CN1764472A - Injectable vaccines against multiple meningococcal serogroups - Google Patents

Abstract

The present invention provides an injectable immunogenic composition comprising capsular saccharides from at least two of serogroups A, C, W135 and Y of Neisseria meningitidis, wherein said capsular saccharides are conjugated to carrier protein(s) and/or are oligosaccharides, and wherein (i) the composition comprises <50 g meningococcal saccharide per dose, and/or (ii) the composition further comprises an antigen from one or more of (a) serogroup B N.meningitidis; (b) Haemophilus influenzae type B; and/or (c) Streptococcus pneumoniae. Saccharide antigens in the compositions are generally conjugated to a carrier.

Description

The syringeability vaccine of anti-multiple meningococcus serum group

All documents that this paper quotes are all included in for reference.

Technical field

The present invention relates to the vaccinology field, especially directed toward bacteria property meningitis.

Background technology

Neisseria meningitidis (Neisseria meningitidis) is the Gram-negative human pathogen [1] that causes bacterial meningitis.(N.gonorrhoeae) is closely related for Neisseria meningitidis and Neisseria gonorrhoeae, is the polysaccharide pod membrane that exists in all pathogenicity meningococcuss though clearly differentiate a meningococcal feature.

On the basis of organism capsular polysaccharide, 12 kinds of Neisseria meningitidis serum group (A, B, C, H, I, K, L, 29E, W135, X, Y and Z) have been identified.Serum group A (MenA) is epiphytotics common reason in Africa, inferior the Sahara.Serum group B and C cause most of cases of developed country, and serum group W135 and Y cause all the other cases of developed country.

Except that being used for classification, capsular polysaccharide has been used to vaccine.Known for many years serum group A, C, the injectable tetravalent vaccine [2,3] of Y and W135 capsular polysaccharide, and approval is used for the mankind.Although effectively, their inductive immunoreation differences and protection persistent period are short in teenager and adult for they, can not be used for baby's [for example 4].Polysaccharide in this vaccine is not puted together, and with 1: 1: 1: there were [5] in 1 weight ratio.In case rebuild Mencevax ACWY again from their lyophilized form ^TMAnd Menomune ^TMThe various polysaccharide that all contain the 50g purification.With serum group A, C, the capsular polysaccharide of W135 and Y with the generation tetravalent vaccine, for example do not contain the Menactra of adjuvant with conjugate form associating [6-9] yet ^TMProduct.

The serum group C oligosaccharide that approved will be puted together is used for the mankind, comprises Menjugate ^TM[10,11], Meningitec ^TMAnd NeisVac-C ^TMProduct Menjugate ^TM, Meningitec ^TMHave and CRM ₁₉₇The oligosaccharide antigen that carrier is puted together, and NeisVac-C ^TMUtilized the complete polysaccharide of puting together with the tetanus toxin carrier (O-is deacetylated).

Yet, also need to improve antiserum group A, the conjugate vaccines of W135 and Y and manufacturing thereof.The product, the methods and applications that disclose in the list of references 7 are devoted to solve this demand, but can also more adjust and improve.

Invention is described

The invention provides a kind of injectable immunogenic composition that contains capsular saccharides, capsular saccharides is from Neisseria meningitidis serum group A, C, among W135 and the Y at least two kinds, wherein said capsular saccharides and carrier protein are puted together and/or are oligosaccharide, and wherein the every dosage of said composition contains≤50 μ g meningococcus sugar.

The present invention also provides a kind of injectable immunogenic composition that contains capsular saccharides, capsular saccharides is from Neisseria meningitidis serum group A, C, among W135 and the Y at least two kinds, wherein said capsular saccharides and carrier protein are puted together and/or are oligosaccharide, and wherein said composition also contains from following one or more antigen: (a) Neisseria meningitidis serum group B; (b) haemophilus influenzae type B; And/or (c) streptococcus pneumoniae.

The antigen that comprises in the preferred 16 kinds of compositionss of the present invention is: (1) Neisseria meningitidis serum group C, W135 and Y; (2) Neisseria meningitidis serum group A, C, W135 and Y; (3) Neisseria meningitidis serum group B, C, W135 and Y; (4) Neisseria meningitidis serum group A, B, C, W135 and Y; (5) haemophilus influenzae type B and Neisseria meningitidis serum group C, W135 and Y; (6) haemophilus influenzae type B and Neisseria meningitidis serum group A, C, W135 and Y; (7) haemophilus influenzae type B and Neisseria meningitidis serum group B, C, W135 and Y; (8) haemophilus influenzae type B and Neisseria meningitidis serum group A, B, C, W135 and Y; (9) streptococcus pneumoniae and Neisseria meningitidis serum group C, W135 and Y; (10) streptococcus pneumoniae and Neisseria meningitidis serum group A, C, W135 and Y; (11) streptococcus pneumoniae and Neisseria meningitidis serum group B, C, W135 and Y; (12) streptococcus pneumoniae and Neisseria meningitidis serum group A, B, C, W135 and Y; (13) haemophilus influenzae type B, streptococcus pneumoniae and Neisseria meningitidis serum group C, W135 and Y; (14) haemophilus influenzae type B, streptococcus pneumoniae and Neisseria meningitidis serum group A, C, W135 and Y; (15) haemophilus influenzae type B, streptococcus pneumoniae and Neisseria meningitidis serum group B, C, W135 and Y; (16) haemophilus influenzae type B, streptococcus pneumoniae and Neisseria meningitidis serum group A, B, C, W135 and Y.

Carbohydrate antigen in the present composition is preferably puted together with carrier.Carbohydrate antigen in the present composition is preferably oligosaccharide.Carbohydrate antigen in the present composition is most preferably puted together with oligosaccharide.

The meningococcus sugar mixture

The present composition contains from Neisseria meningitidis serum group A, C, the capsular saccharides of among W135 and the Y at least two kinds (promptly 2,3 or 4 kind).The present composition preferably comprises at least from serum group C, the Neisseria meningitidis sugar of W135 and Y (being non-MenA sugar).Preferably each carbohydrate antigen combination is not eliminated protection effect separately, though actual immunogenicity (for example ELISA tires) may reduce.

Preferably, for example contain from serum group A+C A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, C+W135+Y, the compositions of the sugar of A+C+W135+Y etc. from the mixture of the sugar of more than one Neisseria meningitidis serum group.Preferred compositions contains the sugar from serum group C and Y.Other preferred compositions contains from serum group C, the sugar of W135 and Y.Only preferably do not contain from serum group A with only from the compositions (referring to list of references 10,13 and 14) of the capsular saccharides of serum group C.

When mixture contains capsular saccharides from serum group A and C, MenA sugar: the ratio of MenC sugar (w/w) can be greater than 1 (for example 2: 1,3: 1,4: 1,5: 1,10: 1 or higher).When mixture contains from one or both the capsular saccharides among serum group Y and serum group C and the W135, MenY sugar: the ratio of MenW135 sugar (w/w) can be greater than 1 (for example 2: 1,3: 1,4: 1,5: 1,10: 1 or higher) and/or MenY sugar: the ratio of MenC sugar (w/w) can be less than 1 (for example 1: 2,1: 3,1: 4,1: 5 or lower).

The typical content of each meningococcus carbohydrate antigen of every dosage is 1 μ g-20 μ g, the about 2.5 μ g of for example about 1 μ g, about 4 μ g, about 5 μ g, or about 10 μ g (being expressed as sugar).

From the sugar of serum group A: C: W135: Y, preferred ratio (w/w) is 1: 1: 1: 1; 1: 1: 1: 2; 2: 1: 1: 1; 4: 2: 1: 1; 8: 4: 2: 1; 4: 2: 1: 2; 8: 4: 1: 2; 4: 2: 2: 1; 2: 2: 1: 1; 4: 4: 2: 1; 2: 2: 1: 2; 4: 4: 1: 2; With 2: 2: 2: 1.From the sugar of serum group C: W135: Y, preferred ratio (W/W) is 1: 1: 1; 1: 1: 2; 1: 1: 1; 2: 1: 1; 4: 2: 1; 2: 1: 2; 4: 1: 2; 2: 2: 1; With 2: 1: 1.Preferably adopt the various sugar of equivalent basically.

The purification of capsular polysaccharide

The method that is commonly used to prepare the meningococcal capsular polysaccharide may further comprise the steps: polysaccharide precipitation (for example utilizing cationic detergent), alcohol grading separates, cold phenol extracting (deproteinize) and ultracentrifugation (removing LPS) [for example list of references 15].

Yet preferred program [7] comprises polysaccharide precipitation, uses the polysaccharide of lower alcohol dissolution precipitation then.Can precipitate for example tetrabutylammonium and cetyltrimethyl ammonium salt (for example bromine salt), or hexadimethrine bromide and myristyl front three ammonium salt with cationic detergent.Especially preferred cetyl trimethyl ammonium bromide (' CTAB ') [16].Can be with lower alcohol methanol for example, the 1-propanol, the 2-propanol, the 1-butanols, the 2-butanols, 2-methyl isophthalic acid-propanol, 2-methyl-2-propanol, glycol waits the material of dissolution precipitation, but ethanol is particularly suited for dissolving the CTAB-polysaccharides compound.Preferably ethanol is added sedimentary polysaccharide, reach the ethanol final concentration between 50% and 95%.

Again after the dissolving, can further handle this polysaccharide to remove pollutant.This under the situation that less contaminants can not be accepted (for example production of people's vaccine) is even more important.This step generally includes for example depth-type filtration of one or more filtration steps, and through active carbon filtration, size is filtered and/or ultra-filtration.

In case removed by filter impurity, can be with polysaccharide precipitation further to handle and/or to process.Available exchange cation (for example adding calcium or sodium salt) is realized this purpose easily.

As the alternative method of purification, available all or part of synthesizing obtains capsular saccharides of the present invention, and it is synthetic that for example list of references 17 has been described Hib, and the MenA of list of references 18 is synthetic.

But chemically modifying polysaccharides.For example, but modified polysaccharide substitutes one or more hydroxyls with blocking groups.This is particularly useful [19] (as follows) to the hydrolysis that reduces serum group A.The O-that also can carry out polysaccharide is deacetylated.

Neisseria meningitidis serum group B

Some compositions of the present invention comprises the antigen from Neisseria meningitidis serum group B.Be different from serum group A, C, W135 and Y, the capsular saccharides of MenB is because have similarity with people's autoantigen, so be not suitable for the immunogen as the people.If therefore a kind of carbohydrate antigen is used for MenB, need with a kind of modified sugar, for example the N-acetyl group in the sialic acid residues is by the alternate sugar of N-acyl group.Suitable N-acyl group is C ₁-C ₈Acyl group, N-propiono [20] for example.Yet, preferably adopt polypeptide antigen but not carbohydrate antigen.

Therefore said composition can comprise one or more polypeptide antigens, protective immunological reaction that the anti-MenB of these antigen inductions infects or the bactericidal properties immunoreation of anti-MenB.More generally, said composition preferably can be induced two or more (for example 2 or 3) high toxicity pedigree A4 of anti-Neisseria meningitidis serum group B, the antibody response of ET-5 and pedigree 3 in this object after giving object.

Publish the genome sequence [21] of MC58 and MenB bacterial strain, can from encoded polypeptides, select suitable antigen [22].List of references 22-32 has described preferred antigen.The preferred present composition comprise 10 kinds or still less (for example 9,8,7,6,5,4,3,2) purifying antigen mixture but not single antigen, especially preferred said composition should not comprise complicated or for example preferred said composition of uncertain antigen mixture in do not contain outer membrane vesicles (OMVs).

Preferred compositions comprises one or more following five kinds of antigens [32]: (1) ' NadA ' albumen, preferred oligomerization form (for example trimeric form); (2) ' 741 ' albumen; (3) ' 936 ' albumen; (4) ' 953 ' albumen; (5) ' 287 ' albumen.

Preferred L enB antigen contains bacterial strain 2996, MC58, the aminoacid sequence of finding among the 95N477 and one of 394/98.Protein 28 7 is preferably from bacterial strain 2996, or more preferably from bacterial strain 394/98.Albumen 741 is preferably from serum group B bacterial strain MC58,2996,394/98 or 95N477, or from serum group C bacterial strain 90/18311.More preferably bacterial strain MC58.Albumen 936,953 and NadA are preferably from bacterial strain 2996.When compositions comprised specific protein antigen (as 741 or 287), said composition can comprise the antigen greater than a kind of variant form, for example from same protein, but from bacterial strain more than a kind.Can be used as series connection or separately albumen add these albumen.

' NadA ' (neisser's coccus adhesin A) from MenB is shown albumen ' 961 ' (SEQ IDs 2943 and 2944) and is shown ' NMB1994 ' (also referring to the GenBank accession number: 11352904 and 7227256) in list of references 21 in list of references 25.Proteic detailed description is found in list of references 33.When used according to the invention, NadA can adopt various ways.Preferred NadA form is truncate or disappearance variant, as shown in list of references 29 to 31.Particularly preferably do not have the NadA (as for bacterial strain 2996, disappearance residue 351-405[SEQ ID NO:1]) of its C-terminal film anchor, use ' C ' subscript to distinguish in this article sometimes, for example NadA ^(C)In escherichia coli (E.coli), express the NadA that does not have its film anchor structure territory and make protein excretion in the culture supernatant, follow the leader peptide (, staying 327 aggressiveness [SEQ ID NO:2]) of removing its 23 aggressiveness as for bacterial strain 2996.The polypeptide of no leader peptide uses ' NL ' subscript to distinguish in this article sometimes, for example NadA ^(NL)Or NadA ^{(C) (NL)}The aminoacid sequence that preferred NadA polypeptide has: (a) have 50% or more homogeneity (for example 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO:2; And/or (b) comprise n at least continuous amino acid from SEQ ID NO:1, wherein n is 7 or more (for example 8,10,12,14,16,18,20,25,30,35,40,50,60,70,80,90,100,150,200,250 or more).For (b) preferred fragment lack one or more (for example 1,2,3,4,5,6,7,8,9,10,15,20,25 or more) from the aminoacid of SEQ ID NO:1C end and/or N-terminal (as NadA ^(C), NadA ^(NL), NadA ^{(C) (NL)}).Other preferred fragment comprises the epi-position from SEQ ID NO:1, and the especially preferred fragment of SEQ ID NO:1 is SEQ ID NO:2.This writes different sequences and comprises NadA variant (as allele variant, congener, lineal congener, side direction congener, mutant etc.).Various NadA sequences are shown in Fig. 9 of list of references 34.

' 741 ' albumen from MenB is shown in list of references 25 (SEQ IDs 2535 and 2536) and is shown ' NMB1870 ' (also referring to GenBank accession number GI:7227128) in list of references 21.Corresponding albumen [35] among the serum group A has GenBank accession number 7379322.The 741st, natural grease albumen.When used according to the invention, 741 albumen can adopt various ways.Preferred 741 forms are truncate or disappearance variant, shown in list of references 29 to 31.Special 741 N-terminal can lack and comprise its polyglycine sequence (promptly for bacterial strain MC58, disappearance residue 1 is to 72[SEQ ID NO:3]), uses sometimes in this article that ' Δ G ' prefix is distinguished.This disappearance can improve expression.Disappearance is also removed 741 fat site.Preferred 741 sequences have aminoacid sequence: (a) have 50% or more homogeneity (for example 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO:3; And/or (b) comprise n at least continuous amino acid from SEQ ID NO:3, wherein n is 7 or more (for example 8,10,12,14,16,18,20,25,30,35,40,50,60,70,80,90,100,150,200,250 or more).Comprise epitope for (b) preferred fragment from 741.Other preferred fragment lacks one or more (for example 1,2,3,4,5,6,7,8,9,10,15,20,25 or more) aminoacid from SEQ ID NO:3C end and/or N-terminal.These sequences comprise 741 variants (as allele variant, congener, lineal congener, side direction congener, mutant etc.).Various 741 sequences are found in the SEQ IDs 1-22 of list of references 31 and the SEQ IDs 1-23 of list of references 36.The SEQ IDs 1-299 of list of references 37.

Albumen 741 is the extremely effectively antigen that causes the meningococcemia antibody response, and it is expressed in all meningococcus serum group.Phylogenetic Analysis shows that albumen splits into 2 groups; these the division in one of once more the division; produce 3 variants [38] altogether; although the serum that anti-specific variants is cultivated is bactericidal in identical variant group; it is to expressing the bacterial strain non-activity of one of 2 variants in addition; cross protection in the variant is promptly arranged, but do not have cross protection between variant [36,38].Therefore, for maximum intersection bacterial strain efficient, compositions preferably should comprise greater than 1 albumen 741 variant.From the exemplary sequence of each variant at the SEQ of this paper ID ^s10, provide in 11 and 12, initial is the N-terminal cysteine residues of lipid covalent attachment in the 741 lipoprotein forms.Thereby, compositions preferably should comprise following at least two kinds: (1) the 1st kind of albumen, and amino acid contained sequence and SEQ ID NO:10 are by a% sequence homogeneity and/or amino acid contained sequence are made up of x at least continuous amino acid fragment from SEQ ID NO:10 at least; (2) the 2nd kinds of albumen, amino acid contained sequence and SEQ ID NO:11 are by b% sequence homogeneity and/or amino acid contained sequence are made up of y at least continuous amino acid fragment from SEQ ID NO:11 at least; (3) the 3rd kinds of albumen, amino acid contained sequence and SEQ ID NO:12 are by c% sequence homogeneity and/or amino acid contained sequence are made up of z at least continuous amino acid fragment from SEQ ID NO:12 at least.The a value is 85 at least, for example 86,87,88,89,90,91,92,93,94,95,96,97,98,99,99.5 or more.The b value is 85 at least, for example 86,87,88,89,90,91,92,93,94,95,96,97,98,99,99.5 or more.The c value is 85 at least, for example 86,87,88,89,90,91,92,93,94,95,96,97,98,99,99.5 or more.A, b and c value are inherent each other uncorrelated.The x value is 7 at least, for example 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,60,70,80,90,100,120,140,160,180,200,225,250.The y value is 7 at least, for example 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,60,70,80,90,100,120,140,160,180,200,225,250.The z value is 7 at least, for example 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,60,70,80,90,100,120,140,160,180,200,225,250.X, y and z value are inherent each other uncorrelated.Any specific 741 aminoacid sequences preferably do not belong to more than one classifications (1), (2) and (3).Therefore, any specific 741 sequences only belong to one of classification (1), (2) and (3).Thereby preferred: albumen (1) and albumen (2) have the sequence homogeneity less than i%; Albumen (1) and albumen (3) have the sequence homogeneity less than j%; Albumen (2) and albumen (3) have the sequence homogeneity less than k%.The i value is 60 or more (for example 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90 etc.) and be a at the most.The j value is 60 or more (for example 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90 etc.) and be b at the most.K value is 60 or more (for example 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90 etc.) and be c at the most.I, j and k value are inherent each other uncorrelated.

' 936 ' albumen from serum group B is shown in list of references 25 (SEQ IDs 2883 and 2884) and is shown ' NMB2091 ' (also referring to GenBank accession number GI:7227353) in list of references 21.Corresponding gene [35] among the serum group A has GenBank accession number 7379093.When used according to the invention, 936 albumen can adopt various ways.Preferred 936 forms are truncate or disappearance variant, as shown in list of references 29 to 31.Particularly 936 N-terminal leader peptide can lack (promptly for bacterial strain MC58, disappearance residue 1 to 23[SEQID NO:4]) to produce 936 ^(NL)Preferred 936 sequences have aminoacid sequence: (a) have 50% or more homogeneity (for example 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO:4; And/or (b) comprise n at least continuous amino acid from SEQ ID NO:4, wherein n is 7 or more (for example 8,10,12,14,16,18,20,25,30,35,40,50,60,70,80,90,100,150,200,250 or more).Comprise epitope for (b) preferred fragment from 936.Other preferred fragment lacks one or more (for example 1,2,3,4,5,6,7,8,9,10,15,20,25 or more) aminoacid from SEQ ID NO:4C end and/or N-terminal.These sequences comprise 936 variants (as allele variant, congener, lineal congener, side direction congener, mutant etc.).

' 953 ' albumen from serum group B is shown in list of references 25 (SEQ IDs 2917 and 2918) and is shown ' NMB1030 ' (also referring to GenBank accession number GI:7226269) in list of references 21.Corresponding albumen [35] among the serum group A has GenBank accession number 7380108.When used according to the invention, 953 albumen can adopt various ways.Preferred 953 forms are truncate or disappearance variant, as shown in list of references 29 to 31.Particularly 953 N-terminal leader peptide can lack (promptly for bacterial strain MC58, disappearance residue 1 to 19[SEQID NO:5]) to produce 953 ^(NL)Preferred 953 sequences have aminoacid sequence: (a) have 50% or more homogeneity (for example 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO:5; And/or (b) comprise n at least continuous amino acid from SEQ ID NO:5, wherein n is 7 or more (for example 8,10,12,14,16,18,20,25,30,35,40,50,60,70,80,90,100,150,200,250 or more).Comprise epitope for (b) preferred fragment from 953.Other preferred fragment lacks one or more (for example 1,2,3,4,5,6,7,8,9,10,15,20,25 or more) aminoacid from SEQ ID NO:5C end and/or N-terminal.These sequences comprise 953 variants (as allele variant, congener, lineal congener, side direction congener, mutant etc.).953 allelic form is found in Figure 19 of list of references 28.

' 287 ' albumen from serum group B is shown in list of references 25 (SEQ IDs 3103 and 3104) and is shown ' NMB2132 ' in list of references 21, is shown ' GNA2132 ' (also referring to GenBank accession number GI:7227388) in list of references 13.Corresponding albumen [35] among the serum group A has GenBank accession number 7379057.When used according to the invention, 287 albumen can adopt various ways.Preferred 287 forms are truncate or disappearance variant, as shown in list of references 29 to 31.Particularly 287 N-terminal can lack and comprise its polyglycine sequence (for example for bacterial strain MC58, the disappearance residue 1 to 24 obtain Δ G287[SEQ ID NO:6]).This disappearance can improve expression.Preferred 287 sequences have aminoacid sequence: (a) have 50% or more homogeneity (for example 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO:6; And/or (b) comprise n at least continuous amino acid from SEQ ID NO:6, wherein n is 7 or more (for example 8,10,12,14,16,18,20,25,30,35,40,50,60,70,80,90,100,150,200,250 or more).Comprise epitope for (b) preferred fragment from 287.Other preferred fragment lacks one or more (for example 1,2,3,4,5,6,7,8,9,10,15,20,25 or more) aminoacid from SEQID NO:6C end and/or N-terminal.These sequences comprise 287 variants (as allele variant, congener, lineal congener, side direction congener, mutant etc.).287 allelic form is found in Fig. 5 and 15 of list of references 28, embodiment 13 and Figure 21 (SEQ IDs 3179 to 3184) of list of references 25.

5 kinds of basic MenB antigen (NadA, 741,953,936 and 287) can be used as 5 kinds of independent albumen is present in the compositions, but preferably at least 2 kinds of antigen presentations are single polypeptide chain (' heterozygosis ' albumen [list of references 29 to 31]), and promptly 5 kinds of antigens form and are less than 5 peptide species.Hybrid protein provides 2 major advantages: at first, the suitable heterozygosis companion who overcomes problem by adding assists albumen unstable or that express difference itself; The second, simplify commodity production, because only need use a kind of expression and purification to be convenient to generate 2 independent useful albumen.The hybrid protein that comprises in the invention compositions can comprise 5 kinds of basic antigens of 2 kinds or more (promptly 2,3,4 or 5).Preferably by 2 kinds of heterozygotes of forming in 5 kinds of basic antigen.

In 5 kinds of antigenic substantially combinations, antigen can be present in greater than a kind of hybrid protein and/or as non-hybrid protein.Yet antigen preferably exists as heterozygote or non-heterozygote, is not both, comes in handy as heterozygosis and non-heterozygosis (preferred lipoprotein) antigen although comprise albumen 741, and specific is when using more than one 741 variants.

Hybrid protein can be by formula NH ₂-A-[-X-L-] _n-B-COOH represents that wherein: X is the aminoacid sequence of one of 5 basic antigens; L is the joint aminoacid sequence of choosing wantonly; A is the N-terminal aminoacid sequence of choosing wantonly; B is the C-terminal aminoacid sequence of choosing wantonly; N is 2,3,4 or 5.

If-X-part has leader peptide sequences in its wild type form, it can comprise or omit in hybrid protein.In some embodiments, will lack leader peptide, except be positioned at the hybrid protein N-terminal-the X-part, promptly keep X ₁Leader peptide, but omit X ₂... X _nLeader peptide.This is equal to all leader peptides of disappearance and uses X ₁Leader peptide conduct-A-part.

For each n[-X-L-] example, joint aminoacid sequence-L-can exist or lack.For example, when n=2, heterozygote can be NH ₂-X ₁-L ₁-X ₂-L ₂-COOH, NH ₂-X ₁-X ₂-COOH, NH ₂-X ₁-L ₁-X ₂-COOH, NH ₂-X ₁-X ₂-L ₂-COOH etc.Joint aminoacid sequence-L-short usually (for example 20 or aminoacid still less, promptly 19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2,1).Example comprises that promotion clone's short peptide sequence, polyglycine joint (promptly comprise Gly _n, n=2,3,4,5,6,7,8,9,10 or more wherein) and the histidine sign (be His _n, n=3,4,5,6,7,8,9,10 or more wherein).Other suitable joint aminoacid sequence it will be apparent to those skilled in the art that.Useful joint is GSGGGG (SEQ ID NO:9), and the Gly-Ser dipeptides forms from the BamHI restriction site, thereby assists clone and operation, (Gly ₄) tetrapeptide is typical polyglycine joint.If X _N+1Be Δ G albumen and L _nBe the glycine joint, this can be equal to X _N+1Not Δ G albumen and shortage L _n

-A-is the N-terminal aminoacid sequence of choosing wantonly.It is short usually (for example 40 or aminoacid still less, promptly 39,38,37,36,35,34,33,32,31,30,29,28,27,26,25,24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2,1).Example comprises that targeting sequencing that instructs albumen transportation or the short peptide sequence that promotes clone or purification are (as histidine sign, i.e. His _n, n=3,4,5,6,7,8,9,10 or more wherein).Other suitable N-terminal aminoacid sequence it will be apparent to those skilled in the art that.If X ₁The N-terminal methionine that lacks himself ,-A-preferably provide the oligopeptide (1,2,3,4,5,6,7 or 8 aminoacid is for example arranged) of N-terminal methionine.

-B-is the C-terminal aminoacid sequence of choosing wantonly.It is short usually (for example 40 or aminoacid still less, promptly 39,38,37,36,35,34,33,32,31,30,29,28,27,26,25,24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2,1).Example comprises that the short peptide sequence of the targeting sequencing, promotion clone or the purification that instruct the albumen transportation (as comprises histidine sign, i.e. His _n, n=3,4,5,6,7,8,9,10 or more wherein) or improve the sequence of protein stability.Other appropriate C terminal amino acid sequence it will be apparent to those skilled in the art that.

Most preferably n is 2.The two antigen heterozygotes that are used to invent comprise: NadA and 741; NadA and 936; NadA and 953; NadA and 287; 741 and 936; 741 and 953; 741 and 287; 936 and 953; 936 and 287; 953 and 287.2 kinds of optimization proteins are: X ₁Be 936 and X ₂Be 741; X ₁Be 287 and X ₂Be 953.

2 kinds of especially preferred hybrid proteins of invention are as follows:

n	A	X 1	L 1	X 2	L 2	B	SEQ ID NO：
								2	MA	ΔG287	GSGGGG	953 (NL)	-	-	7
2	M	936 (NL)	GSGGGG	ΔG741	-	-	8

These 2 kinds of albumen NadA capable of being combined (especially with SEQ ID NO:2) use.Therefore being used for preferred L enB antigen composition of the present invention comprises first polypeptide, and this polypeptide contains aminoacid sequence SEQ ID NO:2, comprises second polypeptide and the 3rd polypeptide that comprises aminoacid sequence SEQ ID NO:8 of aminoacid sequence SEQ ID NO:7.This is to be used for preferred L enB antigen group of the present invention.

As mentioned above, comprise the antigenic invention compositions of MenB and can preferably induce effectively anti-2 or the high toxicity pedigree A4 of 3 kind of MenB, the serum sterilizing antibody response of ET-5 and pedigree 3.They may induce anti-one or more high toxicity pedigree subgroups I in addition, subgroup III, the bactericidin reaction of subgroup IV-1 or ET-37 complex and the anti-for example high invasive pedigree of other pedigree.These antibody responses can detect and be the standard indication postscript 14 of document 22 [for example, see reference] of efficacy of vaccines easily in mice.The antibacterial that serum bactericidal activity (SBA) is measured complement-mediated kills, and can choose or young rabbit complement mensuration.WHO standard requires vaccine to induce SBA to rise at least 4 times in surpassing 90% receptor.

Said composition need not induced each and each MenB bacterial strain in anti-these high toxicity pedigrees of bactericidin; Group that but for any particular group of 4 kinds or more serum group B Neisseria meningitidis bacterial strains in the specific high toxicity pedigree, the inductive antibody of compositions is bactericidal, anti-at least 50% (for example 60%, 70%, 80%, 90% or more).Preferred bacterial strain group is included in strain separated at least 4 following countries: Great Britain, Australia, Canada, Norway, Italy, the U.S., New Zealand, Holland, Belgium and Cuba.Serum preferably has at least 1024 sterilization and tires (for example 2 ¹⁰, 2 ¹¹, 2 ¹², 2 ¹³, 2 ¹⁴, 2 ¹⁵, 2 ¹⁶, 2 ¹⁷, 2 ¹⁸Or more, preferably at least 2 ¹⁴), promptly when dilution 1/1024 time, serum can kill the bacteria tested of at least 50% specific bacterial strain, as described in list of references 22.Preferred compositions can be induced the bactericidal reaction of anti-following serum group B meningococcus bacterial strain: (i) from a bunch A4, and bacterial strain 961-5945 (B:2b:P1.21,16) and/or bacterial strain G2136 (B:-); (ii) from the ET-5 complex, and bacterial strain MC58 (B:15:P1.7,16b) and/or bacterial strain 44/76 (B:15:P1.7,16); (iii) from pedigree 3, bacterial strain 394/98 (B:4:P1.4) and/or bacterial strain BZ198 (B:NT:-).Preferred compositions can be induced antibiotic strain 961-5945,44/76 and 394/98 bactericidal reaction.Bacterial strain 961-5945 and G2136 are neisser's coccus MLST reference strain [ids 638 and 1002 in the list of references 39].Bacterial strain MC58 extensively available (for example ATCC BAA-335) and be in the list of references 21 order-checking bacterial strain.Bacterial strain 44/76 is extensive use of and identifies (for example list of references 40) and be one of neisser's coccus MLST reference strain [id 237 in the list of references 39; The 32nd row of list of references 41 invading the exterior 2].Bacterial strain 394/98 separated in New Zealand in 1998 at first, and what have that some use this bacterial strain delivers research (for example list of references 42 and 43).Bacterial strain BZ198 is the another kind of neisser's coccus MLST reference strain [id 409 in the list of references 39; The 41st row of list of references 41 invading the exterior 2].Compositions can be induced bactericidal reaction antiserum group W135 bacterial strain LNP17592 (W135:2a:P1.5,2) in addition, from the ET-37 complex.This be 2000 at the isolating Haji bacterial strain of France.

Other MenB polypeptide antigen that can comprise in the invention compositions is selected from one of following amino acid sequences: from the SEQ ID NO:650 of list of references 23; SEQ ID NO:878 from list of references 23; SEQ ID NO:884 from list of references 23; SEQ ID NO:4 from list of references 24; SEQ ID NO:598 from list of references 25; SEQ ID NO:818 from list of references 25; SEQID NO:864 from list of references 25; SEQ ID NO:866 from list of references 25; SEQ IDNO:1196 from list of references 25; SEQ ID NO:1272 from list of references 25; SEQ IDNO:1274 from list of references 25; SEQ ID NO:1640 from list of references 25; SEQ IDNO:1788 from list of references 25; SEQ ID NO:2288 from list of references 25; SEQ IDNO:2466 from list of references 25; SEQ ID NO:2554 from list of references 25; SEQ IDNO:2576 from list of references 25; SEQ ID NO:2606 from list of references 25; SEQ IDNO:2608 from list of references 25; SEQ ID NO:2616 from list of references 25; SEQ IDNO:2668 from list of references 25; SEQ ID NO:2780 from list of references 25; SEQ IDNO:2932 from list of references 25; SEQ ID NO:2958 from list of references 25; SEQ IDNO:2970 from list of references 25; From the SEQ ID NO:2988 of list of references 25, or polypeptide, it comprises aminoacid sequence: (a) have 50% or more homogeneity (for example 60%, 70%, 80%, 90%, 95%, 99% or more) with described sequence; And/or (b) from the described sequence fragment of n continuous amino acid at least, wherein n is 7 or more (for example 8,10,12,14,16,18,20,25,30,35,40,50,60,70,80,90,100,150,200,250 or more).The epitope that comprises coming autocorrelation sequence for the preferred fragment of (b).Can comprise these polypeptide greater than a kind (for example 2,3,4,5,6).

Haemophilus influenzae type b (Hib)

When compositions comprised haemophilus influenzae type B antigen, it is Hib capsular saccharides antigen normally.Carbohydrate antigen from hemophilus influenza is known.

Advantageously, Hib sugar is puted together to improve its immunogenicity with the carrier protein covalency, especially the immunogenicity in the child.General polysaccharides conjugate preparation and specific Hib capsular polysaccharide prepare in the literature write up [for example list of references 44 to 52 etc.].Invention can be used any appropriate H ib conjugate.Suitable carrier albumen is described in down, and the preferred vector that is used for Hib sugar is CRM ₁₉₇The outer film composite (' PRP-OMP ') of (' HbOC '), tetanus toxoid (' PRP-T ') and Neisseria meningitidis.

The sugar moieties of conjugate can be polysaccharide (a for example total length polyribosylribitol phosphate (PRP)), but the selective hydrolysis polysaccharide with form oligosaccharide (for example MW from～1 to～5kDa).

Preferred conjugate comprises the Hib oligosaccharide, through the covalently bound CRM of adipic acid joint ₁₉₇[53,54].Tetanus toxoid also is a preferred vector.

Use Hib antigen and preferably make anti-PRP antibody concentration 〉=0.15 μ g/ml, more preferably 〉=1 μ g/ml.

Inventive compositions can comprise more than one Hib antigen.

When compositions comprised the Hib carbohydrate antigen, it did not preferably comprise aluminum hydroxide adjuvant yet.If compositions comprises the aluminum phosphate adjuvant, then the Hib carbohydrate antigen is adsorbable in adjuvant [55] or can not absorb [56].

The lyophilizing of Hib antigen energy is for example with hitchens and Hansen antigen.

Streptococcus pneumoniae

When compositions comprised streptococcus pneumoniae antigen, it is capsular saccharides antigen normally, and this antigen is preferably puted together [for example list of references 57 to 59] with carrier protein.Preferably include sugar from streptococcus pneumoniae serotype more than a kind.For example, be extensive use of polysaccharide mixture, have conjugate vaccines also to be extensive use of [60] from the polysaccharide of 5 to 11 kinds of different serotypes from 23 kinds of different serotypes.For example, PrevNar ^TM[61] comprise antigen from 7 kinds of serotypes (4,6B, 9V, 14,18C, 19F and 23F), each sugar is puted together CRM separately by reduction amination ₁₉₇, with each sugared every 0.5ml dosage of 2 μ g (4 μ g serotype 6B), conjugate is adsorbed on the aluminum phosphate adjuvant.Inventive compositions preferably includes serotype 6B, 14,19F and 23F at least.Conjugate is adsorbable on aluminum phosphate.

Except using from the pneumococcal carbohydrate antigen, compositions can comprise a kind or multiple polypeptides antigen.The genome sequence of some streptococcus pneumoniae bacterial strains is that useful [62,63] also can stand reverse vaccinology [64-67] to identify suitable polypeptide antigen [68-69].For example, compositions can comprise a kind or multiple following antigen: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp130, such as list of references 70 definition.Compositions can comprise more than a kind these antigens of (for example 2,3,4,5,6,7,8,9,10,11,12,13 or 14 kind).

In some embodiments, compositions can comprise from pneumococcal sugar and polypeptide antigen.These can be used for simple mixtures, or the streptococcus pneumoniae carbohydrate antigen can be puted together pneumoprotein.The suitable carrier albumen of these embodiments comprises listed antigen [70] in the earlier paragraphs.

But the PNEUMOVAX-23 lyophilizing is for example with meningococcus and/or Hib antigen.

The Neisseria meningitidis serum group A sugar of modifying

When inventive compositions comprised the MenA carbohydrate antigen, antigen was preferably modified sugar, and wherein one or more hydroxyls on the natural sugar replace [19] by blocking group.This modifies the resistance of improving hydrolysis, means that serum group A antigen can be stored in and be used for liquid preparation, and does not need lyophilizing.

There is the monosaccharide units number of blocking group to change.For example, all or substantially all monosaccharide units can have blocking group.Perhaps, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% monosaccharide units can have blocking group.At least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 monosaccharide units can have blocking group.

Equally, the blocking group number on the monosaccharide units can change.For example, the blocking group number on the monosaccharide units can be 1 or 2.Blocking group generally is in 4 positions of monosaccharide units and/or 3 positions.

Terminal monosaccharide units can maybe can not have blocking group to replace its natural hydroxyl.Preferably keep free different hydroxyl so that the further reaction handle of (for example puting together) to be provided on the monosaccharide units endways.Different hydroxyl can (use for example NaBH by reduction amination ₃CN/NH ₄Cl) be transformed into amino (NH ₂-or-NH-E, wherein E is a nitrogen-protecting group group), can after being transformed into blocking group, other hydroxyl regenerate then.

The blocking group of substituted hydroxy can be directly approaching, and by the derivation reaction of hydroxyl, promptly the hydrogen atom of hydroxyl is replaced by another group.As the suitable hydroxy derivatives of blocking group is for example carbaminate, sulfonate, carbonate, ester, ether (for example silyl ether or alkyl ether) and acetal.Some specific examples of this blocking group are pi-allyl, Aloc, benzyl, BOM, t-butyl, trityl, TBS, TBDPS, TES, TMS, TIPS, PMB, MEM, MOM, MTM, THP etc.Other can not comprise C by blocking group directly approaching and substituted hydroxy fully _1-12Alkyl, C _3-12Alkyl, C _5-12Aryl, C _5-12Aryl-C _1-6Alkyl, NR ¹R ²(R ¹And R ²In the following passage, define), H, F, Cl, Br, CO ₂H, CO ₂(C _1-6Alkyl), CN, CF ₃, CCl ₃Deng.Preferred blocking group is an electron withdraw group.

Preferred blocking group has formula :-O-X-Y or-OR ³, wherein X is C (O), S (O) or SO ₂Y is C _1-12Alkyl, C _1-12Alkoxyl, C _3-12Cycloalkyl, C _5-12Aryl or C _5-12Aryl-C _1-6Alkyl can be chosen wantonly with 1,2 or 3 separately and independently be selected from F, Cl, Br, CO ₂H, CO ₂(C _1-6Alkyl), CN, CF ₃Or Cl ₃Group replace; Or Y is NR ¹R ²R ¹And R ²Independently be selected from H, C _1-12Alkyl, C _3-12Cycloalkyl, C _5-12Aryl, C _5-12Aryl-C _1-6Alkyl; Or R ¹And R ²Can connect to form C _3-12The saturated heterocyclic group; R ³Be C _1-12Alkyl or C _3-12Cycloalkyl can be chosen wantonly with 1,2 or 3 separately and independently be selected from F, Cl, Br, CO ₂(C _1-6Alkyl), CN, CF ₃Or CCl ₃Group replace; Or R ³Be C _5-12Aryl or C _5-12Aryl-C _1-6Alkyl can be chosen wantonly with 1,2,3,4 or 5 separately and be selected from F, Cl, Br, CO ₂H, CO ₂(C _1-6Alkyl), CN, CF ₃Or Cl ₃Group replace.Work as R ³Be C _1-12Alkyl or C _3-12During cycloalkyl, it replaces with 1,2 or 3 group defined above usually.Work as R ¹And R ²Connection is to form C _3-12During the saturated heterocyclic group, mean R ¹And R ²Form the saturated heterocyclic group with nitrogen-atoms, contain the carbon atom (C for example of 3 to 12 any amount ₃, C ₄, C ₅, C ₆, C ₇, C ₈, C ₉, C ₁₀, C ₁₁, C ₁₂).Except nitrogen-atoms, heterocyclic group can comprise 1 or 2 hetero atom (as N, O or S).C _3-12The example of saturated heterocyclic group is pyrrolidinyl, piperidyl, morpholinyl, piperazinyl, imidazolidinyl, azetidinyl and aziridinyl.

Blocking group-O-X-Y and-OR ³Can preparation from-OH group, the reactions such as program such as hydroxyl and acyl halide, alkyl halide, sulfonic acid halide of deriving by standard.Therefore, the oxygen atom the among-O-X-Y is the oxygen atom of hydroxyl preferably, and-among the O-X-Y-hydrogen atom of the preferred substituted hydroxy of X-Y group.

Perhaps, it is approaching that blocking group can be substituted reaction, replaces as the Mitsonobu-type.These and other is known from the method that hydroxyl prepares blocking group.

Blocking group is more preferably-OC (O) CF ₃Or carbaminate group-OC (O) is NR [71], ¹R ², R wherein ¹And R ²Independently be selected from C _1-6Alkyl.R ¹And R ²More preferably all be methyl, promptly blocking group is-OC (O) NMe ₂The carbaminate blocking group has Stabilization to glycosidic bond and can prepare under temperate condition.

The preferred MenA steamed bun stuffed with sugar of modifying contains n monosaccharide units, and wherein the h% monosaccharide units does not all have-the OH group at 3 and 4 at least.The h value is 24 or more (for example 25,26,27,28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,95,98,99 or 100) and preferably 50 or more.Lack-OH group blocking group preferably defined above.

Other is preferably modified the MenA steamed bun stuffed with sugar and draws together monosaccharide units, and wherein the s monosaccharide units does not have at 3-OH and do not have-OH at 4 at least.The s value is at least 1 (for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,60,70,80,90).Lack-OH group blocking group preferably defined above.

The suitable modification MenA sugar that is used to invent has formula:

Wherein n is 1 to 100 integer (preferred 15 to 25 integer);

T has formula (A) or (B):

Each Z group independently is selected from OH or blocking group defined above; With

Each Q group independently is selected from OH or blocking group defined above;

Y is selected from OH or blocking group defined above;

E is H or nitrogen-protecting group group;

Wherein the Q group greater than about 7% (for example 8%, 9%, 10% or more) is a blocking group.

Each n+2Z group each other can be identical or different.Equally, each n+2Q group each other can be identical or different.All Z groups can be OH.In addition, at least 10%, 20%, 30%, 40%, 50% or the 60%Z group can be OAc.Preferred about 70%Z group is OAc, and residue Z group is OH or blocking group defined above.At least about the 7%Q group is blocking group.Preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even the 100%Q group be blocking group.

Preferred invention compositions can be preserved 28 days at 37 ℃, after this stage, puted together non-less than the MenA sugar of puting together of the initial total amount of f%, and wherein f is 20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5 or lower.

Oligosaccharide

Capsular saccharides is generally used with the oligosaccharide form.They easily form, and the fracture (as hydrolysis) of the capsular polysaccharide by purification then is the fragment of the required size of purification usually.

Preferably carry out polysaccharide fracture with produce less than final average degree of polymerization (DP) in 30 the oligosaccharide (as for serum group A at 10 and 20, preferred about 10; For serum group W135 and Y at 15 and 25, preferably about 15-20; For serum group C at 12 and 22; Or the like).DP easily measures [72] by ion-exchange chromatography or colorimetric determination.

Preferred L enC carbohydrate antigen is shown in list of references 10, as Meniugate ^TMUsed.

If be hydrolyzed, hydrolyzate generally is to carry out sizing to take out the short oligosaccharide [73] of length.This can finish with multiple mode, as ultrafiltration, then is ion-exchange chromatography.For serum group A, preferably take out the degree of polymerization and be less than or equal to about 6 oligosaccharide, for serum group W135 and Y, the preferred oligosaccharide that takes out less than about 4.

Covalency is puted together

Capsular saccharides in the invention compositions is puted together carrier protein usually.Generally, put together the immunogenicity that improves sugar because it make they from T-not the dependency antigenic shift become the T-dependence antigen, thereby can the immune stimulatory memory.Put together for the department of pediatrics vaccine and be particularly useful and be the technology of knowing [as list of references 44 and 52 summaries].

Preferred carrier protein is bacteriotoxin or toxoid, as diphtheria toxoid or tetanus toxoid.Preferred especially CRM ₁₉₇Diphtheria toxoid [75-77].Other suitable carriers albumen comprises Neisseria meningitidis outer membrane protein [78], synthetic peptide [79-80], heatshock protein [81-82], pertussis albumen [83-84], cytokine [85], lymphokine [85], hormone [85], somatomedin [85], artificial protein, contains a plurality of from the antigenic people CD4 of Different Kinds of Pathogens syntaxy ⁺T cell antigen epitope [86], protein D [87,88], streptococcus pneumoniae surface protein PspA[89 from hemophilus influenza], ferrum absorbs albumen [90], from the toxin A or the B[91 of clostridium difficile (C.difficile)] etc.Preferred carrier is diphtheria toxoid, tetanus toxoid, hemophilus influenza protein D and CRM ₁₉₇

In inventive compositions, may use carrier protein more than a kind, for example be used to reduce carrier and suppress risk.Therefore, different carriers albumen can be used for different serum group, and for example serum group A sugar may be puted together CRM ₁₉₇, and serum group C sugar may be puted together tetanus toxoid.Also may use the carrier protein more than a kind to be used for specific carbohydrate antigen, for example serum group A sugar may be in 2 groups, and some put together CRM ₁₉₇, other put together tetanus toxoid.Yet, general preferred to all sugar use same vehicle albumen.

Single carrier protein portability is more than a kind carbohydrate antigen [92].For example, single carrier protein can be puted together its sugar from serum group A and C.For reaching this target, can be before conjugation reaction mixed sugar.Yet general preferred each serum group has independent conjugate.

Preferred sugar: the conjugate of albumen ratio (w/w) between 1: 5 (being excessive albumen) and 5: 1 (being excessive glucocorticoid).Preferred 1: 2 and 5: 1 s' ratio, more preferably 1: 1.25 and 1: 2.5 's ratio.Excessive carrier protein is preferred for MenA and MenC.

Conjugate can be used for puting together free carrier protein [93].When specific support albumen free and put together when all existing in the invention compositions of form, do not put together and be no more than carrier protein total amount in 5% the compositions on the form preferred general, more preferably to exist less than 2% weight.

Can use any suitable conjugation reaction, the joint of any appropriate can be arranged in case of necessity.

Sugar is generally being puted together front activating or functionalization.Activation for example can comprise that cyanidization agent (cyanylating) is as CDAP (as 1-cyano group-4-dimethylamino naphthyridine tetrafluoroborate [94,95 etc.]).Other appropriate technology uses carbodiimides, hydrazides, active ester, nor-borine (norborane), p-nitrobenzoic acid, N-hydroxy-succinamide, S-NHS, EDC, TSTU; Also referring to the introduction of list of references 50).

Connect available any known method through the joint group and carry out, for example referring to list of references 96 and 97 described methods.One class connects and comprises the reduction amination polypeptide, an end of coupling gained amino and n diacid joint group, and coupling protein is to another end [48,98,99] of n diacid joint group then.Other joint comprises B-propionamido-[100], nitrobenzophenone-ethamine [101], halogen acyl halide (haloacyl halides) [102], glycosidic bond [103], 6-aminocaprolc acid [104], ADH[105], C ₄To C ₁₂Partly [106] etc.Except using joint, can use direct connection.Directly connect albumen and can comprise oxidation of polysaccharides, then with the albumen reduction amination, as described in list of references 107 and 108.

Preferable methods comprises introduces the amino sugar that arrives (as passing through usefulness-NH ₂Replace end=O group) in, then derive with di adipate (for example adipic acid N-hydroxy-succinamide base diester).Preferably react with carrier protein.Another kind of preferred reaction is used the CDAP activation with protein D carrier, for example is used for MenA or MenC.

After puting together, the separable sugar that dissociates and put together.Many suitable methods are arranged, comprise hydrophobic chromatography, tangential ultrafiltration, diafiltration etc. [also referring to list of references 109 and 110 etc.].

When inventive compositions comprises that when puting together oligosaccharide, preferred oligosaccharides prepares prior to puting together.

The preparation of the present composition

Compositions of the present invention comprises from Neisseria meningitidis serum group A, C, at least 2 kinds capsular saccharides among W135 and the Y.The preferred preparation separately of sugar (comprise any fracture, put together etc.) mixes then to produce inventive compositions.

Yet, when compositions comprises capsular saccharides from serum group A, serum group A sugar preferably not in conjunction with other sugar before using not soon, be used for may dropping to hydrolysis minimum.This easily finishes, and by making serum group A composition (usually with suitable vehicle) be in lyophilized form and making other serum group composition be in liquid form (suitable vehicle is also arranged), liquid component is used to rebuild the MenA composition when preparing to use.When using aluminum salt adjuvant, preferably include adjuvant in the bottle that contains aqueous vaccine and lyophilizing do not have the MenA composition of adjuvant.

Therefore, inventive compositions can prepare from test kit, and test kit comprises: (a) from the capsular saccharides of Neisseria meningitidis serum group A, with lyophilized form; (b) from Neisseria meningitidis serum group C, the capsular saccharides of one or more among W135 and the Y (for example 1,2,3 kinds) is with liquid form.Invention also provides preparation invention method for compositions, comprise mixing from the capsular saccharides of Neisseria meningitidis serum group A with from Neisseria meningitidis serum group C, one or more among W135 and the Y (for example 1,2,3 kinds) capsular saccharides, wherein one or more sugar are with liquid form.

The present invention also provides the invention compositions, comprises from Neisseria meningitidis serum group C, and the capsular saccharides of W135 and Y, wherein sugar is with liquid form.Said composition can be packed with freeze dried serum group A carbohydrate antigen and is used for rebuilding, or said composition can be separately as the compositions use, for example when not needing the immunity inoculation of antiserum group A.

Invention also provides a kind of test kit, and test kit comprises: (a) the 1st container contains the capsular saccharides from 2 or a plurality of Neisseria meningitidis serum group C, W135 and Y, all with lyophilized form; (b) the 2nd container, (i) that contains liquid form is from capsular saccharides non-or 1 Neisseria meningitidis serum group C, W135 and Y, with optional (ii) more antigens (as follows) that do not comprise the Neisseria meningitidis capsular saccharides, wherein the inclusions of rebuilding container (a) of the inclusions by container (b) provides the invention compositions.

The outward appearance of the present composition

The present composition can exist and pack in a different manner.

Compositions can be present in the bottle, or they can be present in the syringe that easily is full of.The syringe that provides can be with or without pin.Syringe comprises unit-dose composition, and bottle comprises single dose or multiple dose.Injectable composition is liquid solution or suspension normally.In addition, they can exist (for example lyophilization) to be used for the solution or the suspension of liquid-carrier by solid form, inject then.

Inventive compositions can unit dosage forms or multi-pharmaceutics packing.For multi-pharmaceutics, bottle is the syringe of prefilled preferably.The effective dose volume energy is conventional to be determined, but the compositions volume of the typical human dosage that is used to inject is 0.5ml.

When existing when inventive compositions preparation (existing with lyophilized form) temporarily before use with as test kit as serum group A sugar, test kit can comprise 2 bottles, maybe can comprise 1 syringe that easily is full of and 1 bottle, the inclusions of syringe is used for the inclusions of reactivate bottle before injection.

In each dosage, independent carbohydrate antigen amount is between 1-50 μ g (as saccharic measurement amount) generally, and each preferably is about 2.5 μ g, 5 μ g or 10 μ g.A: C: W135: the Y part by weight is 1: 1: 1: 1; 1: 1: 1: 2; 2: 1: 1: 1; 4: 2: 1: 1; 8: 4: 2: 1; 4: 2: 1: 2; 8: 4: 1: 2; 4: 2: 2: 1; 2: 2: 1: 1; 4: 4: 2: 1; 2: 2: 1: 2; 4: 4: 1: 2 and 2: 2: 2: 1, therefore, amount shown in Figure 1 preferably is about 2.5 μ g, 5 μ g or 10 μ g.Thereby, for 1: 1: 1: the A of 1 ratio: C: W: Y compositions and the every sugar of 10 μ g, use the every dosage of 40 μ g sugar.Preferred compositions has the every dosage of about following μ g sugar:

A	10	0	0	0	10	5	2.5
								C	10	10	5	2.5	5	5	2.5
W135	10	10	5	2.5	5	5	2.5
								Y	10	10	5	2.5	5	5	2.5

Preferred invention compositions comprises the every dosage of meningococcus sugar less than 50 μ g.Other preferred composition comprises≤the every dosage of 40 μ g meningococcuss sugar.Other preferred composition comprises≤the every dosage of 30 μ g meningococcuss sugar.Other preferred composition comprises≤the every dosage of 25 μ g meningococcuss sugar.Other preferred composition comprises≤the every dosage of 20 μ g meningococcuss sugar.Other preferred composition comprises≤the every dosage of 10 μ g meningococcuss sugar, but inventive compositions comprises the every dosage of at least 10 μ g meningococcuss sugar ideally.

The present composition is preferably aseptic, does not preferably contain thermal source, for example preferably cushions between pH and pH8 general about 7.When compositions comprises aluminium hydroxide salt, preferably use histidine buffering liquid [111].But invention compositions counterpart etc. is oozed.

Adjuvant

Said composition generally comprises one or more adjuvants.Can and/or afterwards adjuvant be added sugar before the adjuvant and the sugar mixing formation present composition, but preferably before with various sugar mixing, adjuvant and carbohydrate antigen be made up.

Yet not necessarily every kind of sugar all must add adjuvant before mixing.Can comprise excessive adjuvant in a kind of preparation of sugar, for example when adding did not more add the carbohydrate antigen of adjuvant, excessive adjuvant was diluted to required final concentration.In a specific embodiments, when the present composition when freeze-dried antigen (for example freeze dried serum group A component) prepares, preferred said composition does not comprise the adjuvant of lyophilized form.

The adjuvant that preferably is contained in the present composition is aluminum salt (vitriol), aluminium hydroxide (comprising oxyhydroxide) for example, aluminum phosphate (comprising subphosphate), aluminum sulfate etc. [list of references 112 the 8th and 9 chapters].Preferred especially hydroxyl aluminum phosphate, especially in the compositions that comprises the hemophilus influenza carbohydrate antigen, typical adjuvant is amorphous hydroxyl aluminum phosphate, PO ₄/ Al mol ratio comprises 0.63mg A1 at 0.84 and 0.92 ³⁺/ ml.Can use the absorption of low dosage aluminum phosphate, for example 50 to 100 μ g Al ³⁺The every dosage of every conjugate.When conjugate was arranged in the compositions more than a kind, not all conjugate all need absorb.Menjugate ^TMAnd NeisVac ^TMThe MenC conjugate adopts the hydroxide adjuvant, and Meningitec ^TMAdopt phosphate.

Calcium phosphate is another kind of preferred adjuvants.

Also can adopt or other adjuvant of substitution of Al salt comprises:

A. the compositions that contains mineral

The compositions that contains mineral is suitable as the invention adjuvant, comprises inorganic salt, as aluminum salt and calcium salt.Invention comprise inorganic salt such as hydroxide (for example oxyhydroxide), phosphate (for example hydroxide phosphate, orthophosphate), sulfate etc. [for example referring to list of references 112 the 8th with 9 chapters] or the mixture of different inorganic compound, chemical compound adopts any suitable form (example gel, crystal, amorphous etc.), the preferred absorption.The compositions that contains mineral can be made into the granule of slaine [113].

B. oil emulsion

The oil emulsion compositions that is suitable as the invention adjuvant comprises zamene-aqueous emulsion, as the 10th chapter of MF59[list of references 112; Also referring to list of references 114] (5% zamene, 0.5% Tween 80 and 0.5%Span 85 make submicron particles with the Micro Fluid machine).Also can use complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA).

C. saponin preparation [oozing the 22nd chapter of examining document 112]

The saponin preparation also can be as the adjuvant of invention.Saponin is the xenogenesis group of steroline and triterpene glucosides, at the floristic bark of wide scope, leaf, stem, root with even spend middle discovery.From the saponin of Quillaia saponaria (Quillaiasaponaria) Molina bark as adjuvant broad research.Saponin also can commercially obtain from beautiful colored Rhizoma Smilacis Chinensis (Smilax ornata) (sarsaparilla), Caulis et folium pavettae hongkongensis (Gypsophilla paniculata) (brides veil) and Saponaria officinalis (Saponaria officianalis) (Radix saponariae).The saponin adjuvant preparation comprises purification preparation such as QS21 and lipid formulations such as ISCOMs.QS21 is with Stimulon ^TMSell.

Saponin preparation HPLC and RP-HPLC purification.Identify the specific purification part that uses these technology, comprised QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C.Saponin is QS21 preferably.The method that generates QS21 is shown in list of references 42.The saponin preparation also can comprise sterol, as cholesterol [116].

The combination of saponin and cholesterol can be used for forming the unique granule [the 23rd chapter of list of references 112] that is called immunostimulation complex (ISCOMs).ISCOMs also comprises phospholipid such as PHOSPHATIDYL ETHANOLAMINE or phosphatidylcholine usually.Any known saponin can be used for ISCOMs.ISCOM preferably includes a kind or multiple QuilA, QHA and QHC.ISCOMs is further described in list of references 116-118.ISCOMs is optional can to lack other detergent [119].

Exploitation can be found in list of references 120 and 121 based on the summary of the adjuvant of saponin.

D. virion and virus-like particle

Virion and virus-like particle (VLPs) also can be used as the adjuvant of invention.These structures generally comprise a kind or multiple albumen from virus, and virus is optional to be made in conjunction with phospholipid or with phospholipid.They are not pathogenic usually, do not duplicate and generally do not comprise any natural viral genome.Virus protein can be recombinated from intact virus and be generated or separation.These are applicable to that the virus protein of virion or VLPs comprises acquisition gravity flow susceptible poison (as HA or NA), hepatitis B virus (as core or capsid protein), hepatitis E virus, Measles virus, the sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, Norwalk virus, the human papillomavirus, HIV, the RNA-phage, Q pnagus beta (as coat protein), the GA-phage, the fr-phage, the albumen of AP205 phage and Ty (as retrotransposon Ty albumen p1).VLPs further is discussed at list of references 122-127.Virion further is discussed at for example list of references 128.

E. antibacterial or microorganism derivant

The adjuvant that is suitable for inventing comprises antibacterial or microorganism derivant, as non-toxic derivative, lipid A derivant, immunostimulatory oligonucleotide and ADP-ribosylation toxin and its detoxification derivant of enterobacteria lipopolysaccharide (LPS).

The non-toxic derivative of LPS comprises monophosphoryl lipid A (MPL) and 3-O-deacylated tRNA MPL (3d MPL).3dMPL is the mixture of 3-O-deacylated tRNA monophosphoryl lipid A and 4,5 or 6 acidylate chains.Preferred " granule " form of 3-O-deacylated tRNA monophosphoryl lipid A is shown in list of references 129.This " granule " 3d MPL is small enough to through 0.22 μ m film aseptic filtration [129].Other non-toxicity LPS derivant comprises the monophosphoryl lipid A analogies, as the aminoalkyl glucosaminide phosphoric acid derivatives, and RC-529[130 for example, 131].

The lipid A derivant comprises from colibacillary lipid A derivant, as OM-174.OM-174 is described in for example list of references 132 and 133.

The immunostimulatory oligonucleotide that is suitable as the invention adjuvant comprises and contains the CpG motif nucleotide sequence of (the dinucleotide sequence contains the cytosine that do not methylate that connects guanosine by phosphate bond).The double-stranded RNA s and the oligonucleotide that contain the palindrome or poly-(dG) sequence also show it is immunostimulating.

CpG can comprise that nucleotide modification/analog such as D2EHDTPA are modified and can be two strands or strand.List of references 61,62 and 63 discloses possible analog and replaces, as replacing guanosine with 2 '-deoxidation-7-denitrogenation guanosine.The adjuvant effect of CpG oligonucleotide further is discussed at list of references 137-142.

The CpG sequence can be guided TLR9 into, as motif GTCGTT or TTCGTT[70].The CpG sequence can be to inducing the Th1 immunoreation special, and as CpG-A ODN, or it can be to inducing the B cell effect more special, as CpG-BODN.CpG-A and CpG-B ODN are discussed at list of references 144-146.CpG is CpG-A ODN preferably.

Preferred formation CpG oligonucleotide makes 5 ' end easily accept receptor identification.2 kinds of CpG oligonucleotide sequences can be chosen wantonly at its 3 ' end and adhere to form " immune aggressiveness " (immunomers).Referring to for example list of references 143 and 147-149.

Antibacterial ADP-ribosylation toxin and its detoxification derivant can be used as the adjuvant of invention.Albumen preferably obtains from escherichia coli (escherichia coli thermal instability enterotoxin " LT "), cholera (" CT ") or pertussis (" PT ").Use detoxification ADP-ribosylation toxin to be described in list of references 150 and to be described in list of references 151 as the parenteral adjuvant as mucosal adjuvants.Toxin or toxoid comprise A and B subunit preferably with the holotoxin form.The A subunit preferably comprises the detoxification sudden change; The B subunit does not preferably suddenly change.Adjuvant is detoxification LT mutant preferably, as LT-K63, LT-R72 and LT-G192.Use ADP-ribosylation toxin and the specific LT-K63 of being of its detoxification derivant and LT-R72 can be found in list of references 152-159 as adjuvant.The A and the B subunit that are preferably based on list of references 160 listed ADP-ribosylation toxin about the digital reference of aminoacid replacement are arranged, and it is for reference that list of references 160 is especially all included this paper in.

F. people's immunomodulator

The people's immunomodulator that is suitable as the invention adjuvant comprises cytokine such as interleukin (for example IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12[161] etc.) [162], interferon (for example interferon-), M-CSF and tumor necrosis factor.

G. biological adhesive and mucoadhesive

Biological adhesive and mucoadhesive also can be used as the adjuvant of invention.Suitable biological adhesive comprises that esterification hyaluronic acid microsphere [163] or mucoadhesive are as gathering the cross-linked derivant of (acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharide and carboxymethyl cellulose.Chitosan and its derivant also can be used as the adjuvant [164] of invention.

H. microgranule

Microgranule also can be used as the adjuvant of invention.Preferably the microgranule that forms from biodegradable and non-toxicant (as poly-(alpha-hydroxy acid), poly butyric, poe, polyanhydride, polycaprolactone etc.) material (promptly～100nm is to the granule of～150nm diameter, more preferably～200nm is to～30 μ m diameters, most preferably～500nm is to～10 μ m diameters), preferably has the poly-glycolide copolymer of polyactide, optional treatment to have the surface (for example using cationic detergent) of electronegative surface (for example using SDS) or positively charged as CTAB.

I. liposome (the 13rd and 14 chapters of list of references 112)

The example that is suitable as the Liposomal formulation of adjuvant is described in list of references 165-167.

J. polyoxyethylene ether and polyoxyethylene ester formulation

The adjuvant that is applicable to invention comprises polyoxyethylene ether and polyoxyethylene ester [168].This preparation further comprises polyoxyethylene sorbitan esters surfactant [169] that makes up octoxinol and polyoxyethylene alkyl ether or the ester surfactant [170] that makes up other non-ionic surface active agent of at least one such as octoxinol.Preferred polyoxyethylene ether is selected from following group: polyoxyethylene-9-Laurel ether (laureth9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-Laurel ether, polyoxyethylene-35-Laurel ether and polyoxyethylene-23-Laurel ether.

K. polyphosphazene (PCPP)

The PCPP preparation is described in for example list of references 171 and 172.

L. muramyl peptide

The example that is suitable as the muramyl peptide of invention adjuvant comprises N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-positive muramyl-L-alanyl-D-isoglutamine (nor-MDP) and the different glutamy of N-acetyl muramyl-L-alanyl-D--L-alanine-2-(1 '-2 '-two palmityls-sn-glycerol-3-hydroxyl phosphorus acyloxy)-ethamine (MTP-PE).

M. imidazoles quinolinones (imidazoquinolone) chemical compound

The example that is suitable as the imidazolone chemical compound of invention adjuvant comprises imiquimod and its congener (for example " resiquimod 3M "), and they are further described in list of references 173 and 174.

Invention also can comprise the combination of a kind or the multiple adjuvant of identifying above.For example, following adjunvant composition can be used for invention: (1) saponin and oil in water emulsion [175]; (2) saponin (for example QS21)+non-toxicity LPS derivant (for example 3dMPL) [176]; (3) saponin (for example QS21)+non-toxicity LPS derivant (for example 3dMPL)+cholesterol; (4) saponin (for example QS21)+3dMPL+IL-12 (optional+sterol) [177]; (5) combination [178] of 3dMPL and for example QS21 and/or oil in water emulsion; (6) SAF contains 10% zamene, 0.4% Tween 80 ^TM, 5%pluronic-block copolymer L121 and thr-MDP, miniflow changes into submicron Emulsion or vortex produces than coarsegrain Emulsion; (7) Ribi ^TMAdjuvant system (RAS) (Ribi Immunochem), contain 2% zamene, 0.2% Tween 80 and a kind or multiple bacteria cell wall composition, preferred MPL+CWS (Detox from monophosphoryl lipid A (MPL), trehalose two mycolic acids (TDM) and cell wall skeleton (CWS) ^TM); (8) 1 kinds or plurality of inorganic salt (as aluminum salt)+non-toxicity LPS derivant (as 3dMPL).

Other material as immunostimulant is shown in the 7th chapter of list of references 112.

When using aluminum sulfate, one or more sugar may be adsorbed onto on the aluminum salt, but preferably do not do like this, and this promotes by comprising free phosphoric acid ion (for example using phosphate buffer) in the solution.When using aluminium hydroxide, preferred sugar is adsorbed onto on the salt.Sugar for serum group A preferably uses aluminium hydroxide as adjuvant.

In inventive compositions, more possible antigens are adsorbed in aluminium hydroxide, but other antigen and aluminum phosphate link.For for example tetravalence Neisseria meningitidis serum group combination, can obtain following arrangement

Serum group	Aluminum salt (H=hydroxide; P=phosphoric acid)
																	A	P	H	P	H	H	H	P	P	P	H	H	H	P	P	P	H
C	P	H	H	P	H	H	P	H	H	P	P	H	P	H	P	P
																	W135	P	H	H	H	P	H	H	P	H	H	P	P	P	P	H	P
Y	P	H	H	H	H	P	H	H	P	P	H	P	H	P	P	P

For the combination of trivalent Neisseria meningitidis serum group, can obtain following arrangement:

Serum group	Aluminum salt (H=hydroxide; P=phosphoric acid)
									C	P	H	H	H	P	P	P	H
W135	P	H	H	P	H	P	H	P
									Y	P	H	P	H	H	H	P	P

Other component of compositions

Except that above-mentioned antigen, the present composition can comprise meningococcal protein matter antigen.

Also can comprise non-meningococcus and non-neisserial antigens, preferably not reduce the immunoreactive antigen of meningococcemia component.For example list of references 179 has been described the combination from oligosaccharide and the Hib sugar of Neisseria meningitidis serum group B and C.Preferably from streptococcus pneumoniae, hepatitis A virus (HAV), hepatitis B virus, Bordetella pertussis (B.pertussis), diphtheria, tetanus, the antigen of poliomyelitis and/or hemophilus influenza.Especially preferred antigen comprises:

-diphtheria antigen is as diphtheria toxoid [for example the 3rd chapter of list of references 180].

-tetanus antigen is as tetanus toxoid [for example the 4th chapter of list of references 180].

-from the pertussis holotoxin (PT) and the filamentous hemagglutinin (FHA) of Bordetella pertussis, choose wantonly and also make up for example list of references 181 and 182 of pertussis adhesin (pertactin) and/or agglutinogen 2 and 3[].

-cell pertussis antigen.

-from the antigen of hepatitis A virus (HAV), as inactivation of viruses [for example 183,184].

-from the antigen of hepatitis B virus, as surface and/or cAg [for example 184,185], surface antigen preferably is adsorbed onto [186] on the aluminum sulfate.

-from the microvesicle [187] of Neisseria meningitidis serum group B, ' natural OMVs ' [188], blister or outer membrane vesicles goods [for example list of references 189-190 191 192 193 194 etc.].These goods can prepare from the antibacterial through genetic manipulation, and for example genetic manipulation is with enhance immunity originality (for example crossing the expression immunogen), and reduced toxicity suppresses capsular polysaccharide and synthesizes, downward modulation PorA expression etc.These goods can be from super foaming bacterial strain [199-200 201 202] preparation.Can comprise vesicle [203] from the avirulence neisser's coccus.Preparation OMVs can not use detergent [204,205].These goods can be at the non-neisserial protein of its surface expression [206].These goods can be that LPS exhausts.These goods can mix [189,207] with recombinant antigen.Can use vesicle from antibacterial with different I class 1 outer-membrane protein matter hypotype, for example with six kinds of different subtypes [208 that respectively present two kinds of different genetic engineering vesicle groups of three kinds of hypotypes, 209], or with nine kinds of hypotypes of three kinds that respectively present three kinds of hypotypes different genetic engineering vesicle groups etc.Useful hypotype comprises: P1.7,16; P1.5-1,2-2; PI.19,15-1; P1.5-2,10; PI.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1; P1.18-1,3,6.

-poliomyelitis antigen [for example 210,211] is as IPV.

Mixture can comprise one or more these antigens, but detoxification when needing (for example pertussis toxin, PT with chemistry and/or genetic method detoxification).

When comprising diphtheria antigen in the compositions, preferably also comprise tetanus antigen and pertussis antigen.Similarly, when comprising tetanus antigen, preferably also comprise diphtheria and pertussis antigen.Similarly, when comprising pertussis antigen, preferably also comprise diphtheria and tetanus antigen.This DTP combination can be used for rebuilding the lyophilizing conjugate.

Antigen in the mixture exists with the concentration of each 1 μ g/ml at least usually.General any given antigenic concentration enough causes at this antigenic immunne response.

Except in the invention compositions, using the proteantigen, can use the nucleic acid of coding for antigens.Therefore, the protein ingredient of invention compositions can replace with the nucleic acid (preferred DNA is as the form with plasmid) of encoding proteins.Similarly, invention compositions can comprise the protein of simulation carbohydrate antigen such as mimic epitope [212] or anti-idiotype antibody.They can replace independent sugared composition, maybe can replenish them.For example, vaccine can comprise MenC[213] or MenA[214] peptide mimics of capsular polysaccharide replaces sugar itself.

Inventive compositions can comprise antibacterial, particularly when with the multiple dose packaged.

Inventive compositions can comprise detergent, and tween (polysorbate) for example is as Tween 80.Detergent generally exists with low-level, as＜0.01%.

Inventive compositions can comprise that sodium salt (for example sodium chloride) is to produce permeability.Typical concentration is 10 ± 2mg/ml NaCl.

Inventive compositions generally comprises buffer.The typical case is a phosphate buffer.

Inventive compositions can comprise sugar alcohol (as mannitol) or disaccharide (as sucrose [215] or trehalose [216]), and for example about 15-30mg/ml (as 25mg/ml) is if if particularly they want lyophilizing or them to comprise from the restorative again material of freeze dried substance.Be used for freeze dried compositions pH can transfer to about 6.1, lyophilizing then.

The invention provides and comprise the compositions of puting together capsular saccharides, put together capsular saccharides from Neisseria meningitidis serum group A, C, W135 and Y at least three kinds, wherein said composition comprises sucrose.These sugar are oligosaccharide preferably.Said composition can comprise≤the every dosage of the total meningococcus sugar of 50 μ g (for example≤40 μ g ,≤30 μ g ,≤20 μ g ,≤10 μ g).Preferred compositions comprises: serum group A, C, W135; Serum group A, C, Y; Serum group C, W135, Y; With serum group A, C, four kinds of W135 and Y.Can adopt modified MenA sugar.Said composition can be liquid or drying (for example lyophilizing) form.When being liquid form, sucrose concentration is for example about 25mg/ml between 5-50mg/ml preferably.When being lyophilized form, preferred said composition does not comprise aluminum salt adjuvant.Said composition also can comprise from following one or more antigen: (a) Neisseria meningitidis serum group B; (b) haemophilus influenzae type B; And/or (c) streptococcus pneumoniae.

Immunogenicity

The invention compositions is immunogenic.Preferred immunogenic composition is a vaccine.Vaccine according to invention can be prevention (promptly protecting from infection) or a therapeutic (promptly treating metainfective disease), but normally preventative.

Except above-mentioned composition, the immunogenic composition of invention and vaccine generally include a kind or multiple ' pharmaceutically acceptable carrier ', and the included carrier of pharmaceutically acceptable carrier itself is not induced and generated accepting the individual deleterious antibody of compositions.Suitable carriers generally is big, slow metabolic macromole, as albumen, polysaccharide, polylactic acid, polyglycolic acid, polymeric amino acid, amino acid copolymer, sucrose, trehalose [216], lactose and lipid aggregation (for example oil droplet or liposome).This carrier is known those of ordinary skills.Vaccine also can comprise diluent, as water, saline, glycerol etc.In addition, can there be auxiliary substance such as wetting agent or emulsifying agent, pH buffer substance etc.Aseptic, pyrogen-free phosphate buffer normal saline is typical carrier.Can obtain self-reference document 217 about talking out of pharmaceutically acceptable excipient.

Immunogenic composition as vaccine comprises immunity upward antigen and required any other composition of effective dose.' effective dose in the immunity ' refers to be administered to individual amount to treating or preventing effectively with single dose or as a series of part.This amount can change, and depends on the health of individuality to be treated and health, age, the sorted group (for example inhuman primate, primate etc.) of individuality to be treated, the ability of individual immunity system synthetic antibody, required degree of protection, bacterin preparation, treatment doctor assessment and other correlative factor to medical condition.Desired amount is in scope relatively widely, and scope can determine by routine test, the typical amount of each meningococcus carbohydrate antigen of every dosage between 1 μ g and 200 μ g, for example about 1 μ g, about 2.5 μ g, about 4 μ g, about 5 μ g or about 10 μ g (being expressed as sugar).

Can determine the immunogenicity of invention compositions, by they being administered to the test subject (as the child or the animal model [218] at 12-16 monthly age) and the parameter that settles the standard then, comprise the serum sterilizing antibody (SBA) of the anti-pod membrane IgG of summation high affinity and ELISA tire (GMT).These immunoreation are generally determined after compositions is used, are used the preceding value of determining with compositions and compare in about 4 weeks.Preferred SBA increases at least 4 times or 8 times.When using above 1 dosage composition, the back of using that can carry out more than 1 time is determined.

Preferred invention compositions can be given patient's antibody titer, tires to be higher than the standard of the serum protection that can accept each antigenic component of percentage ratio people experimenter.Know the antigen that has associated antibodies to tire, think the host to the conversion of antigen serum on it, this tiring announced as The World Health Organization (WHO) by tissue.Be preferably greater than the conversion of statistical significance experimenter's sample generation serum of 80%, more preferably greater than 90%, more preferably greater than 93% and 96-100% most preferably.

Give the present composition

Present composition injectable.

Parenteral injection can be subcutaneous, intraperitoneal, intravenous or intramuscular.Preferred intramuscular is administered to thigh or upper arm.Injection can be through pin (for example hypodermic needle), but can use the injection of needleless in addition.Typical intramuscular dosage is 0.5ml.

Administration can be single dose scheme or multiple dose scheme.Multiple dose can be used for the immunization protocol of preliminary immunization protocol and/or reinforcement.The immunization protocol that preliminary dosage can then be strengthened.(for example 4-16 is between week) and sensitization and the suitable selection of time that adds Qianghian can conventional be determined between priming dose.

Generally give animal, specifically, can treat people's object the present composition.Said composition is particularly useful for vaccination child and youth.

Medical approaches and application

Invention also is provided at and produces immunoreactive method in the mammal, comprises with the present composition and injects the patient.The preferred protectiveness of immunoreation ground meningococcemia disease can comprise humoral immune reaction and/or cell immune response.The preferred child of patient.

This method can cause in the patient who causes anti-Neisseria meningitidis strengthens reaction.

The present invention also provides following material to cause application in the immunoreactive injectable drug in being manufactured on animal: (i) from Neisseria meningitidis serum group A, C, at least two kinds capsular saccharides among W135 and the Y, wherein said capsular saccharides and carrier protein are puted together and/or are oligosaccharide and (ii) from following one or more antigen: (a) Neisseria meningitidis serum group B; (b) haemophilus influenzae type B; With or (c) streptococcus pneumoniae.Medicine is preferred for preventing and/or treating bacterial meningitis.

A kind of method of checking the therapeutic treatment effect comprises the bacterial infection after the invention compositions is used in monitoring.A kind of method of checking preventative processing effect comprises that monitoring is used anti-ly after the compositions needs to handle antigenic immunoreation.

Heterologous host

Can express original host (for example Neisseria meningitidis or streptococcus pneumoniae) although be used for the polypeptide of the present composition, preferably use heterologous host.Heterologous host can be protokaryon (as antibacterial) or eukaryote.It is escherichia coli preferably, but other suitable hosts comprises bacillus subtilis (Bacillus subtilis), vibrio cholera (Vibrio cholerae), salmonella typhi (Salmonella typhi), salmonella typhimurium (Salmonella typhimurium), neisseria lactamica (Neisseria lactamica), Lycoperdon polymorphum Vitt neisser's coccus (Neisseria cinerea), mycobacteria (Mycobacteris) (for example mycobacterium tuberculosis), yeast etc.

Comprehensively

Term " comprise " refer to " comprising " and " by ... form ", other composition, for example X+Y be formed or be comprised to the compositions that for example " comprises " X can by X is unique.

The term " about " that relates to numerical value x refers to for example x ± 10%.

Word " basically " is not got rid of " fully ", and for example, the compositions of " not having substantially " Y can not have Y fully.In case of necessity, word " basically " can omit from the invention definition.

Bacterial strain can be used as subscript to be represented, for example 741 _MC58Be albumen 741 from bacterial strain MC58.Except as otherwise noted, albumen shown in this paper (for example not having subscript) is from Neisseria meningitidis bacterial strain 2996, and this bacterial strain can be used as ' reference ' bacterial strain.Yet, be appreciated that invention is not limited by bacterial strain generally.As implied above, general reference protein (for example ' 287 ', ' 919 ' etc.) can comprise the albumen from any bacterial strain.Usually, it and 2996 sequence homogeneity are 90% or more (for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).When using hybrid protein, the antigen alone in the heterozygote (promptly discrete-the X-part) can be from one or more bacterial strains.For example when n=2, X ₂Can from X ₁Identical bacterial strain or from different strains.When n=3, bacterial strain can be (i) X ₁=X ₂=X ₃(ii) X ₁=X ₂≠ X ₃(iii) X ₁≠ X ₂=X ₃(iv) X ₁≠ X ₂≠ X ₃Or (v) X ₁=X ₂≠ X ₃Deng.

Term " alkyl " refers to the alkyl of direct sum branch form.Alkyl can be interrupted with 1,2 or 3 hetero atom, hetero atom is selected from-O-,-NH-or-S-.Alkyl also can be interrupted with 1,2 or 3 two and/or triple bond.Yet term " alkyl " is often referred to does not have hetero atom to be interrupted or alkyl two or that triple bond is interrupted.When mentioning C _1-12During alkyl, alkyl can comprise the carbon atom (C for example of 1 to 12 any amount ₁, C ₂, C ₃, C ₄, C ₅, C ₆, C ₇, C ₈, C ₉, G ₁₀, C ₁₁, C ₁₂).Similarly, when mentioning _C1-6During alkyl, alkyl can comprise the carbon atom (C for example of 1 to 6 any amount ₁, C ₂, C ₃, C ₄, C ₅, C ₆).

Term " cycloalkyl " comprises the combination of cycloalkyl, poly-cycloalkyl and cycloalkenyl and these and alkyl, as cycloalkyl-alkyl.Cycloalkyl can be interrupted with 1,2 or 3 hetero atom, hetero atom is selected from-O-,-NH-or-S-.Yet term " cycloalkyl " is often referred to the cycloalkyl that does not have hetero atom to be interrupted.The example of cycloalkyl comprises cyclopenta, cyclohexyl, cyclohexenyl group, cyclohexyl methyl and adamantyl.When mentioning C _3-12During cycloalkyl, cycloalkyl can comprise the carbon atom (C for example of 3 to 12 any amount ₃, C ₄, C ₅, C ₆, C ₇, C ₈, C ₉, C ₁₀, C ₁₁, C ₁₂).

Term " aryl " refers to aromatic group, as phenyl or naphthyl.When mentioning C _5-12During aryl, aryl can comprise the carbon atom (C for example of 5 to 12 any amount ₅, C ₆, C ₇, C ₈, C ₉, C ₁₀, C ₁₁, C ₁₂).

Term " C _5-12Aryl-C _1-6Alkyl " refer to group such as benzyl, phenethyl and menaphthyl.

Nitrogen-protecting group group comprises that silicyl (as TMS, TES, TBS, TIPS), acyl derivative are (as phthalimide, trifluoroacetamide, methoxycarbonyl, ethoxy carbonyl, t-butoxy carbonyl (Boc), benzyloxycarbonyl (Z or Cbz), 9-fluorenes methoxycarbonyl (Fmoc), 2-(trimethyl silyl) ethoxy carbonyl, 2; 2,2-trichlorine ethoxy carbonyl (Troc)), sulfonyl derivative (as β-trimethyl silyl second sulphonyl (SES)), sulfenyl derivant, C _1-12Alkyl, benzyl, benzhydryl, trityl, 9-phenyl fluorenes etc.Preferred nitrogen-protecting group group is Fmoc.

Should understand sugared ring can exist with open or closed form, although show closed form in the structural formula of this paper, invention also comprises opening mode.

Be used to promote to clone or the sequence of purification etc. not necessarily helps invention and can omit or remove.

The polypeptide of invention can pass through several different methods (as recombinant expressed, purification, chemosynthesis (to small part) etc. from cell culture) and in a variety of forms (as natural, fusion, non-glycosylated, fatization etc.) prepare.They preferably make the form (promptly not having other Neisseria meningitidis or host cell proteins matter substantially) of basic purification.

Nucleic acid according to invention can prepare (as by chemosynthesis (to small part), from genome or cDNA library, from organism itself etc.) in many ways and can adopt various ways (as strand, two strands, carrier, probe etc.).They preferably make the form (promptly not having other Neisseria meningitidis or host cell nucleic acid substantially) of abundant purification.Term " nucleic acid " comprises DNA and RNA and their analog, as contains and modify main chain (for example D2EHDTPA etc.) and peptide nucleic acid(PNA) (PNA) etc.Invention comprises contained sequence and above-mentioned complementary nucleic acid (for example be used for antisense or survey purpose).

After the serum group, the meningococcus classification comprises serotype, blood serum subtype, is immunologic pattern then, and serum group, serotype, blood serum subtype and immunologic pattern are listed in the standard name, and each is separated by colon, as B:4:P1.15:L3, and 7,9.In serum group B, some pedigrees cause disease frequent (high invasive), and some pedigrees cause the disease form (high toxicity) more serious than other pedigree, other do not cause disease.7 high toxicity pedigrees have been discerned, i.e. subgroup I, III and IV-1, ET-5 complex, ET-37 complex, A4 bunch and pedigree 3.They are determined by multilocus enzyme electrophoresis (MLEE), but multidigit point sequence typing (MLST) also is used for branch parameningococcus [list of references 41].

The mode that carries out an invention

1. the meningococcus sugar that is used for the administration of people's intramuscular

As preparation as described in the list of references 7 from MenC, MenW135, MenY and randomly, the oligosaccharide conjugate of MenA.Prepare each 0.5ml dosage (amount of every 0.5ml dosage) of following 6 kinds of compositionss with these:

Component	A *	B	C *	D *	E *	F
							Serum group A oligosaccharide-CRM 197Conjugate μ g	10	0	10	5	2.5	0
Serum group C oligosaccharide-CRM 197Conjugate μ g	10	10	5	5	2.5	10
							Serum group W135 oligosaccharide-CRM 197Conjugate μ g	10	10	5	5	2.5	0
Serum group Y oligosaccharide-CRM 197Conjugate μ g	10	10	5	5	2.5	0
							Aluminum sulfate adjuvant mg	0.3
Sodium chloride mg	4.5
							Mannitol mg	7.5
Sodium dihydrogen phosphate (pH7.6) mg	0.69
							Potassium dihydrogen phosphate mg	0.34
Tween TM80 mg	0.025

^*Serum group A component is a lyophilized form, obtains final ACWY compositions with the dilution of CWY fluid composition.

Give these vaccines by child's huckle of intramuscular injection at the 12-16 monthly age, single dose is (for Menjugate ^TMEffective in greater than 12 months child) or 4 week back injections for the second time.Can compare before the vaccination and vaccination after the serum BCA and the IgG in (for example in the 4th week, if accepted two doses) then in the 8th week.

2. two bottles of compositionss

The conjugate that is used for the people is prepared as 2 independently bottles.Bottle 1 contains the lyophilized powder of MenA conjugate, also has sucrose and potassium dihydrogen phosphate.Bottle 2 contains MenC, and MenW135 and MenY conjugate also have sodium chloride, polysorbate80, the aluminum phosphate adjuvant of sodium phosphate buffer and optional suspensions.Before the use, rebuild bottle 1 with the 0.6ml liquid of bottle 2, the 0.5ml that obtains can be used for administration.

3 kinds of dosage have been prepared.In the form of rebuilding, vaccine contains following antigen:

Component	The amount of every 0.5ml dosage
		Serum group A conjugate	10 μ g sugar+12.5-33 μ g CRM 197Or 5 μ g sugar+6.25-16.5 μ g CRM 197Or 2.5 μ g sugar+3.125-8.25 μ g CRM 197
Serum group C conjugate	10 μ g sugar+12.5-25 μ g CRM 197Or 5 μ g sugar+6.25-12.5 μ g CRM 197Or 2.5 μ g sugar+3.125-6.25 μ g CRM 197
		Serum group W135 conjugate	10 μ g sugar+6.6-20 μ g CRM 197Or 5 μ g sugar+3.3-10 μ g CRM 197Or 2.5 μ g sugar+1.65-5 μ g CRM 197
Serum group Y conjugate	10 μ g sugar+6.6-20 μ g CRM 197Or 5 μ g sugar+3.3-10 μ g CRM 197Or 2.5 μ g sugar+1.65-5 μ g CRM 197

In the form of rebuilding, vaccine contains following other antigen:

Component	The amount of every 0.5ml dosage
			The phosphate adjuvant	0.3mg AL 3+	0

Sodium dihydrogen phosphate	1mM	2.5mM
			Two hypophosphite monohydrate disodium hydrogens	9mM	7.5mM
Sodium phosphate buffer	10mM
			Potassium dihydrogen phosphate	5mM
Tween TM80 (surfactants)	0.025mg
			Sodium chloride (permeability)	4.5mg
Sucrose (lyophilizing and permeability)	12.5mg
			Water for injection	To final volume

Therefore six kinds of vaccine-3 kind of various dose (10,20 or 40 μ g total sugar) can be arranged, respectively be with or without the aluminum phosphate adjuvant.

The Adjuvanted vaccines that sugared dosage is the highest gives people's object in healthy age 18-45 year.Be used for comparison, the contrast object is accepted (a) bottle 1 (rebuilding) and bottle 2 products in buffer, at identical asynchronism(-nization) arm, or (b) Mencevax ^TMEach patient's group has 30 people.

After preceding 28 days of vaccination and vaccination, collected blood in 28 days, to estimate immunoreation and to collect laboratory safety index (full blood count, blood chemical analysis, hepatic and renal function test and urinalysis).Vaccine is not had unexpected untoward reaction by fine tolerance.Remarkable abnormal change does not take place in the laboratory index in research process.

In blood serum sample, measure serum group A, C, W135, Y specificity SBA and IgG (detecting) with ELISA.SBA tires and is expressed as the inverse of the final serum dilution of kill rate 〉=50% in the time of 60 minutes.Detect for IgG, measure high-affinity antibody with improved ELISA.For the detection of functional antibodies, used the SBA that two different external source complements sources are arranged: young rabbit complement source and people's complement source.

High-affinity IgG result following (average GMC (μ g/ml), 95% confidence interval):

Serum group	Serum	Vaccine group
					ACWY	A+CWY	Mencevax TM
		A	Before	0.67(0.3-1.2)				0.85(0.4-1.5)	0.45(0.2-0.8)
After	10(6.6-16)				14(8.8-22)	9.8(6.2-15)
			C	Before			0.21(0.1-0.3)	0.13(0.07-0.2)	0.16(0.1-0.2)
After	7.7(4.7-13)	5.2(3.19-8.46)			8.5(5.2-14)
				W135		Before	0.21(0.1-0.3)	0.2(0.1-0.3)	0.29(0.19-0.4)
After	12(6.5-21)	9(5.5-18)	6.7(3.7-12)
					Y	Before	0.35(0.2-0.5)	0.31(0.1-0.5)	0.57(0.3-0.9)

After

18(12-29)

21(13-33)

20(12-31)

SBA result's following (% responder and average GMT are 95% confidence interval):

Serum group	Vaccine group	Rabbit complement SBA		People's complement SBA
						(%) tires 〉=1: 128	GMT	(%) tires 〉=1: 4	GMT
		A	ACWY A+CWY Mencevax	93(78-99) 97(83-100) 100(88-100)	989(558-1754) 2566(1448-4549) 3132(1767-5552)					90(73-98) 97(83-100) 83(65-94)	42(23-76) 66(36-119) 28(15-50)
C	ACWY A+CWY Mencevax					97(82-100) 100(88-100) 93(78-99)	4480(2455-8176) 3794(2100-6855) 3829(2119-6918)	100(88-100) 100(88-100) 100(88-100)	213(106-427) 162(80-325) 223(111-448)
		W135	ACWY A+CWY Mencevax	100(88-100) 100(88-100) 100(88-100)	10343(5988-17865) 10376(6007-17923) 6795(3934-11737)					100(88-100) 93(78-99) 97(83-100)	248(123-500) 142(71-287) 99(49-199)
Y	ACWY A+CWY Mencevax					100(88-100) 100(88-100) 100(88-100)	22075(14689-33175) 24034(15993-36120) 14630(9735-21987)	100(88-100) 100(88-100) 100(88-100)	263(151-457) 162(194-588) 198(114-344)

For each serum group, in each vaccine group (ACWY, A+CWY and Mencevax contrast), high-affinity ELISA anti-pod membrane IgG GMC and with the SBA GMT of rabbit and the test determination of people's complement all raises after injection.After vaccine injection 29 days, to people's complement SBA of each serum group tire 〉=1: 4 object percent is 90%-100% in the conjugate vaccine group, be 83%-100% in matched group.With rabbit complement source, to the complement SBA of each serum group tire 〉=1: 128 object percent is 93%-100% in the conjugate vaccine group, be 90%-100% in matched group.

On the whole, the immunoreation in the conjugate group (GMC and GMT) is better than the Mencevax matched group.Especially in serum group W-135, see this improvement.Therefore, conjugate vaccines safety of the present invention, better tolerance, the reaction of inductive functional immunity with identical or better after the tetravalence polysaccharide vaccine immunity inoculation of ratifying.

3. use the MenA sugar of modifying

Purification obtains capsular polysaccharide and hydrolysis generation MenA oligosaccharide from MenA.Polysaccharide (2g) about 4 hours of 50 ℃ of hydrolysis in 50mM sodium acetate buffer pH4.75, polysaccharide concentration is 10mg/mL[73].After the hydrolysis, solution comes dry by rotary evaporation.

Oligosaccharide activates with following reaction process:

Oligosaccharide is dissolved in DMSO to produce the sugared concentration of 10mg/mL.Oligosaccharide according to 1: 20: the CDI mol ratio added 21.262g CDI and reactant mixture stirring at room 16 hours then.By at 80: 20 (v/v) acetone: selective precipitation in the DMSO mixture, then centrifugal purification gained MenA-CDI chemical compound.Calculate priming reaction efficient and be about 67.9% by determining free imidazoles and the ratio that combines imidazoles.

In the 2nd reactions steps, the MenA-CDI oligosaccharide is dissolved in DMSO, and sugared concentration is about 10mg/mL.MenA-CDI unit according to 1: 100: the DMA mol ratio, adding 36.288g 99% Dimethylammonium chloride (is R ¹And R ²=Me) and reactant mixture stirring at room 16 hours.The product lyophilization also is dissolved in the 10mg/mL aqueous solution again.

For from the oligosaccharide goods, removing low-molecular-weight reaction reagent (particularly dimethylamine (DMA)), through 3.5kDaMWCO film (Spectra/Por ^TM) step of dialysing.Finish 4 dialysis steps: (i) 2L 1M sodium chloride dialysis 16 hours (the dialysis factor 1: 20) relatively, (ii) 2L 0.5M sodium chloride dialysis 16 hours (the dialysis factor 1: 20) relatively is (iii) with (iv) relative 2L WFI dialysis 16 hours (the dialysis factor 1: 20).For improving purification, also through 1kDaMWCO film (Centricon ^TM) step of dialysing.

The MenA-CDI-DMA product of purification is at 25mM L-histidine (Fluka ^TM) in be buffered in pH6.5.

Be the MenA glycoconjugate (MenA-CDI-DMA) that preparation is modified, total method is as follows:

-Polysaccharides is to produce oligose fragment

The sizing of-oligose fragment

-reduction amination is according to the terminal aldehyde radical on the oligosaccharide of big minispread

-before the CDI reaction, pass through Fmoc radical protection end-NH ₂Group

-inside remove protection-NH at DMA between the reaction period ₂Group

-by SIDEA (N-hydroxy-succinamide adipic acid) activation end-NH ₂Group

-be covalently attached to CRM ₁₉₇Albumen

The MenA oligosaccharide conjugate of modifying is more many greatly than its natural homologue to the resistance of hydrolysis at elevated temperatures.For example, 37 ℃ after 28 days, the release sugar percentage ratio of modifying oligosaccharide is 6.4%, and by contrast, the release of native antigen sugar percentage ratio is 23.5%.In addition, modifying inductive the tiring of oligosaccharide significantly is not lower than with tiring that the natural sugar structure obtains.

The MenA conjugate of modifying substitutes the oligosaccharide conjugate of unmodified in conjunction with MenC, MenW135 and MenY conjugate.

Add MenB antigen

Before freeze dried MenA conjugate is rebuild as mentioned above, with MenB antigen Δ G287-953 (SEQID NO:7), 936-Δ G741 (SEQ ID NO:8) and NadA (SEQ ID NO:2) join in the maximum dose level liquid C-W135-Y mixture, are 20 μ g/ dosage with each final concentration that reaches this 3 peptide species.Therefore the vaccine of rebuilding contains following antigen:

Composition	The amount of every 0.5ml dosage
		Serum group A conjugate	10 μ g sugar+12.5-33 μ g CRM 197
Serum group C conjugate	10 μ g sugar+12.5-25 μ g CRM 197
		Serum group W135 conjugate	10 μ g sugar+6.6-20 μ g CRM 197
Serum group Y conjugate	10 μ g sugar+6.6-20 μ g CRM 197
		ΔG287-953	20 μ g polypeptide
936-ΔG741	20 μ g polypeptide
		NadA	20 μ g polypeptide

4. add Hib antigen

Freeze dried HbOC conjugate is mixed with freeze dried MenA conjugate, and one reinstates the C-W135-Y mixture rebuilds to produce following vaccine:

Composition	The amount of every 0.5ml dosage
		Serum group A conjugate	10 μ g sugar+12.5-33 μ g CRM 197
Serum group C conjugate	10 μ g sugar+12.5-25 μ g CRM 197

Serum group W135 conjugate	10 μ g sugar+6.6-20 μ g CRM 197
		Serum group Y conjugate	10 μ g sugar+6.6-20 μ g CRM 197
HbOC Hib conjugate	10 μ g sugar+2-5 μ g CRM 197

5. adding streptococcus pneumoniae antigen

Before freeze dried MenA conjugate is rebuild as mentioned above, streptococcus pneumoniae conjugate antigen is joined in the median dose liquid C-W135-Y mixture, be 2 μ g/ dosage (6B doubles for serotype) to reach each serotype final concentration.Therefore the vaccine of rebuilding contains following antigen:

Composition	The amount of every 0.5ml dosage
		Serum group A conjugate	5 μ g sugar+6.25-16.5 μ g CRM 197
Serum group C conjugate	5 μ g sugar+6.25-12.5 μ g CRM 197
		Serum group W135 conjugate	5 μ g sugar+3.3-10 μ g CRM 197
Serum group Y conjugate	5 μ g sugar+3.3-10 μ g CRM 197
		Streptococcus pneumoniae serotype 4 conjugates	2 μ g sugar+2.5 μ g CRM 197
Streptococcus pneumoniae serotype 9V conjugate	2 μ g sugar+2.5 μ g CRM 197
		Streptococcus pneumoniae serotype 14 conjugates	2 μ g sugar+2.5 μ g CRM 197
Streptococcus pneumoniae serotype 18C conjugate	2 μ g sugar+2.5 μ g CRM 197
		Streptococcus pneumoniae serotype 19F conjugate	2 μ g sugar+2.5 μ g CRM 197
Streptococcus pneumoniae serotype 23F conjugate	2 μ g sugar+2.5 μ g CRM 197
		Streptococcus pneumoniae serotype 6B conjugate	4 μ g sugar+5 μ g CRM 197

Should understand the present invention and only be described by embodiment, can make amendment and still scope of the present invention and the design in.

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Sequence table

SEQ ID NO:1-is from the NadA of bacterial strain 2996, and C-terminal lacks

MKHFPSKVLTTAILATFCSGALAATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDATAADVEADDF

KGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDATTNALNKLGENITTFAEETKTNIV

KIDEKLEAVADTVDKRAEAENDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAAD

KAEAVAAKVTDIKADIATNKDNIAKKANSADVYTREESDSKFVRIDGLNATTEKLDTRLASAEKSIADHDTRLNGLDKTVS

DLRKETRQGLAEQAALSGLFQPYNVG

SEQ ID NO:2-is from the NadA of bacterial strain 2996, and C-terminal lacks, and leader peptide is through processing

ATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNV

DAKVKAAESEIEKLTTKLADTDAALADTDAALDATTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDI

ADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKDNI

AKKANSADVYTREESDSKFVRIDGLNATTEKLDTRLASAEKSIADHDTRLNGLDKTVSDLRKETRQGLAEQAALSGLFQPY

NVG

SEQ ID NO:3-is from the Δ G741 of MC58 bacterial strain

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQL

ITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYT

IDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKPHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIG

LAAKQ

SEQ ID NO:4-is from 936 of MC58 bacterial strain, and leader peptide is through processing

VSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQ

IARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVST

TVGVQKVITLYQNYVQR

SEQ ID NO:5-is from 953 of MC58 bacterial strain, and leader peptide is through processing

ATYKVDEYHANARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPIANLQSGSQHFTDHLKSADIFDAAQYQDIR

FVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMEKTEVCGGDFSTTIDRTKWGMDYLVNVGMTKSVRIDIQ

IEAAKQ

SEQ ID NO:6-is from the Δ G287 of MC58 bacterial strain

SPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGSQDMAAVSEENTGNGGAVTADNPKNEDEVAQNDMPQN

AAGTDSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGGQNAGNTAAQGANQAGNNQAAGSSD

PIPASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCKGDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDKF

VGLVADSVQMKGINQYIIFYKPKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYL

TYGAEKLPGGSYALRVQGEPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKF

KAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQD

SEQ ID NO:7-287-953 heterozygosis

MASPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGGQDMAAVSEENTGNGGAAATDKPKNEDEGAQNDMP

QNAADTDSLTPNHTPASNMPAGNMENQAPDAGESEQPANQPDMANTADGMQGDDPSAGGENAGNTAAQGTNQAENNQTAGS

QNPASSTNPSATNSGGDFGRTNVGNSVVIDGPSQNITLTHCKGDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKND

GKNDKFVGLVADSVQMKGINQYIIEYKPKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPE

GNYRYLTYGAEKLPGGSYALRVQGEPSKGEMLAGTAVYNGEVLHFHTENGRPSPSRGRFAAKVDFGSKSVDGIIDSGDGLH

MGTQKFKAAIDGNGFKGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGSGGGGATYKVDEYHA

NARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTDHLKSADIFDAAQYPDIRFVSTKFNFNG

KKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTEVCGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDIQIEAAKQ*

SEQ ID NO:8-936-741 heterozygosis

MVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLLGQVATEGEKQFVG

QIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVS

TTVGVQKVITLYQNYVQRGSGGGGVAADIGAGLADALTAPDDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN

TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLP

EGGRATYRGTAEGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRRAVISGSVLYNQAEKGSYSLGI

FGGKAQEVAGSAEVKTVNGIRHIGLAAKQ ^*

SEQ ID NO:9-joint

GSGGGG

SEQ ID NO:10-is from 741 albumen of bacterial strain MC58

CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQ

IEVDGQLLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDLAGHTSFDKLPEGGRATYRGTAFGSDDA

GGKLTYTIDFAAKQGNGKIEHLKSPELBVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTV

NGIRBIGLAAKQ

SEQ ID NO:11-is from 741 albumen of bacterial strain 2996

CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGHGDSLNTGKLKNDKVSRFDFIRQ

IEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAG

GKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGE

KVHEIGIAGKQ

SEQ ID NO:12-is from 741 albumen of bacterial strain m1239

CSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKI

SRFDFVQKIEVDGDTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGK

AFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAG

SATVKIGEKVHEIGIAGKQ

Claims (25)

1. injectable immunogenic composition that contains capsular saccharides, described capsular saccharides is from Neisseria meningitidis serum group A, C, among W135 and the Y at least three kinds, it is characterized in that, described capsular saccharides and carrier protein are puted together and are oligosaccharide, and the every dosage of wherein said compositions contains the meningococcus sugar less than 50 μ g.

2. injectable immunogenic composition that contains capsular saccharides, described capsular saccharides is from Neisseria meningitidis serum group A, C, among W135 and the Y at least two kinds, it is characterized in that, described capsular saccharides and carrier protein are puted together and/or are oligosaccharide, and wherein said compositions also contains from following one or more antigen: (a) Neisseria meningitidis serum group B; (b) haemophilus influenzae type B; And/or (c) streptococcus pneumoniae.

3. compositions as claimed in claim 1 or 2 is characterized in that, said composition contains anti-Neisseria meningitidis serum group C, the antigen of W135 and Y.

4. compositions as claimed in claim 3 is characterized in that, said composition contains the antigen of anti-Neisseria meningitidis serum group A.

5. compositions as claimed in claim 4 is characterized in that, the antigen of described anti-Neisseria meningitidis serum group A is the serum group A capsular saccharides that one or more hydroxyls have been blocked the alternate modification of group.

6. as claim 3 or 4 described compositionss, it is characterized in that said composition also contains the antigen of influenza haemophilus Type B.

7. as the described compositions of the arbitrary claim of 3-5, it is characterized in that said composition also contains the antigen of one or more anti-Neisseria meningitidis serum group B.

8. compositions as claimed in claim 7, it is characterized in that, described one or more antigens can be induced the bactericidal properties antibody response in this object after giving object, at 2 in high toxicity pedigree A4, the ET-5 of Neisseria meningitidis serum group B and the pedigree 3 or a plurality of.

9. as claim 7 or 8 described compositionss, it is characterized in that described one or more antigens comprise following five kinds of antigens: ' NadA ' albumen of (1) oligomerization form; (2) ' 741 ' albumen; (3) ' 936 ' albumen; (4) ' 953 ' albumen; (5) ' 287 ' albumen.

10. compositions as claimed in claim 9, it is characterized in that, said composition also contains: comprise first polypeptide of aminoacid sequence SEQ ID NO:2, comprise second polypeptide and the 3rd polypeptide that comprises aminoacid sequence SEQ ID NO:8 of aminoacid sequence SEQ ID NO:7.

11., it is characterized in that said composition also contains one or more antigens of anti-streptococcus pneumoniae as the described compositions of the arbitrary claim of 3-6.

12. compositions as claimed in claim 11 is characterized in that, described one or more antigens contain the capsular saccharides from streptococcus pneumoniae.

13. compositions as claimed in claim 12 is characterized in that, said composition contains the capsular saccharides from streptococcus pneumoniae 5-11 kind different serotypes.

14. compositions as claimed in claim 11 is characterized in that, described one or more antigens contain the polypeptide from streptococcus pneumoniae.

15. compositions as claimed in claim 14 is characterized in that, said composition comprises one or more following streptococcus pneumoniae antigen: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp130.

16., it is characterized in that the carbohydrate antigen in the said composition is the oligosaccharide of puting together with carrier protein as the described compositions of above any claim.

17. compositions as claimed in claim 16 is characterized in that, the carbohydrate antigen in the said composition is and CRM ₁₉₇Albumen, the protein D of hemophilus influenza, the oligosaccharide that tetanus toxoid or diphtheria toxoid are puted together.

18., it is characterized in that described conjugate has the sugar between 1: 5 (being excessive protein) and 5: 1 (being excessive glucocorticoid): protein ratio (w/w) as claim 16 or 17 described compositionss.

19., it is characterized in that said composition contains and is less than the every dosage of 25 μ g meningococcuss sugar as the described compositions of above any claim.

20., it is characterized in that said composition also contains aluminum salt adjuvant as the described compositions of above any claim.

21. a test kit is characterized in that, this test kit comprises: (a) capsular saccharides, as the lyophilized form of the Neisseria meningitidis serum group A of the optional chemical modification of claim 5 explaination; (b) Neisseria meningitidis serum group C, the liquid form of the capsular saccharides of W135 and Y.

22. test kit as claimed in claim 21 is characterized in that, the lyophilizing capsular saccharides of described Neisseria meningitidis serum group A does not comprise adjuvant, Neisseria meningitidis serum group C, and the liquid capsular saccharides of W135 and Y comprises adjuvant.

23. one kind prepares as method for compositions as described in the arbitrary claim of 1-20, it is characterized in that, this method comprises lyophilizing capsular saccharides and the Neisseria meningitidis serum group C of the Neisseria meningitidis serum group A of the optional chemical modification of will explain as claim 5, and the capsular saccharides of the multiple liquid form of W135 and Y mixes mutually.

24. one kind causes immunoreactive method in the patient, it is characterized in that, this method comprises with the described compositions injection of the arbitrary claim of 1-20 patient.

25. (i) from Neisseria meningitidis serum group A, C, the pod membrane of W135 and Y put together oligosaccharide and (ii) from following one or more antigen: (a) Neisseria meningitidis serum group B; (b) haemophilus influenzae type B; And/or (c) streptococcus pneumoniae, prevent and/or treat application in the injectable drug of bacterial meningitis in manufacturing.