NOVEL SURFACE ANTIGEN

Abstract

novel surface polypeptide from Neisseria meningitidis as well as nucleic acid and nucleic acid sequence homologues encoding this protein. Pharmaceutical compositions containing the polypeptide and nucleic acids of the invention are also disclosed as well as methods useful in the treatment, prevention and diagnosis of N. meningitidis infection.

Description

[0001]The application is a divisional of U.S. application Ser. No. 11/776,709 filed Jul. 12, 2007, which is a continuation of U.S. application Ser. No. 10/637,659 filed Aug. 11, 2003, which is a continuation of U.S. application Ser. No. 09/797,862 filed Mar. 5, 2001, now U.S. Pat. No. 6,607,729, which is a continuation of U.S. application Ser. No. 09/377,155 filed Aug. 19, 1999, now U.S. Pat. No. 6,197,312, which is a continuation of PCT/AU98/01031 filed Dec. 14, 1998 (published as WO 1999/031132); the entireties of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002]The present invention relates to novel polypeptides as for example obtainable from Neisseria meningitidis, to nucleotide sequences encoding such polypeptides, to the use of these in diagnostics, in therapeutic and prophylactic vaccines and in the design and/or screening of medicaments.

BACKGROUND OF THE INVENTION

[0003]Neisseria meningitidis is a Gram-negative bacterium and the causative agent of meningococcal meningitis and septicemia. Its only known host is the human, and it may be carried asymptomatically by approximately 10% of the population (Caugant, D. et al, 1994, Journal of Clinical Microbiology, 32:323-30).

[0004]N. meningitidis may express a polysaccharide capsule, and this allows classification of the bacteria according to the nature of the capsule expressed. There are at least thirteen serogroups of N. meningitidis: A, B, C, 29-E, H, I, K, L, W135, X, Y and Z, of which serogroups A, B, and C cause 90% of meningococcal disease (Poolman, J. T. et al, 1995, Infectious Agents and Disease, 4:13-28). Vaccines directed against serogroups A and C are available, but the serogroup B capsular polysaccharide is poorly immunogenic and does not induce protection in humans.

[0005]Other membrane and extracellular components are therefore being examined for their suitability for inclusion in vaccines. Examples include the outer membrane proteins of classes 1, 2 and 3 (porins), and classes 4 (Rmp) and 5 (Opacity proteins). However, to date, none of these candidates is able to induce complete protection, particularly in children (Romero, J. D., 1994, Clinical Microbiology Review, 7:559-575; Poolman, J. T. et al, 1995, supra).

[0006]To create an effective vaccine, it is necessary to identify components of N. meningitidis which are present in a majority of strains, and which are capable of inducing a protective immune response (bactericidal antibodies). In this regard, reference may be made to Brodeur et al. (International Publication WO 96/29412) who disclose a 22 kDa surface protein which is highly conserved across 99% of all known strains of N. meningitidis. Injection of purified recombinant 22-kDa surface protein protected 80% of immunized mice against development of a lethal infection by N. meningitidis. Notwithstanding the discovery of this protein, there is still a need to isolate more surface proteins of N. meningitidis which are highly conserved across a plurality of strains, and which have immuno-protective profiles against N. meningitidis, and/or which may be used in combination with other components of N. meningitidis to enhance the efficacy of protection against this organism.

SUMMARY OF THE INVENTION

[0007]The present inventors have discovered a new gene which is present in all tested strains of N. meningitidis and which encodes a novel polypeptide having a predicted molecular weight of about 62 kDa. Based upon its sequence characteristics and homologies, this polypeptide is predicted to be an adhesin and this, together with experimental data suggests that it constitutes a surface protein which may be useful for the production of therapeutic and/or prophylactic vaccines against N. meningitidis as described hereinafter.

[0008]Accordingly, in one aspect of the invention, there is provided an isolated polypeptide or fragment thereof, or variant or derivative of these, said polypeptide selected from the group consisting of:-- [0009](a) a polypeptide according to SEQ ID NO 2; [0010](b) a polypeptide according to SEQ ID NO 5; [0011](c) a polypeptide according to SEQ ID NO 7; [0012](d) a polypeptide according to SEQ ID NO 9; [0013](e) a polypeptide according to SEQ ID NO 11; [0014](f) a polypeptide according to SEQ ID NO 13; [0015](g) a polypeptide according to SEQ ID NO 15; [0016](h) a polypeptide according to SEQ ID NO 17; [0017](i) a polypeptide according to SEQ ID NO 19; and [0018](j) a polypeptide according to SEQ ID NO 21.

[0019]Preferably, said polypeptide, fragment, variant or derivative which elicits an immune response against one or more members selected from the group consisting of:-- [0020](i) N. meningitidis; [0021](ii) said polypeptide; [0022](iii) said fragment; [0023](iv) said variant; and [0024](v) said derivative;

[0025]According to another aspect, the invention provides an isolated nucleic acid sequence encoding a polypeptide or fragment thereof, or variant or derivative of said fragment or polypeptide, according to the first-mentioned aspect. Suitably, said sequence is selected from the group consisting of:-- [0026](1) the nucleotide sequence of SEQ ID NO 1; [0027](2) the nucleotide sequence of SEQ ID NO 3; [0028](3) the nucleotide sequence of SEQ ID NO 4; [0029](4) the nucleotide sequence of SEQ ID NO 6; [0030](5) the nucleotide sequence of SEQ ID NO 8; [0031](6) the nucleotide sequence of SEQ ID NO 10; [0032](7) the nucleotide sequence of SEQ ID NO 12; [0033](8) the nucleotide sequence of SEQ ID NO 14; [0034](9) the nucleotide sequence of SEQ ID NO 16; [0035](10) the nucleotide sequence of SEQ ID NO 18; [0036](11) the nucleotide sequence of SEQ ID NO 20; [0037](12) a nucleotide sequence fragment of any one of SEQ ID NOS 1, 3, 4, 6, 8, 10, 12, 14, 16, 18 and 20; and [0038](13) a nucleotide sequence homologue of any of the foregoing sequences

[0039]Preferably, said sequences encode a product which elicits an immune response against one or more members selected from the group consisting of:-- [0040](i) N. meningitidis; [0041](ii) said polypeptide of the first-mentioned aspect; [0042](iii) said fragment of said first-mentioned aspect; [0043](iv) said variant of said first-mentioned aspect; and [0044](v) said derivative of said first-mentioned aspect.

[0045]In yet another aspect, the invention resides in an expression vector comprising a nucleic acid sequence according to the second-mentioned aspect wherein said sequence is operably linked to transcriptional and translational regulatory nucleic acid.

[0046]In a further aspect, the invention provides a host cell containing an expression vector according to the third-mentioned aspect.

[0047]In yet a further aspect of the invention, there is provided a method of producing a recombinant polypeptide according to the first-mentioned aspect, said method comprising the steps of:-- [0048](A) culturing a host cell containing an expression vector according to the third-mentioned aspect such that said recombinant polypeptide is expressed from said nucleic acid; and [0049](B) isolating said recombinant polypeptide.

[0050]In a still further aspect, the invention provides an antibody or antibody fragment that binds to one or more members selected from the group consisting of: [0051](1) N. meningitidis; [0052](2) said polypeptide of the first-mentioned aspect; [0053](3) said fragment of the first-mentioned aspect; [0054](4) said variant of the first-mentioned aspect; and [0055](5) said derivative of the first-mentioned aspect.

[0056]In yet another aspect, the invention provides a method of detecting N. meningitidis in a biological sample suspected of containing same, said method comprising the steps of:-- [0057](A) isolating the biological sample from a patient; [0058](B) mixing the above-mentioned antibody or antibody fragment with the biological sample to form a mixture; and [0059](C) detecting specifically bound antibody or bound antibody fragment in the mixture which indicates the presence of N. meningitidis.

[0060]According to a further aspect, there is provided a method of detecting N. meningitidis bacteria in a biological sample suspected of containing said bacteria, said method comprising the steps of:-- [0061](I) isolating the biological sample from a patient; [0062](II) detecting a nucleic acid sequence according to the second-mentioned aspect in said sample which indicates the presence of said bacteria.

[0063]The invention further contemplates a method for diagnosing infection of patients by N. meningitidis, said method comprising the steps of:-- [0064](1) contacting a biological sample from a patient with a polypeptide, fragment, variant or derivative of the invention; and [0065](2) determining the presence or absence of a complex between said polypeptide, fragment, variant or derivative and N. meningitidis-specific antibodies in said sample, wherein the presence of said complex is indicative of said infection.

[0066]The invention also extends to the use of the polypeptide according to the first-mentioned aspect, the use of the nucleic acids according to the second-mentioned aspect or the use of the antibody or antibody fragment mentioned above in a kit for detecting N. meningitidis bacteria in a biological sample.

[0067]According to a further aspect of the invention, there is provided a pharmaceutical composition comprising an isolated polypeptide or fragment thereof, or a variant or derivative of these, according to the first mentioned aspect.

[0068]Preferably, said pharmaceutical composition is a vaccine.

[0069]In yet a further aspect, the invention provides a method of preventing infection of a patient by N. meningitidis, comprising the step of administrating a pharmaceutically effective amount of the above-mentioned vaccine.

[0070]In a further aspect, the invention provides a method of identifying an immunoreactive fragment of a polypeptide, variant or derivatives according to the first mentioned aspect, comprising the steps of:-- [0071](a) producing a fragment of said polypeptide, variant or derivative; [0072](b) administering said fragment to a mammal; and [0073](c) detecting an immune response in said mammal which response includes production of elements which specifically bind N. meningitidis and/or said polypeptide, variant or derivative, and/or a protective effect against N. meningitidis infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0074]FIG. 1 depicts plasmid maps and cloning strategy. Primers A3A and A3B (SEQ ID NOS 28 and 29, respectively) were used to amplify from MC58 a region identified in preliminary sequence data as a homologue of AIDA-I (subsequently released by TIGR). PCR product was cloned to give pNMAIDA3. Primers A3C (SEQ ID NO 30) and A3D (SEQ ID NO 31) were used in inverse PCR to amplify a 3 kbp EagI fragment encompassing hiaNm. This product was cloned to give piEAGA3. piEAGA3 was subcloned to give piEagA3.8 and piEagA3.9. Primers HiaNm:M and HiaNm:P (SEQ ID NOS 22 and 23, respectively) were used to amplify the contiguous region from MC58 and the product cloned to create pHiaNm. Primers Hia-MBPA (SEQ ID NO 24) and Hia-MBPB (SEQ ID NO 25) were used to amplify the open reading frame of hiaNm, and the product was cloned into pMALC2 to create pMBP-HiaNm;

[0075]FIG. 2 is a Southern blot of genomic DNA of a number of strains of N. meningitidis. 2A: serogroup B strains. Lane 1 PMC28, Lane 2 PMC27, Lane 3 PMC25, Lane 4 PMC24, Lane 5 PMC16, Lane 6 PMC13, Lane 7 PMC12, Lane 8 MWt standards, Lane 9 2970, Lane 10 1000, Lane 11 528 Lane 12 SWZ107, Lane 13 H41, Lane 14 H38, Lane 15 NGH36, Lane 16 H15, Lane 17 NGG40, Lane 18 NGF26, Lane 19 NGE30, Lane 20 Lane NGE28 2B: Strains of serogroups other than B. Lane 1 PMC3, Lane 2 PMC17, Lane 3 PMC20, Lane 4 PMC23, Lane 5 PMC8, Lane 6 PMC9, Lane 7 PMC11, Lane 8 PMC14, Lane 9 PMC18, Lane 10 PMC21, Lane 11 PMC29, Lane 12 MWt standards, Lane 13 PMC19, Lane 14 PMC1, Lane 15 PMC6, Lane 16 PMC10, Lane 17 PMC22, Lane 18 PMC26, Lane 19 PMC2. Molecular weight markers indicated in kilobase pairs (kb). Genomic DNA was hybridized with a probe corresponding to ntp 276-2054 of SEQ ID NO 1;

[0076]FIG. 3 shows a Coomassie stained gel of MBP-HiaNm. Cells containing pMALC2 (Lane 2) or pMBP-HiaNm (Lane 3) after induction with IPTG. Lane 1 molecular weight standards (kDa). Arrows indicate MBP and MBP-HiaNm;

[0077]FIG. 4 is a western blot of MC58 and MC58ΔHiaNm proteins incubated with rabbit immune sera. Lane 1; molecular weight standards indicated in kDa, Lane 2 total cellular protein of MC58, Lane 3 total cellular protein of MC58ΔHiaNm Lane 4, OMC preparation of MC58, Lane 5 OMC preparation of MC58ΔHiaNm, each lane contained 50 μL of protein suspension of A₂₈₀=3.75;

[0078]FIG. 5 shows a Coomassie stained gel run in parallel to the gel that was Western blotted in FIG. 4. Lanes are the same as for FIG. 4;

[0079]FIG. 6 shows a sequence comparison of polypeptides of HiaNm (SEQ ID NO: 2), Hia (SEQ ID NO: 32), Hsf (SEQ ID NO: 33) using the PILEUP alignment program; and

[0080]FIG. 7 shows a sequence comparison of polypeptide sequences of HiaNm from 10 strains of N. meningitidis using the PILEUP program. FIG. 7 discloses SEQ ID NOS 11, 21, 2, 13, 5, 7, 9, 15, 17 and 19, respectively, in order of appearance.

DETAILED DESCRIPTION OF THE INVENTION

[0081]Throughout this specification and the appendant claims, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0082]Polypeptide Sequences

[0083]The present invention provides an isolated polypeptide according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21, or fragment respectively thereof, or variant or derivative of these. In a preferred embodiment, the polypeptide, fragments, variants and derivatives of the invention elicit an immune response against any one member selected from the group consisting of N. meningitidis, said polypeptide, said fragment, said variant and said derivative.

[0084]SEQ ID NO 2 corresponds to the novel about 62 kDa surface polypeptide of the hiaNm gene obtained from N. meningitidis strain MC58, as described more fully hereinafter. SEQ ID NOS 5, 7, 9, 11, 13, 15, 17, 19, and 21 correspond to homologous polypeptides deduced from nucleotide sequences obtained from N. meningitidis strains BZ10, BZ198, EG327, EG329, H15, H38, H41, P20, and PMC21, respectively.

[0085]For the purposes of this invention, the term the phrase"elicits an immune response" refers to the ability of the aforementioned polypeptide, fragment, variant or derivative to produce an immune response in a mammal to which it is administered, wherein the response includes the production of elements which specifically bind N. meningitidis and/or said polypeptide, fragment, variant or derivative, and/or which provide a protective effect against N. meningitidis infection.

[0086]By "isolated" is meant material that is substantially or essentially free from components which normally accompany it in its native state.

[0087]By "polypeptide" is meant a long chain peptide including a protein.

[0088]As used herein, the term "fragment" includes deletion mutants and small peptides, for example of at least 6, preferably at least 10 and more preferably at least 20 amino acids in length, which comprise antigenic determinants or epitopes. Several such fragments may be joined together. Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcins V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.

[0089]The term "variant" refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions). Exemplary conservative substitutions in the polypeptide may be made according to the following table:

TABLE-US-00001 TABLE 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Len, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile, Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0090]Substantial changes in function are made by selecting substitutions that are less conservative than those shown in TABLE 1. Other replacements would be non-conservative substitutions and relatively fewer of these may be tolerated. Generally, the substitutions which are likely to produce the greatest changes in a polypeptide's properties are those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

[0091]In general, variants will be at least 75% homologous, more suitably at least 80%, preferably at least 85%, and most preferably at least 90% homologous to the basic sequences as for example shown in SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21. Homology is defined as the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table 1. Homology may be determined using sequence comparison programs such as GAP (Deveraux at al. 1984, Nucleic Acids Research 12, 387-395) which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein may be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. What constitutes suitable variants may be determined by conventional techniques. For example, nucleic acids encoding polypeptides according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21 can be mutated using either random mutagenesis for example using transposon mutagenesis, or site-directed mutagenesis. The resultant DNA fragments are then cloned into suitable expression hosts such as E. coli using conventional technology and clones that retain the desired activity are detected. Where the clones have been derived using random mutagenesis techniques, positive clones would have to be sequenced in order to detect the mutation. The term "variant" also includes naturally occurring allelic variants.

[0092]By "derivative" is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. Such derivatives include amino acid deletions and/or additions to polypeptides according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21 or variants thereof wherein said derivatives retain activity eliciting an immune response "Additions" of amino acids may include fusion of the polypeptides or variants thereof with other polypeptides or proteins. In this regard, it will be appreciated that the polypeptides or variants of the invention may be incorporated into larger polypeptides, and such larger polypeptides may also be expected to retain immunological activity against, for example, N. meningitidis. The polypeptides as described above may be fused to a further protein, for example, which is not derived from N. meningitidis. The other protein may, by way of example, assist in the purification of the protein. For instance a polyhistidine tag, or a maltose binding protein may be used in this respect as described in more detail below. Alternatively, it may produce an immune response which is effective against N. meningitidis, or it may produce an immune response against another pathogen. Other possible fusion proteins are those which produce an immunomodulatory response. Particular examples of such proteins include Protein A or glutathione S-transferase (GST). In addition, the polypeptide may be fused to an oligosaccharide based vaccine component where it acts as a carrier protein.

[0093]Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides, fragments and variants of the invention.

[0094]Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; and trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS).

[0095]The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide.

[0096]The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[0097]Sulphydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate; 2-chloromercuri-4-nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

[0098]Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or by oxidation with N-bromosuccinimide.

[0099]Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

[0100]The imidazole ring of a histidine residue may be modified by N-carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

[0101]Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated by the present invention is shown in TABLE 2.

TABLE-US-00002 TABLE 2 Non-conventional amino acid Non-conventional amino acid α-aminobutyric acid L-N-methylalanine α-amino-α-methylbutyrate L-N-methylarginine aminocyclopropane-carboxylate L-N-methylasparagine aminoisobutyric acid L-N-methylaspartic acid aminonorbornyl-carboxylate L-N-methylcysteine cyclohexylalanine L-N-methylglutamine cyclopentylalanine L-N-methylglutamic acid L-N-methylisoleucine L-N-methylhistidine D-alanine L-N-methylleucine D-arginine L-N-methyllysine D-aspartic acid L-N-methylmethionine D-cysteine L-N-methylnorleucine D-glutamate L-N-methylnorvaline D-glutamic acid L-N-methylornithine D-histidine L-N-methylphenylalanine D-isoleucine L-N-methylproline D-leucine L-N-medlylserine D-lysine L-N-methylthreonine D-methionine L-N-methyltryptophan D-ornithine L-N-methyltyrosine D-phenylalanine L-N-methylvaline D-proline L-N-methylethylglycine D-serine L-N-methyl-t-butylglycine D-threonine L-norleucine D-tryptophan L-norvaline D-tyrosine α-methyl-aminoisobutyrate D-valine α-methyl-γ-aminobutyrate D-α-methylalanine α-methylcyclohexylalanine D-α-methylarginine α-methylcylcopentylalanine D-α-methylasparagine α-methyl-α-napthylalanine D-α-methylaspartate α-methylpenicillamine D-α-methylcysteine N-(4-aminobutyl)glycine D-α-methylglutamine N-(2-aminoethyl)glycine D-α-methylhistidine N-(3-aminopropyl)glycine D-α-methylisoleucine N-amino-α-methylbutyrate D-α-methylleucine α-napthylalanine D-α-methyllysine N-benzylglycine D-α-methylmethionine N-(2-carbamylediyl)glycine D-α-methylornithiine N-(carbamylmethyl)glycine D-α-methylphenylalanine N-(2-carboxyethyl)glycine D-α-methylproline N-(carboxymethyl)glycine D-α-methylserine N-cyclobutylglycine D-α-methylthreonine N-cycloheptylglycine D-α-methyltryptophan N-cyclohexylglycine D-α-methyltyrosine N-cyclodecylglycine L-α-methylleucine L-α-methyllysine L-α-methylmethionine L-α-methylnorleucine L-α-methylnorvatine L-α-methylornithine L-α-methylphenylalanine L-α-methylproline L-α-methylserine L-α-methylthreonine L-α-methyltryptophan L-α-methyltyrosine L-α-methylvaline L-N-methylhomophenylalanine N-(N-(2,2-diphenylethyl N-(N-(3,3-diphenylpropyl carbamylmethyl)glycine carbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl-ethyl amino)cyclopropane

[0102]The invention also contemplates covalently modifying a polypeptide, fragment or variant of the invention with dinitrophenol, in order to render it immunogenic in humans

[0103]Preferably the invention comprises a polypeptide selected from any one of the polypeptides according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21.

[0104]Polypeptides of the inventions may be prepared by any suitable procedure known to those of skill in the art. For example, the polypeptides may be prepared by a procedure including the steps of: [0105](a) preparing a recombinant nucleic acid containing a nucleotide sequence encoding a polypeptide according to any one of SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21, or fragment thereof, or variant or derivative of these, which nucleotide sequence is operably linked to transcriptional and translational regulatory nucleic acid; [0106](b) transfecting or transforming a suitable host cell with the recombinant nucleic acid; [0107](c) culturing the host cell to express recombinant polypeptide from said recombinant nucleic acid; and [0108](d) isolating the recombinant polypeptide.

[0109]Suitably said nucleotide sequence is selected from the group consisting of SEQ ID NOS 1, 3, 4, 6, 8, 10, 12, 14, 16, 18 and 20.

[0110]By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

[0111]The term "recombinant nucleic acid" as used herein refers to nucleic acid formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. In this regard, the recombinant nucleic acid preferably comprises an expression vector that may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the said nucleotide sequence.

[0112]By "operably linked" is meant that the transcriptional and translational regulatory nucleic acid is positioned relative to the nucleotide sequence encoding the said polypeptide, fragment, variant or derivative in such a manner that such transcription is initiatable. The transcriptional and translational regulatory nucleic acid will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

[0113]Typically, the transcriptional and translational regulatory nucleic acid may include, but is not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

[0114]Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.

[0115]In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

[0116]The expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant polypeptide of the invention is expressed as a fusion polypeptide with said fusion partner. The main advantage of fusion partners is that they assist identification and/or purification of said fusion polypeptide.

[0117]In order to express said fusion polypeptide, it is necessary to ligate a nucleotide sequence according to the invention into the expression vector so that the translational reading frames of the fusion partner and the nucleotide sequence of the invention coincide.

[0118]Well known examples of fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS₆) (SEQ ID NO: 34), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography. For the purposes of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively. Many such matrices are available in "kit" form, such as the QIAexpress® system (Qiagen) useful with (HIS₆) (SEQ ID NO: 34) fusion.

[0119]Another fusion partner well known in the art is green fluorescent protein (GFP). This fusion partner serves as a fluorescent "tag" which allows the fusion polypeptide of the invention to be identified by fluorescence microscopy or by flow cytometry. The GFP tag is useful when assessing subcellular localization of the fusion polypeptide of the invention, or for isolating cells which express the fusion polypeptide of the invention. Flow cytometric methods such as fluorescence activated cell sorting (FACS) are particularly useful in this latter application.

[0120]Preferably, the fusion partners also have protease cleavage sites, such as for Factor X_a or Thrombin, which allow the relevant protease to partially digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom. The liberated polypeptide can then be isolated from the fusion partner by subsequent chromatographic separation.

[0121]Fusion partners according to the invention also include within their scope "epitope tags", which are usually short peptide sequences for which a specific antibody is available. Well known examples of epitope tags for which specific monoclonal antibodies are readily available include c-myc, influenza virus haemagglutinin and FLAG tags.

[0122]Recombinant polypeptides of the invention may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a polypeptide, fragment, variant or derivative according to the invention. The conditions appropriate for protein expression will vary with the choice of expression vector and the host cell. This is easily ascertained by one skilled in the art through routine experimentation.

[0123]Suitable host cells for expression may be prokaryotic or eukaryotic. One preferred host cell for expression of a polypeptide according to the invention is a bacterium. The bacterium used may be Escherichia coli. Alternatively, the host cell may be an insect cell such as, for example, SF9 cells that may be utilized with a baculovirus expression system.

[0124]The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989), incorporated herein by reference, in particular Sections 16 and 17; Ausubel et al., CURRENT PROTOCOLS 1N MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), incorporated herein by reference, in particular Chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997) which is incorporated by reference herein, in particular Chapters 1, 5 and 6.

[0125]Nucleotide Sequences

[0126]The invention further provides a nucleotide sequence that encodes a polypeptide, fragment, variant or derivative as defined above. Suitably, said sequence is selected from the group consisting of: --SEQ ID NOS 1, 3, 4, 6, 8, 10, 12, 14, 16, 18 and 20; a nucleotide sequence fragment of any one of SEQ ID NOS 1, 3, 4, 6, 8, 10, 12, 14, 16, 18 and 20; and a nucleotide sequence homologue of the foregoing sequences. Suitably, these sequences encode a product that elicits an immune response as defined above.

[0127]As will be more fully described hereinafter, SEQ ID NO 1 corresponds to the hiaNm gene obtained from N. meningitidis strain MC58. This gene encodes the novel 62 kDa (approximately) surface polypeptide of SEQ ID NO 2. SEQ ID NO 3 corresponds to the hiaNm open reading frame sequence of strain MC58, HiaNm.

[0128]SEQ ID NOS 4, 6, 8, 10, 12, 14, 16, 18, and 20 correspond to the homologous hiaNm open reading frame sequences obtained from N. meningitidis strains BZ10, BZ198, EG327, EG329, H15, H38, H41, P20, and PMC21, respectively.

[0129]The term "nucleotide sequence" as used herein designates mRNA, RNA, cRNA, cDNA or DNA.

[0130]The term "nucleotide sequence homologues" generally refers to nucleotide sequences that hybridize with a wild-type nucleotide sequence according to the invention under substantially stringent conditions. Suitable hybridization conditions will be discussed hereinafter.

[0131]The nucleotide sequence homologues of the invention may be prepared according to the following procedure: [0132](i) obtaining a nucleic acid extract from a suitable host; [0133](ii) creating primers which are optionally degenerate wherein each comprises a portion of a wild-type nucleotide sequence of the invention; and [0134](iii) using said primers to amplify, via nucleic acid amplification techniques, one or more amplification products from said nucleic acid extract.

[0135]Suitably, the host may be a bacterium. Preferably, the host is from the genus Neisseria, more preferably from N. meningitidis.

[0136]Preferably, the primers are selected from the group consisting of:--

TABLE-US-00003 (SEQ ID NO 22) (1) 5'-TTAGATTCCACGTCCCAGATT-3'; (SEQ ID NO 23) (2) 5'-CTTCCCTTCAAACCTTCC-3'; (SEQ ID NO 24) (3) 5'-GGTCGCGGATCCATGAACAAAATATACCGCAT-3'; (SEQ ID NO 25) (4) 5'-TCACCCAAGCTTAAGCCCTTACCACTGATAAC-3'; (SEQ ID NO 26) (5) 5'-CCAAACCCCGATTTAACC-3'; (SEQ ID NO 27) (6) 5'-AATCGCCACCCTTCCCTTC-3'; (SEQ ID NO 28) (7) 5'-TTTGCAACGGTTCAGGCA-3'; (SEQ ID NO 29) (8) 5'-TATTCAGCAGCGTATCGG-3'; (SEQ ID NO 30) (9) 5'-TGCCTGAACCGTTGCAAA-3'; and (SEQ ID NO 31) (10) 5'-CCGATACGCTGCTGAATA-3'.

[0137]Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) as for example described in Ausubel et al. (1994-1998, supra, Chapter 15) which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in U.S. Pat. No. 5,422,252 which is incorporated herein by reference; rolling circle replication (RCR) as for example described in Liu et al., (1996, J. Am. Chem. Soc. 118:1587-1594 and International application WO 92/01813) and Lizardi et al., (International Application WO 97/19193) which are incorporated herein by reference; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al., (1994, Biotechniques 17:1077-1080) which is incorporated herein by reference; and Q-β replicase amplification as for example described by Tyagi et al., (1996, Proc. Natl. Acad. Sci. USA 93:5395-5400) which is incorporated herein by reference.

[0138]As used herein, an "amplification product" refers to a nucleic acid product generated by nucleic acid amplification techniques.

[0139]"Hybridize" or "hybridization" is used here to denote the pairing of complementary bases of distinct nucleotide sequences to produce a DNA-DNA hybrid, a DNA-RNA hybrid, or an RNA-RNA hybrid according to base-pairing rules.

[0140]In DNA, complementary bases are: [0141](i) A and T; and [0142](ii) C and G.

[0143]In RNA, complementary bases are: [0144](i) A and U; and [0145](ii) C and G.

[0146]In RNA-DNA hybrids, complementary bases are: [0147](i) A and U; [0148](ii) A and T; and [0149](iii) G and C.

[0150]Typically, substantially complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Southern blotting is used to identify a complementary DNA sequence; northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel et al. (1994-1998, supra) at pages 2.9.1 through 2.9.20.

[0151]According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane bound DNA to a complementary nucleotide sequence labeled radioactively, enzymatically or fluorochromatically. In dot blotting and slot blotting, DNA samples are directly applied to a synthetic membrane prior to hybridization as above.

[0152]An alternative blotting step is used when identifying complementary nucleotide sequences in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridization. A typical example of this procedure is described in Sambrook at al., (1989, supra) Chapters 8-12.

[0153]Typically, the following general procedure can be used to determine hybridization conditions. Nucleotide sequences are blotted/transferred to a synthetic membrane, as described above. A wild type nucleotide sequence of the invention is labeled as described above, and the ability of this labeled nucleotide sequence to hybridize with an immobilized nucleotide sequence analyzed.

[0154]A skilled addressee will recognize that a number of factors influence hybridization. The specific activity of radioactively labeled polynucleotide sequence should typically be greater than or equal to about 10⁸ dpm/mg to provide a detectable signal. A radiolabeled nucleotide sequence of specific activity 10⁸ to 10⁹ dpm/mg can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA, usually 10 μg. Adding an inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000 during hybridization can also increase the sensitivity of hybridization (see Ausubel supra at 2.10.10).

[0155]To achieve meaningful results from hybridization between a nucleotide sequence immobilized on a membrane and a labeled nucleotide sequence, a sufficient amount of the labeled nucleotide sequence must be hybridized to the immobilized nucleotide sequence following washing. Washing ensures that the labeled nucleotide sequence is hybridized only to the immobilized nucleotide sequences with a desired degree of complementarity to the labeled nucleotide sequence.

[0156]"Stringency" as used herein, refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the degree of complementarity between the immobilized nucleotide sequences and the labeled polynucleotide sequence.

[0157]"Stringent conditions" designates those conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridize.

[0158]Typical stringent conditions include, for example, (1) 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl at about 42° C. for at least 30 minutes; or (2) 6.0 M urea/0.4% sodium lauryl sulfate/0.1×SSC at about 42° C. for at least 30 minutes; or (3) 0.1×SSC/0.1% SDS at about 68° C. for at least 20 minutes; or (4) 1×SSC/0.1% SDS at about 55° C. for about 60 minutes; or (5) 1×SSC/0.1% SDS at about 62° C. for about 60 minutes; or (6) 1×SSC/0.1% SDS at about 68° C. for about 60 minutes; or (7) 0.2×SSC/0.1% SDS at about 55° C. for about 60 minutes; or (8) 0.2×SSC/0.1% SDS at about 62° C. for about one hour; or (9) 0.2×SSC/0.1% SDS at about 68° C. for about 60 minutes. For a detailed example, see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra at pages 2.10.1 to 2.10.16 and Sambrook et al. in MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989) at sections 1.101 to 1.104, which are hereby incorporated by reference.

[0159]While stringent washes are typically carried out at temperatures from about 42° C. to 68° C., one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization typically occurs at about 20° C. to 25° C. below the T_m for formation of a DNA-DNA hybrid. It is well known in the art that the T_m is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating T_m are well known in the art (see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra at page 2.10.8). Maximum hybridization typically occurs at about 10° C. to 15° C. below the T_m for a DNA-RNA hybrid.

[0160]Other stringent conditions are well known in the art. A skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

[0161]Methods for detecting labeled nucleotide sequences hybridized to an immobilized nucleotide sequence are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection.

[0162]Antibodies

[0163]The invention also contemplates antibodies against the aforementioned polypeptides, fragments, variants and derivatives. Such antibodies may include any suitable antibodies that bind to or conjugate with a polypeptide, fragment, variant or derivative of the invention. For example, the antibodies may comprise polyclonal antibodies. Such antibodies may be prepared for example by injecting a polypeptide, fragment, variant or derivative of the invention into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons, Inc, 1991) which is incorporated herein by reference, and Ausubel et al., (1994-1998, supra), in particular Section III of Chapter 11.

[0164]In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described in an article by Kohler and Milstein (1975, Nature 256, 495-497) which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al., (1991, supra) by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the polypeptides, fragments, variants or derivatives of the invention.

[0165]The invention also includes within its scope antibodies which comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in U.S. Pat. No. 5,091,513, European Patent No 239,400 or the article by Winter and Milstein (1991, Nature, 349 293) which are incorporated herein by reference.

[0166]The antibodies of the invention may be used for affinity chromatography in isolating natural or recombinant N. meningitidis polypeptides. For example reference may be made to immunoaffinity chromatographic procedures described in Chapter 9.5 of Coligan et al., (1995-1997, supra).

[0167]The antibodies can be used to screen expression libraries for variant polypeptides of the invention. The antibodies of the invention can also be used to detect N. meningitidis infection described hereinafter.

[0168]Detection of N. meningitidis

[0169]The presence or absence of N. meningitidis in a patient may determined by isolating a biological sample from a patient, mixing an antibody or antibody fragment described above with the biological sample to form a mixture, and detecting specifically bound antibody or bound fragment in the mixture which indicates the presence of N. meningitidis in the sample.

[0170]The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a patient. Suitably, the biological sample is selected from the group consisting of whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, skin biopsy, and the like.

[0171]Any suitable technique for determining formation of the complex may be used. For example, an antibody or antibody fragment according to the invention having a label associated therewith may be utilized in immunoassays. Such immunoassays may include, but are not limited to, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic techniques (ICTs) which are well known those of skill in the art. For example, reference may be made to "CURRENT PROTOCOLS IN IMMUNOLOGY" (1994, supra) which discloses a variety of immunoassays that may be used in accordance with the present invention. Immunoassays may include competitive assays as understood in the art.

[0172]The label associated with the antibody or antibody fragment may include the following: [0173]i. direct attachment of the label to the antibody or antibody fragment; [0174]ii. indirect attachment of the label to the antibody or antibody fragment; i.e., attachment of the label to another assay reagent which subsequently binds to the antibody or antibody fragment; and [0175]iii. attachment to a subsequent reaction product of the antibody or antibody fragment.

[0176]The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), a radioisotope and a direct visual label.

[0177]In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

[0178]A large number of enzymes suitable for use as labels is disclosed in U.S. Pat. No. 4,366,241, U.S. Pat. No. 4,843,000, and U.S. Pat. No. 4,849,338, all of which are herein incorporated by reference. Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme that is in solution.

[0179]Suitably, the fluorophore is selected from a group including fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITL) or R-Phycoerythrin (RPE).

[0180]The invention also extends to a method for detecting infection of patients by N. meningitidis, said method comprising the steps of contacting a biological sample from a patient with a polypeptide, fragment, variant or derivative of the invention, and determining the presence or absence of a complex between said polypeptide, fragment, variant or derivative and N. meningitidis-specific antibodies in said serum, wherein the presence of said complex is indicative of said infection.

[0181]In a preferred embodiment, detection of the above complex is effected by detectably modifying said polypeptide, fragment, variant or derivative with a suitable label as is well known in the art and using such modified compound in a suitable immunoassay as for example described above.

[0182]In another aspect, the invention provides a method of detecting N. meningitidis bacteria in a biological sample suspected of containing said bacteria, said method comprising the steps of isolating the biological sample from a patient, detecting a nucleic acid sequence according to the invention in said sample which indicates the presence of said bacteria.

[0183]Detection of the said nucleic acid sequence may be determined using any suitable technique. For example, a labeled nucleic acid sequence according to the invention may be used as a probe in a Southern blot of a nucleic acid extract obtained from a patient as is well known in the art. Alternatively, a labeled nucleic acid sequence according to the invention may be utilized as a probe in a Northern blot of a RNA extract from the patient. Preferably, a nucleic acid extract from the patient is utilized in concert with oligonucleotide primers corresponding to sense and antisense sequences of a nucleic acid sequence according to the invention, or flanking sequences thereof, in a nucleic acid amplification reaction such as PCR, or the ligase chain reaction (LCR) as for example described in International Application WO89/09385 which is incorporated by reference herein. A variety of automated solid-phase detection techniques are also appropriate. For example, very large scale immobilized primer arrays (VLSIPS®) are used for the detection of nucleic acids as for example described by Fodor et al., (1991, Science 251:767-777) and Kazal et al., (1996, Nature Medicine 2:753-759). The above generic techniques are well known to persons skilled in the art.

[0184]Pharmaceutical Compositions

[0185]A further feature of the invention is the use of the polypeptide, fragment, variant or derivative of the invention ("immunogenic agents") as actives in a pharmaceutical composition for protecting patients against infection by N. meningitidis. Suitably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0186]By "pharmaceutically-acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

[0187]Any suitable route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.

[0188]Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

[0189]Pharmaceutical compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more immunogenic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the immunogenic agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0190]The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is immunogenically-effective to protect patients from N. meningitidis infection. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over time such as a reduction in the level of N. meningitidis, or to inhibit infection by N. meningitidis. The quantity of the immunogenic agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the immunogenic agent(s) required to be administered will depend on the judgement of the practitioner. In determining the effective amount of the immunogenic agent to be administered in the treatment or prophylaxis against N. meningitidis, the physician may evaluate circulating plasma levels, progression of disease, and the production of anti-N. meningitidis antibodies. In any event, suitable dosages of the immunogenic agents of the invention may be readily determined by those of skill in the art. Such dosages may be in the order of nanograms to milligrams of the immunogenic agents of the invention.

[0191]The above compositions may be used as therapeutic or prophylactic vaccines. Accordingly, the invention extends to the production of vaccines containing as actives one or more of the immunogenic agents of the invention. Any suitable procedure is contemplated for producing such vaccines. Exemplary procedures include, for example, those described in NEW GENERATION VACClNES (1997, Levine et al., Marcel Dekker, Inc. New York, Basel Hong Kong), which is incorporated herein by reference.

[0192]An immunogenic agent according to the invention can be mixed, conjugated or fused with other antigens, including B or T cell epitopes of other antigens. In addition, it can be conjugated to a carrier as described below.

[0193]When an haptenic peptide of the invention is used (i.e., a peptide which reacts with cognate antibodies, but cannot itself elicit an immune response), it can be conjugated with an immunogenic carrier. Useful carriers are well known in the art and include for example: thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant crossreactive material (CRM) of the toxin from tetanus, diptheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streprococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, a haptenic peptide of the invention can be coupled to a T cell epitope of a bacterial toxin, toxoid or CRM. In this regard, reference may be made to U.S. Pat. No. 5,785,973 which is incorporated herein by reference.

[0194]In addition, a polypeptide, fragment, variant or derivative of the invention may act as a carrier protein in vaccine compositions directed against Neisseria, or against other bacteria or viruses.

[0195]The immunogenic agents of the invention may be administered as multivalent subunit vaccines in combination with antigens of N. meningitidis, or antigens of other organisms inclusive of the pathogenic bacteria H. influenzae, M. catarrhalis, N. gonorrhoeae, E. coli, S. pneumoniae etc. Alternatively or additionally, they may be administered in concert with oligosaccharide or polysaccharide components of N. meningitidis.

[0196]The vaccines can also contain a physiologically acceptable diluent or excipient such as water, phosphate buffered saline and saline.

[0197]The vaccines and immunogenic compositions may include an adjuvant as is well known in the art. Suitable adjuvants include, but are not limited to: surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N',N'bis(2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide or alum; lymphokines, QuilA and immune stimulating complexes (ISCOMS).

[0198]The immunogenic agents of the invention may be expressed by attenuated viral hosts. By "attenuated viral hosts" is meant viral vectors that are either naturally, or have been rendered, substantially avirulent. A virus may be rendered substantially avirulent by any suitable physical (e.g., heat treatment) or chemical means (e.g., formaldehyde treatment). By "substantially avirulent" is meant a virus whose infectivity has been destroyed. Ideally, the infectivity of the virus is destroyed without affecting the proteins that carry the immunogenicity of the virus. From the foregoing, it will be appreciated that attenuated viral hosts may comprise live viruses or inactivated viruses.

[0199]Attenuated viral hosts which may be useful in a vaccine according to the invention may comprise viral vectors inclusive of adenovirus, cytomegalovirus and preferably pox viruses such as vaccinia (see for example Paoletti and Panicali, U.S. Pat. No. 4,603,112 which is incorporated herein by reference) and attenuated Salmonella strains (see for example Stocker, U.S. Pat. No. 4,550,081 which is herein incorporated by reference). Live vaccines are particularly advantageous because they lead to a prolonged stimulus that can confer substantially long-lasting immunity.

[0200]Multivalent vaccines can be prepared from one or more microorganisms that express different epitopes of N. meningitidis (e.g., other surface proteins or epitopes of N. meningitidis). In addition, epitopes of other pathogenic microorganisms can be incorporated into the vaccine.

[0201]In a preferred embodiment, this will involve the construction of a recombinant vaccinia virus to express a nucleic acid sequence according to the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic agent, and thereby elicits a host CTL response. For example, reference may be made to U.S. Pat. No. 4,722,848, incorporated herein by reference, which describes vaccinia vectors and methods useful in immunization protocols.

[0202]A wide variety of other vectors useful for therapeutic administration or immunization with the immunogenic agents of the invention will be apparent to those skilled in the art from the present disclosure.

[0203]In a further embodiment, the nucleotide sequence may be used as a vaccine in the form of a "naked DNA" vaccine as is known in the art. For example, an expression vector of the invention may be introduced into a mammal, where it causes production of a polypeptide in vivo, against which the host mounts an immune response as for example described in Barry, M. et al., (1995, Nature, 377:632-635) which is hereby incorporated herein by reference.

[0204]Detection Kits

[0205]The present invention also provides kits for the detection of N. meningitidis in a biological sample. These will contain one or more particular agents described above depending upon the nature of the test method employed. In this regard, the kits may include one or more of a polypeptide, fragment, variant, derivative, antibody, antibody fragment or nucleic acid according to the invention. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) a nucleic acid according to the invention (which may be used as a positive control), (ii) an oligonucleotide primer according to the invention, and optionally a DNA polymerase, DNA ligase etc depending on the nucleic acid amplification technique employed.

Preparation of Immunoreactive Fragments

[0206]The invention also extends to a method of identifying an immunoreactive fragment of a polypeptide, variant or derivatives according to the invention. This method essentially comprises generating a fragment of the polypeptide, variant or derivative, administering the fragment to a mammal; and detecting an immune response in the mammal. Such response will include production of elements which specifically bind N. meningitidis and/or said polypeptide, variant or derivative, and/or a protective effect against N. meningitidis infection.

[0207]Prior to testing a particular fragment for immunoreactivity in the above method, a variety of predictive methods may be used to deduce whether a particular fragment can be used to obtain an antibody that cross-reacts with the native antigen. These predictive methods may be based on amino-terminal or carboxy-terminal sequence as for example described in Chapter 11.14 of Ausubel et al., (1994-1998, supra). Alternatively, these predictive methods may be based on predictions of hydrophilicity as for example described by Kyte and Doolittle (1982, J. Mol. Biol. 157:105-132) and Hopp and Woods (1983, Mol. Immunol. 20:483-489) which are incorporated by reference herein, or predictions of secondary structure as for example described by Choo and Fasman (1978, Ann. Rev. Biochem. 47:251-276), which is incorporated herein by reference.

[0208]Generally, peptide fragments consisting of 10 to 15 residues provide optimal results. Peptides as small as 6 or as large as 20 residues have worked successfully. Such peptide fragments may then be chemically coupled to a carrier molecule such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) as for example described in Sections 11.14 and 11.15 of Ausubel et al., (1994-1998, supra).

[0209]The peptides may be used to immunize an animal as for example discussed above. Antibody titers against the native or parent polypeptide from which the peptide was selected may then be determined by, for example, radioimmunoassay or ELISA as for instance described in Sections 11.16 and 114 of Ausubel et al., (1994-1998, supra).

[0210]Antibodies may then be purified from a suitable biological fluid of the animal by ammonium sulfate fractionation or by chromatography as is well known in the art. Exemplary protocols for antibody purification is given in Sections 10.11 and 11.13 of Ausubel et al., (1994-1998, supra).

[0211]Immunoreactivity of the antibody against the native or parent polypeptide may be determined by any suitable procedure such as, for example, western blot.

[0212]Functional Blockers

[0213]The polypeptides according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21 are believed to have adhesin properties. They in fact have some similarity to adhesins of Haemophilus influenzae, which are surface antigens. Specifically they are approximately 67% homologous to the Hia protein of H. influenzae (Barenkamp, S. and St. Geme III, J. 1996 Molecular Microbiology 19: 1215-1233), and 74% homologous to the Hsf protein of H. influenzae (St. Geme III, J. et al, 1996, Journal of Bacteriology 178: 6281-6287; and U.S. Pat. No. 5,646,259). For these comparisons, a gap weight of 3, and length weight of 0.01 was used using the GAP program (Deveraux, 1984, supra). Aligned sequences of these proteins are illustrated in FIG. 6. Thus, interruption of the function of these polypeptides would be of significant therapeutic benefit since they would prevent N. meningitidis bacteria from adhering to and invading cells. Interruption of the function may be effected in several ways.

[0214]For example, moieties such as chemical reagents or polypeptides which block receptors on the cell surface which interact with a polypeptides according to SEQ ID NOS 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21 may be administered. These compete with the infective organism for receptor sites. Such moieties may comprise for example polypeptides of the invention, in particular fragments, or functional equivalents of these as well as mimetics.

[0215]The term "mimetics" is used herein to refer to chemicals that are designed to resemble particular functional regions of the proteins or peptides. Anti-idiotypic antibodies raised against the above-described antibodies which block the binding of the bacteria to a cell surface may also be used. Alternatively, moieties which interact with the receptor binding sites in the polypeptides according to SEQ ID NO 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21 may effectively prevent infection of a cell by N. meningitidis. Such moieties may comprise blocking antibodies, peptides or other chemical reagents.

[0216]All such moieties, pharmaceutical compositions in which they are combined with pharmaceutically acceptable carriers and methods of treating patients suffering from N. meningitidis infection by administration of such moieties or compositions form a further aspect of the invention.

[0217]The polypeptides of the invention may be used in the screening of compounds for their use in the above methods. For example, polypeptides of the invention may be combined with a label and exposed to a cell culture in the presence of a reagent under test. The ability of reagent to inhibit the binding of the labeled polypeptide to the cell surface can then be observed. In such a screen, the labeled polypeptides may be used directly on an organism such as E. coli. Alternatively, N. meningitidis itself may be engineered to express a modified and detectable form of the polypeptide. The use of engineered N. meningitidis strains in this method is preferred as it is more likely that the tertiary structure of the protein will resemble more closely that expressed in wild-type bacteria.

[0218]In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

Example 1

Molecular Cloning and Subcloning and hiaNm Mutant Construction

[0219]The hiaNm gene was initially isolated by PCR amplification using standard methods. Briefly, due to our previous work on homologues of the AIDA-I protein of E. coli (Jennings, M. et al, 1995, Microbial Pathogenesis, 19: 391-407, Peak, I. et al, Microbial Pathogenesis, in press) we performed a homology search, identifying a sequence of interest in preliminary data from the project to sequence the genome of MC58 3 (subsequently released by The Institute for Genomic Research, Maryland (ftp://ftp.tigr.org/pub/data/n_meningitidis/) and amplified the region of homology by PCR using oligonucleotides A3A (5'-TTTGCAACGGTTCAGGCA-3', SEQ ID NO 28) and A3B (5'-TATTCAGCAGCGTATCGG-3', SEQ ID NO 29). The resulting 449 base pairs (bp) product was cloned into pT7Blue, to create plasmid pNMAIDA3. To clone the full-length gene, further oligonucleotides were designed and used in an inverse PCR reaction. These oligonucleotides were A3C (SEQ ID NO 30) and A3D (SEQ ID NO 31) and correspond to the complementary sequence of A3A (SEQ ID NO 28) and A3B (SEQ ID NO 31) respectively. The template for this reaction was chromosomal DNA of MC58, which had been restriction digested with EagI, and then self ligated. The resulting 3 kbp PCR product was cloned into the vector pCRII (Invitrogen), producing plasmid piEagA3. This was digested with EagI and EcoRI and the resulting fragments of 1.4 kbp and 1.6 kbp containing cloned DNA were cloned into pBluescriptSKII, M13minus (Stratagene), resulting in piEagA3.8 and piEagA3.9. Plasmid pHiaNm was generated by PCR amplifying hiaNm and sequence 5' and 3' to it using oligonucleotide primers HiaNm:P (5'-TTAGATTCCACGTCCCAGATT-3', SEQ ID NO 22) and HiaNm:M (5'-CTTCCCTTCAAACCTTCC-3', SEQ ID NO 23), corresponding to nucleotide position (ntp) 113-133 and 2102-2085 respectively of SEQ ID NO 1, and cloning the product into pT7Blue. Plasmid pHiaNmΔKan was created by insertion of a kanamycin resistance cassette into the unique BglII site of pHiaNm corresponding to ntp 680 of SEQ ID No 1. The kanamycin resistance cassette was excised from pUC4Kan (Pharmacia) with BamHI. pHiaNmΔKan was transformed into N. meningitidis strain MC58 by incubating bacteria with plasmid DNA for 3 hours on Brain Heart Infusion agar (Acumedia Manufacturer's Inc) supplemented with 10% heated horse blood ("BHI plates") at 37° C. in 5% CO₂. A single colony was picked onto fresh selective media, grown, and used for further studies. This mutant strain is designated MC58ΔHiaNm. Disruption of the hiaNM gene in this strain was confirmed by Southern blot using a probe corresponding to ntp 276-2054 of SEQ ID NO 1.

Example 2

Nucleotide Sequence Analysis

[0220]Nucleotide sequence analysis was performed using the PRISM Dye terminator sequencing Kit with AmpliTaq DNA polymerase FS or BigDye terminator sequencing kit as suggested by the manufacturer's instructions (Perkin Elmer), in conjunction with a model 373a automated sequencer (Applied Biosystems). For each strain, hiaNm was amplified in three independent PCR reactions using primers HiaNm5'A2: 5'-CCAAACCCCGATTTAACC-3' (SEQ ID NO 26) and HiaNm3'A: 5'-AATCGCCACCCTTCCCTTC-3' (SEQ ID NO 27), as indicated on FIG. 1, and corresponding to ntp 230-247 and 2114-2097 of SEQ ID No 1, and the resulting products purified and pooled. This was used as template for direct sequencing on both strands. Data were analysed using the GCG programs (Deveraux et al. (1984) Nucleic Acids Research 12, 387-395) and AssemblyLIGN (Oxford Molecular). Several oligonucleotides were generated as necessary to complete sequences. Sequences of hiaNm of 10 strains are shown in SEQ ID NOS 1, 3, 4, 6, 8, 10, 12, 14, 16, 18, and 20, and the deduced amino acid sequences of those genes are shown in SEQ ID NO 2, 5, 7, 9, 11, 13, 15, 17, 19 and 21.

[0221]Comparison of hiaNm from these strains indicated that they share 90-99% identity with hiaNm of MC58. In addition, hiaNm of MC58 is 62% and 68% homologous to hia and hsf of Haemophilus influenzae. However, in the strains examined, hiaNm is 1770-1800 by long. This is markedly different from the hia and hsf, which are 3294 and 7059 by long respectively. The predicted polypeptide of hiaNm, HiaNm, also exhibits homology to several other bacterial proteins, including AIDA-I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), HMW1, another Haemophilus adhesin, UspA1, a high molecular weight protein of Moraxella catarrthalis, and SepA involved in tissue invasion of Shigella flexneri (Benz, I. and Schmidt, M. A., 1992, Molecular Microbiology 6:1539-1546, Barenkamp, S. J. and Leininger, E. 1992, Infection and Immunity 60: 1302-1313, Aebi, C. et. al 1997, Infection and Immunity 65: 4367-4377, Benjelloun-Touimi, Z et al 1995, Molecular Microbiology 17:123-135). Homology to these (and several other proteins) occurs over the first fifty amino acids of HiaNm. Analysis of this sequence reveals the presence of a predicted signal sequence, with cleavage sites at amino acid 50 in all HiaNm sequences examined. Such long signal sequences are common to proteins located in the outer membrane of Gram-negative bacteria (Henderson, I et al, 1998, Trends in Microbiology 6: 370-8). The proteins mentioned above to which the first fifty amino acids of HiaNm is homologous are all members of the "autotransporter" outer-membrane protein family (Henderson, I, supra). This strongly suggests that HiaNm is located in the outer membrane of N. meningitidis.

Example 3

Southern Blot Analysis

[0222]Southern blot analysis was performed using standard techniques (Sambrook et al., supra, Ausubel et al., supra). Briefly, genomic DNA was prepared from 70 strains of N. meningitidis of several serogroups, restriction digested and separated electrophoretically on an agarose gel prior to capillary transfer to a nylon membrane. These membranes were hybridized with a labeled probe. The probe used corresponded to ntp 276-2054 of SEQ ID NO 1, encompassing the entire open reading frame of hiaNm of strain MC58. This was labeled with DIG (dioxygenin) according to manufacturer's instructions (Boehringer Mannheim). Stringent washes were performed (two washes of 5 minutes at 22° C. in 2×SSC/0.1% SDS followed by two washes of 30 minutes, 68° C., 0.2×SSC/0.1% SDS). Hybridization was detected colorimetrically using nitro-blue-tetrazolium/bromo-chloryl-indolyl-phosphate (NBT/BCIP) as recommended by the manufacturer. Signals were detected in all strains examined. (FIG. 2 for example). In addition to the prototypic strain MC58, the following strains were investigated:--

TABLE-US-00004 TABLE 3 Sero- Sero- Strain Name Source group Strain name Source group PMC 3 (J1079) 2^A A NGF26 1 B PMC17 (K874) 2 A NGG40 1 B PMC 20 ( (H79) 2 A H15 1 B PMC23 (K750) 2 A SWZ107 1 B PMC 12 (K852) 2 B 528 1 B PMC 13 (K859) 2 B 2970 1 B PMC 16 (K873) 2 B 1000 1 B PMC 24 (K782) 2 B MPJB28 3^C B PMC 25 (K791) 2 B MPJB56 3 B PMC 27 (K816) 2 B MPJB88 3 B PMC 28 (K837) 2 B MPJB157 3 B BZ10 1^B B MPJB328 3 B BZ47 1 B MPJB627 3 B BZ83 1 B MPJB820 3 B BZ133 1 B MPJB945 3 B BZ147 1 B PMC 8 (K157) 2 C BZ163 1 B PMC 9 (K497) 2 C BZ169 1 B PMC 11 (K848) 2 C BZ198 1 B PMC 14 (K860) 2 C BZ232 1 B PMC 18 (K879) 2 C NG3/88 1 B PMC 21 (K656) 2 C NG4/88 1 B PMC 29 (K841) 2 C NG6/88 1 B MPJC05 3 C EG327 1 B MPJC14 3 C EG329 1 B MPJC154 3 C DK353 1 B MPJC302 3 C 179/82 1 B MPJC379 3 C 66/84 1 B PMC19 2 W DK24 1 B MPJW025 3 W NGH36 1 B PMC 1 (J603) 2 X H38 1 B PMC 6 (K131) 2 X H41 1 B PMC 10 (K526) 2 Y NGE28 1 B PMC 22 (K685) 2 Y NGE30 1 B PMC 26 (K810) 2 Y NGP20 1 B PMC 2 ( (J1049) 2 Z ^AWorld Health Organization Collaborating Centre for Reference and Research on Meningococci, Oslo, Norway ^BPublic Health Laboratory Service Meningococcal Reference Laboratory, Manchester, UK ^CBrisbane Hospitals, now in strain collection of M.P. Jennings, Department of Microbiology, University of Queensland, Birsbane, Australia.

Example 4

Expression and Partial Purification of MBP-Hi a Nm

[0223]A plasmid vector was constructed which permitted the expression of a protein consisting of a fusion of Maltose Binding Protein and HiaNm (MBP-HiaNm). The plasmid pHiaMBP was generated by amplifying hiaNm from MC58 using primers Hianm-MBPA 5'-GGTCGCGGATCCATGAACAAAATATACCGCAT-3' (SEQ ID NO 24) and HiaNm-MBPB 5'-TCACCCAAGCTTAAGCCCTTACCACTGATAAC-3' (SEQ ID NO 25). These primers encompass the start and stop codons of hiaNm of N. meningitidis strain MC58 and engineered restriction sites for ease of cloning. Plasmid restriction maps and positions of oligonucleotides are shown in FIG. 1. The resultant PCR product was ligated into BamHI/HindIII restriction digested plasmid pMALC2 (New England Biolabs), and the resultant plasmid, pHiaMBP (See FIG. 1) reintroduced to E. coli strain DH5α. This E. coli strain containing pHiaMBP was induced to express the HiaNm-MBP fusion protein under conditions recommended by the manufacturer (New England Biolabs). Cell extracts from cultures containing pHiAMBP were separated by 10% SDS-PAGE, and the fusion protein was partially purified by elution using the Mini-Gel Electro-eluter (BioRad) according to manufacturer's instructions. Fractions containing the HiaNm-MBP fusion protein were detected by Western blot using rabbit anti-MBP sera (New England Biolabs). The purity of the HiaNm-MBP fusion protein was determined by SDS-PAGE followed by Coomassie staining, and the amount of recovered protein estimated by BCA assay (Sigma) or absorbance at a wavelength of 280 nm.

Example 5

Generation of Polyclonal Sera

[0224]The partially purified HiaNm-MBP fusion protein obtained in Example 4 was used to generate polyclonal sera in rabbits. Samples of eluted HiaNmMBP fusion protein were dialyzed against sterile phosphate buffered saline pH 7.4, (PBS) (Sigma). This was then mixed with adjuvant (MPL+TDM+CWS, Sigma), at a concentration of 50-150 μg/mL and inoculated at two weekly intervals into two New Zealand White rabbits. Blood was taken from these rabbits. Serum was extracted by clotting at room temperature for one hour followed by overnight incubation at 4° C. before centrifugation at 4000×rpm at 4° C. The supernatant was removed and re-centrifuged. Serum was stored in aliquots at -80° C. Sera obtained were used in bactericidal assays and Western blots (see below).

[0225]To test the specificity of the sera obtained, Western blot analysis was undertaken. Briefly, proteins of N. meningitidis strains MC58 and MC58ΔHianm were separated electrophoretically on SDS-PAGE before electrophoretic transfer to nitrocellulose membrane using a Semi-Dry Blotter (BioRad). These were then incubated sequentially with sera and alkaline-phosphatase-conjugated anti-Rabbit IgG (Sigma) before colorimetric detection with NBT/BLIP (Sigma). These experiments demonstrated that antibodies were elicited by the HiaNm-MBP fusion protein, which were specific for, and detected a band in, MC58 but not in MC58ΔHiaNm (see FIG. 4). The predicted molecular weight of the deduced polypeptide of HiaNm is 62.3 kDa. The band detected by the sera migrates at an apparent MW in excess of 150 kDa. At least three of the homologous "autotransporter" proteins reported in the literature also display such anomalous migration: the high molecular weight outer membrane proteins UspA1 and UspA2 of Moraxella catarrhalis have predicted molecular weights of 62.5 kDa and 88.3 kDa respectively but migrate at 85 kDa and 120 kDa, respectively and as the UspA complex at between 350 kDa and 720 kDa (Aebi, C. et al., 1997, Infection and Immunity, 65: 4367-4377, Klingman, K. L. and Murphy, T. F., 1994, Infection and Immunity, 62: 1150-1155). Similarly, Hia of Haemophilus influenzae has a predicted molecular weight of 116 kDa but when expressed in a phage, Hia migrates at greater than 200 kDa (Barenkamp, S. and St. Geme III, J. 1996 Molecular Microbiology 19: 1215-1233).

[0226]In order to confirm that HiaNm is associated with the outer membrane of N. meningitidis, outer membrane complexes (omc) were prepared, essentially as previously described (van der Ley, P. et al, 1991, Infection and Immunity, 59:2963-71). Briefly, bacteria were grown overnight on Brain Heart Infusion agar (Acumedia Manufacturer's Inc) supplemented with 10% heated horse blood BHI plates, resuspended in 10 mM Tris pH 8.0 and heat killed, before sonication to disrupt the membrane. Cellular debris were removed by centrifugation at 10,000×g (rcf, relative centrifugal force), and the supernatant recentrifuged at 50,000×g. This pellet was resuspended in 1% sarkosyl/10 mM Tris pH8.4 and centrifuged at 10,000×g. The supernatant was centrifuged at 75,000×g and the pellet resuspended in Tris pH 8.4, before quantification spectrophotometrically at a wavelength of 280 nm. An aliquot of the sarkosyl-insoluble fraction, which contains outer membrane proteins, (50 μl of A₂₈₀=3.75) was subjected to SDS-PAGE and Western blotted as described above. The results, shown in FIG. 4 demonstrate that reactivity with the anti-HiaNmMBP antisera is observed with wild type MC58, but not with MC58ΔHiaNm, in which hiaNm has been inactivated. The increase in reactivity with the anti-HiaMBP sera observed between whole cell samples, and the omc samples containing the same amount of total protein, in MC58 cultures is consistent with the membrane association of HiaNm.

Example 6

Bactericidal Assay

[0227]To determine whether the anti-HiaMBP antisera contained bactericidal antibodies specific for HiaNm, bactericidal assays were performed with wild type MC58 and MC58ΔHiaNm. This assay was performed by a modification of the method described by Hoogerhout et. al. (1995, Infection and Immunity, 63: 3473-3478). Briefly, MC58 and MC58ΔHiaNm were grown overnight on BHI plates at 37° C. in 5% CO₂. Bacteria from this overnight culture were subcultured under the same conditions for 4-6 hours before suspension in 1 mL PBS. Numbers of bacteria were estimated by lysis of a sample in 0.2N NaOH/1% SDS and absorbance at a wavelength of 260 nm, where A₂₆₀=1=10⁹ cfu/mL. The bacterial suspension was adjusted to approximately 10⁵ cfu/mL in PBS. Rabbit sera to be tested was heat inactivated at 56° C. for 45 minutes. Serum from four-week-old, New Zealand White rabbits was pooled and used as a source of complement (Central Animal Breeding House, University of Queensland). The assay was carried out in sterile polystyrene flat-bottomed 96 well microtitre plate. The total volume in each well was 24 μL: 12 μL of twofold serially diluted serum in PBS and 6 μL of bacterial suspension (containing between 300-900 bacteria). Sera and bacteria were incubated at room temperature for 10 minutes before addition of 6 μL of 80% complement in PBS (final concentration 20% vol/vol). Controls were a) PBS, bacteria and complement, b) PBS, bacteria and serum. After addition of all components and mixing, a 7 μL aliquot from each control well was spread on a BHI plate. The microtitre plate was then incubated at 37° C. in 5% CO₂ for 60 minutes. After this incubation, a 7 μL aliquot from each well was spread on BHI plates. All BHI plates were then incubated for 14-18 hours at 37° C. in 5% CO₂, and bacterial colonies counted. Serum bactericidal killing is reported as the highest reciprocal dilution at which at least 90% of bacteria were killed. Serum used was from the same rabbit and the same test bleed as used for Western blot experiments as reported in Example 5 above. These experiments consistently demonstrated reduced titers (approximately 3 fold, Table 4) of killing against MC58ΔHiaNm in comparison to the wild type strain, MC58, indicating that the anti-HiaMBP antisera contained bactericidal antibodies specific for HiaNm.

TABLE-US-00005 TABLE 4 STRAIN TITRE^a MC58 12 (+/-4.6) MC58ΔHiaNm 3.5 (+/-1) ^aMean of four independent experiments

Discussion

[0228]Repetitive DNA has been associated with virulence determinants in some pathogenic bacteria. Southern blots using such a repetitive DNA motif revealed the presence of at least three loci which contained this motif in N. meningitidis strain MC58 (Peak, I. et al., 1996, FEMS Microbiology Letters, 137:109-114). These genes were cloned and sequence analysis of two of these repeat associated loci (nmrep2 and nmrep3) revealed open reading frames of approximately 670 amino acids (Jennings, M. et al, 1995, Microbial Pathogenesis, 19: 391-407, Peak, I. et al, Microbial Pathogenesis, in press). These exhibited homology to each other and homology to the carboxyl-terminal of the adhesin AIDA-I of E. coli. AIDA-I is 1286 amino acids long. The carboxyl-terminal region constitutes a putative outer membrane transport domain and the amino-terminal domain of the mature protein constitutes the adhesin domain. The amino-terminal domain crosses the membrane through the putative transport domain and is designated the passenger domain.

[0229]As Nmep2 and Nmep3 share sequence homology with the transporter domain of AIDA-I, they are thought to form membrane pores. Nmrep2 and Nmrep3 are approximately half the size of AIDA-I, and are homologous to the membrane-spanning domain of AIDA-I. We hypothesized that there existed in N. meningitidis a locus with homology to the amino-terminal domain of AIDA-I. We searched for such a homologue in the data from the project to sequence N. meningitidis strain MC58 3 (TIGR, supra) and found one region with homology to a gene designated AIDA-I in Haemophilus influenzae strain Rd (HI1732) because of its homology to AIDA-I of E. coli, (Fleischmann et. al., 1995 Science 269:496-512,). In view of the homologies noted above, the applicants decided to investigate further.

[0230]The gene was initially isolated by PCR amplification of the DNA corresponding to the 471 base pair fragment, named gnmaa84r, from N. meningitidis MC58 3 and the sequence was confirmed. Further PCR experiments enabled larger fragments to be amplified. These were cloned and sequence analysis undertaken as shown in FIG. 1. The gene exhibited homology to the amino-terminal region of AIDA-I of E. coli and we designated it aida3, as it represented the third AIDA-I homologue in N. meningitidis (with nmrep2 and nmrep2). Since then, the discovery of two further genes, hia and hsf from H. influenzae has been published (Barenkamp, S, and St. Geme III, J. 1996 Molecular Microbiology 19: 1215-1233, St. Geme III, J. et al, 1996, Journal of Bacteriology 178: 6281-6287), to which aida3 is more similar. We have therefore re-designated this gene hiaNm. (HI1732, the H. influenzae gene first identified as an homologue of AIDA-I has also been re-designated hia in light of the reports of Barenkamp and St. Geme III).

[0231]Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appendant claims.

[0232]This application claims priority to GB No. 9726398.2, filed Dec. 12, 1997, which is hereby incorporated by reference.

Sequence CWU 1

3412308DNANeisseria meningitidisCDS(276)..(2054) 1gaaaaaccac aggaatttat cagcaaaaac agaaacccca ccgccgtcat tcccgcaaaa 60gcgggaatcc agacccgtcg gcacggaaaa cttaccgaat aaaacagttt ccttagattc 120cacgtcccag attcccgcct tcgcggggaa tgacgagatt ttaagttggg ggaatttatc 180agaaaacccc caacccccaa aaaccgggcg gatgccgcac catccgcccc caaaccccga 240tttaaccatt caaacaaacc aaaagaaaaa acaaa atg aac aaa ata tac cgc 293 Met Asn Lys Ile Tyr Arg 1 5atc att tgg aat agt gcc ctc aat gcc tgg gtc gtc gta tcc gag ctc 341Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Ser Glu Leu 10 15 20aca cgc aac cac acc aaa cgc gcc tcc gca acc gtg aag acc gcc gta 389Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Lys Thr Ala Val 25 30 35ttg gcg aca ctg ttg ttt gca acg gtt cag gca agt gct aac aat gaa 437Leu Ala Thr Leu Leu Phe Ala Thr Val Gln Ala Ser Ala Asn Asn Glu 40 45 50aga cca aga aag aaa gat tta tat tta gac ccc gta caa cgc act gtt 485Arg Pro Arg Lys Lys Asp Leu Tyr Leu Asp Pro Val Gln Arg Thr Val55 60 65 70gcc gtg ttg ata gtc aat tcc gat aaa gaa ggc acg gga gaa aaa gaa 533Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly Thr Gly Glu Lys Glu 75 80 85aaa gta gaa gaa aat tca gat tgg gca gta tat ttc aac gag aaa gga 581Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr Phe Asn Glu Lys Gly 90 95 100gta cta aca gcc aga gaa atc acc ctc aaa gcc ggc gac aac ctg aaa 629Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala Gly Asp Asn Leu Lys 105 110 115atc aaa caa aac ggc aca aac ttc acc tac tcg ctg aaa aaa gac ctc 677Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser Leu Lys Lys Asp Leu 120 125 130aca gat ctg acc agt gtt gga act gaa aaa tta tcg ttt agc gca aac 725Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu Ser Phe Ser Ala Asn135 140 145 150ggc aat aaa gtc aac atc aca agc gac acc aaa ggc ttg aat ttt gcg 773Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala 155 160 165aaa gaa acg gct ggg acg aac ggc gac acc acg gtt cat ctg aac ggt 821Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr Val His Leu Asn Gly 170 175 180att ggt tcg act ttg acc gat acg ctg ctg aat acc gga gcg acc aca 869Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr 185 190 195aac gta acc aac gac aac gtt acc gat gac gag aaa aaa cgt gcg gca 917Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala 200 205 210agc gtt aaa gac gta tta aac gct ggc tgg aac att aaa ggc gtt aaa 965Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys215 220 225 230ccc ggt aca aca gct tcc gat aac gtt gat ttc gtc cgc act tac gac 1013Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp 235 240 245aca gtc gag ttc ttg agc gca gat acg aaa aca acg act gtt aat gtg 1061Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val 250 255 260gaa agc aaa gac aac ggc aag aaa acc gaa gtt aaa atc ggt gtg aag 1109Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val Lys Ile Gly Val Lys 265 270 275act tct gtt att aaa gaa aaa gac ggt aag ttg gtt act ggt aaa gac 1157Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Asp 280 285 290aaa ggc gag aat ggt tct tct aca gac gaa ggc gaa ggc tta gtg act 1205Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr295 300 305 310gca aaa gaa gtg att gat gca gta aac aag gct ggt tgg aga atg aaa 1253Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys 315 320 325aca aca acc gct aat ggt caa aca ggt caa gct gac aag ttt gaa acc 1301Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr 330 335 340gtt aca tca ggc aca aat gta acc ttt gct agt ggt aaa ggt aca act 1349Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser Gly Lys Gly Thr Thr 345 350 355gcg act gta agt aaa gat gat caa ggc aac atc act gtt atg tat gat 1397Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile Thr Val Met Tyr Asp 360 365 370gta aat gtc ggc gat gcc cta aac gtc aat cag ctg caa aac agc ggt 1445Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly375 380 385 390tgg aat ttg gat tcc aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc 1493Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile 395 400 405agc ggc aat gtt tcg ccg agc aag gga aag atg gat gaa acc gtc aac 1541Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn 410 415 420att aat gcc ggc aac aac atc gag att acc cgc aac ggt aaa aat atc 1589Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile 425 430 435gac atc gcc act tcg atg acc ccg cag ttt tcc agc gtt tcg ctc ggc 1637Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly 440 445 450gcg ggg gcg gat gcg ccc act ttg agc gtg gat ggg gac gca ttg aat 1685Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp Gly Asp Ala Leu Asn455 460 465 470gtc ggc agc aag aag gac aac aaa ccc gtc cgc att acc aat gtc gcc 1733Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg Ile Thr Asn Val Ala 475 480 485ccg ggc gtt aaa gag ggg gat gtt aca aac gtc gca caa ctt aaa ggc 1781Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly 490 495 500gtg gcg caa aac ttg aac aac cgc atc gac aat gtg gac ggc aac gcg 1829Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala 505 510 515cgt gcg ggc atc gcc caa gcg att gca acc gca ggt ctg gtt cag gcg 1877Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu Val Gln Ala 520 525 530tat ttg ccc ggc aag agt atg atg gcg atc ggc ggc ggc act tat cgc 1925Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg535 540 545 550ggc gaa gcc ggt tac gcc atc ggc tac tcc agt att tcc gac ggc gga 1973Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Gly Gly 555 560 565aat tgg att atc aaa ggc acg gct tcc ggc aat tcg cgc ggc cat ttc 2021Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe 570 575 580ggt gct tcc gca tct gtc ggt tat cag tgg taa gggctttatc gcctgtctgc 2074Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 585 590tgttgggaca ggcggaaggt ttgaagggaa gggtggcgat ttgccgcctg agacctttgc 2134aaaatccccc caaaatcccc taaattccca ccaagacatt taggggattt ctcatgagca 2194ccttcttccg gcaaaccgcg caagccatga ttgccaaaca catcaaccgt ttcccgctat 2254tgaagttgga ccaagtgatt gattggcagc cgatcgagca gtacctgaac cgtc 23082592PRTNeisseria meningitidis 2Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Ser Ala Asn Asn Glu Arg Pro Arg Lys Lys Asp Leu Tyr Leu Asp 50 55 60Pro Val Gln Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu65 70 75 80Gly Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val 85 90 95Tyr Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys 100 105 110Ala Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr 115 120 125Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys 130 135 140Leu Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr145 150 155 160Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr 165 170 175Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu 180 185 190Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp 195 200 205Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp 210 215 220Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp225 230 235 240Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys 245 250 255Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu 260 265 270Val Lys Ile Gly Val Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys 275 280 285Leu Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu 290 295 300Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys305 310 315 320Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln 325 330 335Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala 340 345 350Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn 355 360 365Ile Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn 370 375 380Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly385 390 395 400Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys 405 410 415Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr 420 425 430Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe 435 440 445Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val 450 455 460Asp Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val465 470 475 480Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn 485 490 495Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp 500 505 510Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr 515 520 525Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile 530 535 540Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser545 550 555 560Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly 565 570 575Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 59031779DNANeisseria meningitidis 3atgaacaaaa tataccgcat catttggaat agtgccctca atgcctgggt cgtcgtatcc 60gagctcacac gcaaccacac caaacgcgcc tccgcaaccg tgaagaccgc cgtattggcg 120acactgttgt ttgcaacggt tcaggcaagt gctaacaatg aaagaccaag aaagaaagat 180ttatatttag accccgtaca acgcactgtt gccgtgttga tagtcaattc cgataaagaa 240ggcacgggag aaaaagaaaa agtagaagaa aattcagatt gggcagtata tttcaacgag 300aaaggagtac taacagccag agaaatcacc ctcaaagccg gcgacaacct gaaaatcaaa 360caaaacggca caaacttcac ctactcgctg aaaaaagacc tcacagatct gaccagtgtt 420ggaactgaaa aattatcgtt tagcgcaaac ggcaataaag tcaacatcac aagcgacacc 480aaaggcttga attttgcgaa agaaacggct gggacgaacg gcgacaccac ggttcatctg 540aacggtattg gttcgacttt gaccgatacg ctgctgaata ccggagcgac cacaaacgta 600accaacgaca acgttaccga tgacgagaaa aaacgtgcgg caagcgttaa agacgtatta 660aacgctggct ggaacattaa aggcgttaaa cccggtacaa cagcttccga taacgttgat 720ttcgtccgca cttacgacac agtcgagttc ttgagcgcag atacgaaaac aacgactgtt 780aatgtggaaa gcaaagacaa cggcaagaaa accgaagtta aaatcggtgt gaagacttct 840gttattaaag aaaaagacgg taagttggtt actggtaaag acaaaggcga gaatggttct 900tctacagacg aaggcgaagg cttagtgact gcaaaagaag tgattgatgc agtaaacaag 960gctggttgga gaatgaaaac aacaaccgct aatggtcaaa caggtcaagc tgacaagttt 1020gaaaccgtta catcaggcac aaatgtaacc tttgctagtg gtaaaggtac aactgcgact 1080gtaagtaaag atgatcaagg caacatcact gttatgtatg atgtaaatgt cggcgatgcc 1140ctaaacgtca atcagctgca aaacagcggt tggaatttgg attccaaagc ggttgcaggt 1200tcttcgggca aagtcatcag cggcaatgtt tcgccgagca agggaaagat ggatgaaacc 1260gtcaacatta atgccggcaa caacatcgag attacccgca acggtaaaaa tatcgacatc 1320gccacttcga tgaccccgca gttttccagc gtttcgctcg gcgcgggggc ggatgcgccc 1380actttgagcg tggatgggga cgcattgaat gtcggcagca agaaggacaa caaacccgtc 1440cgcattacca atgtcgcccc gggcgttaaa gagggggatg ttacaaacgt cgcacaactt 1500aaaggcgtgg cgcaaaactt gaacaaccgc atcgacaatg tggacggcaa cgcgcgtgcg 1560ggcatcgccc aagcgattgc aaccgcaggt ctggttcagg cgtatttgcc cggcaagagt 1620atgatggcga tcggcggcgg cacttatcgc ggcgaagccg gttacgccat cggctactcc 1680agtatttccg acggcggaaa ttggattatc aaaggcacgg cttccggcaa ttcgcgcggc 1740catttcggtg cttccgcatc tgtcggttat cagtggtaa 177941797DNANeisseria meningitidisCDS(1)..(1797) 4atg aac aaa ata tcc cgc atc att tgg aat agt gcc ctc aat gcc tgg 48Met Asn Lys Ile Ser Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg gcg acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gcg aat gct acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80aaa gaa ggt aca gaa gat tca aat tgg gca gta tat ttc gac gag aaa 288Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys 85 90 95aga gta cta aaa gcc gga gca atc acc ctc aaa gcc ggc gac aac ctg 336Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110aaa atc aaa caa aac acc aat gaa aac acc aat gaa aac acc aat gac 384Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp 115 120 125agt agc ttc acc tac tcc ctg aaa aaa gac ctc aca gat ctg acc agt 432Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser 130 135 140gtt gaa act gaa aaa tta tcg ttt ggc gca aac ggt aat aaa gtc aac 480Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn145 150 155 160atc aca agc gac acc aaa ggc ttg aat ttt gcg aaa gaa acg gct ggg 528Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly 165 170 175acg aac ggc gac ccc acg gtt cat ctg aac ggt atc ggt tcg act ttg 576Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu 180 185 190acc gat acg ctg ctg aat acc gga gcg acc aca aac gta acc aac gac 624Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp 195 200 205aac gtt acc gat gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta 672Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val 210 215 220tta aac gca ggc tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct 720Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala225 230 235 240tcc gat aac gtc gat ttc gtc cgc act tac gac aca gtc gag ttc ttg 768Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu 245 250 255agc gca gat acg aaa aca acg act gtt aat gtg gaa agc aaa gac aac 816Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn 260 265 270ggc aag aga acc gaa gtt aaa atc ggt gcg aag act tct gtt att aaa 864Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys 275 280 285gaa aaa gac ggt aag ttg gtt act ggt aaa ggc aaa ggc gag aat ggt 912Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn Gly 290 295 300tct tct aca gac gaa ggc gaa ggc tta gtg act gca aaa gaa gtg att 960Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile305 310 315 320gat gca gta aac

aag gct ggt tgg aga atg aaa aca aca acc gct aat 1008Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn 325 330 335ggt caa aca ggt caa gct gac aag ttt gaa acc gtt aca tca ggc aca 1056Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr 340 345 350aaa gta acc ttt gct agt ggt aat ggt aca act gcg act gta agt aaa 1104Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys 355 360 365gat gat caa ggc aac atc act gtt aag tat gat gta aat gtc ggc gat 1152Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp 370 375 380gcc cta aac gtc aat cag ctg caa aac agc ggt tgg aat ttg gat tcc 1200Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser385 390 395 400aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg 1248Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser 405 410 415ccg agc aag gga aag atg gat gaa acc gtc aac att aat gcc ggc aac 1296Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn 420 425 430aac atc gag att acc cgc aac ggc aaa aat atc gac atc gcc act tcg 1344Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser 435 440 445atg acc ccg caa ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat gcg 1392Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala 450 455 460ccc act tta agc gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag 1440Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys465 470 475 480gat gcc aac aaa ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa 1488Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys 485 490 495gag ggg gat gtt aca aac gtc gca caa ctt aaa ggt gtg gcg caa aac 1536Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn 500 505 510ttg aac aac cgc atc gac aat gtg gac ggc aac gcg cgc gcg ggt atc 1584Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile 515 520 525gcc caa gcg att gca acc gca ggt ttg gct cag gcc tat ttg ccc ggc 1632Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly 530 535 540aag agt atg atg gcg atc ggc ggc ggt act tat cgc ggc gaa gcc ggt 1680Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly545 550 555 560tac gcc atc ggc tac tcg agc att tct gac act ggg aat tgg gtt atc 1728Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile 565 570 575aag ggc acg gct tcc ggc aat tcg cgc ggt cat ttc ggt act tcc gca 1776Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Thr Ser Ala 580 585 590tct gtc ggt tat cag tgg taa 1797Ser Val Gly Tyr Gln Trp 5955598PRTNeisseria meningitidis 5Met Asn Lys Ile Ser Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys 85 90 95Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp 115 120 125Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser 130 135 140Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn145 150 155 160Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly 165 170 175Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu 180 185 190Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp 195 200 205Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val 210 215 220Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala225 230 235 240Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu 245 250 255Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn 260 265 270Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys 275 280 285Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn Gly 290 295 300Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile305 310 315 320Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn 325 330 335Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr 340 345 350Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys 355 360 365Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp 370 375 380Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser385 390 395 400Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser 405 410 415Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn 420 425 430Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser 435 440 445Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala 450 455 460Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys465 470 475 480Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys 485 490 495Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn 500 505 510Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile 515 520 525Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly 530 535 540Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly545 550 555 560Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile 565 570 575Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Thr Ser Ala 580 585 590Ser Val Gly Tyr Gln Trp 59561785DNANeisseria meningitidisCDS(1)..(1785) 6atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg gcg acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gcg aat gct acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80aaa gaa ggt aca gaa gat tca aat tgg gca gta tat ttc gac gag aaa 288Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys 85 90 95aga gta cta aaa gcc gga gca atc acc ctc aaa gcc ggc gac aac ctg 336Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110aaa atc aaa caa aac acc aat gaa aac acc aat gac agt agc ttc acc 384Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Asp Ser Ser Phe Thr 115 120 125tac tcc ctg aaa aaa gac ctc aca gat ctg acc agt gtt gaa act gaa 432Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Glu Thr Glu 130 135 140aaa tta tcg ttt ggc gca aac ggt aat aaa gtc aac atc aca agc gac 480Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp145 150 155 160acc aaa ggc ttg aat ttt gcg aaa gaa acg gct ggg acg aac ggc gac 528Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp 165 170 175ccc acg gtt cat ctg aac ggt atc ggt tcg act ttg acc gat acg ctg 576Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu 180 185 190ctg aat acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat 624Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp 195 200 205gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gca ggc 672Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly 210 215 220tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt 720Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val225 230 235 240gat ttc gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg 768Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr 245 250 255aaa aca acg act gtt aat gtg gaa agc aaa gac aac ggc aag aaa acc 816Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr 260 265 270gaa gtt aaa atc ggt gcg aag act tct gtt att aaa gaa aaa gac ggt 864Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly 275 280 285aag ttg gtt act ggt aaa ggc aaa gac gag aat ggt tct tct aca gac 912Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly Ser Ser Thr Asp 290 295 300gaa ggc gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac 960Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn305 310 315 320aag gct ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt 1008Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly 325 330 335caa gct gac aag ttt gaa acc gtt aca tca ggc aca aat gta acc ttt 1056Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe 340 345 350gct agt ggt aaa ggt aca act gcg act gta agt aaa gat gat caa ggc 1104Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly 355 360 365aac atc act gtt aag tat gat gta aat gtc ggc gat gcc cta aac gtc 1152Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val 370 375 380aat cag ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca 1200Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala385 390 395 400ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga 1248Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly 405 410 415aag atg gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att 1296Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile 420 425 430acc cgc aac ggt aaa aat atc gac atc gcc act tcg atg gcg ccg cag 1344Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Ala Pro Gln 435 440 445ttt tcc agc gtt tcg ctc ggt gcg ggg gcg gat gcg ccc act ttg agc 1392Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser 450 455 460gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag gat acc aac aaa 1440Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Thr Asn Lys465 470 475 480ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt 1488Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val 485 490 495aca aac gtc gca caa ctt aaa ggc gtg gcg caa aac ttg aac aac cgc 1536Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg 500 505 510atc gac aat gtg gac ggc aac gcg cgt gcg ggc atc gcc caa gcg att 1584Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile 515 520 525gca acc gca ggt cta gtt cag gcg tat ctg ccc ggc aag agt atg atg 1632Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met 530 535 540gcg atc ggc ggc gac act tat cgc ggc gaa gcc ggt tac gcc atc ggc 1680Ala Ile Gly Gly Asp Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly545 550 555 560tac tca agt att tcc gac ggc gga aat tgg att atc aaa ggc acg gct 1728Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala 565 570 575tcc ggc aat tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat 1776Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr 580 585 590caa tgg taa 1785Gln Trp 7594PRTNeisseria meningitidis 7Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys 85 90 95Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Asp Ser Ser Phe Thr 115 120 125Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Glu Thr Glu 130 135 140Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp145 150 155 160Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp 165 170 175Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu 180 185 190Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp 195 200 205Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly 210 215 220Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val225 230 235 240Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr 245 250 255Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr 260 265 270Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly 275 280 285Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly Ser Ser Thr Asp 290 295 300Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn305 310 315 320Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly 325 330 335Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe 340 345 350Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly 355 360 365Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val 370 375 380Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala385 390 395 400Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly 405 410 415Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile 420 425 430Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Ala Pro Gln 435 440 445Phe Ser Ser Val Ser Leu Gly

Ala Gly Ala Asp Ala Pro Thr Leu Ser 450 455 460Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Thr Asn Lys465 470 475 480Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val 485 490 495Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg 500 505 510Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile 515 520 525Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met 530 535 540Ala Ile Gly Gly Asp Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly545 550 555 560Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala 565 570 575Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr 580 585 590Gln Trp81785DNANeisseria meningitidisCDS(1)..(1785) 8atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gcc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg gcg acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gcg agt act acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80aaa gaa gtt aca gaa gat tca aat tgg gga gta tat ttc gac aag aaa 288Lys Glu Val Thr Glu Asp Ser Asn Trp Gly Val Tyr Phe Asp Lys Lys 85 90 95gga gta cta aca gcc gga aca atc acc ctc aaa gcc ggc gac aac ctg 336Gly Val Leu Thr Ala Gly Thr Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110aaa atc aaa caa aac acc aat gaa aac acc aat gcc agt agc ttc acc 384Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr 115 120 125tac tcg ctg aaa aaa gac ctc aca gat ctg acc agt gtt gga act gaa 432Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu 130 135 140aaa tta tcg ttt agc gca aac agc aat aaa gtc aac atc aca agc gac 480Lys Leu Ser Phe Ser Ala Asn Ser Asn Lys Val Asn Ile Thr Ser Asp145 150 155 160acc aaa ggc ttg aat ttc gcg aaa aaa acg gct gag acc aac ggc gac 528Thr Lys Gly Leu Asn Phe Ala Lys Lys Thr Ala Glu Thr Asn Gly Asp 165 170 175acc acg gtt cat ctg aac ggt atc ggt tcg act ttg acc gat acg ctg 576Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu 180 185 190ctg aat acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat 624Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp 195 200 205gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gca ggc 672Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly 210 215 220tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt 720Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val225 230 235 240gat ttc gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg 768Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr 245 250 255aaa aca acg act gtt aat gtg gaa agc aaa gac aac ggc aag aga acc 816Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr 260 265 270gaa gtt aaa atc ggt gcg aag act tct gtt atc aaa gaa aaa gac ggt 864Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly 275 280 285aag ttg gtt act ggt aaa gac aaa ggc gag aat gat tct tct aca gac 912Lys Leu Val Thr Gly Lys Asp Lys Gly Glu Asn Asp Ser Ser Thr Asp 290 295 300aaa ggc gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac 960Lys Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn305 310 315 320aag gct ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt 1008Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly 325 330 335caa gct gac aag ttt gaa acc gtt aca tca ggc aca aat gta acc ttt 1056Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe 340 345 350gct agt ggt aaa ggt aca act gcg act gta agt aaa gat gat caa ggc 1104Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly 355 360 365aac atc act gtt atg tat gat gta aat gtc ggc gat gcc cta aac gtc 1152Asn Ile Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val 370 375 380aat cag ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca 1200Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala385 390 395 400ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga 1248Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly 405 410 415aag atg gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att 1296Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile 420 425 430acc cgc aac ggc aaa aat atc gac atc gcc act tcg atg acc ccg caa 1344Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln 435 440 445ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat gcg ccc act tta agc 1392Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser 450 455 460gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag gat gcc aac aaa 1440Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys465 470 475 480ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt 1488Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val 485 490 495aca aac gtc gca caa ctt aaa ggc gtg gcg caa aac ttg aac aac cac 1536Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn His 500 505 510atc gac aat gtg gac ggc aac gcg cgt gcg ggc atc gcc caa gcg att 1584Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile 515 520 525gca acc gca ggt ctg gtt cag gcg tat ctg ccc ggc aag agt atg atg 1632Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met 530 535 540gcg atc ggc ggc ggc act tat cgc ggc gaa gcc ggt tat gcc atc ggc 1680Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly545 550 555 560tac tca agc att tcc gac ggc gga aat tgg att atc aaa ggc acg gct 1728Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala 565 570 575tcc ggc aat tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat 1776Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr 580 585 590cag tgg taa 1785Gln Trp9594PRTNeisseria meningitidis 9Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80Lys Glu Val Thr Glu Asp Ser Asn Trp Gly Val Tyr Phe Asp Lys Lys 85 90 95Gly Val Leu Thr Ala Gly Thr Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr 115 120 125Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu 130 135 140Lys Leu Ser Phe Ser Ala Asn Ser Asn Lys Val Asn Ile Thr Ser Asp145 150 155 160Thr Lys Gly Leu Asn Phe Ala Lys Lys Thr Ala Glu Thr Asn Gly Asp 165 170 175Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu 180 185 190Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp 195 200 205Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly 210 215 220Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val225 230 235 240Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr 245 250 255Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr 260 265 270Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly 275 280 285Lys Leu Val Thr Gly Lys Asp Lys Gly Glu Asn Asp Ser Ser Thr Asp 290 295 300Lys Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn305 310 315 320Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly 325 330 335Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe 340 345 350Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly 355 360 365Asn Ile Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val 370 375 380Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala385 390 395 400Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly 405 410 415Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile 420 425 430Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln 435 440 445Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser 450 455 460Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys465 470 475 480Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val 485 490 495Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn His 500 505 510Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile 515 520 525Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met 530 535 540Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly545 550 555 560Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala 565 570 575Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr 580 585 590Gln Trp101776DNANeisseria meningitidisCDS(1)..(1776) 10atg aac gaa ata ttg cgc atc att tgg aat agc gcc ctc aat gcc tgg 48Met Asn Glu Ile Leu Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gtt gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg aag acc gcc gta ttg gcg act ctg ttg ttt gca acg gtt cag 144Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gca agt gct aac aat gaa gag caa gaa gaa gat tta tat tta gac ccc 192Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro 50 55 60gtg cta cgc act gtt gcc gtg ttg ata gtc aat tcc gat aaa gaa ggc 240Val Leu Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly65 70 75 80acg gga gaa aaa gaa aaa gta gaa gaa aat tca gat tgg gca gta tat 288Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr 85 90 95ttc aac gag aaa gga gta cta aca gcc aga gaa atc acc ctc aaa gcc 336Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala 100 105 110ggc gac aac ctg aaa atc aaa caa aac ggc aca aac ttc acc tac tcg 384Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser 115 120 125ctg aaa aaa gac ctc aca gat ctg acc agt gtt gga act gaa aaa tta 432Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu 130 135 140tcg ttt agc gca aac ggc aat aaa gtc aac atc aca agc gac acc aaa 480Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys145 150 155 160ggc ttg aat ttt gcg aaa gaa acg gct ggg acg aac ggc gac acc acg 528Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr 165 170 175gtt cat ctg aac ggt att ggt tcg act ttg acc gat acg ctg ctg aat 576Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn 180 185 190acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat gac gag 624Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu 195 200 205aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gct ggc tgg aac 672Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn 210 215 220att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt gat ttc 720Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe225 230 235 240gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg aaa aca 768Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 245 250 255acg act gtt aat gtg gaa agc aaa gac aac ggc aag aaa acc gaa gtt 816Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val 260 265 270aaa atc ggt gcg aag act tct gtt att aaa gaa aaa gac ggt aag ttg 864Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu 275 280 285gtt act ggt aaa gac aaa ggc gag aat ggt tct tct aca gac gaa ggc 912Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly 290 295 300gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac aag gct 960Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala305 310 315 320ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt caa gct 1008Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala 325 330 335gac aag ttt gaa acc gtt aca tca ggc aca aat gta acc ttt gct agt 1056Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser 340 345 350ggt aaa ggt aca act gcg act gta agt aaa gat gat caa ggc aac atc 1104Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile 355 360 365act gtt atg tat gat gta aat gtc ggc gat gcc cta aac gtc aat cag 1152Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln 370 375 380ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca ggt tct 1200Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser385 390 395 400tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga aag atg 1248Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met 405 410 415gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att acc cgc 1296Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg 420 425 430aac ggt aaa aat atc gac atc gcc act tcg atg acc ccg cag ttt tcc 1344Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser 435 440 445agc gtt tcg ctc ggc gcg ggg gcg gat gcg ccc act ttg agc gtg gat 1392Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp 450 455 460ggg gac gca ttg aat gtc ggc agc aag aag gac aac aaa ccc gtc cgc 1440Gly Asp Ala Leu Asn

Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg465 470 475 480att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt aca aac gtc 1488Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val 485 490 495gca caa ctt aaa ggc gtg gcg caa aac ttg aac aac cgc atc gac aat 1536Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn 500 505 510gtg gac ggc aac gcg cgt gcg ggc atc gcc caa gcg att gca acc gca 1584Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala 515 520 525ggt ctg gtt cag gcg tat ttg ccc ggc aag agt atg atg gcg atc ggc 1632Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly 530 535 540ggc ggc act tat cgc ggc gaa gcc ggt tac gcc atc ggc tac tcc agt 1680Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser545 550 555 560att tcc gac ggc gga aat tgg att atc aaa ggc acg gct tcc ggc aat 1728Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn 565 570 575tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat cag tgg taa 1776Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 59011591PRTNeisseria meningitidis 11Met Asn Glu Ile Leu Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro 50 55 60Val Leu Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly65 70 75 80Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr 85 90 95Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala 100 105 110Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser 115 120 125Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu 130 135 140Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys145 150 155 160Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr 165 170 175Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn 180 185 190Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu 195 200 205Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn 210 215 220Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe225 230 235 240Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 245 250 255Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val 260 265 270Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu 275 280 285Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly 290 295 300Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala305 310 315 320Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala 325 330 335Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser 340 345 350Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile 355 360 365Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln 370 375 380Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser385 390 395 400Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met 405 410 415Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg 420 425 430Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser 435 440 445Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp 450 455 460Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg465 470 475 480Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val 485 490 495Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn 500 505 510Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala 515 520 525Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly 530 535 540Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser545 550 555 560Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn 565 570 575Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 590121797DNANeisseria meningitidisCDS(1)..(1797) 12atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg gcg acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gcg aat gct acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80aaa gaa ggt aca gaa gat tca aat tgg gca gta tat ttc gac gag aaa 288Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys 85 90 95aga gta cta aaa gcc gga gca atc acc ctc aaa gcc ggc gac aac ctg 336Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110aaa atc aaa caa aac acc aat gaa aac acc aat gaa aac acc aat gac 384Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp 115 120 125agt agc ttc acc tac tcc ctg aaa aaa gac ctc aca gat ctg acc agt 432Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser 130 135 140gtt gaa act gaa aaa tta tcg ttt ggc gca aac ggt aat aaa gtc aac 480Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn145 150 155 160atc aca agc gac acc aaa ggc ttg aat ttt gcg aaa gaa acg gct ggg 528Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly 165 170 175acg aac ggc gac ccc acg gtt cat ctg aac ggt atc ggt tcg act ttg 576Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu 180 185 190acc gat acg ctg ctg aat acc gga gcg acc aca aac gta acc aac gac 624Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp 195 200 205aac gtt acc gat gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta 672Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val 210 215 220tta aac gca ggc tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct 720Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala225 230 235 240tcc gat aac gtt gat ttc gtc cgc act tac gac aca gtc gag ttc ttg 768Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu 245 250 255agc gca gat acg aaa aca acg act gtt aat gtg gaa agc aaa gac aac 816Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn 260 265 270ggc aag aaa acc gaa gtt aaa atc ggt gcg aag act tct gtt att aaa 864Gly Lys Lys Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys 275 280 285gaa aaa gac ggt aag ttg gtt act ggt aaa ggc aaa gac gag aat ggt 912Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly 290 295 300tct tct aca gac gaa ggc gaa ggc tta gtg act gca aaa gaa gtg att 960Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile305 310 315 320gat gca gta aac aag gct ggt tgg aga atg aaa aca aca acc gct aat 1008Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn 325 330 335ggt caa aca ggt caa gct gac aag ttt gaa acc gtt aca tca ggc aca 1056Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr 340 345 350aaa gta acc ttt gct agt ggt aat ggt aca act gcg act gta agt aaa 1104Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys 355 360 365gat gat caa ggc aac atc act gtt aag tat gat gta aat gtc ggc gat 1152Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp 370 375 380gcc cta aac gtc aat cag ctg caa aac agc ggt tgg aat ttg gat tcc 1200Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser385 390 395 400aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg 1248Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser 405 410 415ccg agc aag gga aag atg gat gaa acc gtc aac att aat gcc ggc aac 1296Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn 420 425 430aac atc gag att acc cgc aac ggc aaa aat atc gac atc gcc act tcg 1344Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser 435 440 445atg acc ccg caa ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat gcg 1392Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala 450 455 460ccc act tta agc gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag 1440Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys465 470 475 480gat gcc aac aaa ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa 1488Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys 485 490 495gag ggg gat gtt aca aac gtc gca caa ctt aaa ggt gtg gcg caa aac 1536Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn 500 505 510ttg aac aac cgc atc gac aat gtg gac ggc aac gcg cgc gcg ggt atc 1584Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile 515 520 525gcc caa gcg att gca acc gca ggt ttg gct cag gcg tat ttg ccc ggc 1632Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly 530 535 540aag agt atg atg gcg atc ggc ggc ggt act tat cgc ggc gaa gcc ggt 1680Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly545 550 555 560tac gcc atc ggc tac tcg agc att tct gac act ggg aat tgg gtt atc 1728Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile 565 570 575aag ggc acg gct tcc ggc aat tcg cgc ggc cat ttc ggt gct tcc gca 1776Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala 580 585 590tct gtc ggt tat cag tgg taa 1797Ser Val Gly Tyr Gln Trp 59513598PRTNeisseria meningitidis 13Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg 50 55 60Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu65 70 75 80Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys 85 90 95Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu 100 105 110Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp 115 120 125Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser 130 135 140Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn145 150 155 160Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly 165 170 175Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu 180 185 190Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp 195 200 205Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val 210 215 220Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala225 230 235 240Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu 245 250 255Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn 260 265 270Gly Lys Lys Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys 275 280 285Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly 290 295 300Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile305 310 315 320Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn 325 330 335Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr 340 345 350Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys 355 360 365Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp 370 375 380Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser385 390 395 400Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser 405 410 415Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn 420 425 430Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser 435 440 445Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala 450 455 460Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys465 470 475 480Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys 485 490 495Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn 500 505 510Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile 515 520 525Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly 530 535 540Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly545 550 555 560Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile 565 570 575Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala 580 585 590Ser Val Gly Tyr Gln Trp 595141800DNANeisseria meningitidisCDS(1)..(1800) 14atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gcc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg aag acc gcc gta ttg gcg acg ctg ttg ttt gca acg gtt cag 144Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gcg aat gct acc gat gaa gat gaa gaa gaa gag tta gaa ccc gta gta 192Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Pro Val Val 50 55

60cgc tct gct ctg gtg ttg caa ttc atg atc gat aaa gaa ggc aat gga 240Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly65 70 75 80gaa aac gaa tct aca gga aat ata ggt tgg agt ata tat tac gac aat 288Glu Asn Glu Ser Thr Gly Asn Ile Gly Trp Ser Ile Tyr Tyr Asp Asn 85 90 95cac aac act cta cac ggc gca acc gtt acc ctc aaa gcc ggc gac aac 336His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn 100 105 110ctg aaa atc aaa caa aac acc aat aaa aac acc aat gaa aac acc aat 384Leu Lys Ile Lys Gln Asn Thr Asn Lys Asn Thr Asn Glu Asn Thr Asn 115 120 125gac agt agc ttc acc tac tcg ctg aaa aaa gac ctc aca gat ctg acc 432Asp Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr 130 135 140agt gtt gaa act gaa aaa tta tcg ttt ggc gca aac ggc aat aaa gtc 480Ser Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val145 150 155 160aac atc aca agc gac acc aaa ggc ttg aat ttc gcg aaa gaa acg gct 528Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala 165 170 175ggg acg aac ggc gac acc acg gtt cat ctg aac ggt att ggt tcg act 576Gly Thr Asn Gly Asp Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr 180 185 190ttg acc gat acg ctg ctg aat acc gga gcg acc aca aac gta acc aac 624Leu Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn 195 200 205gac aac gtt acc gat gac aag aaa aaa cgt gcg gca agc gtt aaa gac 672Asp Asn Val Thr Asp Asp Lys Lys Lys Arg Ala Ala Ser Val Lys Asp 210 215 220gta tta aac gca ggc tgg aac att aaa ggc gtt aaa ccc ggt aca aca 720Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr225 230 235 240gct tcc gat aac gtt gat ttc gtc cac act tac gac aca gtc gag ttc 768Ala Ser Asp Asn Val Asp Phe Val His Thr Tyr Asp Thr Val Glu Phe 245 250 255ttg agc gca gat acg aaa aca acg act gtt aat gtg gaa agc aaa gac 816Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp 260 265 270aac ggc aag aga acc gaa gtt aaa atc ggt gcg aag act tct gtt att 864Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile 275 280 285aaa gaa aaa gac ggt aag ttg gtt act ggt aaa ggc aaa ggc gag aat 912Lys Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn 290 295 300ggt tct tct aca gac gaa ggc gaa ggc tta gtg act gca aaa gaa gtg 960Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val305 310 315 320att gat gca gta aac aag gct ggt tgg aga atg aaa aca aca acc gct 1008Ile Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala 325 330 335aat ggt caa aca ggt caa gct gac aag ttt gaa acc gtt aca tca ggc 1056Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly 340 345 350aca aat gta acc ttt gct agt ggt aaa ggt aca act gcg act gta agt 1104Thr Asn Val Thr Phe Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser 355 360 365aaa gat gat caa ggc aac atc act gtt aag tat gat gta aat gtc ggc 1152Lys Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly 370 375 380gat gcc cta aac gtc aat cag ctg caa aac agc ggt tgg aat ttg gat 1200Asp Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp385 390 395 400tcc aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc agc ggc aat gtt 1248Ser Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val 405 410 415tcg ccg agc aag gga aag atg gat gaa acc gtc aac att aat gcc ggc 1296Ser Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly 420 425 430aac aac atc gag att acc cgc aac ggt aaa aat atc gac atc gcc act 1344Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr 435 440 445tcg atg acc ccg cag ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat 1392Ser Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp 450 455 460gcg ccc act ttg agc gtg gat gac aag ggc gcg ttg aat gtc ggc agc 1440Ala Pro Thr Leu Ser Val Asp Asp Lys Gly Ala Leu Asn Val Gly Ser465 470 475 480aag gat gcc aac aaa ccc gtc cgc att acc aat gtc gcc ccg ggc gtt 1488Lys Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val 485 490 495aaa gag ggg gat gtt aca aac gtc gca caa ctt aaa ggc gtg gcg caa 1536Lys Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln 500 505 510aac ttg aac aac cgc atc gac aat gtg gac ggc aac gcg cgt gcg ggc 1584Asn Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly 515 520 525atc gcc caa gcg att gca acc gca ggt ctg gtt cag gcg tat ctg ccc 1632Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro 530 535 540ggc aag agt atg atg gcg atc ggc ggc ggc act tat cgc ggc gaa gcc 1680Gly Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala545 550 555 560ggt tac gcc atc ggc tac tcc agt att tcc gac ggc gga aat tgg att 1728Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile 565 570 575atc aaa ggc acg gct tcc ggc aat tcg cgc ggt cat ttc ggt gct tcc 1776Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser 580 585 590gca tct gtc ggt tat cag tgg taa 1800Ala Ser Val Gly Tyr Gln Trp 59515599PRTNeisseria meningitidis 15Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Pro Val Val 50 55 60Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly65 70 75 80Glu Asn Glu Ser Thr Gly Asn Ile Gly Trp Ser Ile Tyr Tyr Asp Asn 85 90 95His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn 100 105 110Leu Lys Ile Lys Gln Asn Thr Asn Lys Asn Thr Asn Glu Asn Thr Asn 115 120 125Asp Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr 130 135 140Ser Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val145 150 155 160Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala 165 170 175Gly Thr Asn Gly Asp Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr 180 185 190Leu Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn 195 200 205Asp Asn Val Thr Asp Asp Lys Lys Lys Arg Ala Ala Ser Val Lys Asp 210 215 220Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr225 230 235 240Ala Ser Asp Asn Val Asp Phe Val His Thr Tyr Asp Thr Val Glu Phe 245 250 255Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp 260 265 270Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile 275 280 285Lys Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn 290 295 300Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val305 310 315 320Ile Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala 325 330 335Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly 340 345 350Thr Asn Val Thr Phe Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser 355 360 365Lys Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly 370 375 380Asp Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp385 390 395 400Ser Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val 405 410 415Ser Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly 420 425 430Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr 435 440 445Ser Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp 450 455 460Ala Pro Thr Leu Ser Val Asp Asp Lys Gly Ala Leu Asn Val Gly Ser465 470 475 480Lys Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val 485 490 495Lys Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln 500 505 510Asn Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly 515 520 525Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro 530 535 540Gly Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala545 550 555 560Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile 565 570 575Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser 580 585 590Ala Ser Val Gly Tyr Gln Trp 595161779DNANeisseria meningitidisCDS(1)..(1779) 16atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gcc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg aag acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gcg aat gct acc gat gaa gat gaa gaa gaa gag tta gaa tcc gta caa 192Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Ser Val Gln 50 55 60cgc tct gtc gta ggg agc att caa gcc agt atg gaa ggc agc gtc gaa 240Arg Ser Val Val Gly Ser Ile Gln Ala Ser Met Glu Gly Ser Val Glu65 70 75 80ttg gaa acg ata tca tta tca atg act aac gac agc aag gaa ttt gta 288Leu Glu Thr Ile Ser Leu Ser Met Thr Asn Asp Ser Lys Glu Phe Val 85 90 95gac cca tac ata gta gtt acc ctc aaa gcc ggc gac aac ctg aaa atc 336Asp Pro Tyr Ile Val Val Thr Leu Lys Ala Gly Asp Asn Leu Lys Ile 100 105 110aaa caa aac acc aat gaa aac acc aat gcc agt agc ttc acc tac tcg 384Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr Tyr Ser 115 120 125ctg aaa aaa gac ctc aca ggc ctg atc aat gtt gaa act gaa aaa tta 432Leu Lys Lys Asp Leu Thr Gly Leu Ile Asn Val Glu Thr Glu Lys Leu 130 135 140tcg ttt ggc gca aac ggc aag aaa gtc aac atc ata agc gac acc aaa 480Ser Phe Gly Ala Asn Gly Lys Lys Val Asn Ile Ile Ser Asp Thr Lys145 150 155 160ggc ttg aat ttc gcg aaa gaa acg gct ggg acg aac ggc gac acc acg 528Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr 165 170 175gtt cat ctg aac ggt atc ggt tcg act ttg acc gat atg ctg ctg aat 576Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Met Leu Leu Asn 180 185 190acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat gac gag 624Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu 195 200 205aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gca ggc tgg aac 672Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn 210 215 220att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt gat ttc 720Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe225 230 235 240gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg aaa aca 768Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 245 250 255acg act gtt aat gtg gaa agc aaa gac aac ggc aag aaa acc gaa gtt 816Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val 260 265 270aaa atc ggt gcg aag act tct gtt att aaa gaa aaa gac ggt aag ttg 864Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu 275 280 285gtt act ggt aaa ggc aaa ggc gag aat ggt tct tct aca gac gaa ggc 912Val Thr Gly Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly 290 295 300gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac aag gct 960Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala305 310 315 320ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt caa gct 1008Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala 325 330 335gac aag ttt gaa acc gtt aca tca ggc aca aaa gta acc ttt gct agt 1056Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser 340 345 350ggt aat ggt aca act gcg act gta agt aaa gat gat caa ggc aac atc 1104Gly Asn Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile 355 360 365act gtt aag tat gat gta aat gtc ggc gat gcc cta aac gtc aat cag 1152Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln 370 375 380ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca ggt tct 1200Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser385 390 395 400tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga aag atg 1248Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met 405 410 415gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att acc cgc 1296Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg 420 425 430aac ggc aaa aat atc gac atc gcc act tcg atg acc ccg caa ttt tcc 1344Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser 435 440 445agc gtt tcg ctc ggc gcg ggg gcg gat gcg ccc act tta agc gtg gat 1392Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp 450 455 460gac gag ggc gcg ttg aat gtc ggc agc aag gat gcc aac aaa ccc gtc 1440Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val465 470 475 480cgc att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt aca aac 1488Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn 485 490 495gtc gcg caa ctt aaa ggt gtg gcg caa aac ttg aac aac cgc atc gac 1536Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp 500 505 510aat gtg aac ggc aac gcg cgt gcg ggc atc gcc caa gcg att gca acc 1584Asn Val Asn Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr 515 520 525gca ggt ctg gtt cag gcg tat ctg ccc ggc aag agt atg atg gcg atc 1632Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile 530 535 540ggc ggc ggc act tat ctc ggc gaa gcc ggt tat gcc atc ggc tac tca 1680Gly Gly Gly Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser545 550 555 560agc att tcc gcc ggc gga aat tgg att atc aaa ggc acg gct tcc ggc 1728Ser Ile Ser Ala Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly 565 570 575aat tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat cag tgg 1776Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 590taa 177917592PRTNeisseria meningitidis 17Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Lys Thr Ala Val Leu Ala Thr

Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Ser Val Gln 50 55 60Arg Ser Val Val Gly Ser Ile Gln Ala Ser Met Glu Gly Ser Val Glu65 70 75 80Leu Glu Thr Ile Ser Leu Ser Met Thr Asn Asp Ser Lys Glu Phe Val 85 90 95Asp Pro Tyr Ile Val Val Thr Leu Lys Ala Gly Asp Asn Leu Lys Ile 100 105 110Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr Tyr Ser 115 120 125Leu Lys Lys Asp Leu Thr Gly Leu Ile Asn Val Glu Thr Glu Lys Leu 130 135 140Ser Phe Gly Ala Asn Gly Lys Lys Val Asn Ile Ile Ser Asp Thr Lys145 150 155 160Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr 165 170 175Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Met Leu Leu Asn 180 185 190Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu 195 200 205Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn 210 215 220Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe225 230 235 240Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 245 250 255Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val 260 265 270Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu 275 280 285Val Thr Gly Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly 290 295 300Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala305 310 315 320Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala 325 330 335Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser 340 345 350Gly Asn Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile 355 360 365Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln 370 375 380Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser385 390 395 400Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met 405 410 415Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg 420 425 430Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser 435 440 445Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp 450 455 460Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val465 470 475 480Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn 485 490 495Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp 500 505 510Asn Val Asn Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr 515 520 525Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile 530 535 540Gly Gly Gly Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser545 550 555 560Ser Ile Ser Ala Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly 565 570 575Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 590181770DNANeisseria meningitidisCDS(1)..(1770) 18atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gta gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg gcg acc gcc gta ttg gcg aca ctg ctg tcc gca acg gtt cag 144Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Gln 35 40 45gcg aat gct acc gat acc gat gaa gat gaa gag tta gaa tcc gta gca 192Ala Asn Ala Thr Asp Thr Asp Glu Asp Glu Glu Leu Glu Ser Val Ala 50 55 60cgc tct gct ctg gtg ttg caa ttc atg atc gat aaa gaa ggc aat gga 240Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly65 70 75 80gaa atc gaa tct aca gga gat ata ggt tgg agt ata tat tac gac gat 288Glu Ile Glu Ser Thr Gly Asp Ile Gly Trp Ser Ile Tyr Tyr Asp Asp 85 90 95cac aac act cta cac ggc gca acc gtt acc ctc aaa gcc ggc gac aac 336His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn 100 105 110ctg aaa atc aaa caa agc ggc aaa gac ttc acc tac tcg ctg aaa aaa 384Leu Lys Ile Lys Gln Ser Gly Lys Asp Phe Thr Tyr Ser Leu Lys Lys 115 120 125gag ctg aaa gac ctg acc agt gtt gaa act gaa aaa tta tcg ttt ggc 432Glu Leu Lys Asp Leu Thr Ser Val Glu Thr Glu Lys Leu Ser Phe Gly 130 135 140gca aac ggt aat aaa gtc aac atc aca agc gac acc aaa ggc ttg aat 480Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn145 150 155 160ttt gcg aaa gaa acg gct ggg acg aac ggc gac ccc acg gtt cat ctg 528Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Pro Thr Val His Leu 165 170 175aac ggt atc ggt tcg act ttg acc gat acg ctt gcg ggt tct tct gct 576Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Ala Gly Ser Ser Ala 180 185 190tct cac gtt gat gcg ggt aac caa agt aca cat tac act cgt gca gca 624Ser His Val Asp Ala Gly Asn Gln Ser Thr His Tyr Thr Arg Ala Ala 195 200 205agt att aag gat gtg ttg aat gcg ggt tgg aat att aag ggt gtt aaa 672Ser Ile Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys 210 215 220act ggc tca aca act ggt caa tca gaa aat gtc gat ttc gtc cgc act 720Thr Gly Ser Thr Thr Gly Gln Ser Glu Asn Val Asp Phe Val Arg Thr225 230 235 240tac gac aca gtc gag ttc ttg agc gca gat acg aaa aca acg act gtt 768Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val 245 250 255aat gtg gaa agc aaa gac aac ggc aag aga acc gaa gtt aaa atc ggt 816Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr Glu Val Lys Ile Gly 260 265 270gcg aag act tct gtt att aaa gaa aaa gac ggt aag ttg gtt act ggt 864Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Val Thr Gly 275 280 285aaa ggc aaa ggc gag aat ggt tct tct aca gac gaa ggc gaa ggc tta 912Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu 290 295 300gtg act gca aaa gaa gtg att gat gca gta aac aag gct ggt tgg aga 960Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg305 310 315 320atg aaa aca aca acc gct aat ggt caa aca ggt caa gct gac aag ttt 1008Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe 325 330 335gaa acc gtt aca tca ggc aca aaa gta acc ttt gct agt ggt aat ggt 1056Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser Gly Asn Gly 340 345 350aca act gcg act gta agt aaa gat gat caa ggc aac atc act gtt aag 1104Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile Thr Val Lys 355 360 365tat gat gta aat gtc ggc gat gcc cta aac gtc aat cag ctg caa aac 1152Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln Leu Gln Asn 370 375 380agc ggt tgg aat ttg gat tcc aaa gcg gtt gca ggt tct tcg ggc aaa 1200Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser Ser Gly Lys385 390 395 400gtc atc agc ggc aat gtt tcg ccg agc aag gga aag atg gat gaa acc 1248Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Glu Thr 405 410 415gtc aac att aat gcc ggc aac aac atc gag att acc cgc aac ggc aaa 1296Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys 420 425 430aat atc gac atc gcc act tcg atg acc ccg caa ttt tcc agc gtt tcg 1344Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser Ser Val Ser 435 440 445ctc ggc gcg ggg gcg gat gcg ccc act tta agc gtg gat gac gag ggc 1392Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp Asp Glu Gly 450 455 460gcg ttg aat gtc ggc agc aag gat gcc aac aaa ccc gtc cgc att acc 1440Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val Arg Ile Thr465 470 475 480aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt aca aac gtc gca caa 1488Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val Ala Gln 485 490 495ctt aaa ggt gtg gcg caa aac ttg aac aac cgc atc gac aat gtg aac 1536Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn Val Asn 500 505 510ggc aac gcg cgc gcg ggt atc gcc caa gcg att gca acc gca ggt ttg 1584Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu 515 520 525gct cag gcc tat ttg ccc ggc aag agt atg atg gcg atc ggc ggc ggt 1632Ala Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly Gly Gly 530 535 540act tat ctc ggc gaa gcc ggt tac gcc atc ggc tac tcg agc att tct 1680Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser545 550 555 560gac act ggg aat tgg gtt atc aag ggc acg gct tcc ggc aat tcg cgc 1728Asp Thr Gly Asn Trp Val Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg 565 570 575ggt cat ttc ggt act tcc gca tct gtc ggt tat cag tgg taa 1770Gly His Phe Gly Thr Ser Ala Ser Val Gly Tyr Gln Trp 580 58519589PRTNeisseria meningitidis 19Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Gln 35 40 45Ala Asn Ala Thr Asp Thr Asp Glu Asp Glu Glu Leu Glu Ser Val Ala 50 55 60Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly65 70 75 80Glu Ile Glu Ser Thr Gly Asp Ile Gly Trp Ser Ile Tyr Tyr Asp Asp 85 90 95His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn 100 105 110Leu Lys Ile Lys Gln Ser Gly Lys Asp Phe Thr Tyr Ser Leu Lys Lys 115 120 125Glu Leu Lys Asp Leu Thr Ser Val Glu Thr Glu Lys Leu Ser Phe Gly 130 135 140Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn145 150 155 160Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Pro Thr Val His Leu 165 170 175Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Ala Gly Ser Ser Ala 180 185 190Ser His Val Asp Ala Gly Asn Gln Ser Thr His Tyr Thr Arg Ala Ala 195 200 205Ser Ile Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys 210 215 220Thr Gly Ser Thr Thr Gly Gln Ser Glu Asn Val Asp Phe Val Arg Thr225 230 235 240Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val 245 250 255Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr Glu Val Lys Ile Gly 260 265 270Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Val Thr Gly 275 280 285Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu 290 295 300Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg305 310 315 320Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe 325 330 335Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser Gly Asn Gly 340 345 350Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile Thr Val Lys 355 360 365Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln Leu Gln Asn 370 375 380Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser Ser Gly Lys385 390 395 400Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Glu Thr 405 410 415Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys 420 425 430Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser Ser Val Ser 435 440 445Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp Asp Glu Gly 450 455 460Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val Arg Ile Thr465 470 475 480Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val Ala Gln 485 490 495Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn Val Asn 500 505 510Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu 515 520 525Ala Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly Gly Gly 530 535 540Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser545 550 555 560Asp Thr Gly Asn Trp Val Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg 565 570 575Gly His Phe Gly Thr Ser Ala Ser Val Gly Tyr Gln Trp 580 585201776DNANeisseria meningitidisCDS(1)..(1776) 20atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gca tgg 48Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30acc gtg aag acc gcc gta ttg gcg act ctg ttg ttt gca acg gtt cag 144Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45gca agt gct aac aat gaa gag caa gaa gaa gat tta tat tta gac ccc 192Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro 50 55 60gta caa cgc act gtt gcc gtg ttg ata gtc aat tcc gat aaa gaa ggc 240Val Gln Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly65 70 75 80acg gga gaa aaa gaa aaa gta gaa gaa aat tca gat tgg gca gta tat 288Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr 85 90 95ttc aac gag aaa gga gta cta aca gcc aga gaa atc acc ctc aaa gcc 336Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala 100 105 110ggc gac aac ctg aaa atc aaa caa aac ggc aca aac ttc acc tac tcg 384Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser 115 120 125ctg aaa aaa gac ctc aca gat ctg acc agt gtt gga act gaa aaa tta 432Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu 130 135 140tcg ttt agc gca aac ggc aat aaa gtc aac atc aca agc gac acc aaa 480Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys145 150 155 160ggc ttg aat ttt gcg aaa gaa acg gct ggg acg aac ggc gac acc acg 528Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr 165 170 175gtt cat ctg aac ggt att ggt tcg act ttg acc gat acg ctg ctg aat 576Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn 180 185 190acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat gac gag 624Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu 195 200 205aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gct ggc tgg aac 672Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn 210 215 220att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt gat ttc

720Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe225 230 235 240gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg aaa aca 768Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 245 250 255acg act gtt aat gtg gaa agc aaa gac aac ggc aag aaa acc gaa gtt 816Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val 260 265 270aaa atc ggt gcg aag act tct gtt att aaa gaa aaa gac ggt aag ttg 864Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu 275 280 285gtt act ggt aaa gac aaa ggc gag aat ggt tct tct aca gac gaa ggc 912Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly 290 295 300gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac aag gct 960Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala305 310 315 320ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt caa gct 1008Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala 325 330 335gac aag ttt gaa acc gtt aca tca ggc aca aat gta acc ttt gct agt 1056Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser 340 345 350ggt aaa ggt aca act gcg act gta agt aaa gat gat caa ggc aac atc 1104Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile 355 360 365act gtt atg tat gat gta aat gtc ggc gat gcc cta aac gtc aat cag 1152Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln 370 375 380ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca ggt tct 1200Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser385 390 395 400tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga aag atg 1248Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met 405 410 415gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att acc cgc 1296Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg 420 425 430aac ggt aaa aat atc gac atc gcc act tcg atg acc ccg cag ttt tcc 1344Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser 435 440 445agc gtt tcg ctc ggc gcg ggg gcg gat gcg ccc act ttg agc gtg gat 1392Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp 450 455 460ggg gac gca ttg aat gtc ggc agc aag aag gac aac aaa ccc gtc cgc 1440Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg465 470 475 480att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt aca aac gtc 1488Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val 485 490 495gca caa ctt aaa ggc gtg gcg caa aac ttg aac aac cgc atc gac aat 1536Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn 500 505 510gtg gac ggc aac gcg cgt gcg ggc atc gcc caa gcg att gca acc gca 1584Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala 515 520 525ggt ctg gtt cag gcg tat ttg ccc ggc aag agt atg atg gcg atc ggc 1632Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly 530 535 540ggc ggc act tat cgc ggc gaa gcc ggt tac gcc atc ggc tac tcc agt 1680Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser545 550 555 560att tcc gac ggc gga aat tgg att atc aaa ggc acg gct tcc ggc aat 1728Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn 565 570 575tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat cag tgg taa 1776Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 59021591PRTNeisseria meningitidis 21Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro 50 55 60Val Gln Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly65 70 75 80Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr 85 90 95Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala 100 105 110Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser 115 120 125Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu 130 135 140Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys145 150 155 160Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr 165 170 175Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn 180 185 190Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu 195 200 205Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn 210 215 220Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe225 230 235 240Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 245 250 255Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val 260 265 270Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu 275 280 285Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly 290 295 300Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala305 310 315 320Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala 325 330 335Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser 340 345 350Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile 355 360 365Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln 370 375 380Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser385 390 395 400Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met 405 410 415Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg 420 425 430Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser 435 440 445Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp 450 455 460Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg465 470 475 480Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val 485 490 495Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn 500 505 510Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala 515 520 525Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly 530 535 540Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser545 550 555 560Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn 565 570 575Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 5902221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 22ttagattcca cgtcccagat t 212318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23cttcccttca aaccttcc 182432DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24ggtcgcggat ccatgaacaa aatataccgc at 322532DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25tcacccaagc ttaagccctt accactgata ac 322618DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26ccaaaccccg atttaacc 182719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27aatcgccacc cttcccttc 192818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28tttgcaacgg ttcaggca 182918DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29tattcagcag cgtatcgg 183018DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30tgcctgaacc gttgcaaa 183118DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31ccgatacgct gctgaata 18321098PRTHaemophilus influenzae 32Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Val Thr Gln Thr Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Thr His Thr Lys Cys Ala Ser Ala 20 25 30Thr Val Ala Val Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Glu 35 40 45Ala Asn Asn Asn Thr Pro Val Thr Asn Lys Leu Lys Ala Tyr Gly Asp 50 55 60Ala Asn Phe Asn Phe Thr Asn Asn Ser Ile Ala Asp Ala Glu Lys Gln65 70 75 80Val Gln Glu Ala Tyr Lys Gly Leu Leu Asn Leu Asn Glu Lys Asn Ala 85 90 95Ser Asp Lys Leu Leu Val Glu Asp Asn Thr Ala Ala Thr Val Gly Asn 100 105 110Leu Arg Lys Leu Gly Trp Val Leu Ser Ser Lys Asn Gly Thr Arg Asn 115 120 125Glu Lys Ser Gln Gln Val Lys His Ala Asp Glu Val Leu Phe Glu Gly 130 135 140Lys Gly Gly Val Gln Val Thr Ser Thr Ser Glu Asn Gly Lys His Thr145 150 155 160Ile Thr Phe Ala Leu Ala Lys Asp Leu Gly Val Lys Thr Ala Thr Val 165 170 175Ser Asp Thr Leu Thr Ile Gly Gly Gly Ala Ala Ala Gly Ala Thr Thr 180 185 190Thr Pro Lys Val Asn Val Thr Ser Thr Thr Asp Gly Leu Lys Phe Ala 195 200 205Lys Asp Ala Ala Gly Ala Asn Gly Asp Thr Thr Val His Leu Asn Gly 210 215 220Ile Gly Ser Thr Leu Thr Asp Thr Leu Val Gly Ser Pro Ala Thr His225 230 235 240Ile Asp Gly Gly Asp Gln Ser Thr His Tyr Thr Arg Ala Ala Ser Ile 245 250 255Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Ala Gly 260 265 270Ser Thr Thr Gly Gln Ser Glu Asn Val Asp Phe Val His Thr Tyr Asp 275 280 285Thr Val Glu Phe Leu Ser Ala Asp Thr Glu Thr Thr Thr Val Thr Val 290 295 300Asp Ser Lys Glu Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys305 310 315 320Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Phe Thr Gly Lys Ala 325 330 335Asn Lys Glu Thr Asn Lys Val Asp Gly Ala Asn Ala Thr Glu Asp Ala 340 345 350Asp Glu Gly Lys Gly Leu Val Thr Ala Lys Asp Val Ile Asp Ala Val 355 360 365Asn Lys Thr Gly Trp Arg Ile Lys Thr Thr Asp Ala Asn Gly Gln Asn 370 375 380Gly Asp Phe Ala Thr Val Ala Ser Gly Thr Asn Val Thr Phe Ala Ser385 390 395 400Gly Asn Gly Thr Thr Ala Thr Val Thr Asn Gly Thr Asp Gly Ile Thr 405 410 415Val Lys Tyr Asp Ala Lys Val Gly Asp Gly Leu Lys Leu Asp Gly Asp 420 425 430Lys Ile Ala Ala Asp Thr Thr Ala Leu Thr Val Asn Asp Gly Lys Asn 435 440 445Ala Asn Asn Pro Lys Gly Lys Val Ala Asp Val Ala Ser Thr Asp Glu 450 455 460Lys Lys Leu Val Thr Ala Lys Gly Leu Val Thr Ala Leu Asn Ser Leu465 470 475 480Ser Trp Thr Thr Thr Ala Ala Glu Ala Asp Gly Gly Thr Leu Asp Gly 485 490 495Asn Ala Ser Glu Gln Glu Val Lys Ala Gly Asp Lys Val Thr Phe Lys 500 505 510Ala Gly Lys Asn Leu Lys Val Lys Gln Glu Gly Ala Asn Phe Thr Tyr 515 520 525Ser Leu Gln Asp Ala Leu Thr Gly Leu Thr Ser Ile Thr Leu Gly Thr 530 535 540Gly Asn Asn Gly Ala Lys Thr Glu Ile Asn Lys Asp Gly Leu Thr Ile545 550 555 560Thr Pro Ala Asn Gly Ala Gly Ala Asn Asn Ala Asn Thr Ile Ser Val 565 570 575Thr Lys Asp Gly Ile Ser Ala Gly Gly Gln Ser Val Lys Asn Val Val 580 585 590Ser Gly Leu Lys Lys Phe Gly Asp Ala Asn Phe Asp Pro Leu Thr Ser 595 600 605Ser Ala Asp Asn Leu Thr Lys Gln Asn Asp Asp Ala Tyr Lys Gly Leu 610 615 620Thr Asn Leu Asp Glu Lys Gly Thr Asp Lys Gln Thr Pro Val Val Ala625 630 635 640Asp Asn Thr Ala Ala Thr Val Gly Asp Leu Arg Gly Leu Gly Trp Val 645 650 655Ile Ser Ala Asp Lys Thr Thr Gly Gly Ser Thr Glu Tyr His Asp Gln 660 665 670Val Arg Asn Ala Asn Glu Val Lys Phe Lys Ser Gly Asn Gly Ile Asn 675 680 685Val Ser Gly Lys Thr Val Asn Gly Arg Arg Glu Ile Thr Phe Glu Leu 690 695 700Ala Lys Gly Glu Val Val Lys Ser Asn Glu Phe Thr Val Lys Glu Thr705 710 715 720Asn Gly Lys Glu Thr Ser Leu Val Lys Val Gly Asp Lys Tyr Tyr Ser 725 730 735Lys Glu Asp Ile Asp Leu Thr Thr Gly Gln Pro Lys Leu Lys Asp Gly 740 745 750Asn Thr Val Ala Ala Lys Tyr Gln Asp Lys Gly Gly Lys Val Val Ser 755 760 765Val Thr Asp Asn Thr Glu Ala Thr Ile Thr Asn Lys Gly Ser Gly Tyr 770 775 780Val Thr Gly Asn Gln Val Ala Asp Ala Ile Ala Lys Ser Gly Phe Glu785 790 795 800Leu Gly Leu Ala Asp Glu Ala Asp Ala Lys Arg Ala Phe Asp Asp Lys 805 810 815Thr Lys Ala Leu Ser Ala Gly Thr Thr Glu Ile Val Asn Ala His Asp 820 825 830Lys Val Arg Phe Ala Asn Gly Leu Asn Thr Lys Val Ser Ala Ala Thr 835 840 845Val Glu Ser Thr Asp Ala Asn Gly Asp Lys Val Thr Thr Thr Phe Val 850 855 860Lys Thr Asp Val Glu Leu Pro Leu Thr Gln Ile Tyr Asn Thr Asp Ala865 870 875 880Asn Gly Lys Lys Ile Thr Lys Val Val Lys Asp Gly Gln Thr Lys Trp 885 890 895Tyr Glu Leu Asn Ala Asp Gly Thr Ala Asp Met Thr Lys Glu Val Thr 900 905 910Leu Gly Asn Val Asp Ser Asp Gly Lys Lys Val Val Lys Asp Asn Asp 915 920 925Gly Lys Trp Tyr His Ala Lys Ala Asp Gly Thr Ala Asp Lys Thr Lys 930 935 940Gly Glu Val Ser Asn Asp Lys Val Ser Thr Asp Glu Lys His Val Val945 950 955 960Ser Leu Asp Pro Asn Asp Gln Ser Lys Gly Lys Gly Val Val Ile Asp 965 970 975Asn Val Ala Asn Gly Asp Ile Ser Ala Thr Ser Thr Asp Ala Ile Asn 980 985 990Gly Ser Gln Leu Tyr Ala Val Ala Lys Gly Val Thr Asn Leu Ala Gly 995 1000 1005Gln Val Asn Asn Leu Glu Gly Lys Val Asn Lys Val Gly Lys Arg 1010 1015 1020Ala Asp Ala Gly Thr Ala Ser Ala Leu Ala Ala Ser Gln Leu Pro1025 1030 1035Gln Ala Thr Met Pro Gly Lys Ser Met Val Ala Ile Ala Gly Ser 1040 1045 1050Ser Tyr Gln Gly Gln Asn Gly Leu Ala Ile Gly Val Ser Arg Ile 1055 1060 1065Ser Asp Asn Gly Lys Val Ile Ile Arg

Leu Ser Gly Thr Thr Asn 1070 1075 1080Ser Gln Gly Lys Thr Gly Val Ala Ala Gly Val Gly Tyr Gln Trp 1085 1090 1095332353PRTHaemophilus influenzae 33Met Asn Lys Ile Phe Asn Val Ile Trp Asn Val Met Thr Gln Thr Trp1 5 10 15Val Val Val Ser Glu Leu Thr Arg Thr His Thr Lys Arg Ala Ser Ala 20 25 30Thr Val Glu Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45Ala Asn Ala Thr Asp Glu Asp Glu Glu Leu Asp Pro Val Val Arg Thr 50 55 60Ala Pro Val Leu Ser Phe His Ser Asp Lys Glu Gly Thr Gly Glu Lys65 70 75 80Glu Val Thr Glu Asn Ser Asn Trp Gly Ile Tyr Phe Asp Asn Lys Gly 85 90 95Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu Lys 100 105 110Ile Lys Gln Asn Thr Asp Glu Ser Thr Asn Ala Ser Ser Phe Thr Tyr 115 120 125Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Ala Thr Glu Lys 130 135 140Leu Ser Phe Gly Ala Asn Gly Asp Lys Val Asp Ile Thr Ser Asp Ala145 150 155 160Asn Gly Leu Lys Leu Ala Lys Thr Gly Asn Gly Asn Val His Leu Asn 165 170 175Gly Leu Asp Ser Thr Leu Pro Asp Ala Val Thr Asn Thr Gly Val Leu 180 185 190Ser Ser Ser Ser Phe Thr Pro Asn Asp Val Glu Lys Thr Arg Ala Ala 195 200 205Thr Val Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Ala Lys 210 215 220Thr Ala Gly Gly Asn Val Glu Ser Val Asp Leu Val Ser Ala Tyr Asn225 230 235 240Asn Val Glu Phe Ile Thr Gly Asp Lys Asn Thr Leu Asp Val Val Leu 245 250 255Thr Ala Lys Glu Asn Gly Lys Thr Thr Glu Val Lys Phe Thr Pro Lys 260 265 270Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Phe Thr Gly Lys Glu 275 280 285Asn Asn Asp Thr Asn Lys Val Thr Ser Asn Thr Ala Thr Asp Asn Thr 290 295 300Asp Glu Gly Asn Gly Leu Val Thr Ala Lys Ala Val Ile Asp Ala Val305 310 315 320Asn Lys Ala Gly Trp Arg Val Lys Thr Thr Thr Ala Asn Gly Gln Asn 325 330 335Gly Asp Phe Ala Thr Val Ala Ser Gly Thr Asn Val Thr Phe Glu Ser 340 345 350Gly Asp Gly Thr Thr Ala Ser Val Thr Lys Asp Thr Asn Gly Asn Gly 355 360 365Ile Thr Val Lys Tyr Asp Ala Lys Val Gly Asp Gly Leu Lys Phe Asp 370 375 380Ser Asp Lys Lys Ile Val Ala Asp Thr Thr Ala Leu Thr Val Thr Gly385 390 395 400Gly Lys Val Ala Glu Ile Ala Lys Glu Asp Asp Lys Lys Lys Leu Val 405 410 415Asn Ala Gly Asp Leu Val Thr Ala Leu Gly Asn Leu Ser Trp Lys Ala 420 425 430Lys Ala Glu Ala Asp Thr Asp Gly Ala Leu Glu Gly Ile Ser Lys Asp 435 440 445Gln Glu Val Lys Ala Gly Glu Thr Val Thr Phe Lys Ala Gly Lys Asn 450 455 460Leu Lys Val Lys Gln Asp Gly Ala Asn Phe Thr Tyr Ser Leu Gln Asp465 470 475 480Ala Leu Thr Gly Leu Thr Ser Ile Thr Leu Gly Gly Thr Thr Asn Gly 485 490 495Gly Asn Asp Ala Lys Thr Val Ile Asn Lys Asp Gly Leu Thr Ile Thr 500 505 510Pro Ala Gly Asn Gly Gly Thr Thr Gly Thr Asn Thr Ile Ser Val Thr 515 520 525Lys Asp Gly Ile Lys Ala Gly Asn Lys Ala Ile Thr Asn Val Ala Ser 530 535 540Gly Leu Arg Ala Tyr Asp Asp Ala Asn Phe Asp Val Leu Asn Asn Ser545 550 555 560Ala Thr Asp Leu Asn Arg His Val Glu Asp Ala Tyr Lys Gly Leu Leu 565 570 575Asn Leu Asn Glu Lys Asn Ala Asn Lys Gln Pro Leu Val Thr Asp Ser 580 585 590Thr Ala Ala Thr Val Gly Asp Leu Arg Lys Leu Gly Trp Val Val Ser 595 600 605Thr Lys Asn Gly Thr Lys Glu Glu Ser Asn Gln Val Lys Gln Ala Asp 610 615 620Glu Val Leu Phe Thr Gly Ala Gly Ala Ala Thr Val Thr Ser Lys Ser625 630 635 640Glu Asn Gly Lys His Thr Ile Thr Val Ser Val Ala Glu Thr Lys Ala 645 650 655Asp Cys Gly Leu Glu Lys Asp Gly Asp Thr Ile Lys Leu Lys Val Asp 660 665 670Asn Gln Asn Thr Asp Asn Val Leu Thr Val Gly Asn Asn Gly Thr Ala 675 680 685Val Thr Lys Gly Gly Phe Glu Thr Val Lys Thr Gly Ala Thr Asp Ala 690 695 700Asp Arg Gly Lys Val Thr Val Lys Asp Ala Thr Ala Asn Asp Ala Asp705 710 715 720Lys Lys Val Ala Thr Val Lys Asp Val Ala Thr Ala Ile Asn Ser Ala 725 730 735Ala Thr Phe Val Lys Thr Glu Asn Leu Thr Thr Ser Ile Asp Glu Asp 740 745 750Asn Pro Thr Asp Asn Gly Lys Asp Asp Ala Leu Lys Ala Gly Asp Thr 755 760 765Leu Thr Phe Lys Ala Gly Lys Asn Leu Lys Val Lys Arg Asp Gly Lys 770 775 780Asn Ile Thr Phe Asp Leu Ala Lys Asn Leu Glu Val Lys Thr Ala Lys785 790 795 800Val Ser Asp Thr Leu Thr Ile Gly Gly Asn Thr Pro Thr Gly Gly Thr 805 810 815Thr Ala Thr Pro Lys Val Asn Ile Thr Ser Thr Ala Asp Gly Leu Asn 820 825 830Phe Ala Lys Glu Thr Ala Asp Ala Ser Gly Ser Lys Asn Val Tyr Leu 835 840 845Lys Gly Ile Ala Thr Thr Leu Thr Glu Pro Ser Ala Gly Ala Lys Ser 850 855 860Ser His Val Asp Leu Asn Val Asp Ala Thr Lys Lys Ser Asn Ala Ala865 870 875 880Ser Ile Glu Asp Val Leu Arg Ala Gly Trp Asn Ile Gln Gly Asn Gly 885 890 895Asn Asn Val Asp Tyr Val Ala Thr Tyr Asp Thr Val Asn Phe Thr Asp 900 905 910Asp Ser Thr Gly Thr Thr Thr Val Thr Val Thr Gln Lys Ala Asp Gly 915 920 925Lys Gly Ala Asp Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Asp 930 935 940His Asn Gly Lys Leu Phe Thr Gly Lys Asp Leu Lys Asp Ala Asn Asn945 950 955 960Gly Ala Thr Val Ser Glu Asp Asp Gly Lys Asp Thr Gly Thr Gly Leu 965 970 975Val Thr Ala Lys Thr Val Ile Asp Ala Val Asn Lys Ser Gly Trp Arg 980 985 990Val Thr Gly Glu Gly Ala Thr Ala Glu Thr Gly Ala Thr Ala Val Asn 995 1000 1005Ala Gly Asn Ala Glu Thr Val Thr Ser Gly Thr Ser Val Asn Phe 1010 1015 1020Lys Asn Gly Asn Ala Thr Thr Ala Thr Val Ser Lys Asp Asn Gly 1025 1030 1035Asn Ile Asn Val Lys Tyr Asp Val Asn Val Gly Asp Gly Leu Lys 1040 1045 1050Ile Gly Asp Asp Lys Lys Ile Val Ala Asp Thr Thr Thr Leu Thr 1055 1060 1065Val Thr Gly Gly Lys Val Ser Val Pro Ala Gly Ala Asn Ser Val 1070 1075 1080Asn Asn Asn Lys Lys Leu Val Asn Ala Glu Gly Leu Ala Thr Ala 1085 1090 1095Leu Asn Asn Leu Ser Trp Thr Ala Lys Ala Asp Lys Tyr Ala Asp 1100 1105 1110Gly Glu Ser Glu Gly Glu Thr Asp Gln Glu Val Lys Ala Gly Asp 1115 1120 1125Lys Val Thr Phe Lys Ala Gly Lys Asn Leu Lys Val Lys Gln Ser 1130 1135 1140Glu Lys Asp Phe Thr Tyr Ser Leu Gln Asp Thr Leu Thr Gly Leu 1145 1150 1155Thr Ser Ile Thr Leu Gly Gly Thr Ala Asn Gly Arg Asn Asp Thr 1160 1165 1170Gly Thr Val Ile Asn Lys Asp Gly Leu Thr Ile Thr Leu Ala Asn 1175 1180 1185Gly Ala Ala Ala Gly Thr Asp Ala Ser Asn Gly Asn Thr Ile Ser 1190 1195 1200Val Thr Lys Asp Gly Ile Ser Ala Gly Asn Lys Glu Ile Thr Asn 1205 1210 1215Val Lys Ser Ala Leu Lys Thr Tyr Lys Asp Thr Gln Asn Thr Ala 1220 1225 1230Asp Glu Thr Gln Asp Lys Glu Phe His Ala Ala Val Lys Asn Ala 1235 1240 1245Asn Glu Val Glu Phe Val Gly Lys Asn Gly Ala Thr Val Ser Ala 1250 1255 1260Lys Thr Asp Asn Asn Gly Lys His Thr Val Thr Ile Asp Val Ala 1265 1270 1275Glu Ala Lys Val Gly Asp Gly Leu Glu Lys Asp Thr Asp Gly Lys 1280 1285 1290Ile Lys Leu Lys Val Asp Asn Thr Asp Gly Asn Asn Leu Leu Thr 1295 1300 1305Val Asp Ala Thr Lys Gly Ala Ser Val Ala Lys Gly Glu Phe Asn 1310 1315 1320Ala Val Thr Thr Asp Ala Thr Thr Ala Gln Gly Thr Asn Ala Asn 1325 1330 1335Glu Arg Gly Lys Val Val Val Lys Gly Ser Asn Gly Ala Thr Ala 1340 1345 1350Thr Glu Thr Asp Lys Lys Lys Val Ala Thr Val Gly Asp Val Ala 1355 1360 1365Lys Ala Ile Asn Asp Ala Ala Thr Phe Val Lys Val Glu Asn Asp 1370 1375 1380Asp Ser Ala Thr Ile Asp Asp Ser Pro Thr Asp Asp Gly Ala Asn 1385 1390 1395Asp Ala Leu Lys Ala Gly Asp Thr Leu Thr Leu Lys Ala Gly Lys 1400 1405 1410Asn Leu Lys Val Lys Arg Asp Gly Lys Asn Ile Thr Phe Ala Leu 1415 1420 1425Ala Asn Asp Leu Ser Val Lys Ser Ala Thr Val Ser Asp Lys Leu 1430 1435 1440Ser Leu Gly Thr Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr 1445 1450 1455Lys Gly Leu Asn Phe Ala Lys Asp Ser Lys Thr Gly Asp Asp Ala 1460 1465 1470Asn Ile His Leu Asn Gly Ile Ala Ser Thr Leu Thr Asp Thr Leu 1475 1480 1485Leu Asn Ser Gly Ala Thr Thr Asn Leu Gly Gly Asn Gly Ile Thr 1490 1495 1500Asp Asn Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn 1505 1510 1515Ala Gly Trp Asn Val Arg Gly Val Lys Pro Ala Ser Ala Asn Asn 1520 1525 1530Gln Val Glu Asn Ile Asp Phe Val Ala Thr Tyr Asp Thr Val Asp 1535 1540 1545Phe Val Ser Gly Asp Lys Asp Thr Thr Ser Val Thr Val Glu Ser 1550 1555 1560Lys Asp Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr 1565 1570 1575Ser Val Ile Lys Asp His Asn Gly Lys Leu Phe Thr Gly Lys Glu 1580 1585 1590Leu Lys Asp Ala Asn Asn Asn Gly Val Thr Val Thr Glu Thr Asp 1595 1600 1605Gly Lys Asp Glu Gly Asn Gly Leu Val Thr Ala Lys Ala Val Ile 1610 1615 1620Asp Ala Val Asn Lys Ala Gly Trp Arg Val Lys Thr Thr Gly Ala 1625 1630 1635Asn Gly Gln Asn Asp Asp Phe Ala Thr Val Ala Ser Gly Thr Asn 1640 1645 1650Val Thr Phe Ala Asp Gly Asn Gly Thr Thr Ala Glu Val Thr Lys 1655 1660 1665Ala Asn Asp Gly Ser Ile Thr Val Lys Tyr Asn Val Lys Val Ala 1670 1675 1680Asp Gly Leu Lys Leu Asp Gly Asp Lys Ile Val Ala Asp Thr Thr 1685 1690 1695Val Leu Thr Val Ala Asp Gly Lys Val Thr Ala Pro Asn Asn Gly 1700 1705 1710Asp Gly Lys Lys Phe Val Asp Ala Ser Gly Leu Ala Asp Ala Leu 1715 1720 1725Asn Lys Leu Ser Trp Thr Ala Thr Ala Gly Lys Glu Gly Thr Gly 1730 1735 1740Glu Val Asp Pro Ala Asn Ser Ala Gly Gln Glu Val Lys Ala Gly 1745 1750 1755Asp Lys Val Thr Phe Lys Ala Gly Asp Asn Leu Lys Ile Lys Gln 1760 1765 1770Ser Gly Lys Asp Phe Thr Tyr Ser Leu Lys Lys Glu Leu Lys Asp 1775 1780 1785Leu Thr Ser Val Glu Phe Lys Asp Ala Asn Gly Gly Thr Gly Ser 1790 1795 1800Glu Ser Thr Lys Ile Thr Lys Asp Gly Leu Thr Ile Thr Pro Ala 1805 1810 1815Asn Gly Ala Gly Ala Ala Gly Ala Asn Thr Ala Asn Thr Ile Ser 1820 1825 1830Val Thr Lys Asp Gly Ile Ser Ala Gly Asn Lys Ala Val Thr Asn 1835 1840 1845Val Val Ser Gly Leu Lys Lys Phe Gly Asp Gly His Thr Leu Ala 1850 1855 1860Asn Gly Thr Val Ala Asp Phe Glu Lys His Tyr Asp Asn Ala Tyr 1865 1870 1875Lys Asp Leu Thr Asn Leu Asp Glu Lys Gly Ala Asp Asn Asn Pro 1880 1885 1890Thr Val Ala Asp Asn Thr Ala Ala Thr Val Gly Asp Leu Arg Gly 1895 1900 1905Leu Gly Trp Val Ile Ser Ala Asp Lys Thr Thr Gly Glu Pro Asn 1910 1915 1920Gln Glu Tyr Asn Ala Gln Val Arg Asn Ala Asn Glu Val Lys Phe 1925 1930 1935Lys Ser Gly Asn Gly Ile Asn Val Ser Gly Lys Thr Leu Asn Gly 1940 1945 1950Thr Arg Val Ile Thr Phe Glu Leu Ala Lys Gly Glu Val Val Lys 1955 1960 1965Ser Asn Glu Phe Thr Val Lys Asn Ala Asp Gly Ser Glu Thr Asn 1970 1975 1980Leu Val Lys Val Gly Asp Met Tyr Tyr Ser Lys Glu Asp Ile Asp 1985 1990 1995Pro Ala Thr Ser Lys Pro Met Thr Gly Lys Thr Glu Lys Tyr Lys 2000 2005 2010Val Glu Asn Gly Lys Val Val Ser Ala Asn Gly Ser Lys Thr Glu 2015 2020 2025Val Thr Leu Thr Asn Lys Gly Ser Gly Tyr Val Thr Gly Asn Gln 2030 2035 2040Val Ala Asp Ala Ile Ala Lys Ser Gly Phe Glu Leu Gly Leu Ala 2045 2050 2055Asp Ala Ala Glu Ala Glu Lys Ala Phe Ala Glu Ser Ala Lys Asp 2060 2065 2070Lys Gln Leu Ser Lys Asp Lys Ala Glu Thr Val Asn Ala His Asp 2075 2080 2085Lys Val Arg Phe Ala Asn Gly Leu Asn Thr Lys Val Ser Ala Ala 2090 2095 2100Thr Val Glu Ser Thr Asp Ala Asn Gly Asp Lys Val Thr Thr Thr 2105 2110 2115Phe Val Lys Thr Asp Val Glu Leu Pro Leu Thr Gln Ile Tyr Asn 2120 2125 2130Thr Asp Ala Asn Gly Asn Lys Ile Val Lys Lys Ala Asp Gly Lys 2135 2140 2145Trp Tyr Glu Leu Asn Ala Asp Gly Thr Ala Ser Asn Lys Glu Val 2150 2155 2160Thr Leu Gly Asn Val Asp Ala Asn Gly Lys Lys Val Val Lys Val 2165 2170 2175Thr Glu Asn Gly Ala Asp Lys Trp Tyr Tyr Thr Asn Ala Asp Gly 2180 2185 2190Ala Ala Asp Lys Thr Lys Gly Glu Val Ser Asn Asp Lys Val Ser 2195 2200 2205Thr Asp Glu Lys His Val Val Arg Leu Asp Pro Asn Asn Gln Ser 2210 2215 2220Asn Gly Lys Gly Val Val Ile Asp Asn Val Ala Asn Gly Glu Ile 2225 2230 2235Ser Ala Thr Ser Thr Asp Ala Ile Asn Gly Ser Gln Leu Tyr Ala 2240 2245 2250Val Ala Lys Gly Val Thr Asn Leu Ala Gly Gln Val Asn Asn Leu 2255 2260 2265Glu Gly Lys Val Asn Lys Val Gly Lys Arg Ala Asp Ala Gly Thr 2270 2275 2280Ala Ser Ala Leu Ala Ala Ser Gln Leu Pro Gln Ala Thr Met Pro 2285 2290 2295Gly Lys Ser Met Val Ala Ile Ala Gly Ser Ser Tyr Gln Gly Gln 2300 2305 2310Asn Gly Leu Ala Ile Gly Val Ser Arg Ile Ser Asp Asn Gly Lys 2315 2320 2325Val Ile Ile Arg Leu Ser Gly Thr Thr Asn Ser Gln Gly Lys Thr 2330 2335 2340Gly Val Ala Ala Gly Val Gly Tyr Gln Trp 2345 2350346PRTArtificial SequenceDescription of Artificial Sequence Synthetic 6x His tag 34His His His His His His1 5

Claims

1-34. (canceled)

35. An isolated nucleic acid molecule which encodes a protein comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:21.

36. A nucleic acid molecule according to claim 35, comprising a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:20.

37. An isolated nucleic acid molecule encoding an immunogenic polypeptide having 90% or greater sequence identity to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:21, wherein the immunogenic polypeptide can raise antibodies which specifically bind to SEQ ID NO:2 or SEQ ID NO:21.

38. An isolated nucleic acid molecule comprising a fragment of 25 or more nucleotides of a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:20, wherein the fragment is capable of detecting N. meningitidis nucleic acids through hybridization under high stringency conditions comprising a wash in 0.1.times.SSC, 0.5% SDS solution at 65.degree. C.

39. An isolated nucleic acid molecule encoding an immunogenic polypeptide, wherein the isolated nucleic acid molecule can hybridize to a nucleic acid molecule which encodes a protein comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:21 under high stringency conditions comprising a wash in 0.1.times.SSC, 0.5% SDS solution at 65.degree. C., wherein the isolated nucleic acid molecule is capable of detecting N. meningitidis nucleic acids through hybridization.

40. An isolated nucleic acid molecule that encodes a protein comprising an immunogenic fragment of at least 20 consecutive amino acids of an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:21, wherein the immunogenic fragment can raise antibodies which specifically bind to SEQ ID NO:2 or SEQ ID NO:21.

41. The isolated nucleic acid molecule of claim 37, wherein the immunogenic polypeptide has 99% or greater sequence identity to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:21.

42. The isolated nucleic acid molecule of claim 37, wherein the immunogenic polypeptide has 95% or greater sequence identity to an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:21.

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