Combinations containing serotype 14 pneumococcal saccharide

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and concerns an adjuvant immunogenic composition containing meningococcal lipooligosaccharide (LOS) and serotype 14 pneumococcal capsular saccharide (CS14), wherein CS14 contains tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, while LOS is free from tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc. The group of inventions also concerns a method for inducing the immune response in a mammal involving administering the above composition.

EFFECT: group of inventions provides the stronger immune response to CS14 A as compared to wild-type OMV containing LNnT.

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The present application claims the priority of provisional patent application U.S. No. 61/162996 (filed March 24, 2009), the contents of which are fully incorporated herein by reference.

AREA of TECHNOLOGY

The present invention relates to the field of combined vaccines, in particular vaccines containing capsular saccharide of pneumococcus serotype 14 and lipooligosaccharides component (for example, fromNeisseria meningitis).

Background of the INVENTION

Streptococcus pneumoniaealso known as the pneumococcus , is a gram-positive spherical bacterium. Modern pneumococcal vaccines based on the capsular saccharides. Approved for use in pediatric vaccines represent (a) PREVNAR™, a 7-valent mixture of conjugated saccharides of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, (b) SYNFLORIX™ - desativando a mixture of conjugates, are also effective against serotypes 1, 5 and 7F, and (C) PREVNAR 13™ - 13-valent mixture of conjugates, are also effective against serotypes 3, 6A and 19A. Also known other 9-, 10-, 11 - and 13-valent combination conjugates.

Neisseria meningitisalso known as meningococcus, is a spherical gram-negative bacterium. Modern meningococcal vaccines based on the capsular saccharides. These vaccines include monovalent kouyate vaccine CE�of gruppe C (MENJUGATE™, MENINGITEC™ and NEISVAC-C™) and 4-valent mixture of conjugates against serogroups A, C, W135 and Y (MENACTRA™). There are currently no approved vaccines utility against serogroup B (MenB"). In the current research aimed at the creation of the MenB vaccine, used vesicles of the outer membrane (for example, MENZB™, HEXAMEN™, NONAMEN™) or purified components of the outer membrane, such as lipooligosaccharide and proteins of the outer membrane. Recently in the focus of attention were the MenB vaccine on the basis of the vesicles of the outer membrane, (OMV). So, for example, Novartis Vaccines, GlaxoSmithKline and RIVM/NVI there are products on the basis of the vesicles.

In reference 1 discloses a composition for immunization against pneumococcus and MenB obtained by combining the 13-valent pneumococcal conjugate vaccine ("13vPnC"; PREVNAR 13™ production Wyeth) with 9-valent vaccine on the basis of the outer membrane vesicles MenB (NONAMEN™ production NVI).

There is a need for new and improved combined vaccine to protect against meningococcus serogroup B, and pneumococcus.

Description of the INVENTION

The author has found the drawbacks of the combination vaccine according to the reference 1. The 13vPnC vaccine includes capsular saccharide of pneumococcus serotype 14 (CS14), and vesicles component MenB include lipooligosaccharide component (LOS, also denoted LPS) outer membrane of the bacteria. The author came to the conclusion that LOS(at least LOS of meningococcus immunotypes L2, L3, L4 and L7) and CS14 contain the same tetrasaccharide structure Galβ1-4GlcNAcβ1-3Galβ1-4Glc. This tetrasaccharide, known as lacto-N-neoteris (LNnT), is also present in humans, in particular in breast milk, as well as the terminal portion of the lacto-N-neteraser ceramide (also known as paragonate), which is the biosynthetic precursor of glycosphingolipids of blood group ABH and P1and some of gangliosides. Despite the fact that in human tissuesin vivoLNnT epitope normally closed terminal sialic acid residue (also present in the MenB, but not in CS14), in some cases (e.g. at low temperature) it becomes available for the immune system; the binding of an antibody to this epitope leads to hemolysis. This accessibility leads to autoimmune reactions is known as the disease cold agglutinins or AIHA (autoimmune hemolytic anemia).

The author came to the conclusion that the presence of LNnT structure as in LOS MenB and CS14 means that the introduction of any of these antigens, the patient may potentially lead to increased levels of antibodies Protva LNnT. With the introduction of these two antigens, there is a very strong risk of increasing the level of antibodies against LNnT, especially if the antigen is administered together with the adjuvant of the vaccine. Thus, the introduction of a combination vaccine comprising LOS MenB, and CS14, carries a high risk of induction of AIHA in patients, especially with the introduction of the vaccine in the winter. The invention aims to reduce the risk by limiting the immunogenic effect of LNnT epitope in the vaccine and presents various ways of reducing the risk of producing autoreactive antibodies with the introduction of the MenB antigens and pneumococcus.

In the first aspect destroy LNnT epitope consisting of the LOS, or CS14, or both. Thus, the invention presents immunogenic composition comprising lipooligosaccharide meningococcus (LOS) and capsular saccharide of pneumococcus serotype 14 (CS14), where LOS and/or CS14 does not include (do not include) tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc. Immunogenic composition usually also comprises an adjuvant. If the epitope is present in the composition of one of LOS and CS14, preferably, if it is missing in the composition of LOS.

In a second aspect, the LNnT epitope retain, in both LOS and CS14, but the vaccine does not contain adjuvant. Thus, in the invention presented unadjuvanted immunogenic composition comprising lipooligosaccharide meningococcus (LOS) and capsular saccharide of pneumococcus serotype 14 (CS14), where as LOS and CS14 include tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc. For example, can be used LOS of of meningococcus immunotype L2 or L3 (or their mixture).

In the third aspect LNnT epitope remain in the composition to�to LOS, and CS14, but their dosage is reduced. Thus, the invention presents immunogenic composition comprising lipooligosaccharide meningococcus (LOS) and capsular saccharide of pneumococcus serotype 14 (CS14), where (i) both LOS and CS14 include tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, (ii) the concentration of LOS less than 5 μg/ml, and (iii) the concentration CS14 less than 5 μg/ml.

In a fourth aspect of the vaccine against MenB and pneumococcus type 14 get using LNnT-containing LOS against MenB and protein antigen against pneumococcus. Thus, the present invention presents immunogenic composition comprising lipooligosaccharide meningococcus (LOS) and the polypeptide antigen of pneumococcus, where (i) includes LOS tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, (ii) the polypeptide of pneumococcus can cause an immune response effective against pneumococcus serotype 14, and (iii) the composition does not include the capsular saccharide of pneumococcus.

In the fifth aspect of the vaccine against MenB and pneumococcus type 14 get using the capsular saccharide CS14 containing LNnT, and antigen against MenB non LOS. Thus, the invention presents immunogenic composition comprising a polypeptide of the meningococcus and the capsular saccharide of pneumococcus serotype 14, where (i) the capsular saccharide includes tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, (ii) the polypeptide of meningococcus can cause immune resp�t, effective against meningococcus serogroup B, and (iii) the composition does not include lipooligosaccharide of meningococcus.

In some embodiments of the invention, the immunogenic compositions comprise no more than one seropostive the meningococcal PorA, for example, they may not include the outer membrane protein PorA.

Lipooligosaccharide of meningococcus

LOS meningococcus glucosamine is a phospholipid that is located in the outer layer of the outer membrane of bacteria. It is divided by the lipid A and the crust oligosaccharide, and lipid A is the hydrophobic anchor in the membrane. The heterogeneity of the oligosaccharide chains bark provides structural and antigenic diversity among different strains of meningococcus, based on which strains are divided into 12 immunotypes (L1-L12). Figure 1 shows the crust of saccharides immunotype L3. the α-Chain is associated with HepI(leptosol), contains tetrasaccharide LNnT and cap sialic acid. The same structure is observed in immunotypes L7 and L9. Immunotype L2 and L4 contain the same α-chain, as L3, but other β and/or γ-chain associated with HepII. The residue KDO1(2-keto-3-desoxycholate acid) attached to the lipid A LOS and also often associated with the second KDO residue (figure 1 designated as KDOII).

α-Chain L2 and L3 contain tetrasaccharide LNnT. When used in the invention LOS that do not contain tetrasaccharide LNnT, can use�to take another LOS immunotype (for example, L1, L4, L5, L6 or L8). However, in some embodiments it is desirable to keep the epitopes of L2 and/or L3 (different epitopes LNnT). This can be achieved by using mutant strains unable to synthesize tetrasaccharide LNnT in the composition of the α-chain. It is known that this result can be achieved by using the knockout of enzymes, carrying out appropriate connection in the course of biosynthesis (e.g., see references 2-7). For example, knockout of LgtB enzyme prevents the accession of terminal galactose LNnT, and further attaching the terminal sialic acid to the α-chain. Knockout of the enzyme LgtA prevents the accession of N-acetylglucosamine and LNnT all further accessions. Knockout LgtA may be accompanied by a knockout LgtC, if the strain contains a genelgtC(for example, strain MC58 of meningococcal serogroup In immunotype L3 consists oflgtA,lgtBandlgtEand does not containlgtCandlgtD). Similarly, knockout LgtE and/or enzyme GalE prevents the accession of internal galactose and knockout LgtF prevents glucose connection to the rest of HepI. Any of these knockouts can be used individually or in combination for the destruction of tetrasaccharide LNnT in the strain of immunotype L2, L3, L4, L7 or L9. Preferred knockout at least LgtB, as it turns out LOS, preserving useful immunogenicity, n� not comprising LNnT epitope.

In addition to mutations that Deplete the LNnT epitope, knockout of the genegalEalso leads to the formation of useful modified LOS, and similarly it is possible to knock out a gene transferase fatty acids of lipid A [8]. From LOS to cleave at least one primary O-linked fatty acid [9]. You can also use LOS c a smaller number of secondary acyl chains per molecule LOS [10]. Usually LOS includes at least the structure of GlcNac-Hep2-phosphoethanolamine-KDO2-lipid A [11]. LOS may include trisaharid GlcNAcβ1-3Galβ1-4Glc, but do not include tetrasaccharide LNnT.

The compositions according to the invention can include LOS in various forms. LOS can be used in purified form by itself. It can be conjugated to a carrier protein. It may be present in the composition of the outer membrane vesicles of meningococcus. It can be conjugated to the outer membrane vesicles of meningococcus.

The LOS conjugation can be done via the lipid A or via any other suitable group in its structure, for example, via KDO residues. Such alternative binding is required in the absence of lipid A in the composition of LOS. Techniques conjugation LOS known, for example, from references 9, 11, 12, 13, etc. Preferred carrier proteins for the conjugates are bacterial toxins, such as diphtheria or tetanus toxins, or toxoids, Il� mutants. They are usually used in conjugate vaccines. Suitable mutant CRM197diphtheria toxin [14]. Other suitable carrier proteins include the protein complex of the outer membrane ofN. meningitides[15], synthetic peptides [16, 17], heat shock proteins [18, 19], pertussis proteins [20, 21], cytokines [22], lymphokines [22], hormones [22], growth factors [22], artificial proteins containing multiple epitopes of CD4+ T-cells from different antigens of pathogenic origin [23] such as N19 [24], protein DH. influenzae[25-27], pneumolysin [28] or its non-toxic derivatives [29], the surface protein of pneumococcal PspA [30], protein absorption of iron [31], toxin A or bC. difficile[32], recombinant absoprtion A (rEPA)Pseudomonas aeruginosa[33] etc.

LOS can be in the composition of the vesicles. Such vesicles include any proteoliposome vesicles obtained by breaking or blabbing the outer membrane of meningococci with the formation of vesicles comprising protein components and the LOS outer membrane. Thus, the term includes OMV (sometimes called "bleble"), microvesicles ("MV", [34]) and "native OMV" ("NOMV", [35]).

MV and NOMV are natural membrane vesicles spontaneously formed during the growth of bacteria and released into the culture medium. MV can be obtained by cultivation ofNeisseriain a liquid nutrient medium, separating TSE�'s cells from smaller MV in a liquid medium (for example, by filtering or centrifugation at low speed for the deposition of only cells, but not the smaller vesicles) and the further collection of MV from the medium without cells (e.g., using filtration, differential precipitation or aggregation MV, using centrifugation at high speed for deposition MV). The strains used to obtain MV, can in General be chosen depending on the number of MV produced in culture, for example, in references 36 and 37 describedNeisseriaproducing a large number of MV.

OMV derived artificially from bacteria, can be obtained with the use of a detergent (e.g., deoxycholate) or without (see, for example, reference 38). Technology education OMV include treatment bacterial detergent on the basis of bile salts (e.g., salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, desoxycholic acid, halyevoy acid, euroholiday acid, etc.; for the treatment ofNeisseriapreferred desoxycholate sodium [39 and 40] when the pH is high enough, in order for the detergent not to precipitate [41]. Other techniques can be applied practically in the absence of detergent [38] with the use of such techniques as Sonicare, homogenization, microfluidizer, cavitation, osmotic shock, grinding, French press, mixing, etc. is�olshanii ways without detergent or with a low concentration of detergent may remain useful antigens, such as the NspA [38]. Thus, the method can be used buffer for extraction with OMV 0.5% desoxycholate or less, for example about 0.2%, about 0.1%, < 0.05% or 0.

Useful process of obtaining OMV described in reference 42, includes ultrafiltration of crude OMV, instead of centrifugation at high speed. The process may include the stage after ultracentrifugation ultrafiltration.

Vesicles for use in the present invention can be obtained from any strain of meningococcus. Typically, vesicles prepared from a strain of serogroup B, however, you might get them and from serogroups than B (for example, the link 41 is described a process for serogroup A), such as A, C, W135 or y Strain may belong to any serotype (e.g. 1, 2A, 2b, 4, 14, 15, 16, etc.), any xeropotamou and any immunotype (e.g. L1; L2; L3; L3,3,7; L10, etc.). Meningococci can be any suitable line, including hyperinvasive and hypervirulent, for example, to one of the following seven hypervirulent lines: subgroup I; subgroup III; subgroup IV-1; the compound ET-5; complex ET-37; cluster A4; line 3. These lines are determined using multilocus enzyme electrophoresis (MLEE), but also for the classification of meningococci can be used multilocus sequence typing (MLST) [43], for example, the complex ET-37 is a fun� complex ST-11 according to MLST, complex ET-5 is a complex ST-32 (ET-5), line 3 represents the ST-41/44, etc. Vesicles can be obtained from strains of one of the following subtypes: P1.2; P1.2,5; P1.4; P1.5; P1.5,2; P1.5,c; P1.5c,10; P1.made 7.16 interest; P1.made 7.16 interest b; P1.7h,4; P1.9; P1.15; P1.9,15; P1.12,13; P1.13; P1.14; P1.21,16; P1.22,14.

Vesicles for use in the present invention can be obtained from strains of meningococcus wild-type or mutant strains of meningococcus. For example, in the link 44 describes the preparation of vesicles fromN. meningitidiswith a modified genome offur. In the link 51 is shown simultaneously with a knockoutporAandcpsthe expression ofnspAneeds to be improved. Additional knock-out mutants ofN. meningitidisto obtain OMV disclosed in references 3, 51 and 52. In the link 45 disclosed vesicles with increased fHBP. In the link 46 is disclosed the construction of vesicles from strains, modified to Express different PorA subtypes. In the present invention can be used these or other mutants.

Thus, the strain used in the present invention, in some embodiments may Express more than one PorA subtype. Were previously constructed 6-valent and 9-valent strains against PorA. Strain can Express the 2, 3, 4, 5, 6, 7, 8 or 9 of PorA subtypes: P1.made 7.16 interest; P1.5-1,2-2; P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1 and/or P1.18-1,3,6. However, in other embodiments can be used a strain with reduced expression of PorA, e.g.�measures in which the amount of PorA reduced by at least 20% (e.g., ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95% etc.) relative to the wild-type level (e.g. relative to strain N/76, or PorA can be knocked out, as described in reference 51).

Preferably, it has been shown that vesicles derived from a strain of meningococcus LgtB-veincrease the answer to CS14 in the combined drug.

LOS can be derived from a strain (for example, genetically engineered strain of meningococcus), having a constant (i.e., no phase variation) immunochip LOS, as described in reference 47. For example, the constant can be immunotype L2 and L3 LOS. Such strains can change your immunochip more than 2 times less likely (even 50 times) than the original wild type strain. In the link 47 is disclosed how to obtain this result by modifying the lgtA gene products and/or lgtC.

In some embodiments the strain can be observed increased expression of certain proteins (relative to the corresponding wild-type strain). For example, strain can be observed increased expression of NspA, protein 287 [48], fHBP [45], TbpA and/or TbpB [49], Cu,Zn-dependent superoxide dismutase [49], etc.

In some embodiments, the strain may contain one or more mutations that lead to a knockout or overexpress described in references 3 and 50-52. Genes suitable for down-regulation and/or �La knockout include: (a) Cps, CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc,PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB [50]; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB [51]; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB [52]; and (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB and/or SynC [3].

When using a mutant strain in some embodiments it may have one or more or all of the following characteristics, in addition to optional mutations that Deplete LNnT: (i) increased TbpA; (ii) increased NhhA; (iii) increased Omp85; (iv) increased LbpA; (v) increased the NspA; (vi) knocked out PorA; (vii) reduced or knocked out FrpB; (viii) reduced or knocked out Opa; (ix) reduced or knocked out Opc; (x) deleteriously complex genescpx.

Preferably, if the LOS in the composition of the vesicles can be processed in a certain way for the purpose of binding LOS and protein components in the vesicle ("vnutripuzyrnoy" conjugation [3]).

LOS can be About-acetiminophen on the GlcNAc residue attached to the remainder heptose II, for example, for L3 [53].

The immunogenic composition may include more than one type LOS, for example, the LOS of meningococci immunotypes L2 and L3. For example, can be used in combination LOS described in reference 54.

In some embodiments LOS may be present in the composition at a concentration less than 5 ág/m�, for example, ≤4 µg/ml, ≤3 µg/ml, ≤2 μg/ml, ≤1 µg/ml. Such a low concentration can be used if CS14 remains LNnT epitope.

The LOS antigen may preferably to induce bactericidal antibodies against meningococci after administration to a subject.

Capsular saccharide of pneumococcus serotype 14

The compositions according to the invention comprise capsular saccharide of pneumococcus serotype 14 (CS14). In the link 55 is shown CS14 contains a repeating structure shown in figure 2.

Saccharide CS14 used in the invention typically comprises recurring units of wild type, but in some embodiments can be modified to exclude tetrasaccharide Galβ-4GlcNAcβ1-3Galβ1-4Glc. Such modification can be performed by a knockout of one or more important enzymes in the biosynthesis or chemical and/or enzymatic effects on saccharide for modification of one or more of the four residues in the composition of tetrasaccharide. For example, endo-β-galactosidase, purified from culture supernatants ofCytophaga keratolyticathat catalyzes the hydrolysis of linkages galactose is β(1→4) glucose sensitive polysaccharides, including CS14.

CS14 may be N-acetiminophen. As described in reference 56, for example, it can be N-acetiminophen more than 50%, 60%, 70%, 80% or 90%.

Usually CS14 include in the composition is in the form of a conjugate. Fittingly�e carrier proteins for the conjugates described above, for example, bacterial toxins such as diphtheria or tetanus toxins, or toxoids or mutants, such as CRM197the protein complex of the outer membrane ofN. meningitidis, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple epitopes of CD4+T-cells of different antigens of pathogenic origin, such as N19 protein (DH. influenzaepneumolysin or its non-toxic derivatives, surface protein a pneumococcal PspA, iron digestion proteins, toxin A or bC. difficile, rEPA etc. are Particularly suitable in the role of carrier proteins CS14: CRM197, toxoid tetanus, toxoid diphtheria and protein DH. influenzae. Especially suitable CRM197, as shown in PREVNAR™.

The carrier molecule may be covalently konjugierten to CS14 directly or via a linker. There are various linkers, such as the linker-based adipic acid, which may be formed by mating free group-NH2(for example, introduced into the saccharide by amination) with adipic acid (for example, using diimide activation) and future pairing of the protein with the obtained intermediate product of saccharide-adipic acid [57, 58]. Another preferred type of communication is a carbonyl linker, which may be the way�adhering by reaction of the free hydroxyl group of the modified saccharide with CDI [59, 60], followed by reaction with the protein with the formation of carbamate linkages. Other linkers include β-propionamido [61], nitrophenyl-ethylamine [62], haloacyl of halide [63], glycosidic linkages [64], 6-aminocaproic acid [65], N-Succinimidyl-3-(2-pyridylthio)-propionate (SPDP) [66], adipic acid dihydrazide ADH [67], groups With4-C12[68] etc. can Also be used carbodiimide condensation [69].

Can be used conjugation CS14 by reductive amination. The first saccharide can be oxidized with periodate to introduce aldehyde groups, which then can form a direct covalent bond with a carrier protein by reductive amination, for example, with ε-amino group of lysine. If the saccharide comprises multiple aldehyde groups in one molecule, such a technique of binding can lead to the formation of the product with the cross-linkers, where multiple aldehydes react with multiple amines media.

Saccharide CS14 may contain a full-sized intact saccharide derived from pneumococcus, and/or may contain fragments of the full-sized saccharides, i.e. shorter saccharides than the native capsular saccharides seen bacteria. Thus, the saccharides can be depolymerizer, and depolymerization can occur during or after cleaning Sach�reed, but before conjugation. Depolymerization reduces the length of a chain of sugars. The depolymerization can be used to achieve the chain length, for optimal immunogenicity, and/or to reduce the length of the chain for the physical handling of sugars. As can be seen from PREVNAR™, preferred intact CS14.

In some embodiments CS14 is present in the composition in a concentration of less than 5 μg/ml, e.g., ≤4 µg/ml, ≤3 µg/ml, ≤2 μg/ml, ≤ 1mkg/ml. Such a low concentration can be used if CS14 remains LNnT epitope. A convenient concentration of about 4 μg/ml.

Antigen CS14 may preferably to induce anticapsular antibody that binds to CS14, for example to induce the level of antibodies against CS14 ≥0.20 mg/ml [70]. The level of antibodies can be measured using an enzyme immunoassay (EIA) and/or opsonophagocytic activity (OPA). The effectiveness of the EIA method has been repeatedly confirmed, and there is a relationship between the concentration of antibodies and vaccine efficacy.

Alternative meningococcal antigens

In some embodiments of the present invention, the composition comprises capsular saccharide CS14 containing LNnT, and the antigen against MenB non LOS. Alternatives include LOS polypetide antigens, such as fHBP, 287, NadA, NspA, HmbR, NhhA, App, and/or Omp85. Useful if these antigens are present in the form of treated polypeptid�s, for example, recombinant polypeptides.

Meningococcal antigen may preferably to induce bactericidal antibodies against meningococci after administration to a subject.

Factor fHBP (sugar-binding factor (H)

The composition according to the invention may contain an antigen fHBP. The fHBP antigen is described in detail. It is also known as protein "741" [SEQ ID NO:2535 and 2536 in the link 81], "NMB1870", "GNA1870" [link 71-73], "P2086", "LP2086" or "ORF2086" [74-76]. It is a natural lipoprotein and is expressed by meningococcal disease all serogroups. The structure of the C-terminal immunodominant domain (fHbpC) was determined using NMR [77]. This part of the protein forms an 8-stranded β-barrel, the threads which are connected by loops of various lengths. The barrel is preceded by a short α-helix and a flexible N-terminal tail.

There are three different variants of fHBP antigen [78], and it is known that the serum of a specific family has antibacterial properties against members of this family, but is not active against strains expressing one of two other families, i.e. there is cross-protection within the family, but not between families. In the present invention can have a single fHBP variant, but it is useful to include a fHBP from two or three options.

If the invention uses a single fHBP variant, the composition may VK�ucati polypeptide, containing: (a) amino acid sequence identical to at least a% SEQ ID NO:1, and/or containing the amino acid sequence consisting of a fragment of at least x consecutive amino acids from SEQ ID NO:1; or (b) amino acid sequence identical to at least b% SEQ ID NO:2, and/or containing the amino acid sequence consisting of a fragment of at least y consecutive amino acids from SEQ ID NO:2; or (c) amino acid sequence, identical at least with% SEQ ID NO:3 and/or containing the amino acid sequence consisting of a fragment of at least z consecutive amino acids from SEQ ID NO:3.

If the invention is used fHBP from two or three options, the composition may include a combination of two or three different fHBP selected from: (a) a first polypeptide containing an amino acid sequence identical to at least a% SEQ ID NO:1, and/or containing the amino acid sequence consisting of a fragment of at least x consecutive amino acids from SEQ ID NO:1; (b) a second polypeptide containing an amino acid sequence identical to at least b% SEQ ID NO:2, and/or containing the amino acid sequence consisting of a fragment of at least y consecutive amino acids from SEQ ID NO:2;and/or (c) a third polypeptide, containing the amino acid sequence identical to at least% SEQ ID NO:3 and/or containing the amino acid sequence consisting of a fragment of at least z consecutive amino acids from SEQ ID NO:3.

If the invention is used fHBP from two options, the composition may include: (a) a first polypeptide containing an amino acid sequence identical to at least a% SEQ ID NO:1, and/or containing the amino acid sequence consisting of a fragment of at least x consecutive amino acids from SEQ ID NO:1; and (b) a second polypeptide containing an amino acid sequence identical to at least b% SEQ ID NO:2, and/or containing the amino acid sequence, consisting of a fragment of at least y consecutive amino acids from SEQ ID NO:2. The first and second polypeptides have different amino acid sequence.

If the invention is used fHBP from two options, the composition may include: (a) a first polypeptide containing an amino acid sequence identical to at least a% SEQ ID NO:1, and/or containing the amino acid sequence consisting of a fragment of at least x consecutive amino acids from SEQ ID NO:1; and (b) a second polypeptide containing the amino acid sequence, identico� at least with% SEQ ID NO:3, and/or containing the amino acid sequence consisting of a fragment of at least z consecutive amino acids from SEQ ID NO:3. The first and second polypeptides have different amino acid sequence.

The parameter a is equal to at least 85, for example, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99,5 or more. The parameter b is equal to at least 85, for example, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99,5 or more. The parameter c is equal to at least 85, for example, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99,5 or more. The values a, b and C do not depend on each other.

The parameter x is equal to at least 7, 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 parameter y is equal to at least 7, 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 parameter z is equal to at least 7, 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 values x, y, and z do not depend on each other. Fragments of SEQ ID NO:1, 2 and 3 preferably contain the epitope corresponding to SEQ ID.

A suitable composition may include a polypeptide containing an amino acid sequence identical to at least 90% to SEQ ID NO:3 (e.g., at least 93%), and/or containing the amino acid sequence consisting of a fragment of at more consecutive amino acids from SEQ ID NO:3.

In some embodiments the polypeptide (or polypeptides) fHBP can be to join the lipids, for example, via N-terminal cysteine, usually with the formation of dipalmitoyl-S-glacierization. In other embodiments, the lipids can join.

Introduction fHBP preferably induces antibodies capable of contacting the polypeptide of meningococcus consisting of the amino acid sequence SEQ ID NO:1, 2 or 3. Preferred fHBP antigens for use in the present invention can induce bactericidal antibodies against meningococci after administration to a subject.

Regardless of whether one or more fHBP polypeptides, the total dose fHBP may lie in the range of 60 μg per dose to 200 μg per dose.

287

The composition according to the invention may include an antigen 287. Antigen 287 are included in the published genome sequence of a strain of meningococcus MC58 serogroup In [79] as gene NMB2132 (access number in GenBank GI:7227388; SEQ ID NO:9 herein). To date, the published sequence of the antigen 287 of many strains. For example, allelic forms 287 shown in figures 5 and 15 in the link 80 in example 13 and figure 21 in the link 81 (SEQ ID NO:3179-3184 in it). We also discussed various immunogenic fragments of the antigen 287.

Preferred 287 antigens for use in the present Fig�thenia contain amino acid sequence: (a) identical 50% or more (e.g., on 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more) to SEQ ID NO:9; and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:9, where 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). Preferred fragments of (b) contain the epitope of SEQ ID NO:9.

Most suitable 287 antigens according to the invention can induce antibodies, which after administration to a subject can be contacted with a polypeptide of meningococcus consisting of the amino acid sequence SEQ ID NO:9. Preferred 287 antigens for use in the present invention can induce bactericidal antibodies against meningococci after administration to a subject.

NadA (adhesin And Neisseria)

The composition according to the invention may include a NadA antigen. NadA are included in the published genome sequence of a strain of meningococcus MC58 serogroup In [79] as gene NMB1994 (access number in GenBank GI:7227256; SEQ ID NO:10 herein). To date, the published sequence of the antigen NadA from many strains and described in detail the activity of the protein as Neisseria adhesin. We also discussed various immunogenic fragments NadA.

Preferred NadA antigens for use in the present invention contain amino acid sequence: (a) identical 50% or more (on�reamer, on 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more) to SEQ ID NO:10; and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:10, where 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). Preferred fragments of (b) contain the epitope of SEQ ID NO:10.

Most suitable NadA antigens according to the invention can induce antibodies, which after administration to a subject can be contacted with a polypeptide of meningococcus consisting of the amino acid sequence SEQ ID NO:10. Preferred NadA antigens for use in the present invention can induce bactericidal antibodies against meningococci after administration to a subject. SEQ ID NO:6 represents one such fragment.

NspA (surface protein And Neisseria)

The composition according to the invention may include the NspA antigen. NspA are included in the published genome sequence of a strain of meningococcus MC58 serogroup In [79] as gene NMB0663 (access number in GenBank GI:7225888; SEQ ID NO:11 herein). The antigen has previously been described in the links 82 and 83. To date, the published sequence of NspA antigen from many strains. We also discussed various immunogenic fragments of the NspA.

Preferred NspA antigens for use in the present invention contain amino acid sequence�lnost: (a) identical 50% or more (e.g., on 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more) to SEQ ID NO:11; and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:11, where 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). Preferred fragments of (b) contain the epitope of SEQ ID NO:11.

Most suitable NspA antigens according to the invention can induce antibodies, which after administration to a subject can be contacted with a polypeptide of meningococcus consisting of the amino acid sequence SEQ ID NO:11. Preferred NspA antigens for use in the present invention can induce bactericidal antibodies against meningococci after administration to a subject.

HmbR

The compositions according to the invention can include a HmbR antigen of meningococcus. The sequence of the full-HmbR are included in the published genome sequence of a strain of meningococcus MC58 serogroup In [79] as gene NMB1668 (SEQ ID NO:7 herein). In the link 84 is given a HmbR sequence from another strain (SEQ ID NO:8 herein). SEQ ID NO:7 and SEQ ID NO:8 differ under the length of 1 amino acid and is identical to 94.2 percent.

The invention can be used in the polypeptide corresponding to the full-size HmbR sequence, however, can often be used a polypeptide corresponding to partially� HmbR sequence. Thus, in some embodiments a HmbR sequence used according to the invention may include amino acid sequence identical to at least about i% SEQ ID NO:7, where i is equal 50, 60, 70, 80, 90, 95, 99 or more. In other embodiments a HmbR sequence used according to the invention may contain a fragment of at least j consecutive amino acids from SEQ ID NO:7, where j is equal to 7, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more. In other embodiments a HmbR sequence used according to the invention may include amino acid sequence, (i) is identical to at least about i% SEQ ID NO:7 and/or (ii) containing a fragment of at least j consecutive amino acids from SEQ ID NO:7.

Preferred fragments from j amino acids contain an epitope from SEQ ID NO:7. Such epitopes generally contain amino acids located on the surface of HmbR. Useful epitopes include epitopes with amino acids involved in binding HmbR with hemoglobin as antibodies, binding to these epitopes, can inhibit the binding of bacteria to host hemoglobin. Topology HmbR and its key functional residues were investigated in reference 85.

Most useful HmbR antigens according to the invention can induce antibodies, which after administration to a subject can svjazyvaites� polypeptide with meningococcus, consisting of the amino acid sequence SEQ ID NO:7. Preferred HmbR antigens for use in the invention can induce bactericidal antibodies against meningococci after administration to a subject.

Unlike links 31, HmbR antigens according to the invention is usually not kongugiruut with capsular sharedmem antigen.

NhhA (homologue of Neisseria hia)

The composition according to the invention may include an antigen NhhA. Antigen NhhA are included in the published genome sequence of a strain of meningococcus MC58 serogroup In [79] as gene NMB0992 (access number in GenBank GI:7226232; SEQ ID NO:12 herein). To date, the published sequence of the antigen NhhA from many of the strains, see, for example, the links 80 and 86, and also highlighted various immunogenic fragments of NhhA. It is also known as Hsf.

Preferred antigens NhhA for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:12 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:12, where 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). Preferred fragments of (b) contain the epitope of SEQ ID NO:12.

Most useful antigens NhhA according to Fig�the plants can induce antibodies, which after administration to a subject can be contacted with a polypeptide of meningococcus consisting of the amino acid sequence SEQ ID NO:12. Preferred antigens NhhA for use in the invention can induce bactericidal antibodies against meningococci after administration to a subject.

App (protein adhesion and penetration)

The composition according to the invention may include an antigen App. Antigen App are included in the published genome sequence of a strain of meningococcus MC58 serogroup In [79] as gene NMB1985 (access number in GenBank GI:7227246; SEQ ID NO:13 herein). To date, the published sequence of the antigen App from many strains. We also discussed various immunogenic fragments of App.

Preferred antigens App for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:13 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:13, where 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). Preferred fragments of (b) contain the epitope of SEQ ID NO:13.

Most useful antigens App according to the invention can induce antibodies, which after administration to a subject can communi�to yatsa polypeptide with meningococcus, consisting of the amino acid sequence SEQ ID NO:13. Preferred antigens App for use in the invention can induce bactericidal antibodies against meningococci after administration to a subject.

Omp85 (the outer membrane protein of 85 kDa)

The composition according to the invention may include an Omp85 antigen. The Omp85 antigen included in the published genome sequence of a strain of meningococcus MC58 serogroup In [79] as gene NMB0182 (access number in GenBank GI:7225401; SEQ ID NO:14 herein). To date, the published sequences of the Omp85 antigen from many strains. Further information about Omp85 can be obtained from the links 87 and 88. We also discussed various immunogenic fragments of Omp85.

Preferred Omp85 antigens for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:14 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:14, where 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). Preferred fragments of (b) contain the epitope of SEQ ID NO:14.

Most useful Omp85 antigens according to the invention can induce antibodies, which after administration to the subject can contact the polyp�pcidom meningococcus, consisting of the amino acid sequence SEQ ID NO:14. Preferred Omp85 antigens for use in the invention can induce bactericidal antibodies against meningococci after administration to a subject.

Alternative antigens of pneumococcus

Instead CS14 in the composition in some embodiments, the vaccine includes LOS containing LNnT, for protection against MenB, but includes protein antigen against pneumococcus. Peptide pneumococcus can induce an immune response effective against pneumococcus serotype 14.

The composition may include one or more of: (1) antigen spr0057; (2) antigen spr0286; (3) antigen spr0565; (4) the antigen spr1098; (5) antigen spr1345; (6) the antigen spr1416; (7) antigen spr1418; (8) the antigen spr0867; (9) the antigen spr1431; (10) the antigen spr1739; (11) the antigen spr2021; (12) the antigen spr0096; (13) the antigen spr1433; and/or (14) antigen spr1707.

The composition may include one or more of: (1) PspA polypeptide; (2) the PsaA polypeptide; (3) the PspC polypeptide; (4) a polypeptide LytA; (5) a polypeptide PhtA; (6) a polypeptide PhtA; (7) a polypeptide PhtA; and/or (8) PhtD polypeptide.

The composition may include a subunit of fimbria pneumococcus, such as RrgA, RrgB and/or RrgC.

The polypeptide antigen of pneumococcus may be preferable to induce protective antibodies after administration to a subject.

spr0057

The original sequence "spr0057" was given in the link 89 as "the Precursor of beta-N-acety�-hexosaminidase" (see GI:15902101). For ease of reference, the amino acid sequence of the full-size spr0057 strain R6 provided herein as SEQ ID NO:18.

Preferred polypeptides spr0057 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:18 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:18, where 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). These proteins spr0057 include variants of SEQ ID NO:18. Preferred fragments of (b) contain the epitope of SEQ ID NO:18. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:18, but contain at least one epitope of SEQ ID NO:18. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:32, does not contain the natural sequence of leader peptide and the sequence recognized by sortases.

spr0286

The original sequence "spr0286" was given in the link 89 as the "Predecessor hyaluronate lyase (see GI:15902330). For ease of reference, the amino acid� sequence of the full-sized spr0286 strain R6 provided herein as SEQ ID NO:19.

Preferred polypeptides spr0286 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:19 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:19, where 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). These proteins spr0286 include variants of SEQ ID NO:19. Preferred fragments of (b) contain the epitope of SEQ ID NO:19. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:19, but contain at least one epitope of SEQ ID NO:19. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:33, does not contain the natural sequence of leader peptide and the sequence recognized by sortases. Other suitable fragments are shown in SEQ ID NO:34 and 35.

spr0565

The original sequence "spr0565" was given in the link 89 as "the Precursor of beta-galactosidase (see GI:15902609). For ease of reference, the amino acid sequence of the full-size spr0565 strain R6 contained in this document ka� SEQ ID NO:20.

Preferred polypeptides spr0565 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:20 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:20, where 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). These proteins spr0565 include variants of SEQ ID NO:20 (e.g., SEQ ID NO:66; see below). Preferred fragments of (b) contain the epitope of SEQ ID NO:20. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:20, but contain at least one epitope of SEQ ID NO:20. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:36, does not contain the natural sequence of leader peptide and the sequence recognized by sortases. Other suitable fragments are shown in SEQ ID NO:37 and 38.

Variant form spr0565 shown in SEQ ID NO:39 of this document. The use of this form for immunization described in reference 90 (SEQ ID NO:178). Suitable polypeptides spr0565 may contain amino acid sequence: (a) identification�ing 50% or more of SEQ ID NO:39 (for example, on 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:39, where 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). These polypeptides include variants of SEQ ID NO:39 (for example, SEQ ID NO:66, see below). Preferred fragments of (b) contain the epitope of SEQ ID NO:39. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:39, but contain at least one epitope of SEQ ID NO:39. Other fragments contain one or more domains of the protein.

Immunogenic fragments of SEQ ID NO:39 are listed in table 1 of the link 90.

Because spr0565 is a natural long polypeptide (>2000 amino acids), expression of fragments may be more convenient. So suitable form spr0565 for use in the present invention may be shorter than 1500 amino acids (for example, <1400, <1300, <1200, <1100, etc.). Such short forms spr0565 include "spr0565A" (SEQ ID NO:37) and "spr0565B" (SEQ ID NO:38).

spr1098

The original sequence "spr1098" was given in the link 89 as "sortase' (see GI:15903141). For ease of reference, the amino acid sequence of the full-size spr1098 strain� R6 provided herein as SEQ ID NO:21.

Preferred polypeptides spr1098 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:21 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:21, where 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). These proteins spr1098 include variants of SEQ ID NO:21. Preferred fragments of (b) contain the epitope of SEQ ID NO:21. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:21, but contain at least one epitope of SEQ ID NO:21. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:40, does not contain the natural sequence of leader peptide.

spr1345

The original sequence "spr1345" was given in the link 89 as 'hypothetical protein' (see GI:15903388). For ease of reference, the amino acid sequence of the full-size spr1345 strain R6 provided herein as SEQ ID NO:22.

Preferred polypeptides spr1345 for use in the present invention contain the amino acid�ing sequence: (a) identical 50% or more of SEQ ID NO:22 (e.g., on 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:22, where 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). These proteins spr1345 include variants of SEQ ID NO:22. Preferred fragments of (b) contain the epitope of SEQ ID NO:22. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:22, but contain at least one epitope of SEQ ID NO:22. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:41, does not contain the natural sequence of leader peptide and the sequence recognized by sortases.

spr1416

The original sequence "spr1416" was given in the link 89 as 'hypothetical protein' (see GI:15903459). For ease of reference, the amino acid sequence of the full-size spr1416 strain R6 provided herein as SEQ ID NO:23.

Preferred polypeptides spr1416 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:23 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or �ver); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:23, where 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). These proteins spr1416 include variants of SEQ ID NO:23. Preferred fragments of (b) contain the epitope of SEQ ID NO:23. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:23, but contain at least one epitope of SEQ ID NO:23. Other fragments contain one or more domains of the protein.

spr1418

The original sequence "spr1418" was given in the link 89 as 'hypothetical protein' (see GI:15903461). For ease of reference, the amino acid sequence of the full-size spr1418 strain R6 provided herein as SEQ ID NO:24.

Preferred polypeptides spr1418 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:24 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:24, where 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). These proteins spr1418 options include EQ ID NO:24. Preferred fragments of (b) contain the epitope of SEQ ID NO:24. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:24, but contain at least one epitope of SEQ ID NO:24. Other fragments contain one or more domains of the protein.

spr0867

The original sequence "spr0867" was given in the link 89 as "endo-beta-N-acetylglucosaminidase' (see GI:15902911). For ease of reference, the amino acid sequence of the full-size spr0867 strain R6 provided herein as SEQ ID NO:25.

Preferred polypeptides spr0867 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:25 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:25, where 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). These proteins spr0867 include variants of SEQ ID NO:25. Preferred fragments of (b) contain the epitope of SEQ ID NO:25. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or Bo�its amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:25, but contain at least one epitope of SEQ ID NO:25. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:42, contains a natural sequence leader peptide.

spr1431

The original sequence "spr1431" was given in the link 89 as "1,4-beta-N-acetylmuramic' (see GI:15903474). It is also known as "LytC", and its use for immunization described in reference 104. For ease of reference, the amino acid sequence of the full-size spr1431 strain R6 provided herein as SEQ ID NO:26.

Preferred polypeptides spr1431 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:26 (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:26, where 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). These proteins spr1431 include variants of SEQ ID NO:26. Preferred fragments of (b) contain the epitope of SEQ ID NO:26. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:26, but contain at least one epitope of SEQ ID NO:26. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:43, does not contain the natural sequence of leader peptide.

spr1739

Polypeptide "spr1739" is pneumolysin (see, e.g., GI:15903781). For ease of reference, the amino acid sequence of the full-size spr1739 strain R6 provided herein as SEQ ID NO:27.

Preferred polypeptides spr1739 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:27 (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:27, where 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). These proteins spr1739 include variants of SEQ ID NO:27. Preferred fragments of (b) contain the epitope of SEQ ID NO:27. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:27, but contain at least one epitope of SEQ ID NO:27. Other fragments contain one or more home�new protein.

The prior art discloses a mutant form pneumolysin used for vaccination [29, 91-96], and such mutant forms can be used in the present invention. Detoxification can be achieved by shortening of the C-end (see, e.g., reference 97), for example, by deletion of 34 amino acids, 45 amino acids, 7 amino acids [98], etc. Further mutations are numbered according to SEQ ID NO:27, include Pro325→Leu (for example, SEQ ID NO:44) and/or Trp433→Phe (e.g., SEQ ID NO:45). These mutations can be combined with the shortening of the C-end, for example, to combine the mutation Pro325→Leu and shortening of 7 amino acids (e.g., SEQ ID NO:46).

spr2021

The original sequence "spr2021" was given in the link 89 as "protein General stress GSP-781' (see GI:15904062). For ease of reference, the amino acid sequence of the full-size spr2021 strain R6 provided herein as SEQ ID NO:28.

Preferred polypeptides spr2021 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:28 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:28, where 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). These proteins spr2021 include variants of SEQ ID NO:28. Preferred�tive fragments (b) contain the epitope of SEQ ID NO:28. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:28, but contain at least one epitope of SEQ ID NO:28. Other fragments contain one or more domains of the protein. One suitable fragment is shown in SEQ ID NO:47, does not contain the natural sequence of leader peptide.

In the link 90 spr2021 described as a secreted protein mass of 45 kDa, homologous GpbM, discloses its use as an immunogen (SEQ ID NO:243 in the link; SP2216). Immunogenic fragments spr2021 are shown in table 1 references 90 (p. 73). Another useful fragment spr2021 disclosed as SEQ ID NO:1 in the link 90 (amino acids 28-278 SEQ ID NO:28 reference).

spr0096

The original sequence "spr0096" was given in the link 89 as 'hypothetical protein' (see GI:15902140). For ease of reference, the amino acid sequence of the full-size spr0096 strain R6 provided herein as SEQ ID NO:29.

Preferred polypeptides spr0096 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:29 (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment from minicamera "n" consecutive amino acids from SEQ ID NO:29, where 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). These proteins spr0096 include variants of SEQ ID NO:29 (for example, SEQ ID NO:40; see below). Preferred fragments of (b) contain the epitope of SEQ ID NO:29. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:29, but contain at least one epitope of SEQ ID NO:29. Other fragments contain one or more domains of the protein.

Variant form spr0096 with an insertion in the region of the C-end relative to SEQ ID NO:29 shown herein as SEQ ID NO:48. The use of this fragment for immunization described in reference 90 (SEQ ID NO:150), where it is designated as a protein containing a LysM domain. Thus, spr0096 for use in the present invention may contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:48 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:48, where 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). These polypeptides include variants of SEQ ID NO:48. Preferred fragments of (b) contain the epitope of SEQ ID NO:48. Other preferred�individual fragments do not contain one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:48, but contain at least one epitope of SEQ ID NO:48. Other fragments contain one or more domains of the protein. Immunogenic fragments of SEQ ID NO:48 are shown in table 1 references 90.

Polypeptide spr0096 can be used in the form of a dimer, for example, homodimer.

spr1433

The original sequence "spr1433" was given in the link 89 as 'hypothetical protein' (see GI:15903476). For ease of reference, the amino acid sequence of the full-size spr1433 strain R6 provided herein as SEQ ID NO:30.

Preferred polypeptides spr1433 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:30 (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:30, where 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). These proteins spr1433 include variants of SEQ ID NO:30. Preferred fragments of (b) contain the epitope of SEQ ID NO:30. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acid�t (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:30, but contain at least one epitope of SEQ ID NO:30. Other fragments contain one or more domains of the protein.

spr1707

The original sequence "spr1707" was given in the link 89 as "substrate-binding protein ABC Transporter Oligopeptide transport (see GI:15903749). For ease of reference, the amino acid sequence of the full-size spr1707 strain R6 provided herein as SEQ ID NO:31.

Preferred polypeptides spr1707 for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:31 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:31, where 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). These proteins spr1707 include variants of SEQ ID NO:31 (for example, SEQ ID NO:100; see below). Preferred fragments of (b) contain the epitope of SEQ ID NO:31. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:31, but contain at least one epitope of SEQ ID NO:31. Other fragments contain one Il� more domains of the protein.

Variant form spr1707 different from spr1707 4 amino acids, provided herein as SEQ ID NO:49. The use of SEQ ID NO:49 for immunization described in reference 90 (SEQ ID NO:220). Thus, the polypeptide spr1707 for use in the present invention may contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:49 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:49, where 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). These polypeptides include variants of SEQ ID NO:49. Preferred fragments of (b) contain the epitope of SEQ ID NO:49. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:49, but contain at least one epitope of SEQ ID NO:49. Other fragments contain one or more domains of the protein.

Immunogenic fragments of SEQ ID NO:49 shown in table 1 references 90.

PspA

PspA is a surface protein A. For ease of reference, the amino acid sequence of the full-size PspA provided herein as SEQ ID NO:50. In the R6 genome PspA designated as spr0121 [89].

Preferred PspA polypeptides for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:50 (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:50, where 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). These PspA proteins include variants of SEQ ID NO:50. Preferred fragments of (b) contain the epitope of SEQ ID NO:50. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:50, but contain at least one epitope of SEQ ID NO:50. Other fragments contain one or more domains of the protein.

The use of PspA immunization are described, among others, in reference 100. It may preferably be administered in combination with PspC.

PsaA

PsaA is a surface adhesin pneumococcus. For ease of reference, the amino acid sequence of the full-size PsaA provided herein as SEQ ID NO:51.

Preferred polypeptides PsaA for use in the present invention contain amino acid sequence: (a) identical 50% �more of SEQ ID NO:51 (for example, on 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:51, where 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). These proteins PsaA include variants of SEQ ID NO:51. Preferred fragments of (b) contain the epitope of SEQ ID NO:51. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:51, but contain at least one epitope of SEQ ID NO:51. Other fragments contain one or more domains of the protein. A useful fragment of PsaA disclosed as SEQ ID NO:3 in the link 99 (corresponding to amino acids 21-519 SEQ ID NO:51 in this document).

The use of PsaA for immunization described in reference 101. It can be used in combination with PspA and/or PspC.

PspC

PspC is a surface protein of pneumococcus [102], and it is also known as choline-binding protein A (CbpA). Its use for immunization are described in the links 103 and 104. In the R6 strain he called spr1995, and For ease of reference, the amino acid sequence of the full-size spr1995 provided herein as SEQ ID NO:52.

Preferred PspC polypeptides for use in the present every�the acquisition contain the amino acid sequence: (a) identical 50% or more of SEQ ID NO:52 (e.g., on 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:52, where 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). These proteins spr1995 include variants of SEQ ID NO:52 (e.g., SEQ ID NO:27; see below). Preferred fragments of (b) contain the epitope of SEQ ID NO:52. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:52, but contain at least one epitope of SEQ ID NO:52. Other fragments contain one or more domains of the protein.

Option PspC known as "Hic". As shown in figure 1, reference 105, it is similar to PspC; there it binds to factor H (fH). For ease of reference, the amino acid sequence of a full-sized Hic provided herein as SEQ ID NO:53. Protein Hic can be used in the present invention in addition to or instead of the PspC polypeptide.

Preferred polypeptides Hic for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:53 (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at IU�Isha least "n" consecutive amino acids from SEQ ID NO:53, where 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). These Hic proteins include variants of SEQ ID NO:53. Preferred fragments of (b) contain the epitope of SEQ ID NO:53. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:53, but contain at least one epitope of SEQ ID NO:53. Other fragments contain one or more domains of the protein.

PspC and/or Hic may preferably be used in combination with PspA and/or PsaA.

LytA

LytA is an N-acetylmuramyl-L-alanine-amidase (autolysin). For ease of reference, the amino acid sequence of the full-size LytA provided herein as SEQ ID NO:54. In the R6 genome LytA called spr1754 [89].

Preferred polypeptides LytA for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:54 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:54, where 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). These proteins LytA include variants of SEQ ID NO:54. Preferred frag�patients under stood (b) contain the epitope of SEQ ID NO:54. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:54, but contain at least one epitope of SEQ ID NO:54. Other fragments contain one or more domains of the protein.

The use of LytA for immunization described in the link 106, in particular in the form that includes choline-binding domain of the LytA, fused with a heterologous universal epitope of T-helper cells.

PhtA

PhtA is a protein And histidinol triad of pneumococcus. For ease of reference, the amino acid sequence of a full-sized predecessor PhtA provided herein as SEQ ID NO:55. In the R6 genome is called PhtA spr1061 [89].

Preferred polypeptides PhtA for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:55 (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:55, where 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). These proteins PhtA include variants of SEQ ID NO:55. Preferred fragments of (b) contain the epitope of SEQ ID NO:55. Other preferred fragments n� contain one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:55, but contain at least one epitope of SEQ ID NO:55. Other fragments contain one or more domains of the protein.

Use PhtA for immunization are described in the links 107 and 108.

PhtB

PhtB is a protein In histidinol triad of pneumococcus. For ease of reference, the amino acid sequence of a full-sized predecessor PhtB provided herein as SEQ ID NO:56. Amino acid XAA at position 578 may be a lysine.

Preferred polypeptides PhtB for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:56 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:56, where 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). These proteins PhtB include variants of SEQ ID NO:56. Preferred fragments of (b) contain the epitope of SEQ ID NO:56. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-to�NCA SEQ ID NO:56, but contain at least one epitope of SEQ ID NO:56. Other fragments contain one or more domains of the protein.

The use of PhtB for immunization are described in the links 107, 108 and 109.

PhtD

PhtD is a protein D histidinol triad of pneumococcus. For ease of reference, the amino acid sequence of a full-sized predecessor PhtD provided herein as SEQ ID NO:57. In the R6 genome PhtD called spr0907 [89].

Preferred PhtD polypeptides for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:57 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:57, where 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). These proteins PhtD include variants of SEQ ID NO:57. Preferred fragments of (b) contain the epitope of SEQ ID NO:57. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:57, but contain at least one epitope of SEQ ID NO:57. Other fragments contain one or more domains of the protein.

The use of PhtD for immunization OPIE�ANO in links 107, 108 and 110.

PhtE

PhtE is a protein E histidinol triad of pneumococcus. For ease of reference, the amino acid sequence of a full-sized predecessor PhtE provided herein as SEQ ID NO:58. In the R6 genome PhtE called spr0908 [89].

Preferred polypeptides PhtE for use in the present invention contain amino acid sequence: (a) identical 50% or more of SEQ ID NO:58 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or more); and/or (b) containing a fragment of at least n consecutive amino acids from SEQ ID NO:58, where 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). These PhtE proteins include variants of SEQ ID NO:58. Preferred fragments of (b) contain the epitope of SEQ ID NO:58. Other preferred fragments contain one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-end and/or one or more amino acids (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from N-Terminus SEQ ID NO:58, but contain at least one epitope of SEQ ID NO:58. Other fragments contain one or more domains of the protein.

Use PhtE for immunization are described in the links 107 and 108.

Hybrid polypeptides

If the invention uses a hybrid antigens of pneumococcus or meningococcus, they can once were present�ü in the composition in the form of individual separate polypeptides. However, if there is more than one such antigen, they may not be present as separate polypeptides. Instead, at least two (e.g. 2, 3, 4, 5 or more) antigen can be expressed as a single polypeptide chain (a 'hybrid' polypeptide), as disclosed in reference 111 for antigens of meningococcus. Hybrid polypeptides have two big advantages: first, if a particular polypeptide is unstable or poorly expressed on its own, adding a suitable partner for the hybrid can solve these problems; secondly, the use of one procedure to the expression and purification to obtain the two polypeptides suitable as antigens, simplifies the process of commercial production.

The hybrid polypeptide may comprise two or more sequences of the polypeptides of the meningococcus or pneumococcus, as disclosed above. Suitable hybrids consisting of amino acid sequences of two, three, four, five, six, seven, eight, nine or ten antigens. Particularly preferred hybrids consisting of amino acid sequences of two, three, four or five antigens, such as two or three antigens.

Hybrid polypeptides can be described by the formula NH2-A-{-X-L-}n-B-COOH, where X is the amino acid sequence alternative antigen pneumato�ka or meningococcus, as described above; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; n is an integer equal to or greater than 2 (for example, 2, 3, 4, 5, 6 etc.). Usually n is 2 or 3.

If the group-X - includes the leader sequence of the wild-type peptide, this sequence may be included or not be included in a fusion protein. In some embodiments remove all leader peptides, except those included in group-X-, located at N-end of the hybrid protein, i.e. they preserve the leader peptide of X1and remove the leader peptide of X2...Xn. This is equivalent to deleting all leader peptides and using the leader peptide of X1as a group-A-.

For each n combinations of {-X-L-} linker amino acid sequence may be present or absent. For example, when n=2 the hybrid may be described by the formulas of NH2-X1-L1-X2-L2-COOH, NH2-X1-X2-COOH, NH2-X1-L1-X2-COOH, NH2-X1-X2-L2-COOH, etc. of the Linker amino acid sequence (s) -L - usually short (e.g. 20 or fewer amino acids, i.e. 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide� sequence, to facilitate cloning, polyglycine linkers (i.e. comprising Glynwhere n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more) and histidine tags (i.e. Hisnwhere n=3, 4, 5, 6, 7, 8, 9, 10 or more). Specialist in the field of technology are obvious other suitable linker amino acid sequence. A suitable linker is GSGGGG (SEQ ID NO:15) or GSGSGGGG (SEQ ID NO:16), where the dipeptide Gly-Ser is formed from the restriction site BamHI, which facilitates cloning and manipulation, and a tetrapeptide (Gly)4which is a typical polyglycidol the linker. Other suitable linkers, in particular for use as an end Lnrepresent a dipeptide Leu-Glu or SEQ ID NO:59.

-A - is an optional N-terminal amino acid sequence. It is usually short (e.g. 40 or fewer amino acids, i.e. 40, 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). Examples include leader sequences to direct the traffic of proteins, or short peptide sequences which facilitate cloning or purification (e.g., histidinemia label, i.e., Hisnwhere n=3, 4, 5, 6, 7, 8, 9, 10 or more). Specialist in the field of technology are obvious other suitable N-terminal amino acid sequence. If X1missing its N-terminal methionine, -A - is preferably from�fight Oligopeptide (e.g., consisting of 1, 2, 3, 4, 5, 6, 7 or 8 amino acids), which supplies the N-terminal methionine, for example, Met-Ala-Ser, or a single Met residue.

-Is an optional C-terminal sequence. It is usually short (e.g. 40 or fewer amino acids, i.e. 40, 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). Examples include sequences that guides protein traffic, short protein sequences which facilitate cloning or purification (e.g., containing histidinemia label, i.e., Hisnwhere n=3, 4, 5, 6, 7, 8, 9, 10 or more, such as SEQ ID NO:17), or sequences that enhance protein stability. Specialist in the field of technology are obvious other suitable C-terminal amino acid sequence.

Particularly suitable combination of polypeptide antigens of meningococcus disclosed in the links 111 and 112, and the composition according to the present invention may, therefore, include 1, 2, 3, 4, or 5 of: (1) protein "NadA"; (2) protein "936"; (3) protein "953": (4) protein "287" and (5) fHBP protein. For example, the composition may include: (i) a polypeptide with an amino acid sequence of SEQ ID NO:4; (ii) a second polypeptide with an amino acid sequence of SEQ ID NO:5; and (iii) a third polypeptide with an amino acid sequence of SEQ ID NO:6.

Adjuvant

The compositions according to the invention mohawksoft immunological adjuvant. For example, they can include an adjuvant, such as aluminium salt or emulsion oil-in-water" (for example, an emulsion of squalene in water). Can also be used with other adjuvants.

Suitable aluminium salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g., hydroxyphosphate, orthophosphate) (see, e.g., chapters 8 and 9 of 113 links), or mixtures thereof. Salt can be in any suitable form (e.g. gel, crystals, amorphous substance, etc.), with a typical adsorption of the antigen to salt. The concentration of Al+++in a composition for administration to a patient, preferably below 5 mg/ml, e.g., ≤4 mg/ml, ≤3 mg/ml, ≤2 mg/ml, ≤1 mg/ml, etc. the Preferred range of concentrations is 0.3-1 mg/ml. the Preferred maximum is about 0.85 mg per dose. The preferred adjuvant is aluminum salt for use with CS14 and antigens of meningococcus is aluminum phosphate.

There are various adjuvants-emulsion oil-in-water", usually consisting of at least one oil and at least one surface-active substance, where the oil (oil) and surface-active substance (substances) biodegradable (metabolisable) and biocompatible. Oil droplets in the emulsion composition is preferably less than 5 microns in diameter and may even have a submicron diameter; such small dimensions are achieved using microfluidizer to get�of stable emulsions. The preferred droplet size of less than 220 nm, because they can be sterilized by filtering.

The invention can be used with oils such as oils of animal (such as fish) or vegetable origin. Sources of vegetable oils include nuts, seeds and grains. Examples of nut oils include peanut oil, soybean oil, coconut oil and olive oil as the most common oils. You can use jojoba oil, for example, derived from jojoba beans. Oils from seeds include safflower oil, cottonseed oil, sunflower oil, sesame oil, etc. In the group of crops most prevalent corn oil, however, can be used for oil seeds other cereals, such as wheat, oats, rye, rice, Teff, triticale, etc. are Not contained in the oils from seeds of 6-10 carbon fatty acid esters of glycerol or 1,2-PROPANEDIOL can be obtained by hydrolysis, separation and esterification of suitable materials from oils, nuts and seeds. In the practice of the present invention can be used fats and oils from mammalian milk, because they are digestible. Procedures for the separation, purification, saponification, and any other means necessary to obtain the pure oils from animal sources, are well known in the art. Many fish contain digestible oil that can easily scraps�ing. For example, in a number of examples of fish oils that can be used in the present invention include cod liver oil, shark liver oil and whale oil such as spermaceti. Some oils branched chain synthesized biochemically in 5-carbon isoprene units; these oils are usually referred to as terpenoids. The shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23-HEXAMETHYL-2,6,10,14,18,22-tetragoniaceae, particularly preferred in the present invention. Squalane, the saturated analog of squalene, is also the preferred oil. Fish oils, including squalene and squalane, are commercially available or can be obtained using methods known in the art. Other preferred oils are Tocopherols. Can be used mixtures of oils.

If the composition includes a tocopherol, can be used from any α-, β-, γ-, δ-, ε - or ζ-tocopherol, however, the preferred α-Tocopherols. Tocopherol can take several forms, such as various salts and/or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. Can be used as D-α-tocopherol and DL-α-tocopherol. Preferred tocopherol is a DL-α-tocopherol, and the preferred salt of the tocopherol is a Sook�inat.

Surfactants can be classified according to their HLB (hydrophilic-lipophilic balance). Preferred surfactants according to the invention have a HLB of at least 10, preferably at least 15, and most preferably at least 16. The invention can be used with surfactants including, but not limited to: surface active substances on the basis of polyoxyethylene-sorbitane esters (commonly referred to as tweens), especially Polysorbate 20 and Polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO) and/or butilenica (BO), sold under the trademark DOWFAX™, such as a linear block copolymer EO/PO; octoxynol, which can vary the number of repeated (oxy-1,2-afandiyeva) groups, among which is of particular interest to octoxynol-9 (Triton X-100, tert-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); fatty esters of polyoxyethylene derived from lauryl, catelouge, stearyl and olejowego alcohols (known as surfactants Brij), such as triethylene glycol monolaurate ether (Brij 30); and sorbitane esters (also known as SPAN), such as sorbitan trioleate (Span 85) and the sorbitan monolauric�. Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.

You can use a mixture of surface-active substances, for example, a mixture of Tween 80/Span85. Also suitable combination polyoxyethylene-sorbitnogo ester, such as polyoxyethylene of sorbitan monooleate (Tween 80), and octoxynol, such as tert-octylphenoxypolyethoxyethanol (Triton X-100). Another suitable combination includes Laureth-9, and polyoxyethylene-arbitarily ester and/or octoxynol.

Preferred amounts of surfactants (mass fraction) are: polyoxyethylene-sorbitane esters (such as Tween 80) is 0.01 - 1%, in particular about 0.1%; octyl - or nonylphenolethoxylates (such as Triton X-100 or other detergents of the Triton series) - 0,001-0,1%, in particular from 0.005 to 0.02%; polyoxyethylene ethers (such as Laureth 9) is 0.1 - 20%, preferably 0.1 to 10%, and especially 0.1 to 1% or about 0.5%.

Specific adjuvants-emulsion oil-in-water" suitable for use in the present invention include, but are not limited to, the following:

Submicron emulsion of squalene, Tween 80 and Span 85. Volume fraction in the emulsion can be about 5% squalene, about 0.5% Polysorbate 80 and about 0.5% Span 85. In mass fraction� these ratios amount to 4.3% squalene, 0.5% Polysorbate 80 and 0.48 per cent of Span 85. This adjuvant is known as "MF59" [114-116] and described in more detail in Chapter 10 references 113 and Chapter 12 references 117. Emulsion MF59 may include citrate ions, for example 10 mm sodium-citrate buffer.

Emulsion of squalene, a tocopherol, and Polysorbate 80 (Tween 80). The emulsion may include saline solution in phosphate buffer. It can also include the Span 85 (e.g., 1%) and/or lecithin. Such emulsions may contain from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, where the weight ratio of squalene by weight of tocopherol, preferably ≤1, since in this case the emulsion more stable. The volume ratio of squalene and Tween 80 may be approximately 5:2, or their ratio of mass to be at around 11:5. One such emulsion can be obtained by dissolving Tween 80 in PBS to a concentration of 2%, mix 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml squalene) and then microfluidized mixture. The emulsion obtained may contain submicron oil droplets, for example, with an average diameter of 100-250 nm, preferably about 180 nm. The emulsion can also include 3 de-O-acylated monophosphoryl lipid A (3d-MPL). Other suitable emulsion of this type may contain, per dose for humans is 0.5-10 mg of squalene, 0.5 to 11 mg of tocopherol and 0.1-4 mg of Polysorbate 80 [118].

Emulsion of squalene, a tocopherol, and Triton (for example, Triton X-100). The emulsion can also include 3d-MPL. The emulsion may include a phosphate buffer.

Emulsion comprising a Polysorbate (e.g., Polysorbate 80), the detergent Triton (e.g., Triton X-100) and a tocopherol (e.g., α-tocopherol succinate). The mass ratio of these three components in the emulsion may be about 75:11:10 (e.g. 750 µg/ml Polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml α-tocopherol succinate), and these concentrations should take into account the contribution of these components from antigens. The emulsion may also include squalene. The emulsion can also include 3d-MPL. The aqueous phase may also include a phosphate buffer.

Emulsion of squalane, Polysorbate 80 and poloxamer 401 ("Pluronic™ L121"). The emulsion may be prepared in saline solution in phosphate buffer, pH 7,4. This emulsion is suitable for delivery nuramilovich of dipeptides were used together with threonyl-MDP adjuvant in the composition of the "SAF-1" [119] (0,05-1% Thr-MDP, 5% squalane, 2.5 percent Pluronic L121 and 0.2% Polysorbate 80). It can also be used without Thr-MDP, such as in the adjuvant composition "AF" [120] (5% squalane, 1.25% of Pluronic L121 and 0.2% Polysorbate 80). Preferred microfluidizer.

The emulsion containing squalene, water solvent, polyoxyethylene alkylamine hydrophilic non-ionic surface active substances (e.g., polyoxyethylene (12) cetosteatil ether) and a hydrophobic nonionic surface-Akti�substance (for example, ester sorbitan or mannide, such as sorbitan monolead or "Span 80"). The emulsion is preferably of thermooptical and/or contains at least 90% of the oil droplets (by volume) of less than 200 nm [121]. The emulsion may also include one or more cryoprotective agent (e.g., sugars, such as dodecalactone and/or sucrose and/or alkylpolyglycoside. Such emulsions can be liofilizirovanny.

Emulsion containing 0.5-50% oil, and 0.1-10% of a phospholipid and 0.05-5% non-ionic surfactants. As described in the link 122, the preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidyl, phosphatidic acid, sphingomyelin and cardiolipin. Preferred submicron droplet sizes.

Submicron emulsion oil-in-water" unassimilable oil (such as light mineral oil) and at least one surface active substances such as lecithin, Tween 80 or Span 80). The emulsion may include additives such as QuilA saponin, cholesterol, a saponin-lipophilic conjugate (such as GPI-0100, described in reference 123 received when joining the aliphatic amine to detailspane through the carboxyl group of glucuronic acid), dimethyldioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)p�opendiary.

The emulsion containing mineral oil, non-ionic lipophilic ethoxylated fatty alcohol and a nonionic hydrophilic surface active agents (e.g. ethoxylated fatty alcohol and/or a block copolymer of polyoxyethylene and polyoxypropylene) [124].

The emulsion containing mineral oil, non-ionic lipophilic ethoxylated fatty alcohol and non-ionic lipophilic surfactant (e.g. ethoxylated fatty alcohol and/or a block copolymer of polyoxyethylene and polyoxypropylene) [124].

Emulsion in which a saponin (e.g. QuilA or QS21) and a Sterol (e.g., cholesterol) associated to helical micelles [125].

Emulsion oil-in-water" can be used as adjuvants by themselves or as carriers for further immunostimulatory compounds such as immunostimulatory oligonucleotides, 3d-MPL, etc.

The pharmaceutical composition

The invention presents a pharmaceutical composition for administration to a patient. Such compositions typically include a pharmaceutically acceptable carrier. A detailed discussion of pharmaceutically acceptable carriers are presented in reference 126.

The volumes of effective doses can be determined by conventional methods, however, for intramuscular injection of a normal dose of the composition has a volume of approximately 0.5 ml. TA�Oh dose volume characteristic of the product PREVNAR™, the RIVM vaccine on the basis of OMV and MeNZB™. Such volumes typical doses for intramuscular injection, but similar volumes can be used for other routes of administration, for example intranasal vaccine based on the OMV for atomization may have a volume of about 100 μl or about 130 μl per spray, when to introduce the full dose volume of about 0.5 ml is four spraying.

the pH of the composition is usually in the range from 6 to 8, more preferably from 6.5 to 7.5 (e.g., about 7); pH RIVM vaccine based on the OMV is $ 7,4 [127], and for the compositions according to the present invention, the preferred pH<7.5 is. In the RIVM vaccine on the basis of OMV pH is maintained with 10 mm Tris/HCl buffer, and the stable pH of the compositions according to the invention can be maintained using a buffer, such as Tris-buffer, citrate buffer, phosphate buffer or histidinemia buffer. Thus, the compositions according to the invention typically include a buffer.

The composition may be sterile and/or pyrogen free. The compositions according to the invention may be isotonic to human.

The compositions according to the invention for administration to patients are immunogenic and more preferably are vaccine compositions. Vaccines according to the invention may be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to Leche�Oia infection), but usually they are preventive. Immunogenic compositions used as vaccines comprise immunologically effective amount of antigen (antigens), and, if necessary, any other components. By "immunologically effective amount" refers to that the introduction of this number the subject in a single dose or as part of a series of doses, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the subject treated, age, the taxonomic affiliation of the subject undergoing treatment (for example, the primacy is not a human, Primate, etc.), ability of the immune system of a subject to synthesize antibodies, the desired degree of protection, the composition of the vaccine, the assessment of the medical situation, your doctor and other important factors. It is expected that the number may be in a sufficiently wide interval, which is determined in the standard test. The antigen content in the compositions according to the invention is usually expressed as the amount of protein per dose. Typical dose for intranasal vaccines based on OMV is about 0.9 mg of protein per ml.

Meningococci and pneumococci are striking different areas of the body, and thus, compositions according to the invention can be prepared in the form of various liquid forms. For example, �oppozitsii can be prepared for injection, in the form of solutions or suspensions. The composition may be prepared for administration to the lungs, for example, by using a nebulizer in a fine spray. The composition may be prepared for nasal, aural or ocular administration, for example, in the form of a spray or drops. The commonly used form for intramuscular injection.

The compositions according to the invention may include an antimicrobial agent, especially if you use the package for repeated administration. Vaccines typically contain antimicrobial agents, such as thiomersal and 2-Phenoxyethanol, however, it is preferable to use a preservative, containing mercury, or do not use a preservative.

The compositions according to the invention can contain a detergent, for example Tween (Polysorbate), such as Tween 80. The detergent is typically present in small amounts, for example <0.01%.

The compositions according to the invention may include sodium salt (e.g. sodium chloride) to toychest. A typical concentration is 10±2 mg/ml NaCl, for example about 9 mg/ml.

Methods for the treatment of

The present invention also presents a method of inducing an immune response in a mammal, containing the introduction to a mammal the composition according to the invention. The immune response preferably protects from meningococci, and pneumococci (from at �'ere of immunotype (immunotypes) meningococcus and serotype (serotypes) of pneumococcal including serotype 14 provided in the composition) and preferably includes antibodies. This method can also cause booster effect in a patient previously subjected to initial exposure to antigen.

The mammal is preferably a human. If the vaccine is for prophylactic use, human is preferably a child (e.g. the child is preschool age or infant) or a teenager; if the vaccine is for therapeutic use, human is preferably an adult. A vaccine intended for children may also be given to adults, for example, to evaluate its safety, dosage, immunogenicity, etc.

The invention also provides compositions for use in the form of medication. Drug is preferably used, as described above, for the induction of the immune response in a mammal (i.e. it is an immunogenic composition) and more preferably, if the drug is a vaccine.

The invention also presents the use of: (i) the meningococcal LOS and CS14, where LOS and/or CS14 does not include (do not include) tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc; (ii) the meningococcal LOS and CS14 without adjuvant, where as LOS and CS14 contain tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ14Glc; (iii) the meningococcal LOS and the polypeptide antigen of pneumococcus, where LOS includes tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, and the polypeptide of pneumococcus can cause an immune response effective against pneumococcus serotype 14; or (iv) the polypeptide of meningococcus and CS14, where CS14 contains tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, and a peptide of meningococcus can cause an immune response effective against the meningococcal serogroup B, in the manufacture of a medicinal product for the induction of the immune response in the mammal.

These practices and methods are preferably directed at the prevention and/or treatment of diseases caused byN. meningitidesand/orS. pneumonidaesuch as bacterial (or specifically, meningococcal and/or pneumococcal) meningitis or septicemia.

One way to check the effectiveness of therapy includes tracking meningococcal and/or pneumococcal disease after the introduction of the compositions according to the invention. One way of testing the effectiveness of prevention involves monitoring immune responses after administration of the composition. Immunogenic compositions according to the invention can be determined by its introduction of the test subjects (e.g. children 12-16 months or experimental animals [128]) and then determining standard parameters including levels of bactericidal antibodies in serum (SBA) and the titer�in ELISA (GMT) for the meningococcus. These immune responses are usually analyzed through approximately 4 weeks after administration of the composition, and are compared to values obtained before the introduction of the composition. Preferably, the increased level of SBA at least 4 or 8 times. If you enter more than one dose of the composition, may be more than one analysis after the introduction.

The compositions according to the invention is typically administered directly to the patient. Direct introduction is usually carried out by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or into the interstitial space of a tissue) or in any other convenient way. The invention can be used for induction of systemic immunity and/or mucosal immunity. Preferably intramuscular injection in the thigh or shoulder. The injection can be carried out by means of a needle (e.g., needle for subcutaneous injection), but may alternatively be used in needleless injection. The usual intramuscular dose of 0.5 ml.

The dosage may include one or multiple doses. Multiple doses can be used for primary immunization and/or for booster immunization. Booster immunization may be followed for primary immunization. A suitable time difference between primary doses (e.g., 4-16 weeks), and between primary and booster immune�ization can be determined by standard methods.

In some embodiments of the present invention, the antigens of pneumococcus and the meningococcus can be entered together, but separately, i.e. two antigen can be designed for the simultaneous, separate or sequential administration. Usually these two antigen are mixed for simultaneous combined administration.

Additional antigens

In addition to the antigens of meningococcus and pneumococcus, as described above, the composition may include antigen (antigens) against further pathogen (pathogen). For example, the composition may contain one or more of the following further antigens:

- antigen of hepatitis b virus, such as surface antigen HBsAg;

- antigenBordetella pertussissuch as the pertussis holotoxin (PT) and filamentous hemagglutininB. pertussisoptionally also in combination with pertactin and/or agglûtinogeny 2 and 3;

- a diphtheria antigen, such as diphtheria toxoid;

- tetanus antigen, such as tetanus toxoid;

- charigny antigenHaemophilus influenzaeB (Hib), usually conjugated;

- antigens inactivated poliovirus.

If the composition includes a diphtheria antigen, preferably if it includes the tetanus antigen and pertussis antigen. Similarly, if the composition includes tetanus antigen, preferably, if �to also include diphtheria and pertussis antigens. Similarly, if the composition comprises the pertussis antigen, preferably, if it also includes diphtheria and tetanus antigens. Thus, preferred combinations of CDS.

Furthermore, the composition may include additional antigens of meningococcus or pneumococcus.

In addition to antigen CS14, for example, the composition may include capsular saccharides of one or more other serotypes (serotypes) of pneumococcus. Thus, in addition to serotype 14, the composition may include a capsular saccharide from one or more of the following serotypes of pneumococcus: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. The composition may include multiple serotypes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes. In the art known 7-valent, 9-valent, 10-valent and 11-valent and 13-valent conjugate combinations and 23-valent unconjugated combination. For example, a 10-valent combination may include saccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. 11-Valent combination may further include a saccharide serotype 3. A 12-valent combination for the 10-valent mixture can be added: serotypes 6A and 19A; 6A and 22F; 19A and 22F; 6A and 15B; 19A and 15B; or 22F and 15B. A 13-valent combination to the 11-valent mixture can be added: serotypes 19A and 22F; 8 and 12F; 8 and 15; 8 and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12 f and 2F; 15B and 19A, 15V and 22F; 6A and 19A, etc. Fit the 13-valent combination includes capsular saccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19, 19F and 23F, for example, all conjugated separately with CRM197, obtained as described in the links 129, 130 and 131. One such combination includes a saccharide serotype 6B in a concentration of about 8 μg/ml and 12 other saccharides in concentrations of about 4 μg/ml. Another such combination includes saccharides of serotypes 6A and 6B in concentrations of approximately 8 mg/ml and the remaining 11 sugars in concentrations of about 4 mg/ml.

If the composition comprises more than one pneumococcal conjugates, each conjugate can be used the same protein carrier or different carrier proteins. In the link 132 of the potential benefits of using different carrier proteins in multivalent pneumococcal conjugate vaccines.

If the composition includes sacharine antigens of more than one serotype, preferably, if they receive and kongugiruut separately, and then combine.

In addition to meningococcal LOS or polypeptide antigen composition can include meningococcal capsular saccharide, which is usually kongugiruut with carrier protein. The composition according to the invention may include one or more conjugates of capsular saccharides 1, 2, 3 or 4 serogroups of meningococcal A, C, W135 � Y, for example, A+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, A+W135+Y, A+C+W135+Y, etc. the Ideal components including saccharides from all four of serogroups A, C, W135 and Y.

Capsular saccharide of meningococcal serogroup A is a homopolymer consisting of N-acetyl-D-mannosamine-1-phosphate associated (α1→6), with partial O-acetylation at positions C3 and C4. Acetylation at C-3 can be 70-95%. The cleaning of saccharides can result in de-O-acetylation (e.g., alkaline conditions), but is useful to save the OAc at C-3. In some embodiments at least 50% (e.g., at least 60%, 70%, 80%, 90%, 95% or more residues of mannosamine in saccharides serogroups And On-azetilirovanny in position C-3. Acetyl groups can be replaced with blocking groups to prevent hydrolysis [133], and such modified sugars continue to be considered saccharides serogroups And in the context of the present invention.

Capsular saccharide of serogroup C is a homopolymer of sialic acid (N-acetylneuraminic acid or NeuNAc), affiliated (α2→9). Sahariana structure is written as →9)-NeupNac 7/8 OAc-(α2→. Most strains of serogroup C are O-acetyl group at C-7 and/or C-8 of the sialic acid residues, however, approximately 15% of clinical isolates do not contain these O-acetyl groups [134, 135]. The presence of Il� no groups CCA generates a unique epitopes, and the binding specificity of an antibody to the saccharide may affect its bactericidal activity against O-acetylated (CCA+) and de-O-acetylated (SLA-) strains [136-138]. Saccharides serogroups C, used in the present invention, can be obtained from strains SLA+ or the CCA. Approved conjugate vaccines include MenC saccharides as CCA- (NEISVAC-C™) and SLA+ (MENJUGATE™ and MENINGITEC™). In some embodiments, the strains for the production of conjugate serogroup C are strains SLA+, for example, serotype 16, seropositive P1.7a,1 and Thus, can be used strains SLA+ S:16:P1.7a,1. Also suitable strains SLA+ seropositive P1.1, such as strain C11.

Saccharide of serogroup W135 is a polymer of disaccharide units of sialic acid-galactose. As for saccharide of serogroup C it is characterized by a variable O-acetylation, but by regulations 7 and 9 of sialic acid [139]. The structure is written as follows: →4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Gal-α - (1→.

Saccharide of serogroup Y similar to the saccharide of serogroup W135, except that in the repeating disaccharide unit includes glucose instead of galactose. As for saccharide of serogroup W135, typically it has variable O-acetylation, regulations 7 and 9 of sialic acid [139]. The structure of the saccharide of serogroup Y is written as follows: →4)-D-Ne p5Ac(7/9OAc)-α-(2→6)-D-Glc-α - (1→.

The saccharides used according to the invention may be O-azetilirovanny as described above (i.e. having the same pattern of acetylation, and the native capsular saccharides), or they may be partially or totally de-O-azetilirovanny one or more position sugar-barnacles rings, or they may be Hyper-O-azetilirovanny relative to the native capsular saccharides.

Sacharine groups in the conjugates can contain a full-sized saccharides derived from meningococci, and/or may include fragments of the full-sized saccharides, i.e. saccharides can be shorter than the native capsular saccharides bacteria. Thus, the saccharides can be depolymerizer, and depolymerization can occur before or after purification of sugars, but before conjugation. Depolymerization reduces the length of a chain of sugars. One method of depolymerization involves the use of hydrogen peroxide. Hydrogen peroxide is added to a saccharide (e.g., to a final concentration of H2O21%), and the mixture is then incubated (e.g. at about 55°C) until the desired chain length. Another method of depolymerization involves acidic hydrolysis. The prior art discloses other methods of polymerization. The saccharides used to obtain conjugates for use according to the invention, can be obtained�s by using any of these methods of depolymerization. The depolymerization can be used to achieve the optimum chain length for immunogenicity and/or to reduce the length of the chain for better physical control of sugars. In some embodiments, the saccharides have the following interval average degrees of polymerization (Dp): A=10-20; C=12-22; W135=15-25; Y=15-25. Translated at a molecular weight suitable intervals equal to, for all serogroups: <100 kDa, and 5 kDa-75 kDa; 7 kDa-50 kDa; 8 kDa-35 kDa; 12 kDa-25 kDa; 15 kDa-22 kDa.

In some embodiments the average molecular weight saccharides of each of the serogroups of meningococcal A, C, W135 and Y can be greater than 50 kDa, for example ≥75 kDa, ≥100 kDa, ≥110 kDa, ≥120 kDa, ≥130 kDa, etc. [140], and even up to 1500 kDa, in particular when determining with MALLS. For example: molecular weight of MenA saccharide may be in the range of 50-500 kDa, for example 60-80 kDa; the molecular weight of the MenC saccharide may be in the range of 100-210 kDa; the molecular weight of the MenW135 saccharide may be in the range of 60-190 kDa, for example of 120-140 kDa; and/or molecular weight of MenY saccharide may be in the range of 60-190 kDa, for example of 150-160 kDa.

Mass of meningococcal saccharide each serogroup in a composition is generally from 1 μg to 20 μg, for example between 2 and 10 μg per serogroup, or about 4 μg, about 5 μg, or about 10 μg. If the composition comprises conjugates more than one serogroup, their mass may not practically differ�atsya between serogroups, for example, the mass of each saccharide of serogroup may be within ±10% of each other. Alternatively, equal to the ratio can be used 2 times large mass of the saccharide of serogroup A. Thus, the vaccine may include MenA saccharide in an amount of 10 μg, and saccharides MenC, W135 and Y - 5 mcg.

Above discussed are useful carrier proteins and linkers of different nature. Useful carriers include diphtheria toxoid, tetanus toxoid and CRM197.

Can be used conjugates with a ratio by mass of the saccharide:protein of 1:5 (i.e. excess protein) and 5:1 (i.e. excess saccharide), for example with a ratio of 1:2 to 5:1 and with a ratio from 1:1.25 to 1:2,5. As described in the link 141, the conjugates of different serogroups of meningococcus in the mixture may have a different ratio of the saccharide:protein, for example, one may have a ratio of 1:2 to 1:5, and the other from 5:1 to 1:1,99.

As described in the link 142, the mixture may include one conjugate with a direct link to the saccharide to a protein and another conjugate with communication via the linker. It matters especially when you use conjugates of saccharides of different serogroups of meningococci, for example, saccharides MenA and MenC can be konjugierte via a linker, and saccharides MenW135 and MenY can be konjugierte with the carrier protein directly.

General provisions

In the practice of the present invention are used if not specified� otherwise, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology available to the specialist in the field of technology. Such techniques are fully described in the literature. See, for example, links 143-149, etc.

The term "contain" covers "include" and "comprise," i.e., a composition containing" X may consist entirely of X or may include additional components, such as X+Y.

The term "about" relative numerical value x is optional and means, for example, x±10%.

If in the invention, refers to the epitope, the epitope may be a b-cell epitope and/or T-cell epitope, but is typically a b-cell epitope. Such epitopes determined empirically (e.g. using PEPSCAN [150, 151] or similar methods) or predict (e.g., antigenic index (jameson-wolf [152], matrix approaches [153], MAPITOPE [154], TEPITOPE [155, 156], neural networks [157], PC OPTIMER and EpiMer [158, 159], ADEPT [160], Tsites [161], hydrophilicity [162], antigenic index [163], or methods, disclosed in the references 164-186, etc.). The epitopes are the parts of an antigen that are recognized by and binds to the antigen-binding sites of T-cell receptors, they may also be referred to as "antigenic determinants".

If the invention is used "cleaned" antigen, this antigen is separated from its natural�'s internal environment. For example, the antigen will not contain other components of meningococci, in addition to any other purified antigens that are also present. A mixture of purified antigens usually get by cleaning each antigen separately and their combination, even if the two antigens are naturally present in the mixture.

Links to the percent sequence identity between two amino acid sequences means the percentage of identical amino acids in these sequences when aligning them. This alignment and the percent homology or sequence identity can be determined using software known in the art, such as described in section 7.7.18 of the link 169. The preferred alignment is determined using a search algorithm homology Smith-Waterman using affine search for deletions, the penalty for the opening of deletions is equal to 12, and the penalty for continued deletions equal to 2, and matrix BLOSUM62. The search algorithm of the homology Smith-Waterman disclosed in reference 170.

The term "substantially" does not exclude "completely" e.g. a composition "substantially free" Y may be completely free from Y. where necessary, the term "substantially" in the definition of the invention may be omitted.

BRIEF DESCRIPTION of FIGURES

Figure 1 p�estaline crustal saccharides of immunotype L3.

Figure 2 shows a repetitive structure CS14 from reference 55.

Figure 3 shows antibody titers against LNnT in groups of animals. Similarly, figure 4 shows IgG titers against CP14. On both figures, the values presented GMT, expressed in relative luminescence units per ml.

WAYS of carrying out the INVENTION

Mice were immunized (i) vaccine PREVNAR™ 0.4 μg per dose; (ii) the outer membrane vesicles derived from the wild-type strain MC58 of meningococcal serogroup At 10 µg per dose, in combination with PREVNAR™; or (iii) the outer membrane vesicles derived from the wild-type strain MC58 of meningococcal serogroup In the knockout ΔlgtB 10 mcg per dose, in combination with PREVNAR™. All songs were made with the addition of aluminium hydroxide as an adjuvant.

Serum was analyzed by Luminex test for antibodies against, among others, the capsular saccharide of pneumococcus serotype 14 and LNnT epitope.

Figure 3 presents the answers to LNnT for the three groups on day 34 after immunization at 2 and 21 days. The vaccine PREVNAR™ itself evoked response against LNnT approximately two times higher than the blank control (against 0,2 0,12). Adding OMV containing LOS, increased response (0,7), but for OMV LgtB-vethe gain was not as pronounced (0,5). The gain in the group (iii) relative to group (ii) can be attributed to the ability�m adjuvant effect OMV (cf. with reference 51).

Figure 4 shows the responses against CS14 on day 34. Both drugs OMV increased response to CS14; preferably watched more gain when using OMV LgtB-ve.

Although the data are incomplete and not fully convincing and the fact that the test against LNnT gave only a weak signal, the data show that CS14 and OMV LgtB-vecan be combined to obtain better results than the combination CS14 and OMV wild-type.

It should be understood that the present invention is described solely by way of example and that it can be used modification without changing the essence and scope of the invention.

1. Adjuvant immunogenic composition comprising lipooligosaccharide meningococcus (LOS) and capsular saccharide of pneumococcus serotype 14 (CS14), where CS14 contains tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, a LOS does not contain tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc.

2. A composition according to claim 1, where the LOS is obtained from a strain of meningococcus, not containing LgtB enzyme activity.

3. A composition according to claim 1, where the LOS is obtained from a strain of meningococcus, containing the enzymatic activity of the GalE.

4. A composition according to claim 1, where the LOS is obtained from a strain of meningococcus, not containing enzymatic asset�spine LgtA and/or LgtE.

5. A composition according to any one of claims. 1-4, where LOS contains trisaharid 4GlcNAcβ1-3Galβ1-4Glc and contains tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc.

6. A composition according to claim 1, wherein LOS present in the vesicles of the outer membrane of meningococci.

7. A composition according to claim 6, where LOS is conjugated to proteins of the vesicle.

8. A composition according to claim 6 or 7, where vesicles derived from meningococcus with increased expression of TbpA.

9. A composition according to any one of claims. 1-4, where LOS is conjugated to a carrier protein.

10. A composition according to claim 9, where the conjugation can be carried out using a lipid And in the LOS or via a KDO residue.

11. A composition according to claim 1, wherein the CS14 is conjugated to a carrier protein.

12. A composition according to claim 11, where the protein carrier is a CRM197, tetanus toxoid, diphtheria toxoid or protein D H. influenzae.

13. A composition according to claim 1, further comprising aluminum salt adjuvants.

14. A composition according to claim 13, wherein the aluminum salt is an aluminum phosphate.

15. A method of inducing an immune response in a mammal, comprising administering the composition according to any one of claims. 1-14 mammal.



 

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45 cl, 13 dwg, 5 tbl, 1 ex

FIELD: medicine, pharmaceutics.

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32 cl, 3 ex, 9 tbl, 26 dwg

FIELD: chemistry.

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2 cl, 31 dwg, 3 tbl, 4 ex

FIELD: medicine.

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23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to biospecific antibodies, and can be used in medicine. Constructed is an antibody containing one of the following groups of six hypervariable region (HVR) sequences: (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2, containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NIKDTY; (e) HVR-H2 containing the sequence RIYPTNGYTR; and (f) HVR-H3 containing the sequence WGGDGFYAMD; or (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2 containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NISGTY; (e) HVR-H2 containing the sequence RIYPSEGYTR; and (f) HVR-H3 containing the sequence WVGVGFYAMD. The produced antibody specifically binds human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF) The invention also refers to a recovered Fab fragment of the above antibody, a polynucleotide coding it, to an expression vector, a host cell, a method for producing it, as well as to using it for treating HER2-mediated diseases.

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14 cl, 65 dwg, 16 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biochemistry, particularly to an immunogenic composition, which induces cross-reactive bacterial antibodies to a number of strains Neisseria meningitidis serotype B. As an active ingredient, the immunogenic composition contains variants of the non-lipidised polypeptide ORF2086non-functionalised by pyroracemic acid and consisting of an amino acid sequence specified in a group of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO: 20 and SEQ ID NO: 21, wherein the polypeptide contains N-terminal Cys deletion as compared to the existing non-lipidised wild-type polypeptide ORF2086. What is also disclosed is a plasmid, which expresses the non-lipidised polypeptide ORF2086 non-functionalised by pyroracemic acid. What is also disclosed is a method for producing bactericidal antibodies specific to ORF2086 of the sub-family B of the serogroup Neisseria meningitidis in mammals.What is also disclosed is a method for producing the non-lipidised polypeptide ORF2086 non-functionalised by pyroracemic acid.

EFFECT: invention enables inducing the cross-reactive bacterial antibodies to Neisseria meningitidis serotype B.

32 cl, 8 dwg, 14 tbl, 13 ex

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