Hybrid and tandem expression of neisserial proteins

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically obtaining immunogens from Neisseria meningitides and can be used in medicine. Disclosed is protein which is 80% or more identical to amino acid sequence SEQ ID NO:19, and an immunogenic composition with said protein.

EFFECT: invention enables to use said protein for effective prevention or treatment of bacterial meningitis.

6 cl, 5 dwg, 28 tbl

All cited in this description and the documents incorporated by reference in full.

The technical FIELD

This invention relates to the field of protein expression. In particular, the invention relates to the expression of proteins from Neisseria (e.g., N. gonorrhoeae or, preferably, N. meningitidis).

PRIOR art

In publications 1 and 2 disclosed an alternative and improved methods of protein expression of Neisseria described in the publications 3-6. One such method consists of obtaining the hybrid proteins in which two or more proteins Nasseri expressed as a single polypeptide chain. This method provides two advantages. First, a protein that may be unstable or poorly expressed itself can be obtained by adding a suitable partner in the hybrid, in order to overcome this problem. Secondly, simplified commercial production, because you use only one expression and purification, to obtain the two separately applied protein.

The objective of the invention is a new alternative and improved methods of protein expression of Neisseria.

The INVENTION

Hybrid proteins

Thus, the invention relates to a method for the simultaneous expression of two or more (for example, 3, 4, 5, 6 or more) is elkow of Nasseri, wherein the two or more proteins are linked so that they were broadcast in a single polypeptide chain. In General, the hybrid proteins according to the invention can be represented by the formula: NH₂-A-[-X-L-]_n-B-COOH, where x is the amino acid sequence, L is an optional amino acid sequence of the linker, And indicates an optional N-terminal amino acid sequence, means an optional C-terminal amino acid sequence, and n is an integer greater than 1.

The value of n ranges from 2 to x, and the value of x is normally 3, 4, 5, 6, 7, 8, 9 or 10. Preferably n is 2, 3 or 4; more preferably 2 or 3; most preferably n=2.

Components-X-

There are two main groups of the hybrid proteins according to the invention. These two groups are not mutually exclusive.

In the first group, each component of X represents:

(a) amino acid sequence of orf1, orf4, orf25, orf40, orf46.1, orf83, NMB1343, 230, 233, 287, 292, 594, 687, 736, 741, 907, 919, 936, 953, 961 or 983;

(b) amino acid sequence that has sequence identity with the amino acid sequence according to (a); or

(c) the amino acid sequence containing a fragment of the amino acid sequences according to (a).

Preferred Podger is ppoi (a) are: orf46.1, 230, 287, 741, 919, 936, 953, 961 and 983. The preferred sub-group (a) are: orf46.1, 287, 741 and 961. Figure 3 shows the preferred hybrid proteins.

In the second group hybrid protein contains the first component of X- (-X_and-) and the second component,- X- (-X_b-). Component-X_and- has one of the following amino acid sequences:

(d) 446 even SEQ IDS (i.e. 2, 4, 6, ..., 890, 892), presented in publication 3;

(e) 45 even SEQ IDS (i.e. 2, 4, 6, ..., 88, 90), presented in publication 4;

(f) 1674 even-numbered SEQ ID NO:2-3020, the even-numbered SEQ ID NO:3040-3114 and all of SEQ ID NO:3115-3241 presented in publication 5;

(g) 2160 amino acid sequence with NMB0001 on NMB2160 of publications 7; or

(h) the amino acid sequence presented in publication 1 or publication 2.

Component-X_b- a sister-X_andprovided that: (i) -X_b- has a sequence identity of C-X_andand/or (j) -X_b- contains a fragment-X_and-.

Examples of the specified second type of hybrid protein include proteins in which two or more components of X are identical or in which they are variants of the same protein, for example, two polymorphic forms of the same protein can be expressed in the form X_a-X_b-, and three polymorphic forms can be expressed in the form X_a-X_b-the _cand so on

Components-X_andand X_b- can be placed in any order from N-Terminus to the C-end.

Component-X_and- preferably represents amino acid sequence of orf1, orf4, orf25, orf40, orf46.1, orf83, NMB1343, 230, 233, 287, 292, 594, 687, 736, 741, 907, 919, 936, 953, 961 or 983. More preferably component-X_and- is the amino acid sequence orf46.1, 230, 287, 741, 919, 936, 953, 961 or 983. Most preferably component-X_and- is the amino acid sequence orf46.1, 287, 741 or 961.

In the case of proteins, where each of the n components of X - shows sequence identity with each other component of the X-protein called "tandem protein". The preferred tandem proteins, in which n=2.

The degree of identity of the sequence in (b) and (1) is preferably more than 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more, up to 100%). This applies to mutants, homologues, orthologues, allelic variants, etc. [for example, see reference 8]. The identity is preferably determined using the algorithm of finding the homology of the Smith-Waterman that runs in the MPSRCH program (Oxford Molecular), using search affine gaps with the following parameters: the penalty for opening a gap=12 and the penalty for gap continued=1. Typically, the identity component of 50% or more between the two proteins is still what the motor is functional equivalence.

"Fragment" in relation to (C) and (j) must consist of at least m consecutive amino acids of the amino acid sequence of (a), (d), (e), (f), (g) or (h) and depending on the specific sequence of m is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).

Preferably, the fragment contains an epitope of the amino acid sequence of (a), (d), (e), (f), (g) or (h). Preferred fragments are those fragments described in the publications 9 and 10.

The preferred (s)- and (j)-fragments are truncated at the C - and/or N-end fragments (e.g., Δ1-287, Δ2 287, and so on).

In the preferred sequence (b), (C), (i) and (j) are not included polyglycine sequence. Found that this contributes to the expression [reference 2]. Polyglycine sequence can be represented as (Gly)_gwhere g≥3 (for example, 4, 5, 6, 7, 8, 9 or more). If component X contains polyglycerol consistency in his form of the wild type, it is preferable to exclude the specified sequence in the hybrid proteins according to the invention. This can be done by breaking or removing the (Gly)_g- through deletions (for example, CGGGGS-CGGGS, CGGS, CGS or CS), replacement (for example, CGGGGS-CGXGGS, CGXXGS, CGXGXS etc) and/or insert (e.g., CGGGGS→CGGXGGS, CGXGGGS etc). A preferred deletion (Gly)_{g and the deletion of the N-terminal part of the protein up to and including polyglycine sequence (for example, deletions of residues 1-32 in SEQ ID NO:1) is indicated in the present description, "ΔG". Remove polyglycine, in particular, applicable in the case of proteins 287, 741, 983 and Tbp2 (ΔG287, ΔG741, ΔG983 and ΔGTbp2 - links 1 and 2).}

_{In the preferred (s)- and (j)-fragments excluded entire protein domains. In particular, this applies in the case of protein 961, 287 and ORF46. In that case, if the protein is divided into domains, (C)and (j)-fragments can be excluded one or more of these domains (for example, W, S, WS, ORF461-433, ORF46434-608, S - link 2; figures 4 and 5 in this description).}

Protein 287 is divided into three domains, named a, b and C (see figure 5 in publication 2). Domain is aligned with the IgA protease, domain is aligned with the proteins that bind transferrin, and is not observed strict alignment domain And with sequences in the database. Alignment of polymorphic forms 287 described in publication 8.

ORF46 is divided into two domains - the first domain (amino acids 1-433; ORF46.1), which vysokokonservativnykh among species and serogroups, and a second domain (amino acids 434-608), which is highly conservative. The second domain is preferably delegate, leaving ORF46.1. Alignment of polymorphic forms ORF46 described in publication 8.

Protein 961 is divided into nickolodeon (figure 4).

If component X is the sequence of the leader peptide in his form of the wild type, it can be included or omitted in the hybrid proteins according to the invention. When the leader peptide is excluded, the sequence is a preferred example of the amino acid sequence of (C) and (j). In one embodiment, the leader peptides will be delegated, except in the case when the component X is located on the N-end of the hybrid protein, i.e. the leader peptide X₁to be saved, and leader peptides X₂...X_nwill be deleted. This is equivalent to delegate all of its leader peptides and the use of the leader peptide X₁as part of-And-.

In the case when n=2, the preferred pairs of components of X are: ΔG287 and 230; ΔG287 and 936; ΔG287 and 741; s and 287; s and 230; s and 936; 961cL and 287; 961cL and 230; 961cL and 936; ORF46.1 and 936; ORF46.1 and 230; 230 and 961; 230 and 741; 936 and 961; 936 and 741. In the case when n=2, the preferred pairs of components of X - tandem proteins are: ΔG741 and 741; ΔG287 and 287. More specifically, in the case when n=2, the preferred are the following combinations of X₁and X₂:

In that case, when 287 used in full-size form, it preferably is located at the C-end of the hybrid protein; if you want to use for the N-end, preferably using ΔG-form 287. Similarly, in the case when the 741 is used in full-size form, it preferably is located at the C-end of the hybrid protein; if you want to use for the N-end, preferably using ΔG-form 741.

Components-L-

For each case n [-X-L-] linker amino acid sequence of L may be present or absent. For example, when n=2 the hybrid may be a NHsub> 2-X₁-L₁-X₂-L₂-COOH, NH₂-X₁-X₂-COOH, NH₂-X₁-L₁-X₂-COOH, NH₂-X₁-X₂-L₂-COOH, etc.

Linker amino acid sequence(ti) -L - usually will be short(short) (e.g., 20 or fewer amino acids, ie 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences which facilitate cloning, polyglycine linkers (i.e. Gly_nwhere n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more) and his-tag labels (i.e. HiS_nwhere n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequence will be obvious to specialists in this field. Apply the linker GSGGGG (SEQ ID NO:27), and the dipeptide Gly-Ser formed from the restriction site BamHI, thus facilitating cloning and manipulation, and tetrapeptide Gly₄a typical polyglycine linker.

If X_n+1is a protein ΔG and L_nis glycine linker, it may be equivalent to the case when X_n+1not a ΔG-protein, and L_nis missing.

Component-And-

-A - is an optional N-terminal amino acid sequence. Usually it is short (for example, 40 or fewer amino acids, ie 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). Note the market include a leader sequence for the directed movement of proteins or short peptide sequence, which facilitate cloning or purification (e.g., his-tag labels, i.e. His_nwhere n=3, 4, 5, 6, 1, 8, 9, 10 or more). Other suitable N-terminal amino acid sequence will be obvious to specialists in this field. If X₁contains at its N end of methionine, And may be a methionine residue.

Component-In-

- - Is an optional C-terminal amino acid sequence. Usually it is short (for example, 40 or fewer amino acids, ie 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 the sequence for the directed movement of proteins, short peptide sequences which facilitate cloning or purification (e.g., containing his-tag labels, i.e. His_nwhere n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences that enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to experts in this field.

Polymorphic forms of proteins

In the invention it is possible to use amino acid sequences of all strains of N. meningitidis. Link to a specific protein (e.g., "287" or "ORF46.1") thus includes such protein from any strain. Variations of the sequences between strains included in (b), (C), (i) and (j).

SDA is lilwanye sequence of N. meningitidis serogroup In include:

In publication 8 described polymorphic forms of protein ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953. Polymorphic forms 961 described in publications 11 and 12. Any of these polymorphic forms can be used according to this invention.

Described in this description, the list of sequences includes a polymorphic form of protein 741 (SEQ ID 1-22) and NMB1343 (SEQ ID 23-24)that have been identified.

Serogroups and strains

Preferred proteins according to the invention contain the components of X having the amino acid sequence found in N. meningitidis serogroup C. In one protein according to the invention the individual components of X can be from one or more strains. For example, when n=2, X₂may be from the same strain that X₁or from a different strain. When n=3, the strains can be (i) X₁=X₂=X₃(ii) X₁=the ₂≠X₃, (iii) X₁≠X₂=X₃, (iv) X₁≠X₂≠X₃or (v) X₁=X₃≠X₂etc.

In serogroup In the preferred components of X is obtained from strain 2996, MS, 95N477 or 394/98. Strain 95N477 sometimes herein referred to as "ET", it electrophoretic type. Strain 394/98 sometimes herein referred to as the "nz", as it is a new Zealand strain.

In that case, when use the form 287, it is preferably obtained from strain 2996 or from strain 394/98.

In that case, when use the form 741, preferably it is derived from strains of serogroup IN MS, 2996, 394/98 or 95N477 or from a strain of serogroup With 90/18311.

In that case, when use the form 961, preferably it is derived from strain 2996.

Strains of the indicated subscript index, for example, 741_MSmean protein 741 from strain MS. Unless otherwise noted, are specified in the description of proteins (for example, without a subscript) from strain 2996 N. meningitidis, which can be adopted as the reference strain. However, it will be understood that the invention is not limited to the strain. As stated above, it can be assumed that a General reference to a protein (e.g., "287", "919", etc.) includes such protein from any strain. Usually it will have a sequence identity with 2996 equal to 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Bo or is it).

Based on the expression domains of the protein 961

In publications 1 and 2 are described, as can be divided protein domains and how you can make manipulation of the protein on the basis of the specified domains. In this invention extended the application of this method in relation to protein 961 (also known as "NadA" [11, 12]).

In strain 2996 N. meningitidis serogroup In NadA contains 405 amino acids. This protein is divided into the following nine domains (figure 4):

Listed in the ormatio can be used for localization of similar domains in other forms 961.

These domains have been delegated 961 strain 2996 different ways (figure 5). In a preferred fragments 961 excluded one or more of these nine domains, for example, the following:

- 961-2 on 961-5 ("a")

with 961-6 on 961-9 ("961b")

with 961-1 on 961-8 ("961cL")

- 961-2 on 961-8 ("s")

- 961-2 on 961-6 and amino acids 287-325 from the domain 961-7 ("961d")

- 961-2 on 961-8 and amino acids 351-383 from the domain 961-9 ("961Δ1")

with 961-1 on 961-8 and amino acids 351-383 from the domain 961-9 ("961Δ1L")

with 961-1 on 961-7 and amino acids 331-343 from the domain 961-8 ("961cL-Δaro")

with 961-1 on 961-6 and amino acids 287-315 from the domain 961-7 ("961cL-Δcc")

with 961-1 on 961-5 ("961aL")

with 961-1 on 961-4 ("961aL-Δ1")

with 961-1 on 961-3 ("961aL-Δ2")

with 961-1 on 961-2 ("961aL-Δ3")

These thirteen fragments (and their sub-segments, lacking 1, 2, 3, 4, or 5 amino acids at either end or at both ends) are preferred (C)and (j)-fragments, but they can also be expressed themselves, i.e. not in the form of a hybrid protein according to the invention. Thus, the invention relates to a protein that contains one of these fragments, provided that the protein is not full-961 and is not a protein, specifically described in publications 1 or 2. This protein can be fused protein (e.g., by fusion with GST or merge with His-tag).

Sequence

The invention is also relative to the Xia to the protein, having the amino acid sequence of SEQ ID 1-24. The invention also relates to proteins and nucleic acid having a sequence that is identical to the specified. As described above, the degree of identity of the sequence preferably above 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or above).

The invention also relates to a nucleic acid encoding such proteins.

In addition, the invention relates to nucleic acid, which can gibridizatsiya with this nucleic acid, preferably under conditions of "high stringency" (for example, at 65°C in a solution of 0.1×SSC and 0.5% SDS).

The invention also relates to a nucleic acid that encodes a protein according to the invention.

It should be understood that the invention relates to nucleic acid containing the sequence complementary to the sequences described above (for example, used as antisense or probes).

Nucleic acid according to the invention, of course, can be obtained in a variety of ways (for example, by chemical synthesis, from genomic libraries or cDNA, from the organism itself, and so on), and it can take different forms (for example, single-stranded, Dunaeva, vectors, probes etc).

In addition, the term "nucleic acid" includes DNA and RNA, as well as their analogues, such as analogs, stereoselectively skeletons, and peptidoglycan acid (NCP), etc.

Mixture

The invention also relates to compositions containing two or more (e.g., 2, 3, 4, 5, 6 or 7) of the following proteins:

(1) 287

(2) 741

(3) ORF46.1

(4) 961

(5) NH₂-A-[-X-L-]_n-B-COOH, where n=2, X₁=287, X₂=953

(6) NH₂-A-[-X-L-]_n-B-COOH, where n=2, X₁=287, X₂=919

(7) NH₂-A-[-X-L-]_n-B-COOH, where n=2, X₁=287, X₂=961

The mixture may contain one or both of the following proteins or in combination with two or more proteins (1)to(7), or in combination with one of the proteins(1)-(7):

(8) NH₂-A-[-X-L-]_n-B-COOH, where n=2, X₁=287, X₂=741

(9) NH₂-A-[-X-L-]_n-B-COOH, where n=2, X₁=936, X₂=741

In the case when a mixture of proteins included 287 and 741 (i.e. in the case of protein 1, 2, 5, 6, 7 or 8), they can be in the form of "ΔG". In the case when the protein 961, it is preferably in the form of "s"that lacks the N-terminal leader and C-terminal membrane anchor [for example, see references 1, 2 and 11].

A preferred mixture contains the following three protein:

(1) s, preferably 961_s(for example, SEQ ID NO:31, described in this description);

(2) NH₂-A-[-X-L-]_n-B-COOH, where n is 2, X₁means ΔG287 (preferably ΔG287_NZ), -X₂means 953 (preferably 953₂₉₉₆)that does not contain its learnovation, -L₁means GSGGGG and-A - contains the N-terminal methionine (for example, -A - is M or MA) (e.g., SEQ ID NO:28 and 29 shown in this description); and

(3) NH₂-A-[-X-L-]_n-B-COOH, where n=2, X₁=936 (preferably 936₂₉₉₆), X₂=ΔG741 (preferably ΔG741_MC58), L₁=GSGGGG (for example, SEQ ID NO:30, described in this description).

The mixture can also contain bubbles of the outer membrane of N. meningitidis.

Heterologous host

Although the expression of the proteins according to the invention can take place in Neisseria, in this invention preferably uses a heterologous host. Heterologous host may be prokaryotic (e.g. bacteria) or eukaryotic. Preferred host is E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g., M. tuberculosis), yeast, etc.

Vectors and other

The invention relates to (a) nucleic acid encoding the above proteins, (b) the vectors containing these nucleic acid sequences, (C) cell host containing these vectors, (d) compositions containing proteins or nucleic acids according to the invention, which may be suitable as immunogenic compositions (e.g., vaccines) or as diagnostic reagents, (e) the specified comp is the positions for use as a drug (for example, as vaccines) or as diagnostic reagents, (f) the application of such compositions in the production of (1) a medicinal product for the treatment or prevention of infections caused by bacteria, neisseriae, (2) a diagnostic reagent to detect the presence of bacteria Neisseria or antibodies produced against bacteria Neisseria, and/or (3) reagent, which can cause the formation of antibodies against bacteria Neisseria, and (g) the method of treatment of a patient, introducing a patient a therapeutically effective amount of the above compositions.

The implementation of the invention typically will include basic stages: obtaining a first nucleic acid that encodes a first protein; obtaining a second nucleic acid that encodes a second protein; and ligation of the first and second nucleic acids. The resulting nucleic acid can be embedded in expressing vector, or it may already be part of an expressing vector.

To improve solubility, purification of hybrid proteins may include methods of refolding described in this application.

Immunogenic compositions and medicaments

Compositions according to the invention are preferably immunogenic compositions and more preferably vaccine compositions. The pH value to which the position is preferably from 6 to 7. the pH can be maintained using a buffer. The composition may be sterile.

Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but, as a rule, will be preventive.

The invention also relates to compositions according to the invention for use as a drug. The drug is preferably able to induce an immune response in a mammal (i.e. is immunogenic composition) and most preferably is a vaccine.

The invention also relates to the use of a composition according to the invention in the manufacture of medicines that causes an immune response in a mammal. The drug is preferably a vaccine.

The invention also relates to a method allowing to induce an immune response in a mammal, comprising the stage of introducing an effective amount of the composition according to the invention. The immune response is preferably protective. The method can cause secondary immune response.

Mammal, preferably a human being is. In the case where the vaccine is for prophylactic use, the human is preferably a child (for example, a child who anego or younger); in the case where the vaccine is for therapeutic use, the human is preferably adults. The vaccine is intended for children, you can also enter an adult, for example, to evaluate the safety, dose, immunogenicity, etc.

These applications and methods are preferably for the prevention and/or treatment of a disease caused by Neisseria (e.g., meningitis, blood poisoning, gonorrhoea etc). The preferred prophylaxis and/or treatment of bacterial meningitis.

Additional components of the composition

The composition according to the invention, as a rule, in addition to the above components will contain one or more "pharmaceutically acceptable carriers", which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids, trehalose (WO 00/56365) and lipid aggregate (such as oil droplets or liposomes). Such carriers are well known to specialists in this field. Vaccines may also contain diluents, such as water, saline, glycerol, etc. On the Executive may be present excipients, such as moisturizing or emulsifying agents, buffering agents to maintain pH, and the like. A detailed discussion of pharmaceutically acceptable excipients is available in Remington''s Pharmaceutical Sciences.

Immunogenic compositions used as vaccines contain immunologically effective amount of the antigen, and optionally any other of the above components. By "immunologically effective amount" is meant that the introduction of this quantity to the individual either in a single dose or as part of a series of doses is effective for treatment or prevention. This number varies depending on the health status and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g., Primate, non-human, Primate, etc), the ability of the immune system of an individual to synthesize antibodies, the degree of required protection, prepare drugs, vaccines, evaluation by the attending physician medical case and other factors that have to do with it. It is expected that the number will be in a relatively wide range, which can be determined by the usual tests. The dosage can be a scheme based on a single dose or scheme used is Itanium multiple doses (e.g., including repeated doses). The vaccine can be administered together with other immunoregulatory agents.

The composition may contain other adjuvants in addition to (or instead of) aluminium salts. Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) emulsion preparations of type oil-in-water (in the presence or without other specific immunostimulating agents such as muramylpeptide (see below) or components of the cell wall of bacteria), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in reference 13), containing 5% Squalene, 0.5% tween 80, and 0.5% span 85 (optionally containing MTP-PE), prepared in the form of submicron particles using microfluidizer, (b) SAF, containing 10% Squalane, 0.4% tween 80, 5% of a block copolymer of polyoxyethylene and polyoxypropylene L121, and thr-MDP, either microfluidized to submicron emulsion or subjected to shaking for the formation of an emulsion with particles of a larger size, and (C) adjuvant system Ribi™ (RAS) (Ribi Immunochem, Hamilton, MT)containing 2% Squalene, 0.2% tween 80, and one or more components of the cell wall of bacteria from the group consisting of monophosphorylated A (MPL), dimycolate trehalose (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (2) you can use adjuvants on the basis of saponins such as QS21 or Stimulon™ (Cambridge Bioscience, Worceter, MA), or particles that are created on their basis, such as ISCOM (immunostimulating complexes), and these ISCOM may not contain additional detergent, for example, WO 00/07621; (3) complete adjuvant's adjuvant (CFA) and incomplete adjuvant's adjuvant (IFA); (4) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO 99/44636), and so on), interferons (e.g.gamma-interferon), colony stimulating factor, macrophage (M-CSF), tumor necrosis factor (TNF), etc; (5) monophosphorylated A (MPL) or 3-O-describeany MPL (3dMPL), for example, GB-2220221, EP-A-0689454; (6) combinations of 3dMPL with, for example, QS21 and/or emulsions of the type oil-in-water", for example, EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides containing CpG-motifs [Krieg, Vaccine 2000, 19, 618-622; Krieg Curr. opin. Mol. Ther. 2001, 3: 15-24; Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J. Immunol., 1996, 156, 4570-4575; Halpern et al., Cell. Immunol., 1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messina et al., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998, 160, 5898-5906; international applications for patents WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581], i.e. containing at least Odie the dinucleotide CG, when this optional instead of cytosine using 5-methylcytosine; (8) simple polyoxyethylene ether or complex polyoxyethylene ether, for example, WO 99/52549; (9) surfactant ester of polyoxyethylenesorbitan in combination with octoxynol (for example, WO 01/21207) or surfactant simple or complex broadcast polyoxyethyleneglycol in combination, at least one additional nonionic surface-active agent, such as an octoxynol (for example, WO 01/21152); (10) immune-stimulating oligonucleotide (e.g., a CpG oligonucleotide) and a saponin, for example WO 00/62800; (11) an immunostimulant and a particle of metal salt, for example, WO 00/23105; (12) a saponin and an emulsion of the type oil-in-water", for example, WO 99/11241; (13) a saponin (e.g., QS21)+3dMPL+IL-12 (optionally+a Sterol), for example, WO 98/57659; (14) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

Muramylpeptide include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-[1',2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)ethylamine (MTP-PE), etc.

Additional antigens

Additional antigens that can be included in the composition according to the invention include:

- preparation of bubbles outside of memb the Ana (OMV) of N. meningitidis serogroup B, such as drugs, described in references 14, 15, 16, 17, etc.,

- charigny antigen from N. meningitidis serogroup a, C, W135 and/or Y, such as an oligosaccharide described in the publication 18, of serogroup C [see also reference 19], or oligosaccharides described in the publication 20,

- charigny antigen from Streptococcus pneumoniae [e.g., references 21, 22, 23],

- protein antigen from Helicobacter pylori such as Garden [e.g., 24], VacA [e.g., 24], NAP [e.g. 25], Nor [e.g., 26], HopY [e.g., 26] and/or urease,

- antigen hepatitis a virus, such as inactivated virus [e.g., 27, 28],

- antigen hepatitis b virus, such as surface and/or core antigens [e.g., 28, 29],

- antigen hepatitis C virus [e.g. 30],

- an antigen from Bordetella pertussis, such as holotoxin pertussis (RT) and filament hemagglutinin (FHA) Century pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [for example, the links 31 and 32],

- diphtheria antigen, such as diphtheria toxoid [e.g., Chapter 3, reference 33], for example, the mutant CRM₁₉₇[e.g., 34],

- antigen tetanus, such as toxoid tetanus [for example, Chapter 4 in reference 33],

- charigny antigen from Haemophilus influenzae [e.g., 19],

- an antigen from N. gonorrhoeae [e.g., 3, 4, 5],</ p>

- an antigen from Chlamydia pneumoniae [e.g., 35, 36, 37, 38, 39, 40, 41],</ p>

- an antigen from Chlamydia trachomatis [e.g., 42],

the antigen is C Porphyromonas gingivalis [for example, 43],

antigen(s) of poliovirus [e.g., 44, 45], such as IPV or OPV,

antigen(s) of rabies virus [e.g., 46], such as liofilizovannye inactivated virus [e.g., 47, RabAvert™],

- antigen measles, mumps and/or rubella [e.g., chapters 9, 10 and 11, reference 33],

antigen(s) of influenza [e.g., Chapter 19 in the link 33], such as surface proteins hemagglutinin and/or neuraminidase,

- an antigen from Moraxella catarrhalis [e.g., 48],

- protein antigen from Streptococcus agalactiae (group b Streptococcus) [e.g., 49, 50],

- charigny antigen from Streptococcus agalactiae,

- an antigen from Streptococcus pyogenes (group a Streptococcus) [e.g., 50, 51, 52],</ p>

- an antigen from Staphylococcus aureus [e.g., 53].

The composition may contain one or more of these additional antigens.

In that case, when using charigny or carbohydrate antigen, it is preferable anywhereman with protein carrier in order to increase the immunogenicity [for example, links 54-63]. Preferred protein-carriers are bacterial toxins or toxoid, such as toxoid diphtheria or tetanus. Toxoid diphtheria CRM₁₉₇it is especially preferred. Other suitable carrier proteins include protein outer membrane of N. meningitidis [for example, the link 64], synthetic peptides [e.g., 65, 66], heat shock proteins [e.g., 67], pertussis proteins [e.g. 68, 69], protein D from H. influenzae [for example, 70], toxin a or b from .difficile [e.g. 71], etc. In the case where the mixture contains capsular saccharides of both a-and C-serogroup, it is preferable that the ratio (wt./wt.) saccharide Mopa: saccharide MISP was more than 1 (for example, 2:1, 3:1, 4:1, 5:1, 10:1 or more). The saccharides from different serogroups of N. meningitidis can be konjugierte with the same or with different protein-carriers.

You can use any suitable conjugation reaction with any suitable linker if necessary.

Toxic protein antigens, if necessary, can be subjected to detoxification (e.g., detoxification of pertussis toxin chemical and/or genetic methods [32]).

In that case, when the composition is included diphtheria antigen, it is also preferable to include the antigen tetanus and pertussis antigens. Similarly, when the antigen tetanus, preferably also include antigens diphtheria and pertussis. Similarly, when the pertussis antigen, preferably also include antigens diphtheria and tetanus.

The antigens are preferably mixed (and more preferably adsorb) salt of aluminum (for example, phosphate, hydroxide, hydroxyphosphate, oxyhydroxides, orthophosphate, sulfate). Salt can take any suitable form (e.g. gel, crystalline,amorphous, etc).

Antigens in the composition generally will be present in a concentration of at least 1 μg/ml each. In General, the concentration of any given antigen should be sufficient to induce an immune response against this antigen.

Alternatively, the use of protein antigens in the compositions according to the invention can be used nucleic acid encoding the antigen [for example, links 72-80]. Thus, the protein components of the compositions according to the invention can be replaced by nucleic acid (preferably DNA, for example, in the form of a plasmid, which encodes a protein.

Definition

The term "comprising" means "including"and "comprising", for example, a composition "comprising" X may consist exclusively of X or may include something additional, for example, X+y

The term "about" in relation to a numerical value x means, for example, x±10%.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the alignment of twenty-three sequences of protein 741. Sequences are SEQ ID 1-22 plus a sequence of MS.

Figure 2 shows the sequence alignment NMB1343 of the gonococcus (top; SEQ ID NO:25) and meningococcus (bottom; SEQ ID NO:26).

Figure 3 shows the hybrid and tandem proteins according to the invention.

N the figure 4 shows the 9 domains in 961 ₂₉₉₆and figure 5 shows how the manipulation was subjected domains.

Embodiments of the INVENTIONS

Hybrid proteins - X₁=ΔG287

In addition to the proteins described in publications 1 and 2, designed seven hybrid proteins with ΔG287 from strain 2996 N-end. Also received eight tandem proteins 287 (see below).

To these proteins were added adjuvant or adjuvant's adjuvant (FCA)or 3 mg/ml alum and used for immunization of mice. The resulting sera were tested against different strains of Neisseria using bactericidal assay. Titles when using protein No. 3 were as follows:

In the following experiments using protein No. 3 with the addition of adjuvant aluminum hydroxide, the titers of anti-287 and anti-741 in ELISA, each of which exceeded the titles 984150 and BSA, were as follows:

The following results were obtained after immunization with proteins, are described in publications 1 and 2, tested against the homologous strain:

Hybrid proteins - X₁=S or 961cL

In addition to the proteins described in publications 1 and 2, designed eight hybrid proteins or s or 961cL (i.e. s+leader peptide) at the N-end:

To these proteins were added adjuvant or adjuvant's adjuvant (FCA)or 3 mg/ml alum and used for immunization of mice. The resulting sera were tested against different strains of Neisseria using bactericidal assay. Titles when using protein No. 8 were as follows;

The titers obtained after immunization s-741 [references 1 and 2], were as follows:

These results can be had is locsite by mixing s-741 with ORF46.1 or ΔG287-919.

Got the following results after immunization with proteins, are described in publications 1 and 2, tested against the homologous strain:

Hybrid proteins - X₁=ORF46.1

To these proteins were added adjuvant or adjuvant's adjuvant (FCA)or 3 mg/ml alum and used for immunization of mice. The resulting serum was tested against the homologous strain, using bactericidal analysis and ELISA.

The following results were obtained after immunization with proteins, are described in publications 1 and 2:

Hybrid protein - X₁=230

In addition to the proteins described in publications 1 and 2, designed four hybrid protein from 230 to the N-end:

Hybrid protein - X₁=936

In addition to the proteins described in publications 1 and 2, designed seven hybrid proteins with 936 M-end:

To these proteins were added adjuvant or adjuvant's adjuvant (FCA)or 3 mg/ml alum and used for immunization of mice. The resulting sera were tested against different strains of Neisseria using bactericidal assay. Titles when using protein No. 2 were as follows:

Titles when using protein No. 4 were as follows:

Titles when using protein No. 7 were as follows:

The following results were obtained after immunization with proteins, are described in publications 1 and 2, tested against the homologous strain:

A mixture of hybrid proteins

Mice were immunized three proteins with the addition of adjuvant aluminum hydroxide, either alone or in triple combination: (1) 287_NZ-953; (2) 936-741 and (3) s. The mixture had the ability to induce high bactericidal titers against different strains:

Monitoring individual mice is shown that the mixture induced a high and persistent bactericidal titres:

Tandem proteins

Hybrid proteins according to the invention can be represented by the formula NH₂-[-X-L-]_n-COOH. When-X - all n cases is one and the same basic protein (either identical or the same protein from different strains or species), a protein called "tandem" protein.

Twelve specific tandem proteins:

All proteins No. 1-No. 5 expressibility in a soluble form in E. coli. The expression levels ranged from 0.24 to 0.50 mg of protein per liter of culture. The tandem of the s protein was purified and mixed with aluminum phosphate as adjuvant. Tandem proteins No. 2, No. 4 and No. 5 were easily adsorbiroval on the aluminum phosphate; adsorption was less complete in the case of tandem protein No. 1 and No. 3.

Allelic variants - 741

Found twenty-two polymorphic sequence 741 (SEQ ID 1-22). These sequences and the sequence S aligned in figure 1.

Allelic variants - NMB1343

Using PCR 42 strains of meningococci of different serogroups gene that encodes a protein NMB1343 found in 24/42 strains and absent in 18/42 strains (table 1). Gene NMB1343 sequenced for 10 strains NMB1343⁺(table 1, column 3). The nucleic acid sequence (and hence the amino acid sequence of SEQ ID NO:23; GenBank AAF41718) identical in all 10 strains.

NMB1343 also detected in two strains of N. gonorrhoeae (F62 and SN4). Amino acid sequence of the gonococcus represents SEQ ID NO:24. Alignment with the sequence of meningococcus:

The appropriate alignment of the nucleotide sequences shown in figure 2. The alignment shows that the sequence of gonococci has a 4-dimensional insertions in the 5'region of the gene NMB1343, which causes a shift in the reading frame and, as a consequence, the loss of the 5'-residue of methionine.

Deletion of domain - 961

961 is not present in the genomic sequence of N. meningitidis serogroup a, [81], although the surrounding area is conservative (> 90%) in the serogroups a and B. In publications 11 and 12 described polymorphic forms 961. Detected that the gene is present in 91% of the strains of serogroup specific to hypervirulent lines ET-5, ET-37 and cluster A4, but absent in all tested strains of line 3. Most of the tested strains of serogroup were positive, although not related to hypervirulent lines. The same is true for strains of serogroup b serotype 2A and 2b. In the case of serogroup one strain belonging to subgroup III, was positive, whereas the other two strains belonging to subgroup IV-1 were negative. 961 was absent in N. gonorrhoeae and species-commensals N. lactamica and N. cinerea.

Figs.4 and 5 show the domains in the protein 961.

When the anchor region (domain 9) ("961cL") in the protein 961 delegated and the protein expressed in E. coli, it is exported in periplasm and secreted in adosados culture.

To explore this protein is further designed deletion mutants in the C-terminal region 961 (961cL-Δaro, 961cL-Δcc, 961aL, 961aL-Δ1, 961aL-Δ2 961aL-Δ3) on the basis of structural parameters (deletions aromatic residues in the cases of mutant s-Δaro and regions of the double helix in other cases). The mutants were analyzed in relation to the expression and secretion in periplasm and adosados culture. In all cases, these deletion mutant protein is produced in large quantities,present in periplasmatic faction and released in adosados culture.

ΔG287 - cross-bactericidal activity between strains

Cloned 287 for five different strains of N. meningitidis serogroup b and subjected to manipulation, to delegate the N-end until the end of polyglycerol field and enter C-terminal his-tag. Received five proteins ΔG287. To proteins was added adjuvant FCA and used to stimulate the formation of immune sera in mice, which are then tested against bactericidal activity against all five strains of serogroup B, and also against strains of serogroup a and C. Received the following bactericidal titres:

Refolding

To increase the levels of soluble protein for some hybrid proteins, chose alternative protocols refolding compared to the protocols described in publication 2.

Intracellular Taurus (IB) was allocated as follows:

1. Homogenized cells (5 g wet weight) in 25 ml,1 M Tris-HCl, pH 7, 1 mm EDTA at 4°C using an Ultraturrax homogenizer (10,000 rpm).

2. Added 1.5 mg of lysozyme per gram of cells, quickly mixed using Ultraturrax, and incubated at 4°C for 30 minutes

3. Used sonication or homogenization at high pressure (French press)to destroy the cells.

4. To cleave DNA, was added MgCl₂to a final concentration of 3 mm and Tenkasu to a final concentration of 10 μg/ml and incubated for 30 min at 25°C.

5. To the solution was added 0.5 to about. 60 mm EDTA, 6% Triton X-100, 1.5 m NaCl, pH 7, and incubated for 30 min at 4°C.

6. Besieged intracellular calf by centrifugation at 31000×g (20,000 rpm) for 10 min, 4°C.

7. Resuspendable residue in 40 ml of 0.1 M Tris-HCl, pH 7, 20 mm EDTA, using Ultraturrax.

8. Repeated stage centrifugation 6.

9. The precipitate intracellular Taurus can be used or stored frozen at -20°C.

Hybrid proteins expressed in E. coli as follows:

Precipitation was dissolved, was subjected to refolding, was ultrafiltrable, were dialyzed and then the protein was purified:

ORF46.1-961-His. IB was dissolved in the following way: proteins IB resuspendable in 4 ml of buffer containing 6 M guanidine-HCl, 1 mm EDTA, pH 8.5, to a final protein concentration of 1 mg/ml to expose the protein refolding, 2 ml of the dissolved protein was diluted in 400 ml of buffer for refolding (0.1 M Tris-HCl, 1 M L-arginine, 2 mm EDTA, pH of 8.2) and incubated for 1 hour at 15°C, resulting in the protein concentration of 5 μg/ml was Then added 2 ml of the dissolved protein and incubated for hours at the same temperature, produces the resulting final protein concentration of 10 ág/ml The product was subjected to ultrafiltration using a cell ultrafiltration on Amicon 300 ml (8400), using a pressure of 3 bars on the Amicon membrane with a cut-off of 30 KD (YM30), receiving the final volume of 130 ml of the Product obtained after ultrafiltration, were dialyzed using a membrane made of regenerated cellulose in the form of a tube with a cut-off of 12-14 KD (Cellusep - Step bio), within 24 hours against 10 l of 0.1 M buffer Tris-HCl, pH of 8.2. Performed the second dialysis for 24 h against 10 l of buffer containing 300 mm NaCl, 50 mm sodium phosphate, pH 8.0. Otvetsvennyy product was centrifuged at 22,000 rpm for 45 minutes at 4°C in a centrifuge rotor Beckman JA25.5. Adosados allocated after centrifugation was used for purification on the basis of the His-tag.

orf46.1-961c-His. IB was dissolved in the following way: proteins IB resuspendable in 4 ml of buffer containing 6 M guanidine-HCl, 1 mm EDTA, pH 8.5, to a final protein concentration of 1 mg/ml to expose the protein refolding, 2 ml of the dissolved protein was diluted in 400 ml of buffer for refolding (0.5 M Tris-HCl, 1 M L-arginine, 2 mm EDTA, pH of 8.2) and incubated for 1 hour at 15°C, resulting in the protein concentration of 5 μg/ml was Then added 2 ml of the dissolved protein and incubated for hours at the same temperature, receiving a final protein concentration of 10 ág/ml of the Product was subjected to ultrafiltration using a cell d is I Amicon ultrafiltration 300 ml (8400), using a pressure of 3 bars on the Amicon membrane with a cut-off of 30 KD (YM30), receiving the final volume of 150 ml Product obtained after ultrafiltration, were dialyzed using a membrane made of regenerated cellulose in the form of a tube with a cut-off of 12-14 KD (Cellusep - Step bio), within 24 hours against 10 l of 0.1 M buffer Tris-HCl, pH of 8.2. Performed the second dialysis for 24 h against 10 l of buffer containing 300 mm NaCl, 50 mm sodium phosphate, pH 8.0. Otvetsvennyy product was centrifuged at 22,000 rpm for 45 minutes at 4°C in a centrifuge rotor Beckman JA25.5. Adosados allocated after centrifugation was used for purification on the basis of the His-tag.

961c-orf46.1-His. IB was dissolved in the following way: proteins IB resuspendable in 4 ml of buffer containing 6 M guanidine-HCl, 1 mm EDTA, pH 8.5, to a final protein concentration of 1 mg/ml to expose the protein refolding, 2 ml of the dissolved protein was diluted in 400 ml of buffer for refolding (0.1 M Tris-HCl, 0.5 M L-arginine, 2 mm EDTA, pH of 8.2) and incubated for 1 hour at 15°C, obtaining a protein concentration of 5 μg/ml was Then added 2 ml of the dissolved protein and incubated in an hour at the same temperature, resulting in the final protein concentration of 10 ág/ml of the Product was subjected to ultrafiltration using a cell ultrafiltration on Amicon 300 ml (8400), using a pressure of 3 bars on the Amicon membrane with cut off the eat 30 KD (YM30), receiving the final volume of 150 ml Product obtained after ultrafiltration, were dialyzed using a membrane made of regenerated cellulose in the form of a tube with a cut-off of 12-14 KD (Cellusep - Step bio), within 24 hours against 10 l of 0.1 M buffer Tris-HCl, pH of 8.2. Performed the second dialysis for 24 h against 10 l of buffer containing 300 mm NaCl, 50 mm sodium phosphate, pH 8.0. Otvetsvennyy product was centrifuged at 22,000 rpm for 45 minutes at 4°C in a centrifuge rotor Beckman JA25.5. Adosados allocated after centrifugation was used for purification on the basis of the His-tag.

orf46.1-741-His. IB was dissolved in the following way: proteins IB resuspendable in 4 ml of buffer containing 6 M guanidine-HCl, 1 mm EDTA, pH 8.5, to a final protein concentration of 10 mg/ml to expose the protein refolding, 2 ml of the dissolved protein was diluted in 400 ml of buffer for refolding (0.5 M Tris-HCl, 0.7 M L-arginine, 2 mm EDTA, pH of 7.2), and incubated for 1 hour at 15°C, resulting in the protein concentration of 50 μg/ml was Then added 2 ml of the dissolved protein and incubated for hours at the same temperature, resulting in the final protein concentration of 100 μg/ml of the Product was subjected to ultrafiltration using a cell ultrafiltration on Amicon 300 ml (8400), using a pressure of 3 bars on the Amicon membrane with a cut-off of 30 KD (YM30), receiving the final volume of 10 ml The product obtained after ultrafiltration, were dialyzed using a membrane made of regenerated cellulose in the form of a tube with a cut-off of 12-14 KD (Cellusep - Step bio), within 24 hours against 10 l of 0.1 M buffer Tris-HCl, pH of 8.2. Performed the second dialysis for 24 h against 10 l of buffer containing 300 mm NaCl, 50 mm sodium phosphate, pH 8.0. Otvetsvennyy product was centrifuged at 22,000 rpm for 45 minutes at 4°C in a centrifuge rotor Beckman JA25.5. Adosados allocated after centrifugation was used for purification on the basis of the His-tag.

When compared with the protein, purified as described in publication 2, got the following titles in bactericidal analysis:

Similar methods were used in the case ORF46.1 to purify protein from IB when he expressively without His-tag ("ORF46.1K"):

Proteins IB resuspendable in 4 ml of buffer containing 6 M guanidine-HCl, 1 mm EDTA, pH 8.5, to a final protein concentration of 10 mg/ml to expose the protein refolding, 2 ml of the dissolved protein razbavlyali 400 ml of buffer for refolding (0.5 M Tris-HCl, 0.7 M L-arginine, 2 mm EDTA, pH of 7.2), and incubated for 1 hour at 15°C, resulting in the protein concentration of 50 μg/ml was Then added 2 ml of the dissolved protein and incubated for another hour at the same temperature, receiving a final protein concentration of 100 μg/ml of the Product was subjected to ultrafiltration using a cell ultrafiltration on Amicon 300 ml (8400), using a pressure of 3 bars on the Amicon membrane with a cut-off of 30 KD (YM30), receiving the final volume of 120 ml Product obtained after ultrafiltration, were dialyzed using a membrane made of regenerated cellulose in the form of a tube with a cut-off of 12-14 KD (Cellusep - Step bio), within 12 hours against 10 l of buffer containing 50 mm sodium phosphate, 2 mm EDTA, pH to 7.2. Performed the second dialysis for 24 h against 10 l of the same buffer. Otvetsvennyy product was centrifuged at 22,000 rpm for 45 minutes at 4°C in a centrifuge rotor Beckman JA25.5. Adosados allocated after centrifugation was used for cation exchange chromatography. Purification was carried out in the system of the chromatographic analyzer INSTRUMENT (Amersham-Pharmacia Biotech), using a column of HP separate HiTrap SP a volume of 5 ml (Amersham-Pharmacia Biotech). The applied flow rate was 1.5 ml per minute. The column was washed with 35 ml of 50 mm sodium phosphate buffer, pH 7,2. Created a linear gradient (0-1 M NaCl)using 50 mm sodium-FOSFA the private buffer, pH of 7.2. Protein was elyuirovaniya in two peaks at 92 mm and 380 mm NaCl. Fractions constituting each peak were combined and accordingly called pool 1 and pool 2.

When compared with the protein, purified as described in publication 2, the bactericidal titres analysis when used in the adjuvant aluminum hydroxide was increased from <4 to 1024. The titer when used as adjuvant phosphate of aluminum subjected to refolding protein was 2048. In ELISA titers were as follows:

It will be understood that the invention is described only as examples and can be implemented modifications while preserving the volume and usnot of the invention.

PUBLISHING

(the contents of which are incorporated in this description by reference)

1 - international application for a patent WO01/64 920.

2 - international application for a patent WO01/64922.

3 - international application for a patent W099/24578.

4 - international application for a patent W099/36544.

5 - international application for a patent WO99/57280.

6 - international application for a patent WO00/22430.

7 - Tettelin et al. (2000) Science 287:1809-1815.

8 - international application for a patent WO00/66741.

9 - international application for a patent WO00/71574.

10 - international application for a patent WO01/04316.

11 - international application for a patent PCT/IB02/03396.

12 - Comanducci et al. (2002) J Exp Med 195:1445-1454.

13 - Vaccine Design: the subunit & adjuvant approach (1995) Powell & Newman (ISBN: 030644867X).

14 - international application for a patent WO01/52885.

15 - Bjune et al. (1991) Lancet 338 (8775):1093-1096.

16 - Fukasawa et al. (1999) Vaccine 17:2 951-2 958.

17 - Rosenqvist et al. (1998) Dev Biol. Stand. 92:323-333.

18 - Costantino et al. (1992) Vaccine 10:691-698.

19 - Costantino et al. (1999) Vaccine 17:1251-1263.

20 - international application for a patent PCT/IB02/03191.

21 - Watson (2000) Pediatr Infect Dis J 19:331-332.

22 - Rubin (2000) Pediatr Clin North Am 47:269-285, v.

23 - Jedrzejas (2001) Environ Mol Biol Rev 65:187-207.

24 - international application for a patent WO93/18150.

25 - international application for a patent WO99/53310.

26 - international application for a patent WO98/04702.

27 - Bell (2000) Pediatr Infect Dis J 19:1187-1188.

28 - Iwarson (1995) APMIS 103:321-326.

29 - Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.

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Beloc for use for prevention and/or treatment of bacterial meningitis, characterized by:
(a) amino acid sequence of SEQ ID NO:19; or
(b) amino acid sequence with 80% or more identical to the amino acid sequence of SEQ ID NO:19.

2. Composition for prevention and/or treatment of bacterial meningitis containing protein according to claim 1 and a pharmaceutically acceptable carrier.

3. The composition according to claim 2, additionally containing adjuvant.

4. The composition according to claim 2, where the protein is adsorbed onto aluminium salts.

5. The composition according to claim 4, where the aluminium salt is a phosphate of aluminum.

6. The composition according to claim 2, additionally containing buffer.