Neisseria meningitidis polypeptides

FIELD: medicine.

SUBSTANCE: polypeptide (versions) immunogenic with respect to meningococcal infections contains: an amino acid sequence at least 90 % identical to a sequence presented in the description (SEQ ID NO: 32), or said amino acid sequence, or a fragment of 80 sequenced amino acids of said sequence. What is described is an antibody which contacts with the polypeptide under the invention and which may be used as a drug. What is described is nucleic acid of the preset structure which codes the polypeptide or its versions and which may be used for treating or preventing a disease and/or an infection caused by Neisseria meningitides. The invention provides additional polypeptides applicable in advanced vaccines for preventing and/or treating meningococcal meningitis. The peptides can also find application in diagnosing of the disease and as targets of antibiotics.

EFFECT: higher clinical effectiveness for meningococcal meningitis.

19 cl, 2 tbl

 

All cited in the present description documents are fully incorporated in it by reference.

The technical FIELD

This invention relates to the field of researchNeisseria meningitidis.

BACKGROUND of the INVENTION

Neisseria meningitidis(the meningococcus) is fixed by gram-negative diplococcal pathogenic for humans. These bacteria colonise the mouth and cause meningitis (and, sometimes, septicaemia without meningitis).

All pathogenic meningococci have a polysaccharide capsule. These polysaccharides are the basis of available vaccines against serogroups of meningococcus A, C, W135 and Y, but these vaccines are not suitable for use against serogroup C. Therefore, an important task for researchers is to identify alternative antigens for immunization against meningococcal disease serogroup C. Such alternative antigens are proteins, lipopolysaccharides and outer membrane vesicles.

In references 1-7 disclosed various polypeptides derived from the sequence of the genome of Neisseria meningitides serogroup B, and in these links selected specific sequences for use in vaccines. The genome sequence for strain serogroup disclosed in reference 8.

Object of the invention is the provision of additional polypeptides for use in vaccine development for the prevention and/or is ecene meningococcal infections. In particular, the object of the invention is to provide polypeptides for use in improved vaccines for the prevention and/or treatment of meningococcal meningitis. The peptides can also be used in the diagnosis and be used as targets of action of antibiotics.

Description of the INVENTION

Polypeptides

The invention relates to polypeptides containing the amino acid sequence of meningococcus disclosed in the examples. These amino acid sequences in the list of sequences have even numbers, starting with SEQ ID NO 2 and to SEQ ID NO 78. Therefore, the above 39 amino acid sequences, called B269_nnwherennis a number from 01 to 50 (eleven rooms B269_nnno sequence: 02, 03, 04, 05, 06, 07, 08, 09, 10, 12 and 40). Two sequences (B and B269_37) are preferred.

The invention also relates to polypeptides containing amino acid sequences that are identical in sequence with the amino acid sequences of Neisseria meningitides, disclosed in the examples. Depending on the particular sequence, the degree of sequence identity, preferably is more than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). These polypeptides include d the logs orthologues, allelic variants and functional mutants. It is usually assumed that the functional similarity of proteins indicates the identity of 50% or more between two polypeptide sequences. For each sequence (SEQ ID) the degree of sequence identity, preferably, higher values in column (b), and in column (A), in table II of this description, and, more preferably, above all values in columns (C), (b) and (A) for this sequence.

Compared with meningococcal sequences from examples of these polypeptides can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.) conservative amino acid substitutions, i.e. substitutions of one amino acid by another amino acid, which has a related side chain. In General encoded in the genome of the amino acids are divided into four families: (1) acidic, namely aspartate, glutamate; (2) the principal, namely lysine, arginine, histidine; (3) non-polar, namely alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, namely glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Sometimes in the classification of phenylalanine, tryptophan and tyrosine unite as aromatic amino acids. Usually replacement of single amino acids on the other within these families are not substantially leaet on biological activity. These polypeptides may also be present one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.) single amino acid deletions relative to meningococcal sequences of examples. The polypeptides may also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.) insertions (e.g. each of the insert 1, 2, 3, 4, or 5 amino acids) relative to the meningococcal sequences of examples.

In addition, the invention relates to polypeptides containing fragments of meningococcal amino acid sequences disclosed in the examples. The records must include, at least,nconsecutive amino acids of the sequences, and, depending on the particular sequence,nis 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more).

A fragment may contain at least one T cell or, preferably, In-cell epitope of the sequence. T - and b-cell epitopes can be identified empirically (for example, using PEPSCAN [9,10] or similar methods), or they can be predicted (for example, using antigenic index-jameson-wolf [11], approaches based matrices [12], TEPITOPE [13], neural networks [14], OptiMer & EpiMer [15, 16], ADEPT [17], Tsites [18], hydrophilicity [19], antigenic index [20] or ways calorierestricted in reference 21, and so on). Other preferred fragments are (a) N-terminal signal peptides meningococcal polypeptide according to the invention, (b) meningococcal polypeptide, but without N-terminal signal peptide, (C) meningococcal polypeptide, but without their N-terminal amino acid residue.

The polypeptides according to the invention can be obtained in many ways, for example by chemical synthesis (fully or partially), by cleavage of longer peptides by proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expressing culture), from the organism itself (for example, after the bacterial culture or directly from the patient's body), etc. Preferred method of producing peptides less than 40 amino acids in length includes chemical synthesisin vitro[22, 23]. Especially preferred is a solid-phase peptide synthesis, for example, methods based on the use of reactions using tBoc or Fmoc [24]. You can also use enzymatic synthesis [25], partially or completely. Alternatively, chemical synthesis can be used for biological synthesis, for example, polypeptides can be obtained by translation. The broadcast can bein vitroorin vivo. Biological methods are usually limited to the production of polypetides consisting of L-amino acids, but you can use the with manipulation of the mechanism of translation (for example, the aminoacyl-tRNA molecules), to include D-amino acids (or other unnatural amino acids, such as iodotyrosine or methylphenylene, azithromiacin etc.) [26]. However, for inclusion of D-amino acids, it is preferable to use chemical synthesis. The polypeptides according to the invention can be covalent modification at the C-end and/or N-end.

The polypeptides according to the invention can take different forms (for example, a protein may be native, merged, glycosylated, deglycosylation, modified lipids, unmodified lipids, phosphorylated, nefosfaurilirovanna, monitorowanym, demeritorious, Monomeric, multimeric, disperse, denatured and so on).

Preferably, the polypeptides according to the invention were provided in purified or substantially purified form, i.e. essentially free from other polypeptides (e.g. free from natural polypeptides), particularly from other meningococcal polypeptide or polypeptide in the host cell; and purity which, in General, was, at least about 50% (by weight), and usually at least about 90%, i.e. less than about 50% and, more preferably, less than about 10% (e.g., 5%) composition was downregulation of other polypeptides. Preferred polypeptides of the invention t is Auda meningococcal polypeptide. Preferably, the polypeptides according to the invention have a function that is listed in table I for the corresponding sequence.

The polypeptides according to the invention can be attached to a solid substrate. The polypeptides according to the invention may include detektiruya label (e.g. a radioactive or fluorescent label, or bitenova label).

The term "polypeptide" refers to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and peptide sequences can be insert connections that are not amino acids. The term also encompasses amino acid polymer that has been modified in nature or according to the invention; for example, by the formation of a disulfide bond, glycosylation, addition of lipids, acetylation, phosphorylation, or other manipulation or modification, such as conjugation with bearing the label component. Also included in the definition, for example, polypeptides that contain one or more amino acid analogs (including, for example, unnatural amino acids, etc), as well as other modifications known in this field. The polypeptides may be present in the form of single circuits or associated circuits. The polypeptides according to the invention can be natural chap who kosiorowski or artificially glycosylated (i.e. the polypeptide of the location of sites of glycosylation differs from the location of glycosylation sites in the corresponding natural polypeptide).

The invention relates to polypeptides containing a sequence-X-Y - or-Y-X-, in which-X - represents an amino acid sequence as defined above, and Y is defined above by the sequence, i.e. the invention relates to fused proteins. If encoding the polypeptide sequence N-terminal codon is ATG, then this codon will be broadcast as a standard amino acid that the codon instead Met, if the codon is a start codon.

The invention relates to a method for producing a polypeptide according to the invention, comprising the stage of culturing host cells according to the invention under conditions that induce expression of the polypeptide.

The invention relates to a method for producing a polypeptide according to the invention, in which the synthesis of the polypeptide partially or completely carried out by chemical methods.

The invention relates to compositions comprising two or more polypeptides according to the invention.

The invention also relates to hybrid polypeptide represented by the formula NH2-A-[-X-L-]n-B-COOH, in which X is a polypeptide defined above, L is long the school linker amino acid sequence, A is an optional N-terminal amino acid sequence, b is an optional C-terminal amino acid sequence, andnis an integer greater than one. The value ofnis between 2 andxandxusually is 3, 4, 5, 6, 7, 8, 9 or 10. Preferably,thenis 2, 3 or 4; more preferably 2 or 3; most preferably,n-2. For eachnlink sequence-X-may be the same or different. For eachnlink [-X-L-] linker amino acid sequence of L may be present or absent. For example, whenn=2 the hybrid may be a 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 (amino acid linker sequence) -L - will typically be short (e.g., 20 or fewer amino acids, namely 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. Glynwheren= 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) and his-tag Tagi (i.e. Hisnwheren=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequence will be PTS is visible for specialists in this field. -A - and-B - are optional sequences, which will typically be short (e.g. 40 or fewer amino acids, namely 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, which direct traffic polypeptide, or a short peptide sequences which facilitate cloning or purification (e.g., his-tag tags, namely Hisnwheren= 3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal and C-terminal amino acid sequence will be obvious to specialists in this field.

To assess the immunogenicity of the polypeptides according to the inventionin vivoyou can apply different tests. For example, it is possible to obtain recombinant polypeptides using the expression and use them for screening sera of patients using Western blot turns. The positive reaction of the serum of the patient and of the polypeptide indicates that the patient has previously developed an immune response to the test protein, i.e. the protein is the immunogen. This method can also be used to identify immunodominant proteins.

Antibodies

The invention relates to antibodies that bind polypeptides according to the invention. They can be polyclonal or monoclonal, and may be obtained by any appropriate is ejstvujuschij means (i.e. recombinant expression). To increase compatibility with the human immune system antibodies may be chimeric or humanitarianism (see, for example, references 27 and 28) or you can use human antibodies. Antibodies may include detektiruya label (for example, antibodies for diagnostics). Antibodies according to the invention can be attached to a solid substrate. Preferred antibodies according to the invention are neutralizing antibodies.

In particular, monoclonal antibodies are suitable for identification and purification of individual polypeptides, against which they are directed. Monoclonal antibodies according to the invention can be used as reagents in the immune methods of analysis, radioimmunoassay methods of analysis (RIA) or enzyme-linked immunosorbent method of assay (ELISA), etc. In these applications, the antibodies can be marked analytically detektivami reagent, such as a radioactive isotope, a fluorescent molecule or an enzyme. Monoclonal antibodies obtained in the above manner, can also be used for molecular identification and characterization (epitope mapping) polypeptides according to the invention.

Preferably, antibodies of the invention were purified or substantially purified. Typically, the antibody will be present in the composition, which will, there is the free of other polypeptides, for example, when less than 90% (by weight), typically less than 60% or, most often, less than 50% of the composition consists of other polypeptides.

Antibodies according to the invention can belong to any isotype (e.g., IgA, IgG, IgM, i.e. heavy chain α, γ or µ), but, as a rule, are of IgG. In the framework of the IgG isotype antibodies can be subclasses IgG1, IgG2, IgG3 or IgG4. Antibodies according to the invention may have a light chain κ or λ.

But usually will be IgG. The isotype of IgG antibodies may belong to IgG1, IgG2, IgG3 or IgG4 subclasses. Antibodies according to the invention.

Antibodies according to the invention can have different shapes, including whole antibodies, antibody fragments such as F(ab')2 and F(ab)fragments, Fv fragments (the non-covalent heterodimer), single-chain antibodies such as single-chain Fv molecules (scFv), Manantial, oligoarticular etc. the Term "antibody" does not imply a specific source of origin and includes antibodies obtained in non-standard ways, such as phage display.

The invention relates to a method of detection of the polypeptides according to the invention, comprising the stage of: (a) the stage of contacting the antibody according to the invention with a biological way under conditions suitable for the formation of complexes of antigen-antibody; and (b) the stage of detection of these complexes.

The invention relates to a method of detection of polypeptides on izopet the tion, includes stages: (a) the stage of contacting the antibody according to the invention with a biological way (for example, a sample of blood or serum) under conditions suitable for the formation of complexes of antigen-antibody; and (b) the stage of detection of these complexes.

Nucleic acids

The invention relates to a nucleic acid comprising the nucleotide sequence of meningococcus disclosed in the examples. These nucleotide sequences are SEQ ID NO odd numbers from 1 to 77.

The invention relates to nucleic acid containing a nucleotide sequence homologous meningococcal nucleotide sequences disclosed in the examples.

The invention relates to nucleic acid, which can gibridizatsiya with nucleic acid disclosed in the examples. Hybridization can be performed under conditions with varying degrees of "hardness". Increased stiffness hybridization conditions are well known and published in the literature [for example, see p. 7.52 in reference 29]. Examples of relevant conditions include (in order of increasing stringency): incubation temperature 25°C, 37°C, 50°C, 55°C and 68°C; concentration of the buffer 10 × SSC, 6 × SSC, 1 × SSC with 0.1 × SSC (where SSC is a 0.15 M NaCl and 15 mm citrate buffer) and their equivalents using other buffer systems; is concentratie of formamide 0%, 25%, 50% and 75%; incubation time from 5 minutes to 24 hours; 1, 2 or more stages of washing; laundering and solutions of 6 × SSC, 1 × SSC with 0.1 × SSC, or deionized water. Methods of hybridization and their optimization are well known in this area [e.g., see references 29-32, etc.].

In some embodiments, the implementation of the nucleic acid according to the invention hybridizes with the target according to the invention under mild conditions of hybridization; in other variants of the invention it hybridizes under average conditions of hybridization; in preferred embodiments of the invention it hybridizes under severe conditions. An example of a mild hybridization conditions are 50°C. and 10 × SSC. Example of average conditions for hybridization are 55°C and 1 × SSC. An example of stringent hybridization conditions are 68°C. and 0.1 × SSC.

The invention relates to nucleic acid containing fragments of these sequences. They must contain at leastnconsecutive nucleotides of meningococcal sequences and, depending on the particular sequence,nis 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).

The invention relates to nucleic acid with the formula 5'-X-Y-Z-3', in which-X - is a nucleotide sequence consisting ofxnucleotides; -Z - is nucleate the Noah sequence, consisting ofznucleotides; -Y - is a nucleotide sequence consisting of: or (a) fragment of one of SEQ ID NO odd number from 1 to 77, or (b) sequences complementary to (a); and the specified nucleic acid 5'-X-Y-Z-3' is neither (i) a fragment of one of SEQ ID NO odd number from 1 to 77, or (ii) a sequence complementary to (i). Items-X - and/or-Z - can include a promoter sequence (or she complementary sequence).

The invention relates to a nucleic acid that encodes the polypeptides and fragments of the polypeptides according to the invention.

The invention includes nucleic acid containing a sequence complementary to the sequences disclosed in the list of sequences (for example, for use as antisense sequences or as samples or for use as primers), and the sequence in actually given orientation.

Nucleic acids according to the invention can be used in hybridization reactions (for example, in the methods of the Northern and southern blotting, or microarrays of nucleic acids, or "gene chips") and in the amplification reactions (for example, PCR (polymerase chain reaction), SDA (amplification substitution circuits), SSSR (self-sustaining amplification pic is egovernance), LCR (ligase chain reaction), TMA (transcription amplification), first NASBA (amplification on the basis of the nucleotide sequence), and so on) and other methods of nucleic acids.

Nucleic acid according to the invention may be in any form (for example, single-stranded, double-stranded, in the form of a vector, primers, samples, labeled form etc). Nucleic acids according to the invention can be circular or branched, but, usually, there are linear. If not specified or requires otherwise, in any embodiment of the invention, which uses a nucleic acid, it can be used in double-stranded form and each of the complementary single-stranded forms, which form a double-stranded form. Typically, the primers and samples are single-stranded, because they are antimuslim nucleic acids.

Preferably, the nucleic acid according to the invention were provided in purified or substantially purified form, i.e. essentially free from other nucleic acids (e.g., available from other natural nucleic acids), in particular from other nucleic acidsHaemophilusor host cells, in General, the purity of the nucleic acids according to the invention is at least about 50% (by weight), and usually at least about 90%. Preferred NUS is Inouye acids according to the invention are nucleic acids H. meningitidis.

Nucleic acids according to the invention can be obtained in a variety of ways, for example by chemical synthesis (e.g., phosphoroamidites by DNA synthesis) fully or partially, by cleavage of longer nucleic acids using nucleases (e.g., enzymes), connection of shorter nucleic acids or nucleotides (e.g., using ligase or polymerases), from genomic or cDNA libraries, etc.

You can attach a nucleic acid according to the invention to a solid substrate (e.g., ball, plate, filter, film, slide, the substrate used in the method of microarrays, resin and so on). You can mark the nucleic acid according to the invention, for example, radioactive or fluorescent label, or bitenova label. Tagging is particularly useful when the nucleic acid to be used in the methods of detection, for example, if the nucleic acid is a primer or a probe.

The term "nucleic acid" generally refers to a polymeric form of nucleotides of any length, which include deoxyribonucleotides, ribonucleotides, and/or their analogues. It includes DNA, RNA, and hybrids of DNA/RNA. This term also includes analogs of DNA or RNA, such as those that contain a modified frame (e.g., peptide nucleic acid (PNA) or phosphorothioate) or modi is zirovnice Foundation. Therefore, the invention includes mRNA, tRNA, rRNA, ribozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, samples, primers, etc. If the nucleic acid according to the invention is an RNA, it can be kupirovano at the 5'-end or not.

Nucleic acids according to the invention contain meningococcal sequence, defined above, but they may also contain meminimalkan sequence (for example, in nucleic acids with the formula 5'-X-Y-Z-3'described above). In particular, it is applicable to primers which, therefore, may contain the first sequence, complementary to the nucleic acid target Neisseria, and a second sequence that is not complementary to the nucleic acid target. Preferably, any such complementary sequence in the primer were located in the 5'-region from complementary sequences. Typical complementary sequence containing the restriction sites or promoter sequence.

Nucleic acids according to the invention can be obtained in a variety of ways, for example by chemical synthesis (at least partially), by cleavage of longer nucleic acids using nucleases (e.g., enzymes), the connection is more Corot is such nucleic acids (e.g., using ligase or polymerases), from genomic or cDNA libraries, etc.

Nucleic acids according to the invention can be part of a vector, i.e. a part of the nucleotide constructs created for transduction/transfection of one or more types of cells. The vectors may be, for example, vectors for cloning", created for the selection, amplification and replication built-nucleotides, "expression vectors", created for the expression of the nucleotide sequence in the cell-host, "viral vectors designed to produce recombinant viruses or virus-like particles, or "Shuttle vectors", which include signs of more than one type of the vector. Preferred vectors are plasmids. "A host cell" includes an individual cell or cell culture which can be or is a recipient of exogenous nucleic acid. Cell hosts include progeny of a single host cell, and the progeny should not be completely identical (in morphology or be identical DNA) with the original parent cell due to natural, accidental or specific mutations and/or changes. Cell hosts include cells, transfected or infected within vivoorin vitronucleic acid according to the invention.

If the nucleic acid is DNA, what about the obvious, that "U" in the RNA sequence will be replaced by "T" in DNA. Similarly, if the nucleic acid is RNA, it is clear that ' t ' in the DNA sequence will be replaced by U in RNA.

The terms "complementary" and "complementary" in relation to nucleic acids refers to the formation of base pairs according to the Watson-Crick. Therefore, the base complementary To, is G, base, complementary G is S, base, complementary And that is T (or U), and a base, complementary to T (or U)is A. you can Also use bases, such as I (purine base inosine), for example, complementary binding to pyrimidine (C or T). The terms also imply direction: sequence, complementary to the 5'-ACAGT-3', 5'-ACTGT-3'and 5'-TGTCA-3'.

Nucleic acids according to the invention can be used, for example, to obtain polypeptides; as hybridization samples for detection of nucleic acids in biological samples; to create additional copies of nucleic acids; to generate ribozymes or antisense oligonucleotides, single-stranded DNA primers or probes or oligonucleotides that form a triple chain.

The invention relates to a method of obtaining a nucleic acid according to the invention, in which nukleinovokisly synthesize partially or fully chemical methods.

The invention relates to vectors containing the nucleotide sequence of the invention (for example, vectors for cloning or expression), and to the cell host transformed with such vectors.

The invention also relates to a set containing primers (e.g., PCR primers) for amplifying matrix sequence, which includes meningococcal nucleotide sequence, and the set contains the first primer and the second primer, the first primer is essentially complementary to the specified matrix sequence and the second primer is essentially complementary chain, complementary to the specified matrix sequence, where part of the above mentioned primers essentially complementary matrix, limit ends amplificare matrix sequence. The first primer and/or second primer can include detektiruya label (e.g., fluorescent label).

The invention also relates to a set comprising the first and second single-stranded oligonucleotides which allow to matrix amplify the nucleotide sequence of meningococcal disease, which is contained in single-stranded or double-stranded nucleic acid (or mixture thereof)in which: (a) the first oligonucleotide contains a sequence of primer, which is essentially the complementary specified matrix nucleotide sequence; (b) the second oligonucleotide contains a sequence of primer that is essentially complementary to the sequence complementary to the specified matrix nucleotide sequence; (C) the first oligonucleotide and/or the second oligonucleotide contains a sequence that complementary specified matrix nucleic acid; and (g) specified primernye sequence limit ends amplificare matrix sequence. Preferably, complementary sequence (complementary sequence) of the characteristic (C) is above (i.e. in the 5'-region) primernih sequences. One or both of these (C) sequences may contain a restriction site [e.g., as in the link 33] or promoter sequence [e.g., as in the link 34]. The first oligonucleotide and/or the second oligonucleotide may include detektiruya label (e.g., fluorescent label).

Matrix sequence can be any part of the genomic sequence.

The invention relates to a method of detecting a nucleic acid according to the invention, comprising the stage of: (a) contacting nucleic acid sample according to the invention with a biological way under hybridization conditions to form duplexes; and (b) detection of these duplexes.

Image is eenie relates to a method of detection of meningococcal disease in a biological sample (for example, blood), comprising a stage of contacting the nucleic acid according to the invention with the biological sample under conditions suitable for hybridization. The process may include amplification of nucleic acids (e.g., PCR, SDA, SSSR, LCR, TMA, first NASBA etc) or hybridization (e.g., microarrays, blots, hybridization with the sample in solution, and so on).

The invention relates to a method of obtaining a fragment of a target sequence in which the fragment is obtained by lengthening the nucleotide of the primer. The sequence of the target and/or Primera sequences are nucleic acids according to the invention. The reaction elongation of the primer may include amplification of nucleic acids (e.g., PCR, SDA, SSSR, LCR, TMA, first NASBA and so on).

Amplification of nucleic acid according to the invention can be quantitative and/or take place in real time.

For some embodiments of the invention, the preferred length nucleic acids, at least, is 7 nucleotides (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300 nucleotides or more).

For some embodiments of the invention, the preferred length nucleic acids, at most, 500 nucleotides (e.g., 450, 400, 350, 300, 250, 200, 150, 14, 130, 120, 110, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 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 nucleotides or less).

Preferably, the length of the primers and samples according to the invention and other nucleic acids used in hybridization, were between 10 and 30 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides).

The pharmaceutical composition

The invention relates to compositions that contain (a) a polypeptide, antibody and/or nucleic acid according to the invention and (b) a pharmaceutically acceptable carrier. For example, these compositions are suitable for use as immunogenic compositions, or as diagnostic reagents, or as vaccines. According to the invention of vaccines can be prophylactic (i.e. to prevent infection)or therapeutic (i.e. to treat infection), but the vaccine can usually be preventive.

The term "pharmaceutically acceptable carriers" includes any media that does not in itself cause the production of antibodies harmful to the individual who receives the composition. Suitable carriers are typically large, slowly metabolisable macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids, sucrose, trigal is for, lactose and aggregates of lipids (such as oil droplets or liposomes). The average specialist in this area is well known for such media. Vaccines may also contain diluents, such as water, saline, glycerol, etc. may Optionally be present auxiliary substances, such as moisturizers or emulsifying agents, substances that support pH, etc. Typical carrier is pyrogen-free, phosphate saline. A detailed description of pharmaceutically acceptable excipients are listed in the link 141.

The composition of the invention may include an antimicrobial compound, in particular, if the composition are in mnogochasovoj package.

The composition of the invention may contain a detergent, such as Tween (Polysorbate, such as Tween 80. Usually, the contents present detergent is low, for example < 0,01%.

The composition of the invention may include sodium salt (e.g. sodium chloride), which causes toychest solution. A typical concentration of NaCl is 10±2 mg/ml

Typically, the compositions according to the invention will include a buffer solution. A typical buffer solution is a phosphate buffer solution.

The composition of the invention may contain sugar alcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose), for example, at a concentration of about15-30 mg/ml (e.g., 25 mg/ml), in particular, in the case where the composition must be in lyophilized form, or when the compositions include material that was recovered from the dried material. For lyophilization the pH of the composition can be brought to about 6.1 to lyophilization.

The polypeptides according to the invention can be entered together with other immunoregulatory agents. In particular, the compositions will typically include adjuvant. Adjuvants that can be used in the compositions according to the invention, include, but are not limited to, the following :

A. Mineral composition

Mineral compositions suitable for use as adjuvants in the invention include mineral salts such as aluminum salts and calcium. The invention includes mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (for example, hydroxyphosphate, orthophosphate, sulfate, etc. [for example, see chapters 8 and 9 of the links 35], or mixtures of different mineral compounds; and the form of connection can be any (e.g. gel, crystalline, amorphous, etc); and it is also preferable that the compound had an adsorbing properties. Containing minerals composition can be a composition of particles of metal salts [36].

Particularly preferred compounds are phosphates of aluminum, in particular in compositions, which include diabetes antigenH.influenzaeand a typical adjuvant is amorphous hydroxyphosphate aluminum with a molar ratio of PO4/Al from 0.84 and 0.92, included at a concentration of 0,6mg Al3+/ml. Can be used adsorption with a low dose of phosphate of aluminum, for example, 50-100 µg Al3+the dose of the conjugate. If the composition contains more than one conjugate, then not all the conjugates must be adsorbed.

B. Oil emulsion

Composition oil emulsions for use as adjuvants in the invention include emulsion of squalane in the water, such as MF59 [see Chapter 10 of reference 35; see also reference 37] (5% squalene, 0.5% of Tween 80, and 0.5% Span 85 in the form of submicron particles obtained by using a homogenizer (Microfluidizer). You can also use complete and incomplete adjuvants's adjuvant (CFA and IFA, respectively).

Century structures on the basis of saponin[Chapter 22 of the links 35]

As adjuvants in the invention it is also possible to use mixtures with saponin. Saponins are a heterologous group of Sterol glycosides and triterpene glycosides that are found in the bark, leaves, stems, roots and even flowers large number of plant species. Investigated intensively used as adjuvant saponin from soap barkQuillaia saponariaMolina. Also in p is myshlennyh quantities of saponins can be obtained from Smilax ornata(sarsaparilla plant),Gypsophilla paniculata(gypsophila paniculate) andSaponaria officianalis(soap root). The compositions sapojnikova adjuvants include purified compounds, such as QS21, and lipid compounds, such as ISCOM. QS21 is sold under the trademark Stimulon™.

Saponine composition purified using HPLC and RP-HPLC. Using these techniques were identified individual purified fractions, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferred saponin is QS21. The method of obtaining QS21 is disclosed in reference 38. Saponine compositions can also contain a Sterol, such as cholesterol [39].

Combinations of saponins and cholesterol you can use to get special particles called immunostimulating complexes (ISCOM) [Chapter 23 of the links 35]. ISCOM complexes also usually include a phospholipid, such as phosphatidylethanolamine or phosphatidylcholine. In ISCOM complexes can be used any known saponin. Preferably, ISCOM included one or more of the QuilA, QHA and QHC. ISCOM complexes described in more detail in the links 39-41. Optional, ISCOM complexes may not contain additional detergent [42].

A review of the development of adjuvants on the basis of saponins can be found in references 43 and 44.

G Virosome and virus-like particles

Also as adjuvants in the invention can be used virosome and virusopodobnyh the e particles (the VLP). Usually these structures contain one or more viral proteins, optionally combined or incorporated with phospholipid. They are usually non-pathogenic, not replicated and do not contain any natural viral genome. Recombinant viral proteins can be obtained recombinant methods, or they can be distinguished from the whole virus. Suitable for use in virosome or the VLP viral proteins include proteins derived from influenza virus (such as HA or NA), hepatitis b virus (such as nuclear or capsid proteins), hepatitis E virus, measles virus, virus Sindbis, rotavirus, a virus disease of the hands and feet, virus diseases of the hands, feet and mouth, retrovirus, virus Norwalk, human papilloma virus, HIV, RNA-containing phages, Qβ-phage (such as membrane proteins), GA-phage, fr-phage, phage AP205 and retrotransposon Ty (such as protein p1 retrotransposon Ty). In more detail, the VLP is described in the references 45-50. Virosome described in more detail in, for example, the link 51.

D. Derivative of bacteria or microbes

Adjuvants suitable for use in the invention include derivatives of bacteria or microbes, such as non-toxic derivatives enterobacterial lipopolysaccharide (LPS), a derivative of lipid A, immunostimulatory oligonucleotides and ADP-reboilers toxins and their neutralized derivatives.

Non-toxic derivative of LPS include monophosphoryl-lipid A (MPL) and 3-O-describeany MPL (3dMPL). 3dMPL is a mixture of 3-O-decelerating monophosphoryl-lipid a with 4, 5 or 6 acylated chains. Preferred particulate form 3-O-decelerating monophosphoryl-lipid And disclosed in reference 52. The particle size in such fine form 3dMPL small enough so that the solution can be filtered for sterilization through a membrane with pore size 0.22 μm [52]. Other non-toxic derivative of LPS include mimetics monophosphoryl-lipid A, such as derivative aminoalkylphosphonic phosphate, e.g. RC-529 [53,54].

Derivatives of lipid And include derivatives of lipid a fromEscherichia colisuch as OM-174. For example, the description of OM-174 is in the links 55 and 56.

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence that contains demetilirovanny cytosine connected phosphate linkage with guanosine). Also been shown immunostimulatory properties of double-stranded RNA molecules and oligonucleotides, which contain palindrome or poly(dG)-sequence.

CpG may include modified nucleotides (or analogs), such as phosphothioate modification,and can be double-stranded or single-stranded. In the links 57, 58 and 59 are disclosed possible analog replacement, for example the replacement of guanosine is 2'-deoxy-7-deazaguanosine. In more detail the effect of CpG oligonucleotides as adjuvants described in the references 60-65.

TLR9 can recognize CpG sequence, such as GTCGTT or TTCGTT [66]. CpG sequence, such as CpG-A one, specific can induce Th1-immune response or CpG sequence, such as CpG-B one can be more specific to cause b-cell response. The sequence of CpG-A one and CpG-B one described in more detail in the links 67-69. Preferred CpG CpG is-A one.

The preferred design of the CpG oligonucleotide is designed with an open 5'-end that are available for recognition by the receptor. Optional, two of the CpG oligonucleotide can be linked at their 3'ends, forming a "immunome". See, for example, links 66 and 70-72.

Bacterial ADP-reboilers toxins and their neutralized derivatives can be used as adjuvants in the invention. Preferably, the protein was derived fromE. coli(thermolabile enterotoxin "LT" fromE. coli), V. cholerae ("CT"), or tetanus Bacillus ("PT"). The use of neutralized ADP-reboilers toxins as adjuvants, interacting with mucous, described in the link 73, and as parenteral adjuvants in the link 74. Suppose the equipment, to the toxin or toxoid was holotoxin containing both a and b subunits. Preferably, the a-subunit contained neutralizing mutation; however, it is preferable that In the-subunit was not metirovan. The preferred adjuvant is neutralized LT mutant, such as LT-K63, LT-R72 include and LT-G192. The use of ADP-reboilers toxins and their neutralized derivatives, particularly LT-K63 and LT-R72 include can be found in the links 75-82. Preferably, the numbering of amino acid substitutions was based on the alignments a and b-subunits of ATP-reboilers toxins listed in the link 83, fully incorporated into the present description by reference.

That is, the Modulators of the human immune system

Modulators of the human immune system, which is suitable for use as adjuvants in the invention include cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [84] and so on) [85], interferons (e.g. interferon-γ), the factor stimulating macrophages, and tumor necrosis factor.

J. Bioadhesive and mucoadhesive

Also as adjuvants in the invention can be used bioadhesive and mucoadhesive. Suitable bioadhesive include microspheres esterified hyaluronic acid [86] or mucoadhesive, such as cross-linked derivatives of poly(acrylic KIS is the notes), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose. Also as adjuvants in the invention can be used chitosan and its derivatives [87].

H. Microparticles

Also as adjuvants in the invention can be used microparticles. Microparticles (i.e. a particle of from approximately 100 nm to approximately 150 microns in diameter, more preferably, from about 200 nm to about 30 microns in diameter and most preferably, from about 500 nm to about 10 microns in diameter)made from materials that are biodegradable and non-toxic toxic (e.g. a poly(α-hydroxy acid), polyhydroxybutyric acid, polychaetes, polyanhydride, polycaprolactone etc) with poly(lactide-co-glycolide) are preferred, optionally treated to a negatively charged surface (e.g., LTOs) or positively charged surface (e.g., a cationic detergent, such as CTAB).

I. Liposomes (chapters 13 and 14 of the links 35)

Examples of liposomal formulations for use as adjuvants are described in the references 88-90.

Because the Compositions, including polyoxyethylene ethers and esters

Adjuvants suitable for use in the invention include polyoxyethylene simple and SL is the author esters [91]. In addition, such compositions include surfactants - esters of polyoxyethylenesorbitan in combination with octoxynol [92], and surfactants - esters and ethers of polyoxyethyleneglycol in combination with at least one additional non-ionic surface-active agent, such as an octoxynol [93]. Preferred ethers of polyoxyethylene selected from the following group: polyoxyethylene-9-lauric ether (Laureth-9), polyoxyethylene-9-sterilely ether, polyoxyethylene-8-sterilely ether, polyoxyethylene-4-lauric ether, polyoxyethylene-35-lauric ether and polyoxyethylene-23-lauric ether.

L. Polyphosphate (RSRR)

The PCPP formulations, for example, described in the links 94 and 95.

M Morelove peptides

Examples nuramilovich peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-norbornyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetyl-muramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)-ethylamine MTP-PE.

N. Imidazoquinolines connection

Examples imidazoquinolines compounds suitable for use as adjuvants in the invention include Imiquimod and its homologues (for example, "Resiquimod"), described in more detail in the links 96 and 97.

Also the invention may include combinations of one or more of the above-described adjuvants. For example, the invention can be used with the following composition adjuvants: (1) a saponin and an emulsion oil in water [98]; (2) a saponin (e.g., QS21) and a non-toxic derivative of LPS (such as 3dMPL) [99]; (3) saponin (e.g., QS21), a non-toxic derivative of LPS (such as 3dMPL) and cholesterol; (4) a saponin (e.g., QS21), 3dMPL and IL-12 (optional, + a Sterol) [100]; (5) combinations of 3dMPL with, for example, QS21 and/or oil emulsions in water [101]; (6) SAF, containing squalene, 10%Tween 80™ 0.4%, pluronic block polymer L121 5% and thr-MDP (homogenized to a submicron emulsion or broken on the vortex to obtain emulsions with larger particles); (7) adjuvant Ribi™ adjuvant system (RAS), (Ribi Immunochem)containing squalene, 2%Tween 80™ 0.2% and one or more of the components of the bacterial wall from the group consisting from monophosphoryl-lipid A (MPL), dimycolate trehalose (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); and (8) one or more mineral salts (such as an aluminium salt) and a non-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed in Chapter 7 of reference 35.

Particularly preferable to use aluminum hydroxide adjuvant or FOSFA is aluminum, usually happens sorption antigens on these salts. Another preferred adjuvant is calcium phosphate.

Preferably, the pH value of the compositions was from 6 to 8, preferably about 7. A constant value of pH can be maintained by using the buffer compounds. If the composition includes a salt of aluminum hydroxide, it is preferable to use his-tag buffer [102]. The compositions can be sterile and pyrogen-free. The composition of the invention can be isotonic for people.

The compositions may be presented in vials or they can be presented in ready-to-use filled syringes. Syringes can be supplied with or without needles. In the syringe will be a single dose of the composition, while the bottle may contain a single dose or multiple doses. Compositions for injection will usually be a liquid solution or suspension. Alternatively, they can be represented in the form of solid forms (e.g., lyophilized) for preparation of a solution or suspension in liquid media prior to injection.

The composition of the invention can be packaged in single doses or in mnogochasovoj form. For parenteral multi-dose form preferably, the bottles are not filled syringes. Effective amounts of dosages determine what I routine methods, but the usual dose of the composition for injection of the people is 0.5 ml.

If the composition according to the invention need to be prepared immediately prior to use (for example, if the component is presented in lyophilized form), and the composition is represented in the set, the set may include two bottle or it may contain one ready-filled syringe and one vial, and the contents of the syringe is used to activate the contents of the vial before injection.

Used as vaccines, immunogenic compositions contain immunologically effective amount of the antigen (antigens), and, if necessary, other components. By "immunologically effective amount" is meant that the introduction of this quantity to the individual alone, or as part of the administration scheme is effective for treatment or prevention. This number varies depending on the health status and physical condition of the individual, which must be treated, age, the taxonomic group of individual to be treated (e.g., a human primates, primates etc), the ability of the human immune system to produce antibodies of the desired degree of protection, vaccine composition, evaluation by the attending physician and other important factors. It is expected that this value will find the change in a wide range, which can be determined in routine trials, and a typical quantity of each meningococcal Zaharenko antigen per dose is in the range from 1 μg to 10 mg per one antigen.

Pharmaceutical application

The invention also relates to a method of treating a patient comprising the administration to a patient a therapeutically effective amount of the composition according to the invention. The patient may have the risk of disease or the patient may be pregnant woman ("immunization in the womb").

The invention relates to nucleic acid, polypeptide, the antibody according to the invention for use as a drug (such as immunogenic compositions or vaccines, including use in the treatment or prevention of diseases and/or infections caused by meningococcus) or as a diagnostic reagent. The invention relates to the use of nucleic acid, polypeptide or antibody according to the invention in the manufacture of: (1) a medicinal product for the treatment or prevention of a disease and/or infection caused by meningococcal disease; (ii) a diagnostic reagent for detecting the presence of Neisseria meningitides or antibodies raised against meningococcal disease; and/or (iii) reagent, which may cause the occurrence of antibodies against meningococci. The decree is effective meningococcal disease can belong to any serogroup or strain, but preferably refers to serogroup C. the Specified disease can be, for example, bacterial meningitis (and, in particular, meningococcal meningitis or septicaemia.

Preferably, the patient was a man. If the vaccine is used for prophylactic use, the human, preferably, is a child (for example, a child of kindergarten age or a baby or a teenager, for example, ranging in age from 0 to 18 years; if the vaccine is used for therapeutic applications, people, preferably, is an adult, for example, ranging in age from 18 to 55 years. The vaccine, which is designed for children, you can also enter an adult, for example, to assess the safety, dosage, potency, etc.

One way of checking efficacy of therapeutic treatment involves monitoring meningococcal infection after administration of the composition according to the invention. One way of checking the effectiveness of preventive action involves monitoring immune response against the introduced polypeptide after injection. The immunogenic compositions according to the invention can be defined by introducing their test subjects (e.g., children aged 12 to 16 months or animal models) and subsequent determination of standard indicators, including IgG titers by ELISA method (GMT). These immune responses are usually determined by the t of about 4 week after administration of the composition and compared with the values defined before the introduction of the composition. If you enter more than one dose of the composition, the title can be defined more than once. The standard way to assess the prophylactic efficacy against meningococcal disease is serum bactericidal test (SBA). Preferably, the introduction of the composition has led to an increase in SBA titer tested for serogroup at least 4 times, and preferably at least 8 times, which is measured by the complement system [103]. If SBA titers use the complement system of the rabbit, then, preferably, the titer increased, at least 128 times.

The introduction of polypeptide antigens is the preferred method of exposure for the induction of immunity. Another preferred method of impact is the introduction of antibodies according to the invention. This method of passive immunization is particularly suitable for use in infants or pregnant women. This method is usually used monoclonal antibodies that will be humanitarianism or unmodified antibodies person.

Typically, the compositions according to the invention will be prompted to enter the patient directly. Direct introduction can be performed by parenteral injection (e.g. subcutaneous, intraperitoneally, intravenous, intramuscular or in the interstitial space of the tissue and or rectal, oral, vaginal, local, transdermal, intranasal, ocular, through the ear, through the lungs or other type of insertion through the mucous. Preferably intramuscular injection in the thigh or in the upper part of the shoulder. Injections can be done with a needle (for example, hypodermatidae needles), but alternatively you can apply injections without needles. The typical dose for intramuscular injection of 0.5 ml.

The invention can be used to induce systemic immunity or immunity in the mucosa.

The treatment regimen can be a single injection or multiple. Repeated introduction you can use in the circuit of the primary immunization and/or in the scheme of revaccination. For scheme initial immunization may be followed by revaccination. You can easily determine the appropriate time interval between primary doses (e.g., 4-16 weeks) and between the primary vaccination and re-vaccination.

Bacterial infections affect different parts of the body and, therefore, the shape of the resulting compositions can be different. For example, compositions can be made in the form of drugs for injection or in the form of aqueous solutions or suspensions. You can also make solid forms suitable for solution or resuspendable in liquid media prior to injection (n is an example, lyophilized compositions). The composition can be produced for local administration, for example, in the form of ointment, cream or powder. The composition can be manufactured for oral administration, for example, in the form of tablets or capsules or in the form of syrup (optional, flavored). The composition can be made for insertion through the lungs, for example, in the form of an inhaler with fine powder or aerosol form. The composition can be manufactured in the form of suppositories or pessaries. The composition can be made for insertion through the nose, ear or eye, for example, in the form of a spray, drops, gel or powder (for example, see references 104 and 105).

Additional antigenic components of the compositions according to the invention

The invention also relates to compositions containing a polypeptide according to the invention and one or more of the following additional antigens:

- charigny antigen serogroups A, C, W135 and/or Y (preferably all four)N.meningitidissuch as disclosed in reference 106 oligosaccharide serogroup C [see also link 107] or oligosaccharides of links 108;

- charigny antigenStreptococcus pneumoniae[for example, 109, 110, 111];

- antigen hepatitis a virus, such as inactivated virus [e.g., 112, 113];

- antigen hepatitis b virus, such as surface and/or nuclear antigens [e.g., 113, 114];

- diphtheria and is then, such as diphtheria toxoid [e.g. Chapter 3 of the links 115], for example CRM197-mutant [e.g., 116];

- tetanus antigen, such tetanus toxoid [e.g. Chapter 4 of the links 115];

- antigenBordetella pertussissuch as the pertussis holotoxin (RT) and the filament hemagglutinin (FHA) of theB.pertussisoptionally in combination with pertactin and/or agglutinogens 2 and 3 [for example, links 117 and 118];

- charigny antigenHaemophilus influenzaeB [e.g., 107];

- poliovirus antigen (antigens) [for example, 119, 120], such as IPV;

- antigen measles, mumps and/or rubella [e.g., chapters 9, 10 and 11 of the links 115];

antigens of influenza [for example, Chapter 19 of the links 115], such as surface proteins hemagglutinin and/or neuraminidase;

- antigenMoraxella catarrhalis[for example, 121];

- protein antigenStreptococcus agalactiae(for group B Streptococcus) [e.g., 122, 123];

- charigny antigenStreptococcus agalactiae(for group B Streptococcus);

- antigenStreptococcus pyogenes(group a Streptococcus) [e.g., 123, 124, 125];

- antigenStaphylococcus aureus[for example, 126].

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

If necessary, the toxic protein antigens can be neutralized (for example, tetanus toxin can be neutralized by chemical and/or genetic means [118]).

If the composition contains difthe applications antigen, preferably also include tetanus and pertussis antigens. Similarly, if the composition is included tetanus antigen, it is preferable to include diphtheria and pertussis antigens. Similarly, if included pertussis antigen, it is preferable to include diphtheria and tetanus antigens. That is, it is preferable to use DTP combination (diphtheria/tetanus/pertussis).

Preferably, sacharine antigens were in the form of conjugates. Carrier proteins for conjugates include diphtheria toxin, tetanus toxin, a protein of the outer membraneN.meningitidis[127], synthetic peptides [128,129], heat shock proteins [130,131], pertussis proteins [132,133], protein D fromH.influenzae[134], cytokines [135], lymphokines [135], streptococcal proteins, hormones [135], growth factors [135], toxin a or b fromC.difficile[136]involved in the transfer of iron into the cell proteins [137], etc. Preferred protein carrier is diphtheria toxoid CRM197 [138].

The concentration of antigens in the composition will usually be at least 1 μg/ml of each antigen. In General, the concentration of any of the antigen should be sufficient to induce an immune response against the antigen.

Alternatively, the use of protein antigens in the immunogenic compositions according to the invention can be used n kleinova acid (preferably, DNA, such as plasmids).

Preferred are the antigens adsorbed to an aluminium salt.

Screening methods

The invention relates to a method, which allows you to define links do test the connection, the polypeptide according to the invention. If the test compound binds the polypeptide according to the invention, and as a result of this binding is inhibited the life cycle of the meningococcus, it test the connection, you can use as an antibiotic or as a starting compound in the development of antibiotics. Usually, the method will include the stage of contacting the test compound with the polypeptide according to the invention and definitions, connects test whether the connection to the specified polypeptide. Preferred polypeptides for use in these methods are enzymes (e.g., tRNA-synthetase), membrane transporters and ribosomal polypeptides. Suitable test compounds include polypeptides, carbohydrates, lipids, nucleic acids (e.g. DNA, RNA, and their modified forms), and low molecular weight organic compounds (e.g., with MW between 200 and 2000). Test compounds can be present individually, but usually they will be part of the library (e.g., combinatorial libraries). Methods of detecting interaction of the of conduct when linking include NMR, method binding filters, braking method in the gel, substitution methods, surface plasmon resonance, "reverse" twohybrid system, etc. Can be checked connections, linking the polypeptide according to the invention, on its activity as an antibiotic by contacting connection with meningococcal bacteria, and then observing the inhibition of growth. The invention also relates to the connection identified by these methods.

Preferably, the method includes the stages of: (a) contacting the polypeptide according to the invention with one or more compounds of candidates, resulting in a mixture; (b) incubating the mixture to allow the interaction between the polypeptide and the connection candidate (compounds-candidates); and (C) assessing the links if the connection candidate polypeptide or modulating its activity.

Ifin vitroidentified a compound that binds the polypeptide according to the invention, then it may be desirable to conduct further experiments to confirm the ability of the compound to inhibit the growth and/or survival of bacteriain vivo. Therefore, the method includes the additional step of contacting the compound with meningococcal disease and the assessment of its validity.

Used in the method of screening the polypeptide can náchod shall be free in solution, can be attached to a solid substrate, can be located on the cell surface or inside the cell. It is preferable to detect the binding connection-candidate polypeptide with a label directly or indirectly associated with the connection candidate. The label may be a fluorophore, a radioisotope or other detektiruya tag.

General provisions

The invention relates to compatible media (for example, a flexible disk, hard disk, CD-ROM, DVD etc) and/or memory of the computer and/or computer database that contain one or more sequences in the list of sequences.

The term "comprising" encompasses "including"and "comprising", for example, a composition "comprising" X may consist exclusively of X or may include additional compounds, such as X+Y.

The term "about" in relation to the numeric value ofxmeans, for example,x±10%.

The word "essentially" does not exclude "completely"e.g. a composition that is "essentially free" from Y may be completely free from Y. If necessary, the word "essentially" can be excluded from the definition of invention.

The identity between polypeptides preferably be determined by a search algorithm homology Smith-Waterman, the real is implemented in the MPSRCH program (Oxford Molecular), uses affine search breaks with the following parameters: the penalty for opening a gap = 12 and the penalty for continuation gap = 1. The identities of the sequences also preferably be determined by the search algorithm Smith-Waterman.

N-terminal amino acid residues in the sequence list sequence shown as amino acids encoded by the first codon in the corresponding nucleotide sequence. If the first codon is ATG, it is clear that if this codon is a start that he will be broadcast as methionine; but it will be broadcast as specified amino acid (not Met), if the sequence is at the C-end of the slit with her other sequence. In the invention explicitly disclosed and covered each of the amino acid sequence from a list of sequences that have a methionine residue at the N-end (for example, formyl-methioninol balance) instead of any of the specified residue (not Met).

In biology you can use alternative start codons. Amino acid sequence in the list of sequences obtained on the basis of specific start-codons, but alternatively can be used below the start-codons. Therefore, the invention explicitly disclosed and covered each linakis is now a sequence from a list of sequences, which starts with any of the methionine residue of the sequence below the N-terminal residue in the list (for example, SEQ ID NO: 5 and 10).

As indicated in the above text, nucleic acids and polypeptides according to the invention may include sequences that:

(a) are identical (i.e. 100%identical) with the sequences disclosed in the list of sequences;

(b) have sequence identity with the sequences disclosed in the list of sequences;

(C) have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single nucleotide or amino acid alterations (deletions, insertions, substitutions), which may be located in different places or can be consistently compared with sequences of p.(a) or (b); and

(d) when aligned with a particular sequence from a list of sequences using pairwise alignment algorithm of moving the window from thexmonomers (amino acids or nucleotides), which moves from the start (N-Terminus or 5') to the end (- end or 3') so that alignment, which applies topmonomers (ifR>x), it isp-x+1these Windows, and each window has at leastx*yidentical aligned monomers, wherexchoose from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200;y/i> choose from 0,50, 0,60, 0,70, 0,75, 0,80, 0,85, 0,90, 0,91, 0,92, 0,93, 0,94, 0,95, 0,96, 0,97, 0,98, 0,99; and ifx*yis not an integer then it is rounded up to the nearest whole number. The preferred pairwise alignment algorithm is a General algorithm for alignment of Needlman-Wunsch [139] using the default settings (for example, the penalty for opening a gap = 10,0 and the penalty for extending a gap = 0.5, and using the weight of the EBLOSUM62 matrix). This algorithm is conveniently implemented in the programneedlefrom the EMBOSS software package [140].

Nucleic acids and polypeptides according to the invention may have additional sequences at the N-end/5' and/or C-end/3' sequences on PP.(a)to(g).

In the practice of the present invention, unless otherwise specified, will be used standard chemical, biochemical, molecular biological, immunological and pharmacological techniques that are known in these areas. A full description of these methods can be found in the literature. See, for example, links 141-148, etc.

WAYS of carrying out the INVENTION

In the genome of strain M04-240196 serogroup BN. meningitidiswere identified various encoded amino acid sequence. On the basis of different criteria, 39 of them were selected as suitable antigens, and in the list of sequences shown consistent is Telenesti their genes and their amino acid sequences.

Estimated biological functions are shown in table I, but the exact role of antigens in biology meningococcus is not as important as their ability to act as antigens. Table II also indicates the closest match to sequences in the published genomes of serogroups a and b (references 6 and 8), as well as in the unpublished genome of strain FAM18 serogroup C. If the sequence has more than 95% identity with a known sequence (and in particular if it is the same 100%), the invention was directed to a greater extent on the identification of applicable antigenic properties of the protein and not on the identification of proteinper se.

In addition to what is specified in the notes and comparisons, additional traits of interest include: B269_17 contains a domain intein; B269_34 has a splicing sequence at its C-end; in B269_05, B269_10, B269_18, B269_24 present transmembrane domain; and B269_15 and B269_29 contains five transmembrane domains.

Using the steps in this description and information about the sequences, we can easily Express the proteins in recombinant hosts and use them to generate an immune response, using known in the field of methods.

For example, sequences encoding proteins B269_11, 13, 14, 15, 17, 24, 25, 26, 29, 31, 32, 34, 36, 37, 51, 52, 53, 54, 55,56, 57, 58 and 59, were incorporated into expression vectors with polyhistidine tag at the C-end. Was also obtained protein expression B269_14, 29, 31, 34, 37 with a short domain. Also were obtained fused with GST proteins 37, 54, 55 and 57. Conducted clearing expressed proteins fromE. coli. Without optimization of expression was observed various purification of proteins, for example from 20% purity for domain B269_14 and B269_32 and up to 95% purity for B269_51. The expression of proteins in a soluble form was observed for proteins B269_13, 24, 25, 31domain, proteins B269_32, 51, 53 and 56.

For expressed proteins antibodies were obtained in mice, using injection of complete Freund's adjuvant or aluminum hydroxide. The serum of immunized animals are then used for staining Western blots or in the analysis of binding with meningococci using flow cytofluorimetry. By the method of Western blotting is possible to define the following VBA: 13; 25; 29domain; 31; 34domain; 51; 52; 53. In addition, these proteins can be detected on the membrane with a specific molecular weight: 11 (40 KD); 24 (20 KD); and 26 (28 KD). Using flow cytofluorometry was able to detect the following proteins: 17; 24; 25; 26; 29domain; 34domain; and 53.

It should be understood that the invention is described not only by example, and you can make modifications to the invention without going beyond the scope and existence the spine of the invention.

TABLE I - notes
AA = length of polypeptide
In 269SEQ IDAALocalization *Note
012588Aboutprotein PilC
114320Aboutpossible MafA-like adhesin
136387a protein of the superfamily, copynow
148420Iprotein that provides a fusion of membranes
1510670Ipeptidase from when the family S
1612331Ppossible peptidyl-prolyl CIS-TRANS-isomerase
1714209Sconservative hypothetical protein
1816265Oprotein "turbidity" (PR-protein)
1918680Iconnecting transferrin protein 2
20201370Sthe hemagglutinin-hemolysin-related protein
2122734Sthe hemagglutinin-hemolysin-related protein
2224887 Sthe hemagglutinin-hemolysin-related protein
2326794Sthe hemagglutinin-hemolysin-related protein
2428206Cconservative hypothetical protein
25301502SHlyJ-hemolysin-like protein
2632257Cconservative hypothetical protein TIGR00294
2734237Ipossible thiosulfate-sulfotransferase
2836402Cpossible: possible GlcNAc-transferase 19
38297Oconservative hypothetical protein
3040226Cprotein "turbidity" Or
3142588Ofamily YadA-like C-terminal region
3244201Cconservative hypothetical protein
3346337Ipossible glycosyltransferase
3448529Czhelezonikelevoy protein frpA
3550676CTran is Ferin/lactoferrin-binding protein
3652203Cthe mucin
3754340Cconservative hypothetical protein
3856376Ipossible two-component sensor kinase 196
3958346PATP-binding region, ATPase-like:histidine kinase, N-end
41601026Pprotein PilC
4262333Oconservative hypothetical protein
4364229C glycosyltransferase protein family group 2
4466208Cconservative hypothetical protein
4568476CMafB protein
4670229Cadhesin MafB
4772432Oconnecting transferrin protein, subunit 19
4874809Spossible predecessor hemolysin-hemagglutinin-like protein of Nes
4976783Spossible hemagglutinin (DUF637), family 1
5078 300Sthe hemagglutinin-hemolysin-related protein
Designations *localization: O = outer membrane; C = cytoplasm; I = inner membrane; P = periplasmatic space; S = indirect

TABLE II
Homology with other meningococcal sequences [6,8]
In 269SEQ IDMC58(B)(A)(C)
012NMB004977,678,273,1
114NMB065262,710098,4
136NMB17865148,480,3
14 8NMB009751,159,9100
1510NMB00986428,994,9
1612NMB028185,4to 92.193,5
1714NMB036983,261,791,9
1816NMB044286,481,587,1
1918NMB046071,570,941,8
2020NMB177967,47097,4
2122 NMB177582,687,996
2224NMB177981,980,5for 95.2
2326NMB177579,289,288,9
2428NMB051566,199,593,7
2530NMB058585,59190,9
2632NMB080386,886,886,8
2734NMB08418495,4at 88.1
2836NMB0846 8956,185,1
2938NMB088887,979,8to 85.2
3040NMB163680,481,692
3142NMB099287,490,194,6
3244no41,241,270,9
3346NMB12557395,497,5
3448NMB1415to 89.580,598,7
3550NMB154168,3 78,2of 56.4
3652NMB08914953,478,6
3754no30,733,3100
3856NMB160682,280,187
3958NMB16068385,990,9
4160NMB184780,481,678,2
4262NMB187087,487,474,2
4364NMB192948,248,2 48,2
4466NMB1992to 89.981,592,8
4568NMB210589,788,595,9
4670NMB21058896,7to 89.9
4772NMB213276,568,668,7
4874NMB049385,45568,2
4976NMB177567,282,999,4
5078NMB177951,28483,3
"MC58" = the closest match from reference 6
In columns (a)-(C) presents the percentage of overlap with other sequences: (B) = link 6; (A) = reference 8; (c) = strain FAM18

The LIST of references

(the contents of which are fully incorporated into the present description by reference)

1. A polypeptide that is immunogenic against meningococcal infections containing amino acid sequence that has at least 90% identity in the sequence SEQ ID NO:32.

2. The polypeptide according to claim 1, containing the amino acid sequence of SEQ ID NO:32.

3. A polypeptide that is immunogenic against meningococcal infections, containing a fragment of at least 80 consecutive amino acids of SEQ ID NO:32.

4. The polypeptide according to claim 3, in which the fragment comprises a T-cell or b-cell epitope from SEQ ID NO:32.

5. The polypeptide according to claim 1 for use as a medicine.

6. The polypeptide according to any one of claims 1 to 5 for use in the treatment or prevention of diseases and/or infections caused by Neisseria meningitidis.

7. The antibody, which binds to the polypeptide according to any preceding item.

8. The antibody according to claim 7, where the antibody is the monoclonal antibody.

9. The antibody according to claim 7 for use as a medicine.

10. The antibody according to any one of claims 7 to 9, for use in treating or preventing diseases and/or infections caused by Neisseria meningitidis.

11. Nucleic acid encoding a polypeptide that is immunogenic against meningococcal infections, containing the nucleotide sequence of SEQ ID NO:31.

12. Nucleic acid encoding a polypeptide that is immunogenic against meningococcal infections, which can gibridizatsiya with nucleic acid according to claim 11 under stringent conditions.

13. Nucleic acid encoding a polypeptide that is immunogenic against meningococcal infections, containing a fragment of 80 or more consecutive nucleotides from SEQ ID NO:31.

14. Nucleic acid encoding a polypeptide according to any one of claims 1 to 4.

15. Nucleic acid according to claim 11 for use in the treatment or prevention of diseases and/or infections caused by Neisseria meningitidis.

16. Composition, immunogenic against meningococcal infection, comprising: (a) a polypeptide, antibody and/or nucleic acid according to any preceding paragraph and (b) a pharmaceutically acceptable carrier.

17. The composition according to item 16, further containing vaccine adjuvant.

18. The use of a nucleic acid according to any one of § § 11-14, polypeptide according to any one of claims 1 to 4 or the antibody according to claim 7 or 8 in the research Institute of medicinal product for the treatment or prevention of a disease and/or infection, caused by Neisseria meningitidis.

19. Use p for the prevention of meningococcal meningitis.



 

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43 cl, 20 dwg, 10 ex

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11 cl, 5 tbl, 7 ex

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8 cl, 22 dwg, 8 tbl, 9 ex

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13 cl, 14 ex, 1 tbl, 16 dwg

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Glypican-3 antibody // 2427588

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14 cl, 4 dwg, 1 tbl, 2 ex

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39 cl, 4 tbl

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7 dwg, 2 tbl, 9 ex

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27 cl, 5 dwg

FIELD: medicine.

SUBSTANCE: there are described polypeptides eliciting an immune response on H.influenzae. There is offered an antibody selectively binding specified polypeptides. The invention also concerns an immunogenic composition containing the described polypeptides.

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9 cl, 3 tbl

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17 cl, 7 dwg, 8 tbl, 8 ex

FIELD: medicine.

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EFFECT: antibodies can be used for designing immunobiological systems of cholera toxin detection exceeding available analogues in sensitivity.

2 dwg, 4 ex

FIELD: medicine.

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EFFECT: produced antibodies exceeds the analogues available in sensitivity.

2 dwg, 4 ex

FIELD: medicine.

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11 cl, 5 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: method facilitates linkage of sequences, coding immunoglobulin variable regions, T-cells receptors or B-cells receptors. Method is instrument of higher effectivity for making sequence data libraries. Capability of multiple RT-PCR with chain extension by interruption with employment of matrix, derived from single cell, provides highly effective creation of sister pairs libraries.

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51 cl, 25 dwg, 27 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: disclosed is a polypeptide having the capability of inducing antibodies which are bactericidal towards strains in each of three families of NMB 1870 I-III. The invention discloses an immunogenic composition containing said polypeptide. The invention also discloses nucleic acid which codes said polypeptide.

EFFECT: disclosed polypeptides can induce humoral immune response, which is bactericidal towards meningococcus.

8 cl

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