 μ g/2.5 μ g: below be called 2.5/2.5/2.5
● Tritanrix.-HepB/Hib-MenAC 2.5 μ g/5 μ g/5 μ g: below be called 2.5/5/5
● Tritanrix.-HepB/Hib-MenAC5 μ g/5 μ g/5 μ g: below be called 5/5/5
● Tritanrix.-HepB+Hiberix.: below be called Hiberix
● Tritanrix.-HepB/Hiberix.+Menmgitec: below be called Meningitec
Blood sample is taken from for the first time one month (dosage 3 afterwards) after immunizing dose time (Pre) and the immunizing dose for the third time.
Tritanrix is the commercial DTPw vaccine of GlaxoSmithKlme Biologicals S.A company.
Each group in five groups is used 105 objects, in this research, uses 525 objects altogether.
The content of table 4GSK bacterin preparation
| The composition of each dosage (0.5ml) | 2.5/2.5/2.5 * | 2.5/5/5 | ?5/5/5 |
| The Hib capsular polysaccharide PRP that puts together with tetanus toxoid (TT) | 2.5μg | 2.5μg | ?5μg |
| Neisseria meningitidis (Neisseria meningitidis) the A capsular polysaccharide of puting together with TT (PSA) | 2.5μg | 5μg | ?5μg |
| Neisseria meningitidis (Neisseria meningitidis) the C capsular polysaccharide of puting together with TT (PSC) | 2.5μg | 5μg | ?5μg |
*2.5/2.5/2.5 vaccine is the dose dilution thing of the Hib-MenAC5/5/5 vaccine of GSK Biologicals, it contains every kind of PRP-TT of 2.5 μ g, MenA-TT and MenC-TT.
The Hib-MenAC bacterin preparation is mixed with Tritanirix-HepB immediately.The associating diph/tet of GSK Biologicals company-full cell Boulder pertussis-hepatitis B (DTPw-HB) vaccine (Tritanrix-HepB) contains diphtheria toxoid, the tetanus toxoid that is no less than 60IU that is no less than 30 ius (IU), the deactivation Bo Deshi pertussis that is no less than 4IU and 10 μ g recombination hepatitis B surface antigens.
With reference to treatment, dosage, administering mode, lot number:
Rabbit epidemic disease scheme/position: one group 6,10 and 14 the week ages, accept the Thtanrix-HepB vaccine and accept Hiberix at the left thigh intramuscular at the right thigh intramuscular
TMAnother group is accepted Tritanrix in 6,10 and 14 ages in week at the left thigh intramuscular
TM-HepB/Hiberix
TMVaccine is also accepted the Meningitec vaccine at the right thigh intramuscular.
Vaccine/composition/dosage/lot number: the Tntanrix of use
TM-HepB vaccine, as stated.
B type hemophilus influenza (Haemophilus infiuenzae) conjugate vaccines: the Hiberix of 1 dosage (0.5ml) GSK Biologicals
TMContain the PRP that 10 μ g and tetanus toxoid are puted together.At Hiberix
TMIn the group, it is mixed with sterile diluent, at Meningitec
TMIn the group, with itself and Tritanrix
TM-HepB mixes.
The MENINGITEC of 1 dosage (0.5ml) Wyeth Lederle
TMVaccine contains: 10 μ g C group meningitis cocci pod membrane oligosaccharide and the aluminum salt of puting together with 15 μ g diphtheria corynebacterium (Corynebacterium diphtheria) CRM197 albumen.
The immunne response of anti-Hib.MenA of result-generation and MenC
Table 5a resists-PRP (ug/ml)
| Group | 25/25/25 | ?25/5/5 | ?5/5/5 | ?Hiberix TM | Meningitec TM | ||||||||||
| ％ | 95％CI | ?％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ||||||
| CMC/T | LL | UL | ?GMC/T | LL | ?UL | ?GMC/T | ?LL | ?UL | ?GMC/T | ?LL | UL | GMC/T | LL | UL | |
| ％≥0.15 | 100 | 96.5 | 100 | ?99.0 | 94.8 | ?100 | ?100 | ?96.5 | ?100 | ?100 | ?96.5 | 100 | 100 | 96.5 | 100 |
| GMC | 20.80 | 15.96 | 27.10 | ?22.62 | 17.72 | ?28.88 | ?19.36 | ?15.33 | ?24.46 | ?38.55 | ?29.93 | 49.64 | 10.94 | 8.62 | 13.88 |
Table 5b SBA-MenC
| Group | 25/25/25 | 25/5/5 | 5/5/5 | Hiberix TM | Meningitec TM | ||||||||||
| ％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ||||||
| GMC/T | LL | UL | GMC/T | LL | UL | GMC/T | LL | UL | GMC/T | LL | UI | GMC/T | LL | UL | |
| ％≥1∶8 | 99 | 94.7 | 100 | 100 | 96.5 | 100 | 100 | 96.5 | 100 | 29 | 0.6 | 8.4 | 100 | 96.5 | 100 |
| GMT | 3132 | 2497 | 3930 | 4206 | 3409 | 5189 | 3697 | 3118 | 4384 | 4.7 | 3.9 | 5.6 | 4501 | 3904 | 5180 |
Table 5cSBAMenA
| Group | 25/25/25 | 25/5/5 | 5/5/5 | Hiberix TM | Meningtec TM | ||||||||||
| ％ | 95％CL | ％ | 95％CI | ％ | 95％CI | ％ | 95％I | ％ | 95％CL | ||||||
| GMC/T | ?LL | UL | GMC/T | LL | UL | GMC?/T | LL | UI | GMC/T | LL | LL | GMC/T | LL | UL | |
| ％≥1∶8 | 99.7 | 91.9 | 99.7 | 100 | 95.8 | 100 | 100 | 96.2 | 100 | 6.8 | 2.5 | 14.3 | 9.1 | 4.0 | 17.1 |
| GMT | 316.7 | 251.4 | 398.9 | 418.5 | 358.6 | 488.5 | 363 | 310.5 | 424.4 | 5.6 | 4.3 | 7.4 | 5.6 | 4.4 | 7.2 |
Table 5d resists-PSC (ug/ml)
| Group | 25/25/25 | 25/5/5 | 5/5/5 | Hibeix TM | Meningitec TM | ||||||||||
| ％ | 95％CI | ％ | ?95％CI | ％ | 95％CI | ％ | 95％CI | ％ | ?95％CI | ||||||
| GMC/T | LL | UL | GMC/T | ?LL | ?UL | GMC/T | LL | ?UL | GMC/T | LL | UL | GMC/T | ?LL | ?UL | |
| ?％≥0.3 | 100 | 96.5 | 100 | 100 | ?％4 | ?100 | 100 | 96.5 | ?100 | 8.2 | 3.6 | 15.6 | 100 | ?96.5 | ?100 |
| ?GMC | 49.03 | 43.24 | 55.59 | 71.11 | ?62.49 | ?80.92 | 61.62 | 54.88 | ?69.20 | 0.17 | 0.15 | 0.19 | 58.02 | ?51.42 | ?65.46 |
Table 5e resists-PSA (ug/ml)
| Group | 25/25/25 | 25/5/5 | 5/5/5 | Meningtec TM | Meningitec TM | ||||||||||
| ％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ％ | 95％CI | ||||||
| GMC/T | LL | UL | GMC/T | LL | UL | GMC/T | LL | UL | GMC/T | LL | UL | GMC/T | LL | UL | |
| ?％≥0.3 | 100 | 96.4 | 100 | 100 | 96.5 | 100 | 99.0 | 94.8 | 100 | 1.0 | 0.0 | 5.4 | 5.9 | 22 | 125 |
| ?GMC | 18.10 | 15.34 | 21.35 | 26.51 | 22.93 | 30.79 | 23.40 | 20.05 | 27.30 | 0.15 | 0.15 | 0.15 | 0.17 | 0.15 | 0.18 |
Conclusion
With oligosaccharide MenC-CRM197 conjugate vaccine and three kinds of MenA-TT and MenC-TT conjugate GSK preparations that contain polysaccharide; The immunogenicity result who obtains shows that relatively polysaccharide Men conjugate can produce the good immunogenic response similar with oligosaccharide conjugate vaccine Meningitec.All test formulation are replied MenC in 100% patient.
Embodiment 5-by 2,3 and April scheme give Hib MenCY and follow the II phase clinical experiment that gives Infanrix penta
Research design: II phase, open (part double blinding
*), the multicenter study of control at random, it comprises that 5 windings have received three dosage of following vaccine just to exempt from scheme
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-MenC (5/5)+Infanrix
TMPenta
Group Menjugate:Menjugate
TM*+Infanrix
TMHexa (contrast).
*Hib-MenCY2.5/5/5, Hib-MenCY5/10/10 and Hib-MenC give with the double blinding mode, and Hib-MenCY5/5/5 group and Menjugate group then are open.2.5/5/5,5/10/10 and the 5/5/5Hib-MenCY preparation, contain and have plenty of MenC natural polysaccharide and Micro Fluid MenY polysaccharide.
*Menjugate
TMEvery dosage contains the 10 μ g MenC oligosaccharide of puting together with 12.5-25 μ g CRM197, and it is produced by Chiron company.
+/-2,3, April immunity (research month 0, month 1 and months 2), before blood sample (3.5ml) is taken from and is just exempted from just exempt from all back one month individualities (research month 0 with month 3).
Research vaccine, dosage, administering mode, lot number: with three dosage with January at interval, in about 2,3, April, intramuscular injection is following:
Table 6: vaccine gives (research and contrast), group, scheme/position and dosage
| Group | Scheme (month) | The vaccine dose position that gives: upper left thigh | What give follows the vaccine position: upper right thigh |
| Hib-MenCY2.5/5/5 | 2,3 and 4 | ?Hib(25μg)-MenC-TT(5μg)-MenY-TT(5μg) | DTPa-HBV-IPV(Infanrix TM?penta) |
| Hib-MenCY5/10/10 | 2,3 and 4 | ?Hib(5μg)-MenC-TT(10μg)-MenY-TT(10μg) | DTPa-HBV-IPV(Infanrix TM?penta) |
| Hib-MenCY5/5/5 | 2,3 and 4 | ?Hib(5μg)-MenC-TT(5μg)-MenY-TT(5μg) | DTPa-HBV-IPV(Infanrix TM?penta) |
| Hib-MenC | 2,3 and 4 | ?Hib(5μg)-MenC(5μg) | DTPa-HBV-IPV(Infanrix TMPenta) |
| Menjugate TM | 2,3 and 4 | ?Menjugate TM | DTPa-HBV-IPV/Hib(Infanix TM?hexa) |
Immunogenicity: antibody titer/concentration of measuring anti-every kind of vaccine antigen:
Before first time dosage (month 0) and in dosage for the third time after about 1 month (month 3), to all objects to SBA-MenC and SBA-MenY, anti--SC and anti--PSY, anti--PRP, anti--T, anti--FHA, anti--PRN and resist-PT detects.Use the SBA (SBA-MenC and SBA-MenY cutoff: 1: 8 and 1: 128) of anti-Neisseria meningitidis (N.meningitidis) serogroup C and Y, detect; Elisa assay cutoff: antagonism-Neisseria meningitidis (N.meningitidis) serogroup C and Y polysaccharide (anti--PSC IgG and anti--PSY IgG), >=0.3 μ g/ml and >=2 μ g/ml; The Hib polysaccharide is gathered ribose-ribitol-phosphoric acid (anti--PRP IgG), >=0.15 μ g/ml and >=1.0 μ g/ml; Antagonism-FHA, anti--PRN, anti--PT, 5EL.U/ml; Antagonism-tetanus toxoid (anti--TT), >=0.1IU/ml.In dosage for the third time just after what a month (month 3), antagonism-D, anti--HBs and resist in all objects-poliomyelitis 1,2 and 3 detects.Use following cutoff elisa assay: antagonism-diphtheria (anti--D), 0.1IU/ml; As far as anti-hepatitis B (anti--HBs), >=10mIU/ml; Microneutralization experiment cutoff: antagonism-poliomyelitis 1,2 and 3 types (anti--poliomyelitis 1,2 and 3), 1: 8.
Statistical method:
In 95% confidence interval (95%CI); Every group is calculated serum protection/seroprevalence and geometric average concentration/titre (GMCs/GMTs); As far as SBA-MenC, anti--PSC, SBA-MenY, anti--PSY, anti--PRP, anti--tetanus, anti--PT, anti--FHA and anti--PRN, before immunity, calculated with immune back one month; Antagonism-diphtheria, anti--HBs, anti--poliomyelitis 1, anti--poliomyelitis 2 and anti--poliomyelitis 3 calculated in immunity in back one month.Antagonism-PT, anti--PRN and anti--FHA reply (initial seronegativity individuality has antibody and occurs, and the individual AC of perhaps initial seropositivity is kept at least) at the vaccine of 95%CI, also calculate after one month in immunity.The reverse cumulative curve of every antibody 3 o'clock moons also is provided., in the asymptotic 95%CI of standard, surpass specific cutoff or have vaccine and reply individual percent, Menjugate according to (1)
TMIt is poor that group (deducting) Hib-MenCY and Hib-MenC organize, (2) in 95%CI, Menjugate
TMThe GMC of group and Hib-MenCY and Hib-MenC group or the ratio of GMT, with exploratory mode, assessment Hib-MenCY and Hib-MenC organize and Menjugate to every kind of antibody except that SBA-MenY and anti--PSY
TMDifference between the matched group.Carry out identical comparison, assess every pair of Hib-MenCY preparation, the difference between anti--PRP, SBA-MenC, anti--PSC, SBA-MenY, anti--PSY and anti--TT antibody.
Serum protection/Xue Qingyangxingshuai &GMC/Ts (ATP immunogenicity crowd)
Table 7a resists-PRP (μ g/ml)
| Group | N | ％≥0.15 | LL | UL | ≥1 | LL | UL | GMC | LL | UL |
| HibMenCY2.5/5/5 | 67 | 100.0 | 94.6 | 100.0 | 98.5 | 92.0 | 100.0 | 9.01 | 7.25 | 11.21 |
| HibMenCY5/10/10 | 67 | 100.0 | 94.6 | 100.0 | 98.5 | 92.0 | 100.0 | 9.49 | 7.72 | 11.65 |
| HibMenCY5/5/5 | 70 | 100.0 | 94.9 | 100.0 | 98.6 | 92.3 | 100.0 | 8.08 | 6.53 | 9.98 |
| HibMebC | 74 | 100.0 | 95.1 | 100.0 | 98.6 | 92.7 | 100.0 | 10.44 | 8.49 | 12.83 |
| Menjugate TM | 71 | 100.0 | 94.9 | 100.0 | 80.3 | 69.1 | 88.8 | 2.60 | 1.97 | 3.43 |
Table 7b SBA-MenC (titre)
| Group | N | ％≥0.8 | LL | UL | ≥1∶128 | LL | UL | GMT | LL | UL |
| Hib?MenCY2.5/5/5 | 70 | 100.0 | 94.9 | 100.0 | 95.7 | 88.0 | 99.1 | 1005.8 | 773.5 | 1308.0 |
| Hib?MenCY5/10/10 | 67 | 100.0 | 94.6 | 100.0 | 94.0 | 85.4 | 98.3 | 1029.8 | 799.7 | 1326.0 |
| Hib?MenCY5/5/5 | 71 | 100.0 | 94.9 | 100.0 | 94.4 | 86.2 | 98.4 | 906.9 | 691.3 | 1189.8 |
| Hib?MenC | 74 | 100.0 | 95.1 | 100.0 | 95.9 | 88.6 | 99.2 | 871.0 | 677.3 | 1120.0 |
| Menjugate TM | 71 | 100.0 | 94.9 | 100.0 | 100.0 | 94.9 | 100.0 | 3557.6 | 2978.8 | 4248.8 |
Table 7c resists-PSC (μ g/ml)
| Group | N | ％≥0.3 | LL | UL | ≥2 | LL | UL | GMC | LL | UL |
| HibMenCY2.5/5/5 | 69 | 100.0 | 94.8 | 100.0 | 100.0 | 94.8 | 100.0 | 21.70 | 18.36 | 25.65 |
| HibMenCY5/10/10 | 66 | 100.0 | 94.6 | 100.0 | 100.0 | 94.6 | 100.0 | 27.26 | 23.26 | 31.95 |
| HbMenCY5/5/5 | 70 | 100.0 | 94.9 | 100.0 | 100.0 | 94.9 | 100.0 | 19.02 | 16.49 | 21.93 |
| HibMenC | 74 | 100.0 | 95.1 | 100.0 | 100.0 | 95.1 | 100.0 | 21.08 | 18.24 | 24.35 |
| Menjugate TM | 71 | 100.0 | 94.9 | 100.0 | 100.0 | 94.9 | 100.0 | 38.49 | 33.64 | 44.05 |
Table 7d SBA-MenY (titre)
| Group | N | ％≥1∶8 | LL | UL | ≥1∶128 | LL | UL | GMT | LL | UL |
| HibMenCY2.5/5/5 | 69 | 97.1 | 89.9 | 99.6 | 92.8 | 83.9 | 976 | 470.7 | 351.1 | 631.2 |
| HibMenCY5/10/10 | 66 | 97.0 | 89.5 | 99.6 | 86.4 | 75.7 | 93.6 | 437.1 | 322.0 | 593.4.8 |
| HibMenCY5/5/5 | 71 | 98.6 | 92.4 | 100.0 | 95.8 | 88.1 | 99.1 | 635.3 | 501.5 | 804.8 |
| HibMenC | 74 | 21.6 | 12.9 | 32.7 | 13.5 | 6.7 | 23.5 | 9.3 | 6.3 | 13.7 |
| Menjugate TM | 71 | 19.7 | 11.2 | 30.9 | 9.9 | 4.1 | 19.3 | 7.5 | 5.4 | 10.4 |
Table 7e resists-PSY (μ g/ml)
| Group | N | ％≥0.3 | LL | UL | ≥2 | LL | UL | GMC | LL | UL |
| HibMenCY2.5/5/5 | 69 | 100.0 | 94.8 | 100.0 | 100.0 | 94.8 | 100.0 | 26.86 | 22.86 | 31.56 |
| HibMenCY5/10/10 | 66 | 100.0 | 94.6 | 100.0 | 100.0 | 94.6 | 100.0 | 37.02 | 31.84 | 43.04 |
| HibMenCY5/5/5 | 70 | 100.0 | 94.9 | 100.0 | 100.0 | 94.9 | 100.0 | 23.57 | 19.94 | 27.86 |
| HibMenC | 74 | 8.1 | 3.0 | 16.8 | 4.1 | 0.8 | 11.4 | 0.19 | 0.15 | 0.25 |
| Menjugate TM | 71 | 5.6 | 1.6 | 13.8 | 1.4 | 0.0 | 7.6 | 0.17 | 0.15 | 0.19 |
Table 7f resists-tetanus (IU/ml)
| Group | N | ％≥0.1 | LL | UL | GMC | LL | UL |
| HibMenCY2.5/5/5 | 68 | 100.0 | 94.7 | 100.0 | 3.06 | 2.63 | 3.55 |
| HibMenCY5/10/10 | 67 | 100.0 | 94.6 | 100.0 | 3.25 | 2.88 | 3.68 |
| HibMenCY5/5/5 | 70 | 100.0 | 94.9 | 100.0 | 2.97 | 2.59 | 3.41 |
| HibMenC | 74 | 100.0 | 95.1 | 100.0 | 3.15 | 2.73 | 3.64 |
| Menjugate TM | 71 | 100.0 | 94.9 | 100.0 | 1.66 | 1.39 | 1.97 |
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)+lnfanrix
TMPenta
Group Hib-MenC:Hib-Men (5/5)+Infanrix
TMHexa
Group Menjugate:Menjugate
TM+ Infanrix
TMPenta
N=can obtain result's individual amount; The individual percent of %=particular range concentration/titre
GMC/T: geometric average concentration/titre; The 95%CI=95% confidence interval; The LL=lower limit; The UL=upper limit
Conclusion
MenC and Y polysaccharide conjugates have all produced good immunne response in all individualities, wherein 100% individuality has produced MenC and MenY are surpassed replying of 0.3 μ g/ml.
Three kinds of MenACWY-TT preparations of embodiment 6-and Meningitec MenC-CRM197 oligosaccharide are sewed
The clinical comparative experiments of II phase of compound vaccine
Present embodiment has been reported II the phase; Open (part double blinding), research at random, the controlling agent weight range; When assessment gives 12-14 month child with single dose; Meningococcus serogroups A, C, W-135, Y tetanus toxoid conjugate (MenACWY-TT) bacterin preparation of three kinds of different GlaxoSmithKlne BIological companies are with the relative immunity originality of MenC oligosaccharide-CRM197 conjugate vaccine preparation (Meningitec).
Clinical experiment is open (part double blinding
*), control, multicenter study, wherein qualified 12-14 month object by at random (1: 1: 1: 1) assign in one of four parallel-group of 50 objects, it was following to accept single dosage of just exempting from the 1st access time:
Preparation 1T: the MenACWY-TT that contains the MenY polysaccharide dosage that MenA polysaccharide that 2.5 μ g put together tetanus toxoid (TT), MenC polysaccharide that 2.5 μ g put together TT, MenW polysaccharide that 2.5 μ g put together TT and 2.5 μ g put together TT
Preparation 2T: the MenACWY-TT that contains the MenY polysaccharide dosage that MenA polysaccharide that 5 μ g put together TT, MenC polysaccharide that 5 μ g put together TT, MenW polysaccharide that 5 μ g put together TT and 5 μ g put together TT
Preparation 3T: the MenACWY-TT that contains the MenY polysaccharide dosage that MenA polysaccharide that 2.5 μ g put together TT, MenC polysaccharide that 10 μ g put together TT, MenW polysaccharide that 2.5 μ g put together TT and 2.5 μ g put together TT
Contrast T: the 10 μ g MenC oligosaccharide (Meningitec that put together with 12.5-25 μ g CRM197
TM).
*Three kinds of different MenACWY-TT preparations give with the double blinding form.
The vaccination regimen position gives single vaccine by specifying in the 1st access time (the research moon 0) at random at LD, intramuscular.All candidate vaccines provide (being 0.5ml after using the reconstruct of complementarity salt diluent) with freeze dried bead with single dose vial.
Immunogenicity: January, (month 1) institute obtained in the blood sample antibody titer/concentration of measurement meningococcemia vaccine antigen composition approximately behind (month 0) and the research vaccine dose before the research vaccine dose in all objects.Measure the bactericidal properties antibody titer (SBA-MenA, SBA-MenC, SBA-MenW and SBA-MenY) of anti-Neisseria meningitidis (N.meningitidis) serogroups A, C, W-135 and Y through sterilization experiment (analysis cutoff: 1: 8 and 1: 128 dilution factor); The antibody that ELISA measures anti-Neisseria meningitidis (N.meningitidis) serogroups A, C, W-135 and Y (resists-PSA, resists-PSC, resists-PSW and anti--PSY; Analyze cutoff >=0.3 μ g/ml and >=2 μ g/ml), and the anatoxic antibody of tetanus (anti--tetanus is analyzed cutoff 0.1IU/ml).
The result
Inoculate one month (just exempting from terminal point), the antibody response of adding up with SBA-MenA, SBA-MenC, SBA-MenW and SBA-MenY respondent percent is displayed in the table 8.Reply, to the seropositivity object, being defined as more than or equal to 4-doubly increases; Individual to seronegativity before inoculating, be defined as seroconversion.
Table 8: inoculate after one month, the SBA antibody mediated immunity is replied
| Antibody | Group | N | ％ | LL | UL |
| SBA-MenA | Preparation 1T preparation 2T preparation 3T Meningitec TM | 42 39 40 36 | 61.9 82.1 62.5 11.1 | 45.6 66.5 45.8 3.1 | 76.4 92.5 77.3 26.1 |
| SBA-MenC | Preparation 1T preparation 2T preparation 3T Meningitec TM | 46 43 44 49 | 97.8 100.0 95.5 91.8 | 88.5 91.8 84.5 80.4 | 99.9 100.0 99.4 97.7 |
| SBA-MenW | Preparation 1T preparation 2T | 45 43 | 100.0 97.7 | 92.1 87.7 | 100.0 99.9 |
| Preparation 3T Meningitec TM | 45 46 | 100.0 15.2 | 92.1 6.3 | 100.0 28.9 | |
| SBA-MenY | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 44 45 49 | 97.9 88.6 93.3 4.1 | 88.7 75.4 81.7 0.5 | 99.9 96.2 98.6 14.0 |
Table 9 has shown that the SBA titre surpasses number of objects and the GMTs value of cutoff 1: 8 and 1: 128.
Table 9: the seroprevalence and the GMTs that inoculate SBA antibody after one month
| Antibody | Group | N | ％ | ≥1∶8 LL | UL | ％ | ≥1∶128LL | UL | GMT |
| SBA-MenA | Preparation 1T preparation 2T preparation 3T Meningitec TM | 46 45 48 41 | 100 100 97.9 51.2 | 92.3 92.1 88.9 35.1 | 100 100 99.9 67.1 | 100 97.8 97.9 43.9 | 92.388.288.928.5 | 100 99.9 99.9 60.3 | 1457.3 1776.9 1339.5 42.8 |
| SBA-MenC | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 45 47 50 | 97.9 100 95.7 94.0 | 88.7 92.1 85.5 83.5 | 99.9 100 99.5 98.7 | 78.7 84.4 85.1 62.0 | 64.370.571.747.2 | 89.3 93.5 93.8 75.3 | 281.3 428.6 478.4 200.1 |
| SBA-MenW | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 45 48 48 | 100 100 100 27.1 | 92.5 92.1 92.6 15.3 | 100 100 100 41.8 | 100 100 97.9 6.3 | 92.592.188.91.3 | 100 100 99.9 17.2 | 2529.1 2501.6 2300.2 9.4 |
| SBA-MenY | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 45 48 49 | 100 100 100 49.0 | 92.5 92.1 92.6 34.4 | 100 100 100 63.7 | 100 100 97.9 28.6 | 92.592.188.916.6 | 100 100 99.9 43.3 | 1987.4 2464.8 2033.7 25.0 |
The good SBA that adopts all three kinds of ACWY-TT polysaccharide conjugates preparations immunity can produce MenA, MenC, MenW and MenY replys, and wherein the object titre of 95-100% was greater than 1: 8.Especially, 5/5/5/5 compares with oligosaccharide Meningitec vaccine with 2.5/10/2.5/2.5 polysaccharide conjugates preparation, can produce higher MenC and reply, and this point can be found out greater than 1: 128 object more at high proportion and GMT reading by having titre.
After table 10 is inoculated 1 month, the seroprevalence of antipolysaccharide antibody and GMCs
| Group | N | ％ | ≥0.3μg/ml LL | ?UL | ％ | ≥2μg/ml LL | UL | GMC μg/ml | |
| Anti--MenA | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 45 48 50 | 93.6 100 95.8 10.0 | 82.5 92.1 85.7 3.3 | 98.7 100 99.5 21.8 | 68.1 64.4 37.5 2.0 | 52.9 48.8 24.0 0.1 | 80.9 78.1 52.6 10.6 | 2.35 3.11 1.65 0.18 |
| Anti--MenC | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 45 47 49 | 100 100 100 98.0 | 92.5 92.1 92.5 89.1 | 100 100 100 99.9 | 100 100 97.9 93.9 | 92.5 92.1 88.7 83.1 | 100 100 99.9 98.7 | 9.57 12.53 19.29 7.95 |
| Anti--MenW | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 45 48 50 | 100 100 93.8 0.0 | 92.5 92.1 82.8 0.0 | 100 100 98.7 7.1 | 80.9 93.3 72.9 0.0 | 66.7 81.7 58.2 0.0 | 90.9 98.6 84.7 7.1 | 4.56 6.83 2.88 0.15 |
| Anti--MenY | Preparation 1T preparation 2T preparation 3T Meningitec TM | 47 45 47 50 | 100 100 97.9 2.0 | 92.5 92.1 88.7 0.1 | 100 100 99.9 10.6 | 97.9 100 87.2 0.0 | 88.7 92.1 74.3 0.0 | 99.9 100 95.2 7.1 | 8.90 12.78 5.67 0.15 |
All three kinds of ACWY-TT polysaccharide conjugates bacterin preparations all can produce the good immunne response to MenA, MenC, MenW and MenY, and wherein the individuality of 93%-100% has titre greater than 0.3 μ g/ml.With MeningiteC
TMCompare, 5/5/5/5 with 2.5/10/2.5/2.5ACWY-TT polysaccharide conjugates bacterin preparation can obtain higher GMC reading.
The natural immunogenicity with the MenY polysaccharide conjugates adjustment size of embodiment 7-compares
Mice (the female DBA/2 mice in 6-8 week) is accepted the PSY-TT injection in 2 weeks of two minor ticks through subcutaneous route.Blood sample collection after injecting 14 days for the second time resists-PSYELISA and SBA with the S1975menY strain.Per injection lets mice accept 1 μ gPSY-TT (adjuvant formulation is not added in lyophilizing).
Use the conjugate of table 11 record.
Table 11
| Conjugate | ENYTT012 | ENYTT014 | ENYTT015bis |
| The PSY Micro Fluid | Not | Be (40 circulations | Be (20 circulations |
| The TT/PS ratio | 1/1 | 1/1 | 1/1 |
The result
Result (Fig. 1) shows, adopts the conjugate of the PSY preparation of adjustment size to tend to have high immunogenicity.Figure 1A has shown to the GMC result from the prepared antiserum ELISA that conjugate produced of natural MenY (ENYTT012), 40 circulation Micro Fluid MenY (ENYTT014) and 20 circulation Micro Fluid MenY (ENYTT015bis).When MenY-TT be from the MenY of Micro Fluid preparation the time, can obtain higher GMCs.
Through SBA analysis and evaluation antiserum, obtained similar result (Figure 1B).Equally, adopt, obtained higher GMT value from the conjugate of the MenY preparation of Micro Fluid.
Claims (32)
1. comprise immunogenic composition from Neisseria meningitidis (N.meningitidis) capsular polysaccharide of at least a serogroups A, C, W135 and Y; Said capsular polysaccharide comprises the Neisseria meningitidis serogroup C capsular polysaccharide with the above mean size of 100kDa; Said capsular polysaccharide and carrier protein are puted together to produce the Neisseria meningitidis capsular polysaccharide conjugates, and wherein the mean size of each Neisseria meningitidis polysaccharide is more than the 50kDa.
2. the immunogenic composition of claim 1; It comprises the Neisseria meningitidis capsular polysaccharide from least a serogroups A, C, W135 and Y; Said capsular polysaccharide and carrier protein are puted together and are formed the Neisseria meningitidis conjugate, and wherein each Neisseria meningitidis polysaccharide is that natural polysaccharide or quilt are big or small with the coefficient adjustment that is no more than x10.
3. the immunogenic composition of claim 1, wherein each Neisseria meningitidis capsular polysaccharide is a natural polysaccharide.
4. the immunogenic composition of claim 1 is wherein adjusted size through Micro Fluid at least a Neisseria meningitidis capsular polysaccharide.
5. the immunogenic composition of claim 1, wherein each Neisseria meningitidis capsular polysaccharide is by to be no more than the coefficient adjustment size of x10.
6. the immunogenic composition of claim 1, wherein the Neisseria meningitidis conjugate is by natural polysaccharide and made by the mixture with the polysaccharide of the coefficient adjustment size that is no more than x10.
7. the immunogenic composition of claim 6 is wherein big or small with the coefficient adjustment that is no more than x10 from the capsular polysaccharide quilt of sero-group Y.
8. the immunogenic composition of claim 6, wherein the capsular polysaccharide from serogroups A and C is natural, and from the glycocalix of sero-group W135 and Y to be no more than the coefficient adjustment size of x10.
9. the immunogenic composition of claim 1, wherein the mean size of each Neisseria meningitidis capsular polysaccharide is 50kDa-200kDa.
10. the immunogenic composition of claim 1, it comprises the MenA capsular polysaccharide of the mean size with 50-100kDa.
11. the immunogenic composition of claim 1, it comprises the MenA capsular polysaccharide of the mean size with 60-80kDa.
12. the immunogenic composition of claim 1, it comprises the MenC capsular polysaccharide of the mean size with 100-200kDa.
13. the immunogenic composition of claim 1, it comprises the MenC capsular polysaccharide of the mean size with 160-200kDa.
14. the immunogenic composition of claim 1, it comprises the MenY capsular polysaccharide with the above mean size of 100kDa.
15. the immunogenic composition of claim 1, it comprises the MenY capsular polysaccharide with 120-140kDa mean size.
16. the immunogenic composition of claim 1, it comprises the MenW capsular polysaccharide of the mean size with 60-190kDa.
17. the immunogenic composition of claim 1, it comprises the MenW capsular polysaccharide of the mean size with 150-170kD.
18. the immunogenic composition of claim 1, it comprises the MenW capsular polysaccharide of the mean size with 110-140kDa.
19. the immunogenic composition of claim 1, wherein each Neisseria meningitidis capsular polysaccharide is puted together with being independently selected from by the carrier protein of the following group of forming: the fragment C of TT, DT, CRM197, TT and protein D.
20. the immunogenic composition of claim 1, wherein each Neisseria meningitidis capsular polysaccharide is puted together with being selected from by the same vehicle albumen of the following group of forming: the fragment C of TT, DT, CRM197, TT and protein D.
21. the immunogenic composition of claim 1, it further comprises hemophilus influenza (H.influenzae) the b capsular polysaccharide of puting together with carrier protein.
22. the immunogenic composition of claim 21, wherein hemophilus influenza b capsular polysaccharide and arbitrary being selected from by the carrier protein of the following group of forming are puted together: the fragment C of TT, DT, CRM197, TT and protein D.
23. the immunogenic composition of claim 21, it comprises Hib glycoconjugate and at least two kinds of other antibacterial glycoconjugates, and wherein the Hib conjugate exists with the dosage of the mean dose that is lower than two kinds of other antibacterial glycoconjugates at least.
24. the immunogenic composition of claim 23, wherein the Hib conjugate exists with the dosage lower than every kind of dosage of two kinds of other antibacterial glycoconjugates at least.
25. the immunogenic composition of claim 21 wherein is used for same vehicle albumen in two or more of Hib conjugate and two kinds of other antibacterial glycoconjugates at least.
26. the immunogenic composition of claim 1, it comprises the outer membrane vesicles prepared product or the capsular saccharides of Neisseria meningitidis serogroup B.
27. the immunogenic composition of claim 21, it comprises the outer membrane vesicles prepared product or the capsular saccharides of Neisseria meningitidis serogroup B.
28. comprise each immunogenic composition and the vaccine of pharmaceutically acceptable carrier among the claim 1-27.
29. be used to follow or the vaccine kit of order administration; It comprises that two kinds of multivalent immunogenic compositionss are to protect host's opposing by the disease due to Bordetella pertussis (Bordetella pertussis), clostridium tetani (Clostridium tetani), diphtheria corynebacterium (Corynebacterium diphtheriae), hemophilus influenza (Haemophilus influenzae) and the Neisseria meningitidis; Wherein said test kit comprises first container and second container, and first container comprises:
Tetanus toxoid (TT),
Diphtheria toxoid (DT) and
Full cell or acellular pertussis composition,
Second container comprises:
Each immunogenic composition among the claim 1-27.
30. be used to prepare the method for the vaccine of claim 28, it comprises each immunogenic composition and the blended step of pharmaceutically acceptable carrier among the claim 1-27.
31. the immunogenic composition of claim 1-27 is used for treating or preventing the application by the vaccine of the disease due to the Neisseria meningitidis infection in preparation.
32. each immunogenic composition is used for treating or preventing the application by the medicine of the disease due to the Neisseria meningitidis infection in preparation among the claim 1-27.
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| GBGB0513071.1A GB0513071D0 (en) | 2005-06-27 | 2005-06-27 | Immunogenic composition |
| GB0513071.1 | 2005-06-27 | ||
| GB0513069.5 | 2005-06-27 | ||
| GB0515556.9 | 2005-07-28 | ||
| GB0515556A GB0515556D0 (en) | 2005-07-28 | 2005-07-28 | Immunogenic composition |
| GB0524204.5 | 2005-11-28 | ||
| GB0524204A GB0524204D0 (en) | 2005-11-28 | 2005-11-28 | Immunogenic composition |
| GB0526040A GB0526040D0 (en) | 2005-12-21 | 2005-12-21 | Immunogenic composition |
| GB0526041A GB0526041D0 (en) | 2005-12-21 | 2005-12-21 | Immunogenic composition |
| GB0526041.9 | 2005-12-21 | ||
| GB0526040.1 | 2005-12-21 | ||
| PCT/EP2006/006188 WO2007000314A2 (en) | 2005-06-27 | 2006-06-23 | Immunogenic composition |
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| WO2003007985A2 (en) * | 2001-06-20 | 2003-01-30 | Chiron Srl. | Capsular polysaccharide solubilisation and combination vaccines |
| WO2003080678A1 (en) * | 2002-03-26 | 2003-10-02 | Chiron Srl | Modified saccharides having improved stability in water |
| WO2004103400A2 (en) * | 2003-05-07 | 2004-12-02 | Aventis Pasteur,Inc. | Multivalent meningococcal derivatized polysaccharide-protein conjugates and corresponding vaccines |
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| WO2003007985A2 (en) * | 2001-06-20 | 2003-01-30 | Chiron Srl. | Capsular polysaccharide solubilisation and combination vaccines |
| WO2003080678A1 (en) * | 2002-03-26 | 2003-10-02 | Chiron Srl | Modified saccharides having improved stability in water |
| WO2004103400A2 (en) * | 2003-05-07 | 2004-12-02 | Aventis Pasteur,Inc. | Multivalent meningococcal derivatized polysaccharide-protein conjugates and corresponding vaccines |
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