Meningococcus Adhesins Nada, App And Orf 40

Abstract

NadA, App and ORF40 function as adhesins in N. meningitidis. Adhesion can be modulated by targeting these three proteins. NadA allelic variants are disclosed. Autoproteolytic cleavage of App is disclosed, as is removal of the activity by mutagenesis. App is processed and secreted into culture medium when expressed in E. coli. Mature App proteins are disclosed. Knockout mutants are disclosed. Vesicles from non-Neisserial hosts with heterologous adhesin expression are disclosed.

Description

[0001] All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] This invention is in the field of biochemistry and, in particular, the biochemistry of the pathogenic bacteria in the genus Neisseria (e.g. N. meningitidis and N. gonorrhoeae).

BACKGROUND ART

[0003] International patent applications WO99/24578, WO99/36544, WO99/57280 and WO00/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhoeae. The complete genome sequence of serogroup B N. meningitidis has been published [Tettelin et al. (2000) Science 287:1809-1815] and has been subjected to analysis in order to identify vaccine antigens [Pizza et al. (2000) Science 287:1816-1820]. Approaches to expression of the proteins are disclosed in WO01/64922. The complete genome sequence of serogroup A N. meningitidis is also known [Parkhill et al. (2000) Nature 404:502-506].

[0004] Sequence data alone, however, does not reveal everything about this pathogen. Objects of the present invention include: (a) to provide ways of intervening in Neisseria biochemistry; (b) to provide new uses for known Neisseria proteins; (c) to provide alternative and improved forms of known Neisseria proteins, such as enzymatically inactive forms of known proteins or proteolytic products of known proteins; and (d) to provide materials useful for studying and modulating Neisserial adhesion.

DISCLOSURE OF THE INVENTION

Nomenclature Used Herein

[0005] `ORF40` is disclosed in example 1 of WO99/36544. Sequences from serogroups A and B of N. meningitidis are disclosed (SEQ IDs 1 to 6 therein). Other forms of the protein are disclosed in WO99/31132 and WO99/58683, and can also be found in GenBank (see gi accession numbers: 11352902, 7228562, 14578015, 12958107, 7228586, 7228572, 7228594, 7228588, 14578013, 7228568, 7228546, 7228548, 7228592, 14578009, 7228558, 7228600, 7228596, 7228542, 7228574, 7228552, 7228554, 14578023, 14578021, 11354080, 7228584 & 7228590).

[0006] `App` (adhesion and penetration protein) is disclosed as `ORF1` in example 77 of WO99/24578. Sequences from serogroups A and B of N. meningitidis and from N. gonorrhoeae are disclosed (SEQ IDs 647 to 654 therein). Other forms of the protein are disclosed in WO99/55873, and can also be found in GenBank (see gi accession numbers: 11280386, 7227246, 11071865, 6977941, 11071863, 11280387, 7379205).

[0007] `NadA` (Neisserial adhesin A) from serogroup B of N. meningitidis is disclosed as protein `961` in WO99/57280 (SEQ IDs 2943 & 2944) and as `NMB1994` by Tettelin et al. (see also GenBank accession numbers: 11352904 & 7227256) and in FIG. 9 herein.

[0008] These proteins are preferably expressed other than as a fusion protein (e.g. without GST, MBP, his-tag or similar).

[0009] Preferred proteins for use according to the invention are those of serogroup B N. meningitidis strain MC58, strain 2996 or strain 394/98 (a New Zealand strain). It will be appreciated, however, that the invention is not in general limited by strain--references to a particular protein (e.g. `ORF40`, `App` etc.) may be taken to include that protein from any strain. In general, therefore, reference to any particular protein includes proteins which share sequence identity with one of the sequences disclosed above. The degree of `sequence identity` is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants. In the context of the present invention, sequence identity is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.

[0010] The naming conventions used in WO99/24578, WO99/36544 and WO99/57280 are also used herein (e.g. `ORF4`, `ORF40`, `ORF40-1` etc. as used in WO99/24578 and WO99/36544; `m919`, `g919` and `a919` etc. as used in WO99/57280).

Secreted App

[0011] It has been found that, when expressed in E. coli without a GST or his-tag fusion partner, App is exported to the outer membrane as a precursor of about 160 kDa, where it is processed and secreted into the culture.

[0012] The invention therefore provides a method for purifying processed App protein, comprising the steps of: expressing a gene encoding App protein in a non-Neisserial host cell; and purifying processed App protein from the culture medium.

[0013] The invention also provides purified protein obtainable by this process.

[0014] The App protein preferably includes its wild-type 42 residue signal peptide at the N-terminus i.e. no N-terminus fusion partner is used. It is also preferred not to include a C-terminus fusion partner.

[0015] To purify the protein from the culture medium, the culture can be centrifuged and the protein can be recovered from the supernatant.

[0016] The non-Neisserial host cell is preferably a bacterium and is most preferably E. coli.

[0017] Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

[0018] Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (tip) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.

[0019] In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

[0020] In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34]. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' and of E. coli 16S rRNA [Steitz et al. (1979) "Genetic signals and nucleotide sequences in messenger RNA." In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular Cloning: A Laboratory Manual].

[0021] A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EP-A-0219237).

[0022] Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.

[0023] Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

[0024] Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0127328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

[0025] Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

[0026] Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

[0027] Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

[0028] Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl.sub.2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973)Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) "An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].

Adherence Proteins

[0029] Example 22 of international patent application WO01/64922 discloses that E. coli which expresses protein NadA can adhere to human epithelial cells. This adherence activity has been further studied and it has also been found for App and ORF40.

[0030] The invention provides methods for preventing the attachment of Neisserial cells to epithelial cells.

[0031] References to a "Neisserial cell" in this section include any species of the bacterial genus Neisseria, including N. gonorrhoeae and N. lactamica. Preferably, however, the species is N. meningitidis. The N. meningitidis may be from any serogroup, including serogroups A, C, W135 and Y. Most preferably, however, it is N. meningitidis serogroup B.

[0032] References to an "epithelial cell" in this section include any cell found in or derived from the epithelium of a mammal. The cell may be in vitro (e.g. in cell culture) or in vivo. Preferred epithelial cells are from the nasopharynx. The cells are most preferably human cells.

Blocking the Neisseria-Epithelium Interaction

[0033] The invention provides a method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein the ability of one or more App, ORF40 and/or NadA to bind to the epithelial cell is blocked.

[0034] The ability to bind may be blocked in various ways but, most conveniently, an antibody specific for App, ORF40 and/or NadA is used. The invention also provides antibody which is specific for App, ORF40 or NadA. This antibody preferably has an affinity for App, ORF40 and/or NadA of at least 10.sup.-7 M e.g. 10.sup.-8 M, 10.sup.-9 M, 10.sup.-10 M or tighter.

[0035] Antibodies for use in accordance with the invention may be polyclonal, but are preferably monoclonal. It will be appreciated that the term "antibody" includes whole antibodies (e.g. IgG, IgA etc), derivatives of whole antibodies which retain the antigen-binding sites (e.g. F.sub.ab, F.sub.ab', F.sub.(ab')2 etc.), single chain antibodies (e.g. sFv), chimeric antibodies, CDR-grafted antibodies, humanised antibodies, univalent antibodies, human monoclonal antibodies [e.g. Green (1999) J. Immunol Methods 231:11-23; Kipriyanov & Little (1999) Mol Biotechnol 12:173-201 etc.] and the like. Humanised antibodies may be preferable to those which are fully human [e.g. Fletcher (2001) Nature Biotechnology 19:395-96].

[0036] As an alternative to using antibodies, antagonists of the interaction between App, ORF40 or NadA and its receptor on the epithelial cell may be used. As a further alternative, a soluble form of the epithelial cell receptor may be used as a decoy. These can be produced by removing the receptor's transmembrane and, optionally, cytoplasmic regions [e.g. EP-B2-0139417, EP-A-0609580 etc.].

[0037] The antibodies, antagonists and soluble receptors of the invention may be used as medicaments to prevent the attachment of a Neisserial cell to an epithelial cell.

Inhibiting Expression of the Neisserial Gene

[0038] The invention provides a method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein protein expression from one or more of App, ORF40 and/or NadA is inhibited. The inhibition may be at the level of transcription and/or translation.

[0039] A preferred technique for inhibiting expression of the gene is antisense [e.g. Piddock (1998) Curr Opin Microbiol 1:502-8; Nielsen (2001) Expert Opin Investig Drugs 10:331-41; Good & Nielsen (1998) Nature Biotechnol 16:355-358; Rahman et al. (1991) Antisense Res Dev 1:319-327; Methods in Enzymology volumes 313 & 314; Manual of Antisense Methodology (eds. Hartmann & Endres); Antisense Therapeutics (ed. Agrawal) etc.]. Antibacterial antisense techniques are disclosed in, for example, international patent applications WO99/02673 and WO99/13893.

[0040] The invention also provides nucleic acid comprising a fragment of x or more nucleotides from nucleic acid which encodes App, ORF40 or NadA, wherein x is at least 8 (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30 or more). The nucleic acid will typically be single-stranded.

[0041] The nucleic acid is preferably of the formula 5'-(N).sub.a-(X)-(N).sub.b-3', wherein 0.gtoreq.a.gtoreq.15, 0.gtoreq.b.gtoreq.15, N is any nucleotide, and X is a fragment of a nucleic acid which encodes App, ORF40 or NadA. X preferably comprises at least 8 nucleotides (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30 or more). The values of a and b may independently be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. Each individual nucleotide N in the -(N).sub.a- and -(N).sub.b-portions of the nucleic acid may be the same or different. The length of the nucleic acid (i.e. a+b+length of X) is preferably less than 100 (e.g. less than 90, 80, 70, 60, 50, 40, 30 etc.).

[0042] It will be appreciated that the term "nucleic acid" includes DNA, RNA, DNA/RNA hybrids, DNA and RNA analogues such as those containing modified backbones (with modifications in the sugar and/or phosphates e.g. phosphorothioates, phosphoramidites etc.), and also peptide nucleic acids (PNA) and any other polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases etc. Nucleic acid according to the invention can be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes etc.).

[0043] The antisense nucleic acids of the invention may be used as medicaments to prevent the attachment of a Neisserial cell to an epithelial cell.

Knockout of the Neisserial Gene

[0044] The invention provides a method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein one or more of App, ORF40 and/or NadA is knocked out.

[0045] The invention also provides a Neisseria bacterium in which one or more of App, ORF40 and/or NadA has been knocked out.

[0046] Techniques for producing knockout bacteria are well known, and knockout Neisseria have been reported [e.g. Moe et al. (2001) Infect. Immun. 69:3762-3771; Seifert (1997) Gene 188:215-220; Zhu et al. (2000) J. Bacteriol. 182:439-447 etc.].

[0047] The knockout mutation may be situated in the coding region of the gene or may lie within its transcriptional control regions (e.g. within its promoter).

[0048] The knockout mutation will reduce the level of mRNA encoding App, ORF40 and/or NadA to <1% of that produced by the wild-type bacterium, preferably <0.5%, more preferably <0.1%, and most preferably to 0%.

[0049] The knockout mutants of the invention may be used as immunogenic compositions (e.g. as vaccines) to prevent Neisserial infection. Such a vaccine may include the mutant as a live attenuated bacterium.

Mutagenesis of the Neisserial Gene

[0050] The invention provides a method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein one or more of App, ORF40 and/or NadA has a mutation which inhibits its activity.

[0051] The invention also provides a mutant protein, wherein the mutant protein comprises the amino acid sequence of App, ORF40 and/or NadA, or a fragment thereof, but wherein one or more amino acids of said amino acid sequence is/are mutated (e.g. see below for App).

[0052] The amino acids which is/are mutated preferably result in the reduction or removal of an activity of App, ORF40 and/or NadA which is responsible directly or indirectly for adhesion to epithelial cells. For example, the mutation may inhibit an enzymatic activity or may remove a binding site in the protein.

[0053] The invention also provides nucleic acid encoding this mutant protein.

[0054] The invention also provides a method for producing this nucleic acid, comprising the steps of: (a) providing source nucleic acid encoding App, ORF40 or NadA, and (b) performing mutagenesis (e.g. site-directed mutagenesis) on said source nucleic acid to provide nucleic acid encoding a mutant protein.

[0055] Mutation may involve deletion, substitution, and/or insertion, any of which may be involve one or more amino acids. As an alternative, the mutation may involve truncation.

[0056] Mutagenesis of virulence, factors is a well-established science for many bacteria [e.g. toxin mutagenesis described in WO93/13202; Rappuoli & Pizza, Chapter 1 of Sourcebook of Bacterial Protein Toxins (ISBN 0-12-053078-3); Pizza et al. (2001) Vaccine 19:2534-41; Alape-Giron et al. (2000) Eur J Biochem 267:5191-5197; Kitten et al. (2000) Infect Immun 68:4441-4451; Gubba et al. (2000) Infect Immun 68:3716-3719; Boulnois et al. (1991) Mol Microbiol 5:2611-2616 etc.] including Neisseria [e.g. Power et al. (2000) Microbiology 146:967-979; Forest et al. (1999) Mol Microbiol 31:743-752; Cornelissen et al. (1998) Mol Microbiol 27:611-616; Lee et al. (1995) Infect Immun 63:2508-2515; Robertson et al. (1993) Mol Microbiol 8:891-901 etc.].

[0057] Mutagenesis may be specifically targeted to nucleic acid encoding App, ORF40 and/or NadA. Alternatively, mutagenesis may be global or random (e.g. by irradiation, chemical mutagenesis etc.), which will typically be followed by screening bacteria for those in which a mutation has been introduced into App, ORF40 and/or NadA. Such screening may be by hybridisation assays (e.g. Southern or Northern blots etc.), primer-based amplification (e.g. PCR), sequencing, proteomics, aberrant SDS-PAGE gel migration etc.

[0058] The mutant proteins and nucleic acids of the invention may be used as immunogenic compositions (e.g. as vaccines) to prevent Neisserial infection.

Screening Methods

[0059] The invention also provides methods for screening compounds to identify those (antagonists) which inhibit the binding of a Neisserial cell to an epithelial cell.

[0060] Potential antagonists for screening include small organic molecules, peptides, peptoids, polypeptides, lipids, metals, nucleotides, nucleosides, polyamines, antibodies, and derivatives thereof. Small organic molecules have a molecular weight between 50 and about 2,500 daltons, and most preferably in the range 200-800 daltons. Complex mixtures of substances, such as extracts containing natural products, compound libraries or the products of mixed combinatorial syntheses also contain potential antagonists.

[0061] Typically, App, ORF40 and/or NadA protein is incubated with an epithelial cell and a test compound, and the mixture is then tested to see if the interaction between the protein and the epithelial cell has been inhibited.

[0062] Inhibition will, of course, be determined relative to a standard (e.g. the native protein/cell interaction). Preferably, the standard is a control value measured in the absence of the test compound. It will be appreciated that the standard may have been determined before performing the method, or may be determined during or after the method has been performed. It may also be an absolute standard.

[0063] The protein, cell and compound may be mixed in any order.

[0064] For preferred high-throughput screening methods, all the biochemical steps for this assay are performed in a single solution in, for instance, a test tube or microtitre plate, and the test compounds are analysed initially at a single compound concentration. For the purposes of high throughput screening, the experimental conditions are adjusted to achieve a proportion of test compounds identified as "positive" compounds from amongst the total compounds screened.

[0065] Other methods which may be used include, for example, reverse two hybrid screening [e.g. Vidal & Endoh (1999) TIBTECH 17:374-381] in which the inhibition of the Neisseria:receptor interaction is reported as a failure to activate transcription.

[0066] The method may also simply involve incubating one or more test compound(s) with App, ORF40 and/or NadA and determining if they interact. Compounds that interact with the protein can then be tested for their ability to block an interaction between the protein and an epithelial cell.

[0067] The invention also provides a compound identified using these methods. These can be used to treat or prevent Neisserial infection. The compound preferably has an affinity for App, ORF40 and/or NadA of at least 10.sup.-7 M e.g. 10.sup.-8 M, 10.sup.-9 M, 10.sup.-10 M or tighter.

[0068] The invention also provides a composition comprising (a) an E. coli bacterium which expresses App and/or ORF40 (and, optionally, NadA) and (b) an epithelial cell (e.g. a human epithelial cell).

Expression in Outer Membrane Vesicles (OMVs)

[0069] International patent application WO01/52885 discloses that the addition of further defined components to OMV vaccines significantly broadens their efficacy.

[0070] The preparation of OMVs from NmB is well-known in the art. Methods for obtaining suitable preparations are disclosed in, for instance: Claassen et al. [Vaccine (1996) 14:1001-1008]; Cartwright et al. [Vaccine (1999) 17:2612-2619]; Peeters et al. [Vaccine (1996) 14:1009-1015]; Fu et al. [Biotechnology NY (1995) 12:170-74]; Davies et al. [J. Immunol. Meth. (1990) 134:215-225]; Saunders et al. [Infect. Immun. (1999) 67:113-119]; Draabick et al. [Vaccine (2000) 18:160-172]; Moreno et al. [Infect. Immun. (1985) 47:527-533]; Milagres et al. [Infect. Immun. (1994) 62:4419-4424]; Naess et al. [Infect. Immun. (1998) 66:959-965]; Rosenqvist et al. [Dev. Biol. Stand. (1998) 92:323-333]; Haneberg et al. [Infect. Immunn. (1998) 66:1334-41]; Andersen et al. [Vaccine (1997) 15:1225-34]; Bjune et al. [Lancet (1991) 338:1093-96] etc.

[0071] It has now been found that OMVs prepared from E. coli which express a heterologous Neisseria gene can give better results in standard immunogenicity tests than the antigens in purified form.

[0072] The invention therefore provides a method for preparing an OMV from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding App, ORF40 or NadA protein.

[0073] The invention also provides (a) OMVs obtainable by this process, and (b) an outer membrane vesicle from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding App, ORF40 or NadA protein.

[0074] The non-Neisserial host cell is preferably a bacterium and is most preferably E. coli.

[0075] More generally, the invention provides a method for preparing an OMV from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding one or more of the following proteins: [0076] (A) Even SEQ IDs 2-892 from WO99/24578; [0077] (B) Even SEQ IDs 2-90 from WO99/36544; [0078] (C) Even SEQ IDs 2-3020 from WO99/57280; [0079] (D) Even SEQ IDs 3040-3114 from WO99/57280; [0080] (E) SEQ IDs 3115-3241 from WO99/57280; [0081] (F) The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [supra]; [0082] (G) A protein comprising the amino acid sequence of one or more of (A) to (F); [0083] (H) A protein sharing sequence identity with the amino acid sequence of one or more of (A) to (F); and [0084] (I) A protein comprising a fragment of one or more of (A) to (F).

[0085] Similarly, the invention also provides (a) OMVs obtainable by this process, and (b) an outer membrane vesicle from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding one or more of proteins (A) to (I) described above.

[0086] The degree of `sequence identity` referred to in (H) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more) and this includes mutants and allelic variants

[0087] The `fragment` referred to in (I) should comprise at least n consecutive amino acids from one or more of (A) to (F) and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragment comprises an epitope from one or more of (A) to (F). Preferred fragments are those disclosed in WO00/71574 and WO01/04316.

[0088] Preferred proteins for (A) to (F) are found in N. meningitidis serogroup B.

Mutants of App

[0089] Amino acid 267 of SEQ ID 650 of WO99/24578 (SEQ ID 32 herein) is a serine. App is believed to be a serine protease and this serine is believed to be a catalytic residue at its active site. It will be appreciated that standard sequence alignment techniques will reveal the amino acid corresponding to this Ser-267 for any other App sequence (e.g. Ser-260 in SEQ ID 652 of WO99/24578, Ser-267 in SEQ ID 654 etc.).

[0090] The invention provides a protein comprising the amino acid sequence of App, except that one or more of amino acids Ser-267, Asp-158 and His-115 (numbered according to SEQ ID 32) is/are mutated. The mutation may be a deletion, an insertion or, preferably, a substitution. The substitution is preferably with one of the 19 other naturally-occurring amino acids and is more preferably with glycine, alanine, tyrosine or lysine.

[0091] App is believed to cleaved at a site between amino acids 1063 and 1171 (numbered according to SEQ ID 32). It will be appreciated that standard sequence alignment techniques will reveal the amino acids corresponding to these two residues for any other App sequence.

[0092] The invention provides a protein comprising the amino acid sequence of App, except that one or more amino acid(s) between Ser-1064 and Arg-1171 (numbered according to SEQ ID 32) is mutated. The mutation may be a deletion, an insertion, truncation or, preferably, a substitution. The substitution is preferably with one of the 19 other naturally-occurring amino acids. The residue which is mutated is preferably S-1064, D-1065, K-1066, L-1067, G-1068, K-1069, A-1070, E-1071, A-1072, K-1073, K-1074, Q-1075, A-1076, E-1077, K-1078, D-1079, N-1080, A-1081, Q-1082, S-1083, L-1084, D-1085, A-1086, L-1087, I-1088, A-1089, A-1090, G-1091, R-1092, D-1093, A-1094, V-1095, E-1096, K-1097, T-1098, E-1099, S-1100, V-1101, A-1102, E-1103, P-1104, A-1105, R-1106, Q-1107, A-1108, G-1109, G-1110, E-1111, N-1112, V-1113, G-1114, I-1115, M-1116, Q-1117, A-1118, E-1119, E-1120, E-1121, K-1122, K-1123, R-1124, V-1125, Q-1126, A-1127, D-1128, K-1129, D-1130, T-1131, A-1132, L-1133, A-1134, K-1135, Q-1136, R-1137, E-1138, A-1139, E-1140, T-1141, R-1142, P-1143, A-1144, T-1145, T-1146, A-1147, F-1148, P-1149, R-1150, A-1151, R-1152, R-1153, A-1154, R-1155, R-1156, D-1157, L-1158, P-1159, Q-1160, L-1161, Q-1162, P-1163, Q-1164, P-1165, Q-1166, P-1167, Q-1168, P-1169, Q-1170 and/or R-1171.

[0093] App is alternatively believed to cleaved at amino acid 956 and/or amino acid 1178 (numbered according to SEQ ID 32). It will be appreciated that standard sequence alignment techniques will reveal the amino acids corresponding to these residues for any other App sequence.

[0094] The invention provides a protein comprising the amino acid sequence of App, except that one or more of amino acids Phe-956, Asn-957, Ala-1178 & Asn-1179 (numbered according to SEQ ID 32) is mutated. The mutation may be a deletion, an insertion, truncation or, preferably, a substitution. The substitution is preferably with one of the 19 other naturally-occurring amino acids.

[0095] The invention also provides nucleic acid encoding these mutant proteins.

[0096] The invention also provides a method for producing this nucleic acid, comprising the steps of: (a) providing source nucleic acid encoding App, ORF40 or NadA, and (b) performing mutagenesis (e.g. site-directed mutagenesis) on said source nucleic acid to provide nucleic acid encoding a mutant protein.

[0097] The invention provides mature App.

[0098] The invention also provides a protein comprising the amino acid sequence of a processed App, wherein said processed App does not comprise the C-terminus domain which is downstream of an autoproteloytic cleavage site in full-length App. For example, based on SEQ ID 32 as full-length App, the invention provides SEQ IDs 33 to 36. C-terminus domains which may be removed during autoproteolysis are SEQ IDs 38 and 39.

[0099] The invention also provides a protein comprising the amino acid sequence of a processed App, wherein the C-terminus of said processed. App is Phe-956 (numbered according to SEQ ID 32). For example, the invention provides SEQ IDs 33 and 35. The amino acid corresponding to Phe-956 in other App sequences can be identified by standard sequence alignment techniques.

[0100] The invention also provides a protein comprising the amino acid sequence of a processed App, wherein the C-terminus of said processed App is Ala-1178 (numbered according to SEQ ID 32). For example, the invention provides SEQ IDs 34 and 36. The amino acid corresponding to Ala-1178 in other App sequences can be identified by standard sequence alignment techniques.

[0101] The invention also provides a protein comprising the amino acid sequence of a processed App, wherein said processed App does not comprise SEQ ID 37, 38 or 39.

[0102] The invention also provides a protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 33, 34, 35, 36, 37, 38 & 39.

[0103] The invention also provides a protein comprising an amino acid sequence with at least p % sequence identity to one or more of SEQ IDs 33, 34, 35, 36, 37, 38 & 39. Depending on the particular sequence, the value of p is preferably 50 or more (e.g. 60, 70, 80, 90, 95, 99 or more). These proteins include homologs, orthologs, allelic variants and functional mutants. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

[0104] The invention further provides proteins comprising a fragment of one or more of SEQ IDs 33, 34, 35, 36, 37, 38 & 39. The fragments should comprise at least q consecutive amino acids from the sequences and, depending on the particular sequence, q is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragments comprise one or more epitopes from the sequence.

[0105] The invention also provides nucleic acid encoding these proteins of the invention.

Alleles of NadA

[0106] The invention provides a protein comprising the amino acid sequence of one or more of SEQ IDs 1 to 14.

[0107] The invention also provides a protein comprising an amino acid sequence having at least x % sequence identity to one or more of SEQ IDs 1 to 14. The value of x is at least 50% (e.g. 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more). This includes variants e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.

[0108] A preferred allele of NadA for use with the present invention is SEQ ID 3 (or SEQ ID 6).

[0109] The invention also provides a protein comprising a fragment of one or more of SEQ IDs 1 to 14. These should comprise at least n consecutive nucleotides from one or more of SEQ IDs 1 to 14, wherein n is 6 or more (e.g. 7, 8, 9, 10, 11, 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 or more). The fragment may comprise a sequence which is common to SEQ IDs 1 to 14, or may comprise a sequence which is not common to SEQ IDs 1 to 14.

[0110] Preferred fragments comprise one or more epitopes from SEQ IDs 1 to 14. Other preferred fragments are (a) the N-terminal leader peptides of SEQ IDs 1 to 14, (b) SEQ IDs 1 to 14, but without k N-terminal amino acid residue(s), wherein k is 1 or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 etc.), and (c) SEQ Ms 1 to 14, but without l C-terminal amino acid residue(s), wherein l is 1 or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 etc.). Preferred fragments fall within both (b) and (c) i.e. truncation at both C- and N-termini.

[0111] Preferred fragments within category (b) lack the N-terminal leader peptide. For SEQ IDs 1, 2, 3, 7, 9, 11 & 13 the value of k is thus 23; for SEQ IDs 4, 5, 6, 8, 10, 12 & 14 the value of k is 25. The leader peptide may be replaced with the leader peptide from another protein, by another protein (i.e. to form a fusion protein) or by an alternative N-terminus sequence to allow efficient expression.

[0112] Preferred fragments within category (c) lack the C-terminal membrane anchor. The value of l is thus 54. Minor variants of this C-terminal deletion may be used (e.g. where l is 45, 46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66).

[0113] Proteins with the N-terminus sequence MKH or MQH are preferred to those with N-terminus sequence MSM.

[0114] The protein of the invention may include the heptad sequence (AA.sub.1AA.sub.2AA.sub.3AA.sub.4AA.sub.5AA.sub.6AA.sub.7).sub.r wherein: AA.sub.1 is Leu, Be, Val or Met; each of AA.sub.2 AA.sub.3 AA.sub.4 AA.sub.5 AA.sub.6 and AA.sub.7 may independently be any amino acid; r is an integer of 1 or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.). Where r is 2 or more, the meaning of each AA.sub.1 AA.sub.2 AA.sub.3 AA.sub.4 AA.sub.5 AA.sub.6 and AA.sub.7 may be the same or different in each of the r heptad repeats. The heptad(s) can form a leucine-zipper domain.

[0115] Proteins of the invention can be prepared in many ways e.g. by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself (e.g. isolation from prostate tissue), from a cell line source, etc.

[0116] Proteins of the invention can be prepared in various forms e.g. native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated etc.

[0117] The protein is preferably in the form of an oligomer.

[0118] Proteins of the invention may be attached or immobilised to a solid support.

[0119] Proteins of the invention may comprise a detectable label e.g. a radioactive label, a fluorescent label, or a biotin label. This is particularly useful in immunoassay techniques.

[0120] Proteins of the invention are preferably in isolated or substantially isolated form.

[0121] In general, the proteins of the invention are provided in a non-naturally occurring environment e.g. they are separated from their naturally-occurring environment. In certain embodiments, the subject protein is present in a composition that is enriched for the protein as compared to a control. As such, purified protein is provided, whereby purified is meant that the protein is present in a composition that is substantially free of other expressed proteins, where by substantially free is meant that less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed proteins.

[0122] The term "protein" refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example proteins containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Proteins can occur as single chains or associated chains.

[0123] Mutants can include amino acid substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid substituted. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the polypeptide (e.g. a functional domain and/or, where the polypeptide is a member of a polypeptide family, a region associated with a consensus sequence). Selection of amino acid alterations for production of variants can be based upon the accessibility (interior vs. exterior) of the amino acid, the thermostability of the variant polypeptide, desired disulfide bridges, desired metal binding sites etc.

[0124] The invention also provides nucleic acid encoding a protein of the invention as defined above. The invention also provides nucleic acid comprising a fragment of at least n consecutive nucleotides from said nucleic acid, wherein n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or more).

[0125] Furthermore, the invention provides nucleic acid which can hybridise to nucleic acid encoding a protein of the invention, preferably under "high stringency" conditions (eg. 65.degree. C. in a 0.1.times.SSC, 0.5% SDS solution).

[0126] Nucleic acids of the invention can be used in hybridisation reactions (e.g. Northern or Southern blots, or in nucleic acid microarrays or `gene chips`) and amplification reactions (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) and other nucleic acid techniques.

[0127] Nucleic acids of the invention can be prepared in many ways e.g. by chemical synthesis in whole or part, by digesting longer polynucleotides using nucleases (e.g. restriction enzymes), from genomic or cDNA libraries, from the bacterium itself, etc.

[0128] Nucleic acids of the invention can take various forms e.g. single-stranded, double-stranded, vectors, primers, probes, labelled, unlabelled, etc.

[0129] Nucleic acids of the invention are preferably in isolated or substantially isolated form.

[0130] The invention includes nucleic acid comprising sequences complementary to those described above e.g. for antisense or probing, or for use as primers.

[0131] The term "nucleic acid" includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

[0132] Nucleic acid according to the invention may be labelled e.g. with a radioactive or fluorescent label. This is particularly useful where the nucleic acid is to be used in nucleic acid detection techniques e.g. where the nucleic acid is a primer or as a probe for use in techniques such as PCR, LCR, TMA, NASBA, etc.

[0133] The invention also provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors, such as those suitable for nucleic acid immunisation) and host cells transformed with such vectors.

Immunisation

[0134] The invention provides an immunogenic composition comprising (a) a Neisserial NadA protein and/or (b) nucleic acid encoding a NadA protein.

[0135] The invention also provides a method for raising an antibody response in a mammal, comprising administering an immunogenic composition of the invention to the mammal. The antibody response is preferably a protective antibody response. The protective antibody preferably blocks the attachment of NadA and/or App to epithelial cells.

[0136] The invention also provides a method for protecting a mammal against a Neisserial infection, comprising administering to the mammal an immunogenic composition of the invention.

[0137] The invention also provides Neisserial NadA protein for use as a medicament.

[0138] The invention also provides the use of a NadA protein in the manufacture of a medicament for preventing Neisserial infection in a mammal

[0139] The invention also provides the use of nucleic acid encoding a NadA protein in the manufacture of a medicament for preventing Neisserial infection in a mammal.

[0140] The mammal is preferably a human. The human may be an adult or, preferably, a child.

[0141] The NadA protein is preferably a N. meningitidis NadA. It preferably comprises the amino acid sequence of one or more of SEQ IDs 1 to 14, or an amino acid sequence having sequence identity thereto or comprising a fragment thereof (see above). The NadA protein is preferably in the form of an oligomer (e.g. a dimer, trimer, tetramer or higher). Within SEQ IDs 1 to 14, SEQ IDs 1 to 12 are preferred, as antibodies against these NadA proteins are bactericidal across the various hypervirulent alleles. Where an immune response against a non-hypervirulent NadA.sup.+ strain is desired, however, SEQ IDs 13 & 14 are preferred. Of course, NadA mixtures are also possible, particularly mixtures containing more than one NadA allele.

[0142] Immunogenic compositions of the invention may be used therapeutically (i.e. to treat an existing infection) or prophylactically (i.e. to prevent future infection).

[0143] The uses and methods of the invention are particularly useful for treating/protecting against infections of Neisseria meningitidis, including serogroups A, B, and C. They are particularly useful against strains of N. meningitidis from hypervirulent lineages ET-5, EY-37 and cluster A4.

[0144] The uses and methods are particularly useful for preventing/treating diseases including, but not limited to, meningitis (particularly bacterial meningitis) and bacteremia.

[0145] Efficacy of therapeutic treatment can be tested by monitoring Neisserial infection after administration of the composition of the invention. Efficacy of prophylactic treatment can be tested by monitoring immune responses against NadA after administration of the composition.

[0146] The composition of the invention may additionally comprise an antigen which, when administered to a mammal, elicits an immune response which is protective against a lineage III strain of N. meningitidis.

[0147] Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, or pulmonary administration.

[0148] The invention may be used to elicit systemic and/or mucosal immunity.

[0149] Dosage treatment can be a single dose schedule or a multiple dose schedule.

[0150] The immunogenic composition of the invention will generally include a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly-metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Liposomes are suitable carriers. A thorough discussion of pharmaceutical carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

[0151] Neisserial infections affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops.

[0152] The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7.

[0153] Immunogenic compositions comprise an immunologically effective amount of immunogen, as well as any other of other specified components, as needed. By `immunologically effective amount`, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses). The composition may be administered in conjunction with other immunoregulatory agents.

[0154] The immunogenic composition may include an adjuvant. Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (A) aluminium compounds (e.g. an aluminium hydroxide such as oxyhydroxide, or an aluminium phosphate such as hydroxyphosphate or orthophosphate, aluminium sulphate etc.), or mixtures of different aluminium compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous etc.), and with adsorption being preferred; (B) MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer); (C) liposomes; (D) ISCOMs, which may be devoid of additional detergent; (E) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion; (F) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (G) saponin adjuvants, such as QuilA or QS21, also known as Stimulon.TM.; (H) chitosan; (I) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-.gamma.), macrophage colony stimulating factor, tumor necrosis factor, etc.; (K) microparticles (i.e. a particle of .about.100 nm to .about.150 .mu.m in diameter, more preferably .about.200 nm to .about.30 .mu.m in diameter, and most preferably .about.500 nm to .about.10 .mu.m in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(.alpha.-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone etc.); (L) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL); (M) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions; (N) oligonucleotides comprising CpG motifs i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (O) a polyoxyethylene ether or a polyoxyethylene ester; (P) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol; (Q) an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin; (R) an immunostimulant and a particle of metal salt; (S) a saponin and an oil-in-water emulsion; (T) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol); (U) E. coli heat-labile enterotoxin ("LT"), or detoxified mutants thereof, such as the K63 or R72 mutants; (V) cholera toxin ("CT"), or detoxified mutants thereof; (W) microparticles (i.e. a particle of .about.100 nm to .about.150 .mu.m in diameter, more preferably .about.200 nm to .about.30 .mu.m in diameter, and most preferably .about.500 nm to .about.10 .mu.m in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(.alpha.-hydroxy acid) such as poly(lactide-co-glycolide), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone etc.); and (X) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Aluminium salts (aluminium phosphates and particularly hydroxyphosphates, and/or hydroxides and particularly oxyhydroxide) and MF59 are preferred adjuvants for parenteral immunisation. Toxin mutants are preferred mucosal adjuvants.

[0155] Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s- n-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.

[0156] Compositions of the invention may comprise antigens (e.g. protective antigens against N. meningitidis or against other organisms) in addition to NadA e.g. DTP antigens, Hib antigen etc.

[0157] Immunogenic compositions of the invention may be used therapeutically (i.e. to treat an existing infection) or prophylactically (i.e. to prevent future infection). Therapeutic immunisation is particularly useful for treating Candida infection in immunocompromised subjects.

[0158] As an alternative to using proteins antigens in the immunogenic compositions of the invention, nucleic acid (preferably DNA e.g. in the form of a plasmid) encoding the antigen may be used.

Disclaimers

[0159] The invention preferably excludes: (a) amino acid and nucleic acid sequences available in public sequence databases (e.g. GenBank or GENESEQ) prior to 26 Jul. 2002 and, more preferably, prior to 27 Jul. 2001; (b) amino acid and nucleic acid sequences disclosed in patent applications having a filing date or, where applicable, a priority date prior to 26 Jul. 2002 and, more preferably, prior to 27 Jul. 2001. In particular, SEQ ID entries in the following patent applications may be excluded: WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66741; WO00/66791; WO00/71574; WO00/71725; WO01/04316; WO01/31019; WO01/37863; WO01/38350; WO01/52885; WO01/64920; WO01/64922.

DEFINITIONS

[0160] The term "comprising" means "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X+Y.

BRIEF DESCRIPTION OF DRAWINGS

[0161] FIGS. 1 to 3 show expression data for (1) ORF40 (2) App (3) NadA.

[0162] FIGS. 4 to 6 show FACS analysis of proteins involved in adhesion to human cells. In FIGS. 4 and 5 (FIG. 6), the data are for, from left to right, ORF40 (.tangle-solidup.), App ( ), NadA (.diamond-solid.) and GNA2132 (.box-solid.).

[0163] FIGS. 7 and 8 show homologies of (7) ORF40 and (8) App.

[0164] FIG. 9 shows an alignment of NadA alleles, and FIG. 10 shows the relationship of alleles 1 to 3.

[0165] FIG. 11 shows predicted secondary structure for NadA.

[0166] FIG. 12 shows analysis of sequences upstream and downstream of NadA.

[0167] FIG. 13 shows PCR analysis of NadA expression in different strains of N. meningitidis.

[0168] FIG. 14 shows immunoblot analysis of NadA expression in different strains of N. meningitidis.

[0169] FIG. 15 shows variation of NadA expression with culture time.

[0170] FIG. 16 shows NadA FACS of isogenic capsulated and non-capsulated N. meningitidis cells.

[0171] FIG. 17 shows immunofluorescence results obtained using anti-NadA against Chang cells (17A to 17C) or HeLa cells (17D).

[0172] FIG. 18 shows immunofluorescence results obtained using anti-NadA against Chang cells after incubation at (A) 37.degree. C. or (B) 4.degree. C.

[0173] FIG. 19 shows immunofluorescence results for Chang cells treated with saponin.

[0174] FIG. 20 shows immunofluorescence results obtained using monocytes.

[0175] FIG. 21 shows immunofluorescence results obtained using macrophages.

[0176] FIG. 22 shows IL-.alpha. secretion by monocytes in response to NadA treatment.

[0177] FIG. 23 shows the effect of anti-CD14 on IL-.alpha. secretion by monocytes.

[0178] FIG. 24 shows immunofluorescence results obtained using anti-NadA against E. coli transformed to express NadA.

[0179] FIG. 25 shows staining of the transformed E. coli using (A) anti-NadA (B) anti-E. coli or (C) both.

[0180] FIG. 26 is a schematic representation of App features. The N-terminal leader peptide, the passenger domain and the C-terminal .beta.-domain are indicated. The positions of the serine protease active site, the ATP/GTP binding site, the two Arginine-rich sites and the Proline-rich region are shown. In BOX 1, cleavage sites are shown. In BOX 2 a comparison of known proteolytic sites of different autotransporters is shown and a consensus signature is derived. Arrows identify the cleavages; X=any amino acid; hyd=hydrophobic residues; (A,S)=Alanine or Serine.

[0181] FIG. 27 is a schematic representation of the constructs used for studying App.

[0182] FIG. 28 shows a western blot of outer membrane and extracellular proteins in E. coli.

[0183] FIG. 29 shows FACS analysis of outer membrane and extracellular proteins in E. coli.

[0184] FIG. 30 shows immunofluorescence of outer membrane and extracellular proteins in E. coli.

[0185] FIG. 31 shows total E. coli proteins analysed by SDS-PAGE.

[0186] FIG. 32 shows an immunoblot of crude precipitated culture supernatants using mouse antiserum against App-his.

[0187] FIG. 33 shows FACS adhesion data using rabbit antiserum against E. coli. Percentages of cells positive to adhesion are shown near the fluorescence profiles.

[0188] FIG. 34 shows immunofluorescence microscopy data showing bacterial adherence and aggregation.

[0189] FIG. 35 shows concentration-dependent binding of App-His (.diamond-solid.), App.alpha.-His (.box-solid.) and NMB2132 (.tangle-solidup.) expressed as net Mean Fluorescence Intensity (MFI).

[0190] FIG. 36 shows the effect on binding of App-His (100 .mu.g/ml) of pre-incubation with pronase (left-hand columns) or phospholipase A2 (right-hand columns) with increasing concentration of enzyme. Pronase was tested at 0, 250, 500, 1000 .mu.g/ml; phosholipase A2 was tested at 0, 50, 200, 800 .mu.g/ml.

[0191] FIG. 37 is a comparison of cellular binding specificity of App-His protein at 100, 25 or 6.25 .mu.g/ml against various different cells.

[0192] FIG. 38 shows association of wild-type or App-knockout N. meningitidis MC58 bacteria.

[0193] FIG. 39 shows a western blot analysis of total lysates from N. meningitidis MC58 harvested at 0.5 or 0.8 OD.sub.620 nm. Lanes 1 & 3 show wild-type MC58 and lanes 2 & 4 show the App knockout.

[0194] FIG. 40 shows a western blot analysis of supernatants in parallel to FIG. 39.

MODES FOR CARRYING OUT THE INVENTION

NadA Homology

[0195] NadA shows homology to (a) YadA of enteropathogenic Yersinia, a non-pilus associated adhesin implicated in virulence [Cornelis (1998) Microbiol. Mol. Biol. Rev. 62:1315-1352.] and (b) UspA2 of Moraxella catarrhalis, a protein involved in serum resistance and a protective antigen [Chen et al. (1999) Infect. Immun. 67:1310-1316.]. Sequence similarity is mainly clustered in the carboxyl terminal region (56-63% identity in the last 70 amino acids). Outside this region the level of identity drops to 23-25%.

[0196] YadA and UspA2 have been identified as adhesins [Hoiczyk et al. (2000) EMBO J 19:5989-5999]. Both proteins form very stable and difficult-to-dissociate high molecular weight oligomers (150-200 kDa) anchored to the outer membrane. NadA has also been found to form very stable high molecular weight aggregates on the outer membrane of meningococcus.

[0197] The amino acid sequence of NadA was analysed [Nielsen et al. (1997) Protein Engineering 10:1-6; Levin & Garner (1988) Biochim. Biophys. Acta 955:283-295; Berger et al. (1995) PNAS USA 92:8259-8263; Bornberg-Bauer et al. (1998) Nucleic Acids Res. 26:2740-2746]. Secondary structure analysis is shown in FIG. 11. The globular N-terminus and amphipathic C-terminus are indicated, as are the positions of the leader peptide (LP) and a membrane anchor. The carboxyl-terminal region (aa 310-362) has a predicted amphipatic .beta.-structure (.beta.-strands shown in black) and a terminal aromatic amino acid, which are typical features of outer membrane anchoring domains. The amino terminal region (aa 23-90) has no defined secondary structure, but the rest of the protein has mainly .alpha.-helix propensity (84.6%). Within this region, residues 90-146 and 183-288 have high probability of forming coiled coils. In addition, residues 122-143 contain four leucine residues in the "a" positions of the heptad repeats (L-x(6)-L-x(6)-L-x(6)-L) that may form a leucine zipper domain ( ). It is known that both coiled coils and leucine zipper sequences are involved in dimerization and may mediate oligomerisation of monomers via association of two or more alpha helices.

[0198] Even though primary structure similarity between NadA, YadA and UspA2 is clustered at the C-terminus, therefore, the overall similarity between the three proteins is conserved at secondary structure level. Putative leucine zippers are present in both NadA and UspA2. NadA, YadA and UspA2 have a carboxyl terminal membrane anchor made by four amphipathic .beta.-strands and an internal .alpha.-helical region with propensity to form coiled-coils. In YadA and UspA2 these .alpha.-helices have been shown to form coiled-coils regions, which mediate oligomerisation of monomers [Hoiczyk et al. (2000) EMBO J 19:5989-5999; Cope et al. (1999) J. Bacteriol. 181:4026-4034].

[0199] The absence of cysteine residues in the mature forms of NadA is another feature shared with its homologues.

The Genomic Environment of NadA

[0200] The 1086 bp nadA coding region is flanked at the 3' end by a terminator sequence while at the 5' end (FIG. 12A) it shows a putative ribosome-binding site (RBS; 5'-AAGG-3') and a putative promoter region located 8 and 47 base pairs, respectively, upstream the ATG start codon.

[0201] 130 by upstream the coding region are nine repeats of the tetranucleotide TAAA (shaded black in FIG. 12A), preceded by a second putative promoter with -10 and -35 regions. Because of the presence of the TAAA repeats, the gene had been listed as one of those that may undergo phase variation, even though the repeats are not in the coding region [Tettelin et al.]. The homologous gene UspA2 has a tetranucleotide repeat (AGAT) located in the same position as in nadA, which varies in different strains [Cope et al. (1999) J. Bacteriol. 181:4026-4034].

[0202] The G+C content of the nadA gene and its upstream region is lower than average (45% against an average of the rest of the genome, 51.5%), suggesting acquisition of the gene by horizontal transfer.

[0203] The NadA gene and its upstream region are not present in the published sequence of the genome of serogroup A, strain Z2491 [Parkhill et al. (2000) Nature 404:502-506]. In the MenA genome, a short sequence of 16 nucleotides with no homologies in the database, replaces the nadA gene (FIG. 12B), whereas the upstream and downstream genes (nmb1993 and nmb1995) are well conserved (91% and 97% identity). Analysis of the sequences immediately adjacent to the nadA region and absent in the Z2491 serogroup A strain shows that the segment is flanked by the TCAGAC direct repeats. This may indicate a mechanism of recombination. In the A strain the stretch of 16 nucleotides has a disrupted pair of TCAGAC repeats flanking it.

Variation in NadA Genotype

[0204] Given the difference in nadA expression between serotypes A and B, 175 different strains of N. meningitidis were chosen for analysis--150 isolates representative of the five disease-associated serogroups (A, B, C, Y and W-135) and 25 strains isolated from healthy carriers. The analysis also included one strain each of N. gonorrhoeae, N. cinerea and N. lactamica.

[0205] Bacteria were grown overnight at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in air on gonococcus (GC) medium agar (Difco) supplemented with Kellogg's supplement solution (0.22 M D-glucose, 0.03 M L-glutamine, 0.001 M ferric nitrate, and 0.02 M cocarboxylase) (Sigma-Aldrich Chemical Co., St. Louis, Mo.) as previously described [Knapp et al. (1988) Antimicrob. Agents Chemother. 32:765-767; Roberts et al. (1977) J. Bacteriol. 131:557-563]. One loopful of bacteria was dissolved in 500 .mu.l of PBS and chromosomal DNA was prepared as previously described [Tinsley et al. (1996) PNAS USA 93:11109-11114].

[0206] The bacteria were screened by PCR and/or dot blot hybridization.

[0207] PCR amplification of the nadA genes was performed on 10 ng of chromosomal DNA using primers, mapping 350 nt upstream and downstream from the coding region (forward primer: SEQ ID 16; reverse primer: SEQ ID 17), and Platinum Hifi Taq Polymerase (GIBCO). PCR conditions were: 30 cycles of denaturation at 95.degree. C. for 30 s, annealing at 60.degree. C. for 30 s, and extension at 68.degree. C. for 1 min. PCR products were analysed on 1% agarose gel and the sizes were determined using a molecular weight marker 1 Kb Plus DNA Ladder (GIBCO). The amplified fragments were purified on a Qiaquick column (Qiagen) and then automated cyclo-sequenced (Applied Biosystems model 377) by primer walking on both strands of the amplified fragment.

[0208] For dot blotting, the probe used was the whole nadA gene, as amplified from 2996 strain and labelled with digoxigenin using the Roche DIG High-Prime DNA Labelling and Detection Kit. 10 .mu.l aliquot of cell suspension of each strain were boiled for 10 min. and spotted on nylon membrane (Boehringer). The membranes underwent cross-linking of DNA by 2' exposure to UV light and other standard procedures for preparation and signal detection as reported by the manufacturer.

[0209] The nadA gene was absent in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. In N. meningtidis, however, 47% of isolates were positive for its presence.

[0210] PCR generated (FIG. 13) a product of 1800 by in NadA.sup.+ strains MC58 (lane 1), 90/18311 (lane 2) and 2996 (lane 3). It gave a product of 400 by in NadA.sup.- strain Z2491 and NG3/88 (lane 5). Some strains (e.g. 93/4286, C4678, 2022, ISS1113) gave a PCR product of 2500 by (lane 4: L93/4286).

[0211] The presence/absence of NadA in N. meningitidis was correlated with strain lineage. Strains isolated from invasive meningococcal disease have been classified by multilocus enzyme electrophoresis (MLEE) into a small number of hypervirulent lineages: Electrophoretic Types ET37, ET5, cluster A4, lineage III, subgroups I, III and IV-1 [Achtman (1995) Global epidemiology of meningococcal disease. In Meningococcal disease (Cartwight, ed). John Wiley and Sons, Chichester, England. 159-175; Caugant (1998) APMIS 106:505-25]. Recently, a sequence-based classification, multilocus sequence typing (MLST), has been introduced, which classifies the above strains into Sequence Types ST11, ST32, ST8, ST41, ST1, ST5, ST4, respectively [Maiden et al. (1998) PNAS USA 95:3140-3145]. Strains isolated from healthy carriers fall into many different ET and ST types.

[0212] The nadA gene was present in 51 out of 53 strains (96%) of the hypervirulent lineages ET-5, ET-37 and cluster A4, whereas it was absent in all the tested lineage III strains. Seven of the 25 carrier strains were positive. Most of the serogroup C strains tested were positive even if not belonging to hyper-virulent lineages. The same was true for the serogroup B strains with serotype 2a and 2b. For serogroup A, one strain belonging to subgroup III was positive whereas the other two strains belonging to subgroup IV-1 were negative.

[0213] Lineage III has only recently been introduced in Europe and USA and the geographic segregation in New Zealand for many years could have impaired its ability to acquire novel genes. For instance, mutations may have occurred in the surrounding chromosomal regions preventing Lineage III from further recombination events. Another possible explanation is that ET-5, ET-37 and Cluster A4 strains need nadA to achieve peak fitness whereas Lineage III isolates cannot derive any significant benefit from nadA insertion, thus undergoing a negative selection.

[0214] NadA is thus over-represented in three hypervirulent N. meningitidis lineages. It appears to be a foreign gene present in a subset of hypervirulent strains.

[0215] NadA Alleles

[0216] As PCR products were differently sized (FIG. 13) and most of the NadA.sup.+ strains could be grouped in three different sizes, genes were sequenced for 36 strains representative of each size: 26 positive strains, 4 strains with a long PCR product, and 6 NadA.sup.- strains.

[0217] In the negative strains, a 16 bp sequence was found which was identical to the sequence present in the published serogroup A genome sequence.

[0218] Analysis of the sequence of the four long PCR product strains revealed an interruption by a single copy of IS1301, interrupting the protein after 162 amino acids with a stop codon. The insertion site was identical in all four strains, but the orientation of IS1301 differed, indicating independent events. The target consensus for IS1301, 5'-AYTAG-3' was found within the NadA gene at nucleotide 472, generated by, an A->G mutation, and was accompanied by a TA duplication.

[0219] In nadA.sup.+ strains, gene size ranged from 1086 to 1215 bp, with consequent variation of the amino acid sequences of the encoded proteins from 362 to 405 amino acids. It was possible to cluster 22 of the 26 NadA genes into three well-defined alleles (FIGS. 9 & 10; Table I). The sequence of the gene within each allele is identical and overall identity between the alleles ranges from 96% to 99%. This level of conservation is surprising and suggests weak selective pressure and/or a very recent acquisition of the nadA gene. The latter possibility is consistent with the low G+C content of the genome in this region (see above).

TABLE-US-00001 Allele Found in strains SEQ IDs 1 MC58, BZ83, BZ169, NM066, NM119, CU385, 1, 4 ISS832, ISS1071, ISS1104 2 90/18311, NGP165, PMC8, M986, ISS838 and 2, 5 961-5945 3 C11, 973-1720, ISS759, F6124, 2996, 8047, NMB 3, 6

[0220] The sequences shown in FIG. 9A assume that the N-terminus amino acid is the first Met in the open reading frame (SEQ IDs 4 to 6), but the second Met (residue 3 in SEQ IDs 4 to 6) has a better-positioned Shine-Dalgarno motif (FIG. 9B). Sequences starting from the second Met codon are thus preferred (SEQ IDs 1 to 3).

Allele 1 codes for a protein of 362 amino acids (SEQ ID 1) and includes strain MC58 and all the ET-5 positive strains sequenced. The other five strains belonging to allele 1 were very recent isolates and they have not been ET-typed yet, although serotype and serosubtype classification (B:15:P1.7 and B:4:P1.15) of these strains suggests affiliation of these strains to the ET-5 complex. Allele 2 codes for a protein of 398 amino acids (SEQ ID 2) resulting from the addition of 2 aa after residue 268 (numbering according to SEQ ID 1), addition of 41 aa after residue 271, and deletion of 7 aa after residue 122, resulting in the deletion of the first heptad repeat of the leucine zipper domain. Leucine residues at a fixed spacing of seven residues commonly identify leucine zippers. One leucine in the repeats has frequently been replaced mostly by Met, Val or Ile. In this case allele 2 could use the Ile upstream or downstream to form the leucine zipper motif. Allele 3 codes for a protein of 405 amino acids (SEQ ID 3) and, like allele 2, contains 43 extra amino acids at residues 268 and 271 but differs from allele 2 by not having the 7aa deletion after residue 122. Allele 3 is found in serogroup A, B and C strains.

[0221] The remaining 4/26 positive strains (ISS1024, ISS759, 973-1720, 95330; marked with * in Table 1) contain minor variants of alleles 1 to 3: [0222] Serogroup C strain ISS1024 has a variant of allele 2 with a single heptad repeat deletion at residues 229-235 (SEQ IDs 7/8). This sequence was originally classified as a fourth allele but has been re-classified as a variant of allele 2. Allele 2 is thus found in all ET-37 strains, one strain of cluster A4 and three additional non-ET-typed serogroup C strains. [0223] Serogroup C strains ISS759 and 973-1720 both contain a variant of allele 3 with a single amino acid mutation in the leader peptide (SEQ IDs 9/10) resulting from a single nucleotide mutation. Among all allele 3 strains, only 973-1720 belongs to a hypervirulent strain (cluster A4). [0224] Serogroup B strain 95330 contains a recombinant (chimera) of alleles 1 and 2 (SEQ IDs 11/12), with nadA being a fusion between the N-terminal portion of allele 2 and the C-terminal segment of allele 1. The putative site of recombination is located approximately between residues 141 and 265 of the protein.

[0225] All insertions and deletions happen in the coiled-coil region and involve 7 or 41 amino acids which, representing 2 or 6 turns of the .alpha.-helix, allows for variations in length of the coiled coil region without disturbing the overall structure. Furthermore, the deletion in ISS1024 results in the loss of the first heptad repeat of the leucine zipper domain but does not destroy the domain because leucine residues at a fixed spacing of seven residues can be replaced mostly by Met, Val or Ile. In this case allele 2 could use the Ile upstream or downstream to form the leucine zipper motif (FIG. 11).

[0226] Any of these various NadA sequences and alleles can be used in accordance with the invention.

[0227] When sequence analysis was extended to the putative promoter and terminator regions (50 bp upstream, 350 bp downstream), variations were found only in the in the 5' region. Three Italian strains (ISS1071, ISS832 and ISS1104) differed for a single base mutation while in strain 961-5945 there was a 7 base differences (indicated with * in FIG. 10). Variations were also found in the 5' regions where the TAAA tetranucleotide was repeated from 4 to 12 times in different strains (Table 1). The number of repeats was variable also within each allele (Table 1).

[0228] Further work was performed on carrier strains isolated from healthy individuals by oro-pharyngeal swab. Some strains, even if described as carriers, belong to hypervirulent clusters, and NadA was found in all such carrier strains as described above (i.e. allele 1 in the ET-5 strains and allele 2 in the ET-37 strains).

[0229] NadA was also found in five carrier strains (NGE28, 65/96, 149/96, 16269, 16282) which do not belong to a hypervirulent cluster. These five strains shared a sequence (SEQ IDs 13 & 14) which was not found in strains isolated from patients. This allele is referred to as `allele C` (carrier).

[0230] An alignment of allele C with alleles 1 to 3 is shown in FIG. 9C. Disruption in the coiled-coil segments of the protein is evident.

[0231] Unlike alleles 1 to 3, allele C protein does not readily form a high molecular aggregate when expressed in E. coli. Like alleles 1 to 3, however, allele C is exposed on the surface of N. meningitidis, because it is a target for bactericidal antibody raised against itself. However, these antibodies are not bactericidal against strains carrying alleles 1 to 3; similarly, antibodies raised against alleles 1 to 3 are not bactericidal against allele C strains.

NadA Oligomers on the Cell Surface

[0232] WO01/64922 reports that NadA forms oligomeric structures. To study NadA oligomers in more detail, whole cell lysates of N. meningitidis were probed by Western blot.

[0233] Bacterial colonies [strains MC58 (allele 1), 90/18311 (allele 2), 2996 (allele 3), L93/4286 (IS1301 insertion) and NG3/88 (nadA.sup.-)] were grown to stationary phase in GC broth supplemented with 0.3% glucose. Samples were taken at different times, pelleted by centrifugation at 3000.times.g for 10 min, and resuspended in PBS and thawed/frozen up to bacterial lysis. Equal amounts of proteins were subjected to SDS-PAGE on 12.5% polyacrylamide gels and electrotransferred onto nitrocellulose membranes.

[0234] To prepare anti-NadA polyclonal serum, recombinant NadA was expressed and purified. Sequences encoding the three nadA alleles (allele 1: aa 24-362; allele 2: aa 24-343; allele 3: aa 24-350), were amplified by PCR on chromosomal DNA and cloned into pET21b+ vector (Novagen). The plasmids were transformed in E. coli BL21 (DE3) to express the proteins as C-terminal histidine fusions. Protein expression was induced at 30.degree. C. by adding 1 mM IPTG at OD.sub.600 nm 0.3 and growing the bacteria for an additional 3 h; expression was evaluated by SDS-PAGE. Recombinant fusion proteins were purified by affinity chromatography on Ni.sup.2+-conjugated chelating fast-flow Sepharose 4B resin. 20 .mu.g of purified protein was used to immunise six-week-old CD1 female mice (4 to 6 per group). Proteins were given intraperitoneally, with complete Freund's adjuvant (CFA) for the first dose and incomplete Freund's adjuvant (IFA) for the second (day 21) and third (day 35) booster doses. Bleed out samples were taken on day 49 and used for the serological analysis.

[0235] The blots showed a high molecular weight reactive band in strains MC58 (FIG. 14, lane 1), 90/18311 (lane 2) and 2996 (lane 3). The band was absent in strain NG3/88 (lane 5). Boiling of the sample buffer up to 40 minutes did not change the pattern. The different size of the proteins was consistent with the size of the alleles. Given the expected size ranging from 35 to 40 kDa of monomeric proteins, the high MW of the observed band could be explained by the presence of an oligomeric form of NadA. This possibility is supported by the fact that in a strain containing the IS1301 insertion, coding for a shorter protein of 162 amino acids and lacking most of the coiled-coil region, the high MW reactive band was absent and replaced by a band of 14.5 kDa (FIG. 14, lane 4), consistent with the predicted molecular weight of the processed monomeric protein.

[0236] Although the oligomeric protein was found in all strains containing a functional gene, expression levels varied from strain to strain (Table I). Moreover, the amount of NadA protein varied within the same strain during growth.

[0237] Four different strains (MC58, 2996, C11, F6124), chosen as representative of diverse overall NadA expression level, were followed during growth up to stationary phase. FIG. 15 shows growth of two of the tested strains (15A: MC58, with low NadA expression; 15B: 2996, with high NadA expression), with the curve showing OD.sub.600. Western blots of samples taken at each point of the OD.sub.600 growth curve showed that the NadA band was barely visible at the beginning of the growth and became more intense during growth, up to its maximum, at stationary phase. All strains analysed showed the same growth-phase dependent behaviour.

[0238] High MW NadA was also seen in western blots of outer membrane vesicles, consistent with NadA being anchored to the outer membrane.

[0239] Similarly, FACS analysis on live bacteria during log-phase growth showed that NadA was available for antibody binding on the surface of the bacteria. FACS intensity in a strain with a poylsaccharide capsule (strain NMB) was reduced 1 log in comparison to an isogenic non-encapsulated mutant strain (M7), but the protein was surface-exposed and available for binding in both strains (FIG. 16).

[0240] NadA forms surface-exposed oligomers, which are stable to heat, SDS and reduction with .beta.-mercaptoethanol. As the mature form of the lacks cysteine residues, disulphide bond formation cannot be involved in this phenomenon; rather this is consistent with the predicted coiled-coil structure and the presence of leucine zipper motifs that might mediate intermolecular interactions between monomers [Lupas (1996) Trends Biochenz. Sci. 21:375-382; O'Shea et al. (1991) Science 254:539-544]. The size of the oligomers is approximately 170 kDa, suggesting a tetrameric structure [WO01/64922]. However, a rigid coiled-coil structure is likely to have an anomalous migration is SDS PAGE and therefore the 170 kDa form may be a trimer.

Protective Immunogenicity

[0241] Polyclonal anti-NadA serum was tested for bactericidal activity as previously described [Pizza et al. (2000); Peeters et al. (1999) Vaccine 17:2702-2712], with pooled baby rabbit serum (CedarLane) used as complement source. Serum bactericidal titer, was defined as the serum dilution resulting in a 50% decrease in colony forming units (CFU) per ml after 60 minutes incubation of bacteria in the reaction mixture, compared to control CFU per ml at time 0. Typically, bacteria incubated with the negative control antibody in the presence of complement showed a 150 to 200% increase in CFU/ml during the 60 min. of incubation.

[0242] Results were as follows:

TABLE-US-00002 Strain NadA expression Allele Bactericidal titre 2996 +++ 3 32768 C11 +++ 3 16384 F6124 + 3 4096 MC58 + 1 8192 BZ232 - - <4 NGH38 - - <4

[0243] As shown, the serum induced complement-mediated killing of all strains that have the nadA gene, and was inactive against the strains that do not have the gene. However, bactericidal titres varied between strains. Titres were higher against strains expressing higher amounts of protein. This result was confirmed when titres were determined in the early and late phase of growth (FIG. 15).

[0244] To check whether the differences in the bactericidal activity were due to different allele sequences, immune sera, raised against the three NadA types, were produced and used in a cross bactericidal assay. The results obtained with the antisera were similar to those shown above, suggesting that the bactericidal activity is not influenced by the allele diversity but rather to the antigen expression level.

[0245] The ability of immune sera to protect animals from bacteremia during infection was also tested, using the infant rat model. The sera used were obtained by immunising guinea pigs with 50 .mu.g purified rNadA (allele 3). Immunisation of outbred Wistar rats (5 to 7 days old) was performed subcutaneously together CFA for the first dose and IFA for the further three doses (days 28, 56, 84). Bleed out samples were taken on day 105 and used for the animal protection assay.

[0246] Two experiments were performed using two different MenB strains (8047 and 2996). Each strain has been serially passaged three times in infant rats. In experiment 1, groups of four rats were challenged intraperitoneally with 100 .mu.l of a mix of (a) bacteria from strain 8047 (7.times.10.sup.3 CFU per rat) and (b) heat inactivated guinea pig antiserum or anti-capsule control mAb (SEAM 3 [Van Der Ley et al. (1992) Infect. Immun. 60:3156]). In experiment 2, group of six rats were treated with the control mAb or with different dilutions of guinea pig antiserum at time 0. Two hours later, they were challenged with the 2996 bacteria (5.6.times.10.sup.3 CFU per rat). In both experiments, blood cultures were obtained 18 h after the challenge by puncturing the heart with a syringe and needle containing approximately 25 U of heparin without preservative. Bacteria numbers in the blood cultures were obtained by plating out 1, 10, and 100 .mu.l of blood onto chocolate agar overnight. For calculation of geometric mean CFU/ml, animals with sterile cultures were assigned a value of 1 CFU/ml.

[0247] Results were as follows:

TABLE-US-00003 Blood culture at 18 hours CFU/ml Exp.sup.t Treatment Positive/Total (10.sup.3) 1 Anti-capsular mAb (2 .mu.g/rat) 0/4 <0.001 Anti-NadA antiserum (1:5 dilution) 0/4 <0.001 PBS + 1% BSA 5/5 40.17 2 Anti-capsular mAb (20 .mu.g/rat) 1/6 0.003 Anti-NadA antiserum (1:5 dilution) 1/6 0.002 Anti-NadA antiserum (1:25 dilution) 3/6 0.035 Pre-immune NadA serum 6/6 1.683

[0248] Thus anti-NadA antiserum is highly protective in this assay.

[0249] Overall, therefore, NadA has several attributes of being a good vaccine antigen: (i) it is a surface-exposed molecule, potentially involved in bacterial adhesion; (ii) it is present in at least 50% of the disease-associated strains and in almost 100% of three hypervirulent lineages; (iii) it elicits protective and bactericidal antibodies in laboratory animals; and (iv) each allele induces cross-bactericidal antibodies.

ORF40

[0250] ORF40 shows homology to Hsf and its allelic variant Hia, both adhesins of Haemophilus influenzae. The different size among Hia, Hsf and ORF40 is in part explained by the presence of three copies of a large repeated domain in Hsf, which is present in single copy in Hia and only partially in ORF40 (FIG. 7). In MenB, ORF40 is found on the outer membrane as a protein of about 200 kDa (cf. predicted MW of 59 kDa for mature protein).

App

[0251] App shows homology (FIG. 8) to the adhesion and penetration protein Hap of H. influenzae, which is an adhesin with a serine-protease activity that undergoes autoproteolytic cleavage and extracellular release [Hendrixson et al. (1997) Mol Microbiol 26:505-518]. Uncleaved surface-associated Hap mediates adherence to epithelial cells and promotes bacterial aggregation and colonisation.

[0252] In N. meningitidis, App is exported to the outer membrane, processed and secreted. Both Hap and App belong to the autotransporter family which comprises proteins from gram-negative bacteria characterized by a distinct mechanism of secretion. This system was first described for IgA1 protease of N. gonorrhoeae, which is considered the prototype of this family. Proteins of the autotransporter family have been implicated in the virulence of many gram-negative pathogens [Henderson & Nataro (2001) Infect Immun 69:1231-1243]. They are synthesized as large precursor proteins comprising at least three functional domains: a typical N-terminal leader sequence, an internal domain (passenger domain) and a C-terminal domain (translocator domain or .beta.-domain). The leader sequence mediates the export (sec-dependent) of the protein to the periplasm. Subsequently the translocator domain inserts into the outer membrane forming a .beta.-barrel pore to allow the export of the passenger domain. Once at the bacterial surface, the passenger domain can be cleaved and released into the environment. Cleavage can occur by an autoproteolytic event directed by protease activity in the passenger domain itself. Passenger domains of autotransporters are widely divergent, reflecting their remarkably disparate roles. On the contrary the .beta.-domains display high degree of conservation consistent with their conserved function.

[0253] App possesses the prevailing domains of the autotransporter proteins as well as the conserved serine protease motif (GDSGSP). It has been shown that this motif is responsible for cleavage of human IgA by the Neisseria IgA1 proteases and for autoproteolytic cleavage of Hap protein of H. influenzae. App has been shown to be a conserved antigen among meningococci, to be expressed during infection and carriage, to stimulate B cells and T cells, and to induces a bactericidal antibody response [Hadi et al. (2001) Mol. Microbiol. 41:611-623; Van Ulsen et al. (2001) FEMS Immunol Med Microbiol 32:53-64].

[0254] In serogroup B strain 2996, App has 1454 amino acids and a predicted MW of 159,965 Da. FIG. 26 shows the protein's predicted structural features. Three domains can be seen: domain 1 (amino acids 1-42) is the signal peptide; domain 2 is the passenger domain, which is the functionally active protein; domain 3 is the C-terminal translocator domain with .beta. barrel structure.

[0255] At the N-terminus of the passenger domain, His-115, Asp-158 and Ser-267 correspond to the serine protease catalytic triad His-98, Asp-140 and Ser-243 from Hap [Fink et al. (2001) J Biol Chem 276:39492-39500]. Residues 285-302 are a putative ATP/GTP-binding site (P loop), which suggests a mechanism of energy coupling for outer membrane translocation. Towards the C-terminus of the passenger domain, two Arg-rich regions are present. The first (RRSRR) is residues 934-938 and the second (RRARR) begins at residue 1149. These motifs are reminiscent of known targets for trypsin-like proteolytic cleavage sites such as the one in diphtheria toxin and those upstream of the auto-cleavage sites of H. influenzae Hap, N. gonorrhoeae IgA-protease and B. pertussis FhaB (FIG. 26, box 1). Downstream of the Arg-rich regions are motifs .sup.954NTL.sup.956 and .sup.1176NSG.sup.1178, which are identical or similar to the cleavage sites in autotransporters Ssp (Serratia marcescens), Prn (Bordetella bronchiseptica), Brka (Bordetella pertussis) [Jose et al. (1995) Mol. Microbiol. 18:378-380] and Hap (H. influenzae) (FIG. 26, box 2). Together, these sequence motifs suggest that the two motifs .sup.954NTL.sup.956 and .sup.1176NSG.sup.1178 and the RR(A,S,R).sub.2RR pattern could act as signals for correct localisation of downstream processing sites.

[0256] Further analysis of the App sequence shows a proline-rich region, where the dipeptide motif PQ is repeated four times beginning at residue 1156. A search for homology to known protein sequences reveals some similarity to the surface proteins of S. pneumonie PspA and PspC and to a proline-rich region of the B. pertussis outer membrane protein p69 pertactin, where the (PQP).sub.5 motif is located in a loop containing the major immunoprotective epitope.

[0257] Finally, the last three amino acids of App (YRW) are identical to those of Hap where they have been described as crucial for outer membrane localisation and protein stability [Hendrixson et al., 1997].

Expression in E. coli without Fusion Partners

[0258] ORF40, App and NadA full-length genes were cloned in pET21b+ vector and the plasmids were transformed in E. coli BL21(DE3) in order to express the genes under control of T7 promoter. Expression was achieved activating the promoter with IPTG or under non-induced conditions. Localisation and surface-exposure of the proteins were assayed by cell-fractionation experiments (SDS-PAGE and Western blot), FACS analysis and whole-cell immunoblot. As shown in FIGS. 1 to 3, all the three proteins are translocated to the surface of E. coli: [0259] ORF40 is expressed as monomeric form and possibly forms also multimers (FIG. 1). [0260] App is exported to E. coli outer membrane as a precursor of about 160 kDa, that is processed and secreted in the culture supernatant (FIG. 2). [0261] NadA is found to the be present in the outer membrane fraction as a single high molecular weight band of approximately 180 kDa. This probably corresponds to an oligomeric form of the protein. Such a band is absent in E. coli expressing intracellular NadA (FIG. 3).

[0262] App expression was studied in more detail.

[0263] N. meningitidis strain 2996 genomic DNA was prepared as previously described [Tinsley & Nassif (1996) PNAS USA 93:11109-11114[. DNA devoid of the sequence coding for the signal peptide (amino acids 1 to 42) and of the STOP codon was amplified using PCR primers SEQ IDs 18 & 19 followed by digestion with NheI and XhoI and insertion into the NheI/XhoI sites of the pET-21b expression vector, to give `pET-App-His` (FIG. 27). This plasmid was introduced into E. coli BL21(DE3) and used for the expression of a C-terminal His-tagged fusion protein which was purified and used to raise antibodies. The full-length app gene was amplified and cloned in a similar way, using PCR primers SEQ IDs 20 & 21, to give plasmid `pET-App`

[0264] Plasmids were introduced into E. coli BL21(DE3) and expression induced by addition of 1 mM IPTG. The expressed protein was detected by western blotting (FIG. 28, lane 1). To verify that the protein was exported to the E. coli surface, FACS (FIG. 29) and immunofluorescence microscopy (FIG. 30) were used. The FACS analysis showed positive surface expression on the pET-App transformants (full-length gene) but no surface expression with App-His (no signal peptide) or with the empty vector. The immunofluorescence results agreed with FACS. Therefore expression of the full-length app gene resulted in the export of App to the surface of E. coli, but deletion of the first 42 amino acids abolished surface-localisation.

[0265] Western blot analysis of outer membrane proteins from pET-App transformants revealed a specific reactive band of .about.160 kDa (FIG. 28, lane 1), corresponding to the predicted molecular weight of the full-length protein. A corresponding band was missing in the outer membrane fraction from untransformed controls (lane 3). Western blot analysis of culture supernatants revealed a secreted protein of .about.100 kDa with pET-App (lane 2) that was absent with the untransformed controls (lane 4). Sometimes a very weak band was also detected at .about.140 kDa in pET-App transformants.

[0266] Therefore the full length app gene when introduced into E. coli induces expression of an App protein which is exported to the outer membrane, cleaved and released into the culture supernatant.

Native Expression can Influence the Quality of the Immune Response

[0267] To evaluate the role of protein conformation on induction of an immune response, outer membrane vesicles from E. coli expressing ORF40, App or NadA were isolated and used to immunise mice. Sera were tested for bactericidal activity and results compared with those obtained with the fusion proteins. The bactericidal response (strain 2996) was improved 5-10 fold when the proteins are produced in their "native" form in OMVs:

TABLE-US-00004 Bactericidal titres* Antigen Fusion protein E. coli OMV ORF40 256 2048 App 64 1024 NadA 32768 >65536 *Titres expressed as the reciprocal of the serum dilution yielding ~50% bacteria killing

App Autoproteolytic Cleavage

[0268] E. coli pET-App transformants secrete a 100 kDa product into culture supernatant and show a 160 kDa surface product. To test whether the secreted App product derives from an autoproteolytic process, one of the putative catalytic residues (Ser-267) was replaced with Ala.

[0269] The pET-AppS267A mutant was obtained by site-directed mutagenesis using the QuikChange kit (Stratagene) and primers SEQ IDs 22 & 23.

[0270] SDS-PAGE analysis of total proteins from pET-AppS267A transformants (FIG. 31, lane 2) showed a protein similar in size to pET-App transformants (lane 1). The protein was shown to be surface exposed by FACS analysis (FIG. 29). Western blot analysis of culture supernatants showed App in pET-App transformants (FIG. 32, lane 1) but not in pET-AppS267A transformants (lane 2).

[0271] Mutation of Ser-267 to Ala thus abolishes processing and secretion of the App precursor, which remains cell-associated. These data suggest that App has a serine protease activity that is responsible for autoproteolytic processing and release in the supernatant of the secreted App domain.

[0272] Cleavage at .sup.954NTL.sup.956 would leave a fragment with predicted molecular weight of 104190 Da. Cleavage at .sup.176NSG.sup.1178 would give a 128798 Da fragment. These two predicted fragments may match the two bands of .about.140 and .about.100 kDa observed in culture supernatants. Cleavage may occur first to give the .about.140 kDa fragment and then second to give the 100 kDa fragment. The .beta. domain of App would thus begin at residue 1177.

NadA, ORF40 and App Function as Adhesins

[0273] Example 22 of international patent application WO01/64922 discloses that NadA expression in E. coli makes the transformed bacterium adhere to human epithelial cells. The adherent phenotype has been further studied for NadA and also for App and ORF40.

[0274] E. coli BL21(DE3) bacteria (10.sup.8 CFU), grown under non-induced or induced conditions, were inoculated onto Chang human epithelial monolayers (10.sup.5 cells) and incubated at 37.degree. C. for 1 or 2 hours. Cells were then incubated with rabbit anti-E. coli and PE-conjugate secondary antibody. Adhesion was detected by FACS as specific fluorescence intensity associated to Chang cells. Positive controls were E. coli DH5 expressing hsf (DH5/pDC601)); negative controls were BL21(DE3)/pET21b and DH5a/pT7-7. The results in FIG. 4 show that the ability of the recombinant E. coli strains to adhere to cultured epithelial cells is associated with expression of these three proteins.

[0275] To confirm that these three proteins are able to promote interaction with host cells, the recombinant proteins themselves were investigated for binding to epithelial cells. 10.sup.5 Chang human epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, human conjunctiva) were incubated at 4.degree. C. for 30 minutes with medium alone or with different concentration of ORF40 (150 .mu.g/ml), App (150 .mu.g/ml) or NadA (300 .mu.g/ml), or with GNA2132 (300 .mu.m/ml) as negative control [see Pizza et al. (2000)]. Binding was detected by FACS using polyclonal antisera against the single recombinant proteins and a secondary PE-conjugate antibody. The FACS signal shifts (FIG. 5) show that the three proteins are able to bind to human epithelial cells, whereas purified GNA2132 (negative control) does not.

[0276] FIG. 6A shows that binding increases in a dose-dependent manner. Binding of NadA reaches a plateau at around 200 .mu.g/ml. GNA2132 fails to bind even at 400 .mu.g/ml (FIG. 6B). Data in FIG. 6 are mean fluorescent intensity (MFI) values plotted against protein concentration (.mu.g/ml).

[0277] Using FACS, binding of NadA to cells was also seen with Hep-2 and MOLT-4 cells, but not with HeLa, A549, Hec-1B, Hep-G2, CHO or HUVEC cells. Adhesion to Chang cells could be abolished by treating the cells with pronase, indicating that the human receptor for NadA is a protein.

[0278] Adhesion of purified NadA protein to Chang conjunctiva cells was also observed using immunofluorescence microscopy. The protein (lacking its C-terminal anchor domain) was incubated with Chang cells at 37.degree. C. in complete culture medium for 3 hours at various concentrations. Cells were then washed, fixed, and analysed by laser confocal microscopy after staining with anti-NadA mouse polyclonal antibodies and secondary Texas-red coupled anti-mouse IgG antibodies. No binding was seen at 0 nM (FIG. 17A), but binding was evident at 170 nM (17B) and 280 nM (17C), with clustering evident at higher concentrations. In contrast, no binding of NadA was seen with HeLa cells, even at 280 nM protein (17D).

[0279] Binding was much more evident at 37.degree. C. (FIG. 18A) than at 4.degree. C. (FIG. 18B). The dot-like structures seen at 4.degree. C., compared to clusters at 37.degree. C., suggest that lateral interactions between NadA monomers are temperature-dependent (influenced by membrane fluidity).

[0280] To distinguish surface and endocytosed protein, saponin detergent was added during the staining procedure. Intracellular clusters having the size of endosomes were more evident (arrow) when saponin was used, but a high proportion of protein remained on the cell surface (FIG. 19).

[0281] Immunofluorescence also revealed that NadA binds to monocytes (FIG. 20A). NadA alone (no staining antibody; 20B) and NadA stained with pre-immune serum (20C) were not visible. At high magnification, evidence of uptake into vesicles (either endosomes or phagosomes) was seen.

[0282] FIG. 21 shows that murine macrophages (raw 264.7) bind and endocytose NadA (125 nM, 3 hours, 37.degree. C.; cells cultured in DMEM).

[0283] Heating NadA at 95.degree. C. for 15 minutes prior to incubation removed its ability to bind to monocytes, as measured by secretion of IL-.alpha. by the cells (FIG. 22). The stimulatory activity of NadA preparations is thus heat-labile. Stimulatory activity was also blocked by the use of anti-CD14 (FIG. 23) or by the removal of NadA from the preparations using bead-immobilised anti-NadA.

[0284] Immunofluorescence microscopy was also used to detect binding of E. coli expressing NadA. Transformed E. coli bound strongly (FIG. 24A) whereas untransformed bacteria did not (24B). IL-.alpha. release by monocytes was over 1.5.times. higher using the transformed E. coli than the untransformed bacteria at a bacteria/monocyte ratio of 40:1.

[0285] Transformed E. coli were bound to glass cover slips, fixed and double-stained with anti-NadA (FIG. 25A) and anti-E. coli antibodies (25B). When both were used, patches of anti-NadA were visible, suggesting that NadA tends to form aggregates on the bacterial surface, which hamper the interaction of antibodies with other surface antigens.

[0286] Looking at App, recombinant E. coli strains were incubated with monolayers of Chang conjunctiva epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4 [human conjunctiva], ATCC CCL 20.2) and adhesion was analysed using FACS. Cells obtained from confluent monolayers were seeded at 10.sup.5 cells per well in 12-well tissue culture plates and incubated for 24 hours. Cultures of bacteria after IPTG induction were washed twice in PBS and resuspended in DMEM+1% FBS to a concentration of 5.times.10.sup.8 bacteria per ml. Aliquots of 1 ml of each strain were added to monolayer cultures of Chang cells and incubated for 3 hours at 37.degree. C. in 5% CO.sub.2. Non-adherent bacteria were removed by washing three times with PBS, and 300 .mu.l of cell dissociation solution (Sigma) were added to each microtitre well. Incubation was continued at 37.degree. C. for 10 minutes. Cells were harvested and then incubated for 1 hour at 4.degree. C. with rabbit polyclonal anti-E. coli antiserum (DAKO). Cells were washed twice in PBS+5% FBS and incubated for 30 minutes at 4.degree. C. with R-Phycoerythrin-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Cells were then washed in PBS+5% FBS and resuspended in 100 .mu.l PBS. Fluorescence was measured with FACSCalibur flow cytometer (Becton Dickinson). For each of fluorescence profile, 10000 cells were analysed.

[0287] The results reported in FIG. 33 show pET-App transformants were able to adhere to Chang cells, giving a fluorescence shift of 90.3%. S267A transformants were also able to adhere (91.0%). Untransformed E. coli were unable to adhere to Chang cells (bottom FACS plot).

[0288] As for NadA, FACS results were in agreement with immunofluorescence microscopy data. As shown in FIGS. 34A & 34B, pET-App transformants incubated with monolayers demonstrated high levels of adhesion to epithelial cells and visible bacteria-bacteria aggregation. For the S267A mutant, adhesion and bacterial aggregation were increased (34C & 34D). Untransformed controls showed no adhesion (34G). Deletion of the first 42 amino acids also abolished adhesion.

[0289] In contrast to Chang epithelial cells, no adhesion was seen when HUVEC endothelial cells were tested with pET-App transformants. To cause sepsis and meningitis, N. meningitidis has to interact with human endothelial cells. App may thus be involved in the first step of colonisation at the level of human respiratory epithelial mucosa, rather than in pathological endothelial colonisation.

Localization and Specificity of App Binding Activity.

[0290] To identify the binding region of App, a chimeric protein named App.beta. was used. This protein consists of the C-terminal domain of App (amino acids 1077 to 1454) fused to the leader peptide of IgA1 protease of N. gonorrhoeae. The gonococcal leader sequence was chosen because it has been well characterized and is functional in E. coli. Plasmid pET-App.beta. contains a 1.1 kbp DNA fragment amplified by PCR using SEQ IDs 26 & 27.

[0291] The pET-App.beta. construct was introduced into E. coli BL21(DE3). FACS localisation studies confirmed that App.beta. was localized on the E. coli surface. The in vitro adhesion assay using Chang epithelial cells showed adhesion by immunofluorescence (FIGS. 34E & 34F). FACS analysis showed that the pET-App.beta. transformants were still able to adhere to epithelial cells but at lower levels (74.2% shift) than pET-App transformants.

[0292] These results indicate that the App binding domain is located in its C-terminal region, in the 100mer fragment between residues 1077 and 1176.

[0293] Purified recombinant proteins were also studied. App-.alpha.-His consists of the N-terminal portion of App (amino acids 43-1084) fused to a poly-His tag. Plasmid pET-App.alpha.-His contains a NheI/XhoI 3.1 kbp fragment amplified by PCR with SEQ IDs 24 & 25. The binding activity of the purified recombinant App-.alpha.-His was compared to that of App-His by FACS binding assays. Chang cells were incubated with increased concentrations of recombinant App proteins or lipoprotein NMB2132-His (negative control). Binding of App-His (.diamond-solid.) increased in a dose-dependent manner and reached a plateau at a concentration of .about.50 .mu.g/ml whereas the binding of App.alpha.-his (.box-solid.) was very low (FIG. 35). The control NMB2132-His (.tangle-solidup.) failed to bind Chang cells.

[0294] To explore the biochemical nature of the molecule involved in interaction with App, the Chang cells were treated with pronase or phospholipase A2 before the binding experiments. 10.sup.5 cells per well were placed in microplates and incubated in FCS-free DMEM at 37.degree. C. in 5% CO.sub.2 for 30 minutes with (a) pronase at 250, 500, or 1000 .mu.g/ml or (b) phospholipase A2 at 50, 200, or 800 .mu.g/ml. After enzymatic incubation, an equal volume of complete medium was added to each well to stop the reaction. Cells were subsequently mixed with 100 .mu.g/ml App-His or medium alone and incubated for 1 hours at 4.degree. C. As shown in FIG. 36, pronase treatment (left-hand columns) markedly reduced the binding of App-His protein to Chang cells, while treatment with phospholipase A2 (right-hand columns) did not reduce the binding. The receptor for App on Chang cells is thus proteinaceous.

[0295] Adhesion to different cell lines were also tested (FIG. 37). After incubation of cultured cells with three different concentrations of App-His (100, 25 & 6.25 .mu.g/ml) high level binding to Chang cells and HepG2 cells was seen, a moderate level of binding to A-549 cells, and minimal binding to HeLa cells. No binding was observed to Hec-1-B, Hep-2, 16HBE14o epithelial cell lines or to HUVEC endothelial cells.

App Knockout

[0296] After the work on E. coli suggesting an adhesin role for App, an isogenic mutant strain of N. meningitidis was constructed. The starting strain was MC58. Its app gene was truncated and replaced with an antibiotic cassette by transforming the parent strain with the plasmid pBSUDAppERM, which contains a truncated app gene and the ermC gene (erythromycin resistance) for allelic exchange. Briefly, 600 bp of the upstream flanking region including the start codon and 700 by downstream flanking region including the stop codon were amplified from MC58 using primers SEQ IDs 28 to 31. Fragments were cloned into pBluescript and transformed into E. coli DH5 using standard techniques. Once all subcloning was complete, naturally competent N. meningitidis strain MC58 was transformed by selecting a few colonies grown overnight on GC agar plates and mixing them with 20 .mu.l of 10 mM TrisHCl pH8.5 containing 1 .mu.g of plasmid DNA. The mixture was spotted onto a GC agar plate, incubated for 6 hrs at 37.degree. C., 5% CO2 then diluted in PBS and spread on GC agar plates containing 5 .mu.g/ml erythromycin. The deletion app gene in the genome of MC58 was confirmed by PCR. Lack of App expression was confirmed by Western blot analysis.

[0297] Adhesion of wildtype MC58 and the isogenic MC58.DELTA.app mutant strain was evaluated on Chang cells. There was a .about.10 fold reduction (ranging from 3- to 27-fold in different experiments) of the association of the knockout mutant compared with the wild type strain (FIG. 38). No difference was observed between the app.sup.- mutant and the parental strain with Hep2 and 16HBE14o cell lines and with HUVEC endothelial cells, confirming that App does not mediate adhesion to these cells.

[0298] No non-pilus adhesins which contribute to adhesion of N. meningitidis in a capsulated background have previously been reported.

[0299] App expression was studied in N. meningitidis MC58. Colonies from plates grown overnight were diluted in GC broth and incubated at 37.degree. C. with 5% CO.sub.2. Samples were taken when OD.sub.620=0.5 (mid log phase) and 0.8 (stationary phase) and analysed by western blot. Two bands with apparent molecular weights .about.160 and .about.140 kDa were detected in whole cells lysates of log phase bacteria (FIG. 39, lane 1), while stationary phase bacteria showed only a faint band at .about.140 kDa (lane 3). As expected, no App was observed in the .DELTA.App mutant (lanes 2 & 4).

[0300] In marked contrast, supernatant samples of wild-type MC58 showed a band at .about.140 kDa and its amount was higher in stationary phase than in log phase (FIG. 40, lanes 3 & 1). The stationary phase sample also showed a reactive band at .about.100 kDa.

[0301] It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

TABLE-US-00005 TABLE I Characteristics of 26 N. meningitidis strains and their nadA gene allele Serogroup Clonal nadA (TAAA) NadA Strain type: subtype group allele repeats expression 64/69 NG: 15: P1.7, 16 ET-5 1 4 + BZ83 B: 15 ET-5 1 5 +++ CU385 B: 4: P1.15 ET-5 1 6 ++ MC58 B: 15: P1.7, 16b ET-5 1 9 + BZ169 B: 15: P1.16 ET-5 1 12 ++ 95330* B: 4: P1.15 ET-5 1 9 nd ISS1104 B: 15: P1.7, 16 nd 1 4 + ISS1071 B: 15: P1.7, 16 nd 1 5 +++ ISS832 B: 15: P1.7 nd 1 5 ++ NM119 B.4.P1.15 nd 1 6 nd NM066 B: 15: P1.7, 16 nd 1 12 nd 90/18311 C: NT: P1.5 ET-37 2 9 ++ NGP165 B: NT: P1.2 ET-37 2 9 ++ FAM18 C: 2a: P1.5, 2 ET-37 2 9 nd M986 B: 2a: P1.5, 2 ET-37 2 12 ++ ISS1024* C: 2b: P1.5 nd 2 9 ++ ISS838 C: 2a: P1.5, 2 nd 2 6 ++ PMC8 C: nd 2 10 ++++ 961-5945 B: 2b: P1.21, 16 A4 2 12 +++ ISS759* C: 2b: P1.2 nd 3 8 ++++ F6124 A Subgroup 3 9 + III NMB B: 2b: P1.5, 2 nd 3 12 ++ 8047 B: 2b: P1.2 nd 3 12 +++ 2996 B: 2b: P1.5-1, 2 nd 3 12 +++ C11 C: NT: P1.1 nd 3 12 +++ 973-1720* C: 2b: P1.2 A4 3 12 +++ *indicates that the strain carriers a minor variant of the relevant allele nd = not done

TABLE-US-00006 TABLE II Characteristics of N. meningitidis strains analysed for NadA expression ST ET Strain Year Serogroup: type: subtype Country Disease NadA gene 74 ET5 MC58 1985 B: 15: P1.7, 16b UK case + 32 ET5 H44/76 1976 B: 15: P1.7, 16 Norway case + 32 ET5 BZ169 1985 B: 15: P1.16 Netherlands case + 32 ET5 30/00 2000 B: 15: P1.7, 16 Norway case + 33 ET5 N44/89 1989 B: 4, 7: P1.19, 15 Brazil case + 34 ET5 BZ83 1984 B: 15 Netherlands case + -- ET5 72/00 2000 B: 15: P1.7, 13 Norway case + -- ET5 39/99 1999 C: 15: P1.7, 16 Norway case + -- ET5 M4102 1996 B: ND USA case + -- ET5 95330 1995 B: 4: P1, 15 Canada case + -- ET5 220173I 1993 NG: 4: P1.15 Iceland carrier + -- ET5 64/96 1996 NG: 15: P1.7, 16 Norway carrier + -- ET5 CU385 1980 B: 4: P1.15 Cuba case + -- ET5 8680 1987 B Chile case + -- ET5 204/92 1992 B Cuba case + -- ET5 EG329 1985 B Germany case + -- ET5 NG080 1981 B Norway case + -- ET5 NG144/82 1982 B Norway case + -- ET5 NG PB24 1985 B Norway case + -- ET5 196/87 1987 C Norway case + -- ET5 Mk521/99 1999 B Ivory Coast case + -- ET5 GR 4/00 2000 -- Greece case + 11 ET37 FAM18 1983 C: 2a: P1.5, 2 USA case + 11 ET37 L93/4286 1993 C UK case + -- ET37 NGP165 1974 B: NT: P1.2 Norway -- + -- ET37 M986 1963 B: 2a: P1.5, 2 USA case + -- ET37 C4678 1998 C: 2a: P1.5, 2 Germany case + -- ET37 95N477 1995 B: 2a: P1.2 Australia ease - -- ET37 BRAZ10 1976 C Brazil case + -- ET37 F1576 1984 C Ghana case + -- ET37 M597 1988 C Israel case + -- ET37 500 1984 C Italy case + -- ET37 D1 1989 C Mali case + -- ET37 NG P20 1969 B Norway case + -- ET37 MA-5756 1985 C Spain case + -- ET37 38VI 1964 B USA carrier + -- ET37 N1/99 1999 C: 2a Norway case + -- ET37 N28/00 2000 W-135: 2a Norway case + 66 A4 973-1720 1997 C: 2b: P1.2 Australia case + 153 A4 961-5945 1996 B: 2b: P1.21, 16 Australia case + -- A4 5/99 1999 B: 2b: P1.5, 2 Norway case + -- A4 312294 1995 C: 2b: P1.5, 2 UK case + -- A4 96217 1996 B: 2b: P1.5, 10 Canada case + -- A4 G2136 1986 B UK case + -- A4 312 901 1996 C UK case + -- A4 AK22 1992 B Greece case + -- A4 BZ10 1967 B Holland case + -- A4 BZ163 1979 B Holland case + -- A4 B6116/77 1977 B Iceland case + -- A4 94/155 1994 C New Zealand case + -- A4 SB25 1990 C South Africa case + -- A4 N53/00 2000 C: 2b: P1.5, 2 Norway case + -- A4 N62/00 2000 C: 2b: P1.5, 2 Norway case + 41 Lin.III BZ198 1986 B: NT Netherlands case - 42 Lin.III M198/254 1998 B: 4: P1.4 New Zealand case - 158 Lin.III 972-0319 1997 B: NT: P1.4 Australia case - 159 Lin.III 980-2543 1998 B: NT: P1.4 Australia case - 1127 Lin.III 67/00 2000 B: 4, 7 Norway case - -- Lin.III 93/114 1993 C: 4: P1.4 Belgium case - -- Lin.III M198/172 1998 B: 4: P1.4 New Zealand case - -- Lin.III 347/97 1997 B: 4: P1.4 New Zealand case - -- Lin.III 386/98 1998 B: 4: P1.4 New Zealand case - -- Lin.III 389/98 1998 B: 4: P1.4 New Zealand case - -- Lin.III 392/98 1998 B: 4: P1.4 New Zealand case - -- Lin.III 394/98 1998 B: 4: P1.4 New Zealand case - -- Lin.III 400 1991 B Austria case - -- Lin.III M40/94 1994 B Chile case - -- Lin.III AK50 1992 B Greece case - -- Lin.III M-101/93 1993 B Iceland case - -- Lin.III 931905 1993 B Netherlands case - -- Lin.III 91/40 1991 B New Zealand case - -- Lin.III 50/94 1994 B Norway case - -- Lin.III N45/96 1996 B Norway case - -- Lin.III 88/03415 1988 B Scotland case - 1 s I BZ133 1977 B: NT Netherlands case - 5 s III F6124 1988 A Chad case + 4 s IV-1 205900 1990 A 4, 21: P1.7: 1 Mali case - 4 s IV-1 Z2491 1983 A Gambia case - 12 other NG3/88 1988 B: 8(2): P1.1 Norway case - 13 other NG6/88 1988 B: NT: P1.1 Norway case - 14 other NGF26 1988 B: NT: P1.16 Norway carrier - 15 other NGE31 1988 B: NT Norway carrier - 18 other 528 1989 B: nd Russia case - 20 other 1000 1988 B: NT: P1.5 Russia case - 22 other A22 1986 W-135 Norway carrier - 26 other NGE28 1988 B: 4 Norway carrier + 29 other 860800 1986 Y Netherlands case - 31 other E32 1988 Z Norway carrier - 35 other SWZ107 1986 B: 4: P1.2 Switzerland case - 36 other NGH38 1988 B: NT: P1.3 Norway carrier - 38 other BZ232 1964 B: NT: P1.2 Netherlands case - 39 other E26 1988 X Norway carrier - 43 other NGH15 1988 B: 8: P1.15 Norway carrier - 47 other NGH36 1988 B: 8: P1.2 Norway carrier - 48 other BZ147 1963 B: NT Netherlands case - 49 other 297-0 1987 B: 4: P1.15 Chile carrier - 540 other 2996 1975 B: 2b: P1.5-1, 2 UK case + 1034 other 96/1101 1996 C: 14: P.1.1, 7 Belgium case - -- other 15 1990 B: 14, 19: P1.9, 15 Slovenia case - -- other M1090 1996 B: 4 Israel case - -- other M1096 1996 C: NT: P1.5 Israel case - -- other B3937 1995 B: 22: P1.16 Germany case + -- other 31 1993 B: 4 Finland case - -- other 95074 1995 B: NT: P1.13 Canada case + -- other 660/94 1994 B: 4: P1.6 Algeria case - -- other 30/93 1993 B: 14: P1.14 Argentina case - -- other 24370 1996 B: ND South Africa case - -- other 2411751 1993 NG: 21: P1.16 Iceland carrier - -- other 171274I 1993 NG: 15: -- Iceland carrier - -- other 65/96 1996 B: 4: P1.14 Norway carrier + -- other 66/96 1996 B: 17: P1.15 Norway carrier - -- other 149/96 1996 B: 1, 19: P1.5, 2 Belgium carrier + -- other 16060 1991 B: 4: P1.14 Belgium carrier - -- other 16489 1991 NG: 21: P.1.1 Norway carrier - -- other 16990 1991 NG: 14: P1.5, 2, 6 Norway carrier - -- other 2022 1991 NG: 4: P1.10 Norway carrier + -- other M136 1968 B: 11: P1.15 USA case - -- other 860060 1988 X Holland case - -- other NG H41 1986 B Norway carrier - -- other NG G40 1988 B Norway carrier - -- other NG4/88 1988 B Norway case - -- other EG 327 1985 B DDR case - -- other EG 328 1985 B DDR case - -- other 3906 1977 B China case - -- other NG E30 1988 B Norway carrier - -- other 71/94 1994 Y Norway case - -- other DK24 1940 B Denmark case - -- -- C11 1965 C: 16: P1.7a, 1 Germany -- + -- -- pmc8 -- C -- -- + -- -- NMB 1968 B: 2b: P1.5, 2 USA case + -- -- 8047 1978 B: 2b: P1.2 USA case + -- -- S3446 1972 B: 14: P1.23, 14 USA case - -- -- ISS 749 1996 B: 14: P1.13 Italy case - -- -- ISS 759 1996 C: 2b: P1.2 Italy case + -- -- ISS 832 1997 B: 15: P1.7 Italy case + -- -- ISS 838 1997 C: 2a: P1.5, 2 Italy case + -- -- ISS1001 1999 B: 14: P1.13 Italy case - -- -- ISS1024 2000 C: 2b: P1.5 Italy case + -- -- ISS1026 2000 B: 4: P1.13 Italy case - -- -- ISS1071 2000 B: 15: P1.7, 16 Italy case + -- -- ISS1102 2000 B: 15: P1.4 Italy case - -- -- ISS1104 2000 B: 15: P1.7, 16 Italy case + -- -- ISS1106 2000 B: 4: P1.4 Italy case - -- -- ISS1113 2000 C: 2a: P1.5 Italy case + -- -- N1002/90 -- -- Brazil -- + -- -- IMC2135 -- -- Brazil -- + -- -- NM001 -- B: 4: P1.4 UK case - -- -- NM002 -- B: NT: P1.16 UK case - -- -- NM004 -- B: NT: P1.14 UK case - -- -- NM008 -- B: 4: P1.4 UK case - -- -- NM009/10 -- B: 4: P1.3, 6 UK case - -- -- NM021 -- B: 4: P1.16 UK case - -- -- NM036 -- C: 2a: P1.10 UK case + -- -- NM037 -- B: 2b: P1.10 UK case + -- -- NM050 -- B: NT: P1.9 UK case - -- -- NM058 -- B: NT: NST UK case - -- -- NM066 -- B: 15: P1.7, 16 UK case + -- -- NM067 -- C: 2a: NST UK case + -- -- NM069 -- B: 15: P1.7, 16 UK case + -- -- NM081 -- C: 2a: P1.5, 2 UK case + -- -- NM088 -- C: 2a: P1.5, 2 UK case + -- -- NM092 -- B: 4: P1.4 UK case - -- -- NM106 -- B: NT: P1.4 UK case - -- -- NM107/8 -- B: 4: P1.4 UK case - -- -- NM117 -- B: 21: P1.9 UK case - -- -- NM119 -- B: 4: P1.15 UK case + -- -- NM131 -- B UK case - -- -- NM145 -- C UK case + -- -- NM154 -- C: NT: P1.5, 2 UK case + -- -- NM156 -- B: 15: P1.16 UK case + -- -- NM167 -- B UK case - -- -- NM184 -- B: NT: P1.5, 2 UK case - -- -- NM186 -- B UK case - -- -- NM188 -- B UK case + -- -- NM200 -- B: 4: P1.4 UK case -

TABLE-US-00007 TABLE III SEQUENCE LISTING SEQ ID NO: Description 1 allele 1 of 961 2 allele 2 of 961 3 allele 3 of 961 4 allele 1 of 961 (first-ATG start) 5 allele 2 of 961 (first-ATG start) 6 allele 3 of 961 (first-ATG start) 7 variant allele 2 of 961 in strain ISS1024 8 variant allele 2 of 961 (first-ATG start) in strain ISS1024 9 variant allele 3 of 961 in strains 973-1720 and ISS759 10 variant allele 3 of 961 (first-ATG start) in strains 973-1720 and ISS759 11 961 allele 1/2 chimera (strain 95330) 12 961 allele 1/2 chimera (strain 95330) (first-ATG start) 13 961 allele C 14 961 allele C (first-ATG start) 15 coding sequence for SEQ ID 13 16-31 PCR primers 32 SEQ ID 650 from WO99/24578 33-39 Domain derivatives of SEQ ID 32

Sequence CWU 1

1

511362PRTNeisseria speciesMISC_FEATUREallele 1 of NadA 1Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr1 5 10 15Phe Cys Ser Gly Ala Leu Ala Ala Thr Ser Asp Asp Asp Val Lys Lys20 25 30Ala Ala Thr Val Ala Ile Val Ala Ala Tyr Asn Asn Gly Gln Glu Ile35 40 45Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Gly Glu Asp Gly50 55 60Thr Ile Thr Gln Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp Asp65 70 75 80Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr Lys Thr85 90 95Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu100 105 110Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala115 120 125Leu Ala Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala Leu Asn130 135 140Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr Asn145 150 155 160Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr Val Asp165 170 175Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu Thr180 185 190Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala Lys Gln195 200 205Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala210 215 220Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn Thr225 230 235 240Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp Ile Lys245 250 255Ala Asp Ile Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Ser Ala Arg260 265 270Ile Asp Ser Leu Asp Lys Asn Val Ala Asn Leu Arg Lys Glu Thr Arg275 280 285Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr290 295 300Asn Val Gly Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser305 310 315 320Glu Ser Ala Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe325 330 335Ala Ala Lys Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala340 345 350Ala Tyr His Val Gly Val Asn Tyr Glu Trp355 3602398PRTNeisseria speciesMISC_FEATUREallele 2 of NadA 2Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr1 5 10 15Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val Lys Lys20 25 30Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln Glu Ile35 40 45Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu Asp Gly50 55 60Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp Asp65 70 75 80Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr Lys Thr85 90 95Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu100 105 110Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala115 120 125Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn Ile Thr130 135 140Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp Glu Lys145 150 155 160Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala Phe Asn165 170 175Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp Glu Ala180 185 190Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr Lys Gln195 200 205Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly Lys Ala210 215 220Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala Ala Asp Lys Ala Glu Ala225 230 235 240Val Ala Ala Lys Val Thr Asp Ile Lys Ala Asp Ile Ala Thr Asn Lys245 250 255Asp Asn Ile Ala Lys Lys Ala Asn Ser Ala Asp Val Tyr Thr Arg Glu260 265 270Glu Ser Asp Ser Lys Phe Val Arg Ile Asp Gly Leu Asn Ala Thr Thr275 280 285Glu Lys Leu Asp Thr Arg Leu Ala Ser Ala Glu Lys Ser Ile Thr Glu290 295 300His Gly Thr Arg Leu Asn Gly Leu Asp Arg Thr Val Ser Asp Leu Arg305 310 315 320Lys Glu Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu325 330 335Phe Gln Pro Tyr Asn Val Gly Arg Phe Asn Val Thr Ala Ala Val Gly340 345 350Gly Tyr Lys Ser Glu Ser Ala Val Ala Ile Gly Thr Gly Phe Arg Phe355 360 365Thr Glu Asn Phe Ala Ala Lys Ala Gly Val Ala Val Gly Thr Ser Ser370 375 380Gly Ser Ser Ala Ala Tyr His Val Gly Val Asn Tyr Glu Trp385 390 3953405PRTNeisseria speciesMISC_FEATUREallele 3 of NadA 3Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr1 5 10 15Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val Lys Lys20 25 30Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln Glu Ile35 40 45Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu Asp Gly50 55 60Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp Asp65 70 75 80Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr Lys Thr85 90 95Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu100 105 110Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala115 120 125Leu Ala Asp Thr Asp Ala Ala Leu Asp Ala Thr Thr Asn Ala Leu Asn130 135 140Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr Asn145 150 155 160Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr Val Asp165 170 175Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu Thr180 185 190Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala Lys Gln195 200 205Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala210 215 220Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn Thr225 230 235 240Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp Ile Lys245 250 255Ala Asp Ile Ala Thr Asn Lys Asp Asn Ile Ala Lys Lys Ala Asn Ser260 265 270Ala Asp Val Tyr Thr Arg Glu Glu Ser Asp Ser Lys Phe Val Arg Ile275 280 285Asp Gly Leu Asn Ala Thr Thr Glu Lys Leu Asp Thr Arg Leu Ala Ser290 295 300Ala Glu Lys Ser Ile Ala Asp His Asp Thr Arg Leu Asn Gly Leu Asp305 310 315 320Lys Thr Val Ser Asp Leu Arg Lys Glu Thr Arg Gln Gly Leu Ala Glu325 330 335Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly Arg Phe340 345 350Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala Val Ala355 360 365Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly370 375 380Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His Val Gly385 390 395 400Val Asn Tyr Glu Trp4054364PRTNeisseria speciesMISC_FEATUREallele 1 of NadA (first-ATG start) 4Met Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu1 5 10 15Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Ser Asp Asp Asp Val20 25 30Lys Lys Ala Ala Thr Val Ala Ile Val Ala Ala Tyr Asn Asn Gly Gln35 40 45Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Gly Glu50 55 60Asp Gly Thr Ile Thr Gln Lys Asp Ala Thr Ala Ala Asp Val Glu Ala65 70 75 80Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr85 90 95Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala100 105 110Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp115 120 125Ala Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala130 135 140Leu Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys145 150 155 160Thr Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr165 170 175Val Asp Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp180 185 190Glu Thr Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala195 200 205Lys Gln Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys210 215 220Ala Ala Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala225 230 235 240Asn Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp245 250 255Ile Lys Ala Asp Ile Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Ser260 265 270Ala Arg Ile Asp Ser Leu Asp Lys Asn Val Ala Asn Leu Arg Lys Glu275 280 285Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln290 295 300Pro Tyr Asn Val Gly Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr305 310 315 320Lys Ser Glu Ser Ala Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu325 330 335Asn Phe Ala Ala Lys Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser340 345 350Ser Ala Ala Tyr His Val Gly Val Asn Tyr Glu Trp355 3605400PRTNeisseria speciesMISC_FEATUREallele 2 of NadA (first-ATG start) 5Met Ser Met Lys His Phe Pro Ser Lys Val Ile Thr Thr Ala Ile Leu1 5 10 15Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val20 25 30Lys Lys Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln35 40 45Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu50 55 60Asp Gly Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala65 70 75 80Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr85 90 95Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala100 105 110Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp115 120 125Ala Ala Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn130 135 140Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp145 150 155 160Glu Lys Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala165 170 175Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp180 185 190Glu Ala Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr195 200 205Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly210 215 220Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala Ala Asp Lys Ala225 230 235 240Glu Ala Val Ala Ala Lys Val Thr Asp Ile Lys Ala Asp Ile Ala Thr245 250 255Asn Lys Asp Asn Ile Ala Lys Lys Ala Asn Ser Ala Asp Val Tyr Thr260 265 270Arg Glu Glu Ser Asp Ser Lys Phe Val Arg Ile Asp Gly Leu Asn Ala275 280 285Thr Thr Glu Lys Leu Asp Thr Arg Leu Ala Ser Ala Glu Lys Ser Ile290 295 300Thr Glu His Gly Thr Arg Leu Asn Gly Leu Asp Arg Thr Val Ser Asp305 310 315 320Leu Arg Lys Glu Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser325 330 335Gly Leu Phe Gln Pro Tyr Asn Val Gly Arg Phe Asn Val Thr Ala Ala340 345 350Val Gly Gly Tyr Lys Ser Glu Ser Ala Val Ala Ile Gly Thr Gly Phe355 360 365Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly Val Ala Val Gly Thr370 375 380Ser Ser Gly Ser Ser Ala Ala Tyr His Val Gly Val Asn Tyr Glu Trp385 390 395 4006407PRTNeisseria speciesMISC_FEATUREallele 3 of NadA (first-ATG start) 6Met Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu1 5 10 15Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val20 25 30Lys Lys Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln35 40 45Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu50 55 60Asp Gly Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala65 70 75 80Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr85 90 95Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala100 105 110Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp115 120 125Ala Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Ala Thr Thr Asn Ala130 135 140Leu Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys145 150 155 160Thr Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr165 170 175Val Asp Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp180 185 190Glu Thr Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala195 200 205Lys Gln Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys210 215 220Ala Ala Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala225 230 235 240Asn Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp245 250 255Ile Lys Ala Asp Ile Ala Thr Asn Lys Asp Asn Ile Ala Lys Lys Ala260 265 270Asn Ser Ala Asp Val Tyr Thr Arg Glu Glu Ser Asp Ser Lys Phe Val275 280 285Arg Ile Asp Gly Leu Asn Ala Thr Thr Glu Lys Leu Asp Thr Arg Leu290 295 300Ala Ser Ala Glu Lys Ser Ile Ala Asp His Asp Thr Arg Leu Asn Gly305 310 315 320Leu Asp Lys Thr Val Ser Asp Leu Arg Lys Glu Thr Arg Gln Gly Leu325 330 335Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly340 345 350Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala355 360 365Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys370 375 380Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His385 390 395 400Val Gly Val Asn Tyr Glu Trp4057391PRTNeisseria speciesMISC_FEATUREvariant allele 2 of NadA in strain 1881024 7Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr1 5 10 15Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val Lys Lys20 25 30Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln Glu Ile35 40 45Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu Asp Gly50 55 60Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp Asp65 70 75 80Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr Lys Thr85 90 95Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu100 105 110Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala115 120 125Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn Ile Thr130 135 140Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp Glu Lys145 150 155 160Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala Phe Asn165 170 175Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp Glu Ala180 185 190Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr Lys Gln195 200 205Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly Thr Ala210 215 220Asn Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp225 230 235 240Ile Lys Ala Asp Ile Ala Thr Asn Lys Asp Ile Asp Ala Lys Lys Ala245

250 255Asn Ser Ala Asp Val Tyr Thr Arg Glu Glu Ser Asp Ser Lys Phe Val260 265 270Arg Ile Asp Gly Leu Asn Ala Thr Thr Glu Lys Leu Asp Thr Arg Leu275 280 285Ala Ser Ala Glu Lys Ser Ile Thr Glu His Gly Thr Arg Leu Asn Gly290 295 300Leu Asp Arg Thr Val Ser Asp Leu Arg Lys Glu Thr Arg Gln Gly Leu305 310 315 320Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly325 330 335Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala340 345 350Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys355 360 365Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His370 375 380Val Gly Val Asn Tyr Glu Trp385 3908393PRTNeisseria speciesMISC_FEATUREvariant allele 2 of NadA (first-ATG start) in strain 1881024 8Met Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu1 5 10 15Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val20 25 30Lys Lys Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln35 40 45Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu50 55 60Asp Gly Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala65 70 75 80Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr85 90 95Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala100 105 110Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp115 120 125Ala Ala Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn130 135 140Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp145 150 155 160Glu Lys Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala165 170 175Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp180 185 190Glu Ala Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr195 200 205Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly210 215 220Thr Ala Asn Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val225 230 235 240Thr Asp Ile Lys Ala Asp Ile Ala Thr Asn Lys Asp Asn Ile Ala Lys245 250 255Lys Ala Asn Ser Ala Asp Val Tyr Thr Arg Glu Glu Ser Asp Ser Lys260 265 270Phe Val Arg Ile Asp Gly Leu Asn Ala Thr Thr Glu Lys Leu Asp Thr275 280 285Arg Leu Ala Ser Ala Glu Lys Ser Ile Thr Glu His Gly Thr Arg Leu290 295 300Asn Gly Leu Asp Arg Thr Val Ser Asp Leu Arg Lys Glu Thr Arg Gln305 310 315 320Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn325 330 335Val Gly Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu340 345 350Ser Ala Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala355 360 365Ala Lys Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala370 375 380Tyr His Val Gly Val Asn Tyr Glu Trp385 3909404PRTNeisseria speciesMISC_FEATUREvariant allele 3 of NadA in strains 973-1720 and 188759 9Met Gln His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr1 5 10 15Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val Lys Lys20 25 30Ala Ala Thr Val Ala Ile Ala Met Tyr Asn Asn Gly Gln Glu Ile Asn35 40 45Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu Asp Gly Thr50 55 60Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp Asp Phe65 70 75 80Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr Lys Thr Val85 90 95Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu Ser100 105 110Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala Leu115 120 125Ala Asp Thr Asp Ala Ala Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys130 135 140Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile145 150 155 160Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr Val Asp Lys165 170 175His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn180 185 190Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala Lys Gln Thr195 200 205Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu210 215 220Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala225 230 235 240Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp Ile Lys Ala245 250 255Asp Ile Ala Thr Asn Lys Asp Asn Ile Ala Lys Lys Ala Asn Ser Ala260 265 270Asp Val Tyr Thr Arg Glu Glu Ser Asp Ser Lys Phe Val Arg Ile Asp275 280 285Gly Leu Asn Ala Thr Thr Glu Lys Leu Asp Thr Arg Leu Ala Ser Ala290 295 300Glu Lys Ser Ile Ala Asp His Asp Thr Arg Leu Asn Gly Leu Asp Lys305 310 315 320Thr Val Ser Asp Leu Arg Lys Glu Thr Arg Gln Gly Leu Ala Glu Gln325 330 335Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly Arg Phe Asn340 345 350Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala Val Ala Ile355 360 365Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly Val370 375 380Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His Val Gly Val385 390 395 400Asn Tyr Glu Trp10407PRTNeisseria speciesMISC_FEATUREvariant allele 3 of NadA (first-ATG start) in strains 973-1720 and 188759 10Met Ser Met Gln His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu1 5 10 15Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val20 25 30Lys Lys Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln35 40 45Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu50 55 60Asp Gly Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala65 70 75 80Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr85 90 95Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala100 105 110Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp115 120 125Ala Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Ala Thr Thr Asn Ala130 135 140Leu Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys145 150 155 160Thr Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr165 170 175Val Asp Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp180 185 190Glu Thr Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala195 200 205Lys Gln Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys210 215 220Ala Ala Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala225 230 235 240Asn Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp245 250 255Ile Lys Ala Asp Ile Ala Thr Asn Lys Asp Asn Ile Ala Lys Lys Ala260 265 270Asn Ser Ala Asp Val Tyr Thr Arg Glu Glu Ser Asp Ser Lys Phe Val275 280 285Arg Ile Asp Gly Leu Asn Ala Thr Thr Glu Lys Leu Asp Thr Arg Leu290 295 300Ala Ser Ala Glu Lys Ser Ile Ala Asp His Asp Thr Arg Leu Asn Gly305 310 315 320Leu Asp Lys Thr Val Ser Asp Leu Arg Lys Glu Thr Arg Gln Gly Leu325 330 335Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly340 345 350Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala355 360 365Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys370 375 380Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His385 390 395 400Val Gly Val Asn Tyr Glu Trp40511355PRTNeisseria speciesMISC_FEATURENadA allele 1/2 chimera (strain 95330) 11Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr1 5 10 15Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val Lys Lys20 25 30Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln Glu Ile35 40 45Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu Asp Gly50 55 60Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp Asp65 70 75 80Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr Lys Thr85 90 95Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu100 105 110Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala115 120 125Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn Ile Thr130 135 140Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp Glu Lys145 150 155 160Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala Phe Asn165 170 175Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp Glu Ala180 185 190Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr Lys Gln195 200 205Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly Lys Ala210 215 220Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala Ala Asp Lys Ala Glu Ala225 230 235 240Val Ala Ala Lys Val Thr Asp Ile Lys Ala Asp Ile Ala Thr Asn Lys245 250 255Ala Asp Ile Ala Lys Asn Ser Ala Arg Ile Asp Ser Leu Asp Lys Asn260 265 270Val Ala Asn Leu Arg Lys Glu Thr Arg Gln Gly Leu Ala Glu Gln Ala275 280 285Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly Arg Phe Asn Val290 295 300Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala Val Ala Ile Gly305 310 315 320Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly Val Ala325 330 335Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His Val Gly Val Asn340 345 350Tyr Glu Trp35512357PRTNeisseria speciesMISC_FEATURENadA allele 1/2 chimera (strain 95330) (first-ATG start) 12Met Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu1 5 10 15Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Val20 25 30Lys Lys Ala Ala Thr Val Ala Ile Ala Ala Ala Tyr Asn Asn Gly Gln35 40 45Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Asp Glu50 55 60Asp Gly Thr Ile Thr Lys Lys Asp Ala Thr Ala Ala Asp Val Glu Ala65 70 75 80Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr85 90 95Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala100 105 110Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp115 120 125Ala Ala Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn130 135 140Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp145 150 155 160Glu Lys Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala165 170 175Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp180 185 190Glu Ala Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr195 200 205Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly210 215 220Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala Ala Asp Lys Ala225 230 235 240Glu Ala Val Ala Ala Lys Val Thr Asp Ile Lys Ala Asp Ile Ala Thr245 250 255Asn Lys Ala Asp Ile Ala Lys Asn Ser Ala Arg Ile Asp Ser Leu Asp260 265 270Lys Asn Val Ala Asn Leu Arg Lys Glu Thr Arg Gln Gly Leu Ala Glu275 280 285Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly Arg Phe290 295 300Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala Val Ala305 310 315 320Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly325 330 335Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His Val Gly340 345 350Val Asn Tyr Glu Trp35513323PRTNeisseria speciesMISC_FEATURENadA allele C 13Met Lys His Phe Pro Ser Lys Val Leu Thr Ala Ala Ile Leu Ala Ala1 5 10 15Leu Ser Gly Ser Ala Met Ala Asp Asn Ala Pro Thr Ala Asp Glu Ile20 25 30Ala Lys Ala Ala Leu Val Asn Ser Tyr Asn Asn Thr Gln Asp Ile Asn35 40 45Gly Phe Thr Val Gly Asp Thr Ile Tyr Asp Ile Lys Asn Asp Lys Ile50 55 60Thr Lys Lys Glu Ala Thr Glu Ala Asp Val Glu Ala Asp Asp Phe Lys65 70 75 80Gly Leu Gly Leu Lys Glu Val Val Ala Gln His Asp Gln Ser Leu Ala85 90 95Asp Leu Thr Glu Thr Val Asn Glu Asn Ser Glu Ala Leu Val Lys Thr100 105 110Ala Ala Val Val Asn Asp Ile Ser Ala Asp Val Lys Ala Asn Thr Ala115 120 125Ala Ile Gly Glu Asn Lys Ala Ala Ile Ala Thr Lys Ala Asp Lys Thr130 135 140Glu Leu Asp Lys Val Ser Gly Lys Val Thr Glu Asn Glu Thr Ala Ile145 150 155 160Gly Lys Lys Ala Asn Ser Ala Asp Val Tyr Thr Lys Ala Glu Val Tyr165 170 175Thr Lys Gln Glu Ser Asp Asn Arg Phe Val Lys Ile Ser Asp Gly Ile180 185 190Gly Asn Leu Asn Thr Thr Ala Asn Gly Leu Glu Thr Arg Leu Ala Ala195 200 205Ala Glu Gln Ser Val Ala Asp His Gly Thr Arg Leu Ala Ser Ala Glu210 215 220Lys Ser Ile Thr Glu His Gly Thr Arg Leu Asn Gly Leu Asp Arg Thr225 230 235 240Val Ser Asp Leu Arg Lys Glu Thr Arg Gln Gly Leu Ala Glu Gln Ala245 250 255Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly Arg Phe Asn Val260 265 270Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala Val Ala Ile Gly275 280 285Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly Val Ala290 295 300Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His Val Gly Val Asn305 310 315 320Tyr Glu Trp14325PRTNeisseria speciesMISC_FEATURENadA allele C (first-ATG start) 14Met Ser Met Lys His Phe Pro Ser Arg Val Leu Thr Ala Ala Ile Leu1 5 10 15Ala Ala Leu Ser Gly Ser Ala Met Ala Asp Asn Ala Pro Thr Ala Asp20 25 30Glu Ile Ala Lys Ala Ala Leu Val Asn Ser Tyr Asn Asn Thr Gln Asp35 40 45Ile Asn Gly Phe Thr Val Gly Asp Thr Ile Tyr Asp Ile Lys Asn Asp50 55 60Lys Ile Thr Lys Lys Glu Ala Thr Glu Ala Asp Val Glu Ala Asp Asp65 70 75 80Phe Lys Gly Leu Gly Leu Lys Glu Val Val Ala Gln His Asp Gln Ser85 90 95Leu Ala Asp Leu Thr Glu Thr Val Asn Glu Asn Ser Glu Ala Leu Val100 105 110Lys Thr Ala Ala Val Val Asn Asp Ile Ser Ala Asp Val Lys Ala Asn115 120 125Thr Ala Ala Ile Gly Glu Asn Lys Ala Ala Ile Ala Thr Lys Ala Asp130 135 140Lys Thr Glu Leu Asp Lys Val Ser Gly Lys Val Thr Glu Asn Glu Thr145 150 155 160Ala Ile Gly Lys Lys Ala Asn Ser Ala Asp Val Tyr Thr Lys Ala Glu165 170 175Val Tyr Thr Lys Gln Glu Ser Asp Asn Arg Phe Val Lys Ile Ser Asp180

185 190Gly Ile Gly Asn Leu Asn Thr Thr Ala Asn Gly Leu Glu Thr Arg Leu195 200 205Ala Ala Ala Glu Gln Ser Val Ala Asp His Gly Thr Arg Leu Ala Ser210 215 220Ala Glu Lys Ser Ile Thr Glu His Gly Thr Arg Leu Asn Gly Leu Asp225 230 235 240Arg Thr Val Ser Asp Leu Arg Lys Glu Thr Arg Gln Gly Leu Ala Glu245 250 255Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro Tyr Asn Val Gly Arg Phe260 265 270Asn Val Thr Ala Ala Val Gly Gly Tyr Lys Ser Glu Ser Ala Val Ala275 280 285Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly290 295 300Val Ala Val Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His Val Gly305 310 315 320Val Asn Tyr Glu Trp32515971DNANeisseria speciesmisc_featurecoding sequence for 8EQ ID 13 15atgaaacact ttccatccaa agtactgacc gcagccatcc ttgccgccct cagcggcagc 60gcaatggcag acaacgcccc caccgctgac gaaattgcca aagccgccct agttaactcc 120tacaacaata cccaagacat caacggattc acagtcggag acaccatcta cgacattaaa 180aatgacaaga ttaccaaaaa agaagcaaca gaagccgatg ttgaagctga cgactttaaa 240ggtctgggtc tgaaagaagt cgtggctcaa cacgaccaaa gccttgccga cctgaccgam 300ccgtcaatga aaacagcgaa gcattggtaa aaaccgccgc ggttgtcaat gacatcagtg 360ccgatgtcaa agccaacaca gcagctatcg gggaaaacaa agctgctatc gctacaaaag 420cagacaaaac cgaactggat aaagtgtccg gcaaagtaac cgagaacgag actgctatcg 480gtaaaaaagc aaacagtgcc gacgtgtaca ctaaagctga ggtgtacacc aaacaagagt 540ctgacaacag atttgtcaaa attagtgacg gaatcggtaa tctgaacact actgccaatg 600gattggagac acgcttggcc gctgccgaac aatccgttgc agaccacggt acgcgcttgg 660cttctgccga aaaatccatt accgaacacg gtacgcgcct gaacggtttg gatagaacag 720tgtcagacct gcgtaaagaa acccgccaag gccttgcaga acaagccgcg ctctccggtc 780tgttccaacc ttacaacgtg ggtcggttca atgtaacggc tgcagtcggc ggctacaaat 840ccgaatcggc agtcgccatc ggtaccggct tccgctttac cgaaaacttt gccgccaaag 900caggcgtggc agtcggcact tcgtccggtt cttccgcagc ctaccatgtc ggcgtcaatt 960acgagtggta a 9711622DNAArtificialmisc_featureforward primer 16gtcgacgtcc tcgattacga ag 221722DNAArtificialmisc_featurereverse primer 17cgaggcgatt gtcaaaccgt tc 221832DNAArtificialmisc_featureforward primer 18cgcggatccg ctagcggaca cacttatttc gg 321929DNAArtificialmisc_featurereverse primer 19cccgctcgag ccagcggtag cctaatttg 292033DNAArtificialmisc_featureforward primer 20cgcggatccg ctagcaaaac aaccgacaaa cgg 332132DNAArtificialmisc_featurereverse primer 21cccgctcgag ttaccagcgg tagcctaatt tg 322241DNAArtificialmisc_featuremutagenesis primer 22ctcatttggc gacgctggct caccaatgtt tatctatgat g 412341DNAArtificialmisc_featuremutagenesis primer 23catcatagat aaacattggt gagccagcgt cgccaaatga g 412432DNAArtificialmisc_featureforward prmer 24cgcggatccg ctagcggaca cacttatttc gg 322526DNAArtificialmisc_featurereverse primer 25cccgctcgag cagcgcgtca aggctt 2626124DNAArtificialmisc_featureforward primer 26gggaattcca tatgaaagcc aaacgtttta aaattaacgc catatcctta tccatctttc 60ttgcctatgc ccttacgcca tactcagaag cggctagcga caacgcgcaa agccttgacg 120cgct 1242732DNAArtificialmisc_featurereverse primer 27cccgctcgag ttaccagcgg tagcctaatt tg 322824DNAArtificialmisc_featureknockout primer 28gctctagagg aggctgtcga aacc 242926DNAArtificialmisc_featureknockout primer 29tcccccgggc ggttgtccgt ttgtcg 263027DNAArtificialmisc_featureknockout primer 30tcccccgggg cgggcatcaa attaggc 273127DNAArtificialmisc_featureknockout primer 31cccgctcgag cgcaaccgtc cgctgac 27321455PRTNeisseria speciesMISC_FEATURESEQ ID 650 from WO99/24578 32Met Lys Thr Thr Asp Lys Arg Thr Thr Glu Thr His Arg Lys Ala Pro1 5 10 15Lys Thr Gly Arg Ile Arg Phe Ser Pro Ala Tyr Leu Ala Ile Cys Leu20 25 30Ser Phe Gly Ile Leu Pro Gln Ala Trp Ala Gly His Thr Tyr Phe Gly35 40 45Ile Asn Tyr Gln Tyr Tyr Arg Asp Phe Ala Glu Asn Lys Gly Lys Phe50 55 60Ala Val Gly Ala Lys Asp Ile Glu Val Tyr Asn Lys Lys Gly Glu Leu65 70 75 80Val Gly Lys Ser Met Thr Lys Ala Pro Met Ile Asp Phe Ser Val Val85 90 95Ser Arg Asn Gly Val Ala Ala Leu Val Gly Asp Gln Tyr Ile Val Ser100 105 110Val Ala His Asn Gly Gly Tyr Asn Asn Val Asp Phe Gly Ala Glu Gly115 120 125Arg Asn Pro Asp Gln His Arg Phe Thr Tyr Lys Ile Val Lys Arg Asn130 135 140Asn Tyr Lys Ala Gly Thr Lys Gly His Pro Tyr Gly Gly Asp Tyr His145 150 155 160Met Pro Arg Leu His Lys Phe Val Thr Asp Ala Glu Pro Val Glu Met165 170 175Thr Ser Tyr Met Asp Gly Arg Lys Tyr Ile Asp Gln Asn Asn Tyr Pro180 185 190Asp Arg Val Arg Ile Gly Ala Gly Arg Gln Tyr Trp Arg Ser Asp Glu195 200 205Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr His Ile Ala Ser Ala Tyr210 215 220Ser Trp Leu Val Gly Gly Asn Thr Phe Ala Gln Asn Gly Ser Gly Gly225 230 235 240Gly Thr Val Asn Leu Gly Ser Glu Lys Ile Lys His Ser Pro Tyr Gly245 250 255Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp Ser Gly Ser Pro Met Phe260 265 270Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu Ile Asn Gly Val Leu Gln275 280 285Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn Gly Phe Gln Leu Val Arg290 295 300Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala Gly Asp Thr His Ser Val305 310 315 320Phe Tyr Glu Pro Arg Gln Asn Gly Lys Tyr Ser Phe Asn Asp Asp Asn325 330 335Asn Gly Thr Gly Lys Ile Asn Ala Lys His Glu His Asn Ser Leu Pro340 345 350Asn Arg Leu Lys Thr Arg Thr Val Gln Leu Phe Asn Val Ser Leu Ser355 360 365Glu Thr Ala Arg Glu Pro Val Tyr His Ala Ala Gly Gly Val Asn Ser370 375 380Tyr Arg Pro Arg Leu Asn Asn Gly Glu Asn Ile Ser Phe Ile Asp Glu385 390 395 400Gly Lys Gly Glu Leu Ile Leu Thr Ser Asn Ile Asn Gln Gly Ala Gly405 410 415Gly Leu Tyr Phe Gln Gly Asp Phe Thr Val Ser Pro Glu Asn Asn Glu420 425 430Thr Trp Gln Gly Ala Gly Val His Ile Ser Glu Asp Ser Thr Val Thr435 440 445Trp Lys Val Asn Gly Val Ala Asn Asp Arg Leu Ser Lys Ile Gly Lys450 455 460Gly Thr Leu His Val Gln Ala Lys Gly Glu Asn Gln Gly Ser Ile Ser465 470 475 480Val Gly Asp Gly Thr Val Ile Leu Asp Gln Gln Ala Asp Asp Lys Gly485 490 495Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu Val Ser Gly Arg Gly Thr500 505 510Val Gln Leu Asn Ala Asp Asn Gln Phe Asn Pro Asp Lys Leu Tyr Phe515 520 525Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn Gly His Ser Leu Ser Phe530 535 540His Arg Ile Gln Asn Thr Asp Glu Gly Ala Met Ile Val Asn His Asn545 550 555 560Gln Asp Lys Glu Ser Thr Val Thr Ile Thr Gly Asn Lys Asp Ile Ala565 570 575Thr Thr Gly Asn Asn Asn Ser Leu Asp Ser Lys Lys Glu Ile Ala Tyr580 585 590Asn Gly Trp Phe Gly Glu Lys Asp Thr Thr Lys Thr Asn Gly Arg Leu595 600 605Asn Leu Val Tyr Gln Pro Ala Ala Glu Asp Arg Thr Leu Leu Leu Ser610 615 620Gly Gly Thr Asn Leu Asn Gly Asn Ile Thr Gln Thr Asn Gly Lys Leu625 630 635 640Phe Phe Ser Gly Arg Pro Thr Pro His Ala Tyr Asn His Leu Asn Asp645 650 655His Trp Ser Gln Lys Glu Gly Ile Pro Arg Gly Glu Ile Val Trp Asp660 665 670Asn Asp Trp Ile Asn Arg Thr Phe Lys Ala Glu Asn Phe Gln Ile Lys675 680 685Gly Gly Gln Ala Trp Ser Arg Asn Val Ala Lys Val Lys Gly Asp Trp690 695 700His Leu Ser Asn His Ala Gln Ala Val Phe Gly Val Ala Pro His Gln705 710 715 720Ser His Thr Ile Cys Thr Arg Ser Asp Trp Thr Gly Leu Thr Asn Cys725 730 735Val Glu Lys Thr Ile Thr Asp Asp Lys Val Ile Ala Ser Leu Thr Lys740 745 750Thr Asp Ile Ser Gly Asn Val Asp Leu Ala Asp His Ala His Leu Asn755 760 765Leu Thr Gly Leu Ala Thr Leu Asn Gly Asn Leu Ser Ala Asn Gly Asp770 775 780Thr Arg Tyr Thr Val Ser His Asn Ala Thr Gln Asn Gly Asn Leu Ser785 790 795 800Leu Val Gly Asn Ala Gln Ala Thr Phe Asn Gln Ala Thr Leu Asn Gly805 810 815Asn Thr Ser Ala Ser Gly Asn Ala Ser Phe Asn Leu Ser Asp His Ala820 825 830Val Gln Asn Gly Ser Leu Thr Leu Ser Gly Asn Ala Lys Ala Asn Val835 840 845Ser His Ser Ala Leu Asn Gly Asn Val Ser Leu Ala Asp Lys Ala Val850 855 860Phe His Phe Glu Ser Ser Arg Phe Thr Gly Gln Ile Ser Gly Gly Lys865 870 875 880Asp Thr Ala Leu His Leu Lys Asp Ser Glu Trp Thr Leu Pro Ser Gly885 890 895Thr Glu Leu Gly Asn Leu Asn Leu Asp Asn Ala Thr Ile Thr Leu Asn900 905 910Ser Ala Tyr Arg His Asp Ala Ala Gly Ala Gln Thr Gly Ser Ala Thr915 920 925Asp Ala Pro Arg Arg Arg Ser Arg Arg Ser Arg Arg Ser Leu Leu Ser930 935 940Val Thr Pro Pro Thr Ser Val Glu Ser Arg Phe Asn Thr Leu Thr Val945 950 955 960Asn Gly Lys Leu Asn Gly Gln Gly Thr Phe Arg Phe Met Ser Glu Leu965 970 975Phe Gly Tyr Arg Ser Asp Lys Leu Lys Leu Ala Glu Ser Ser Glu Gly980 985 990Thr Tyr Thr Leu Ala Val Asn Asn Thr Gly Asn Glu Pro Ala Ser Leu995 1000 1005Glu Gln Leu Thr Trp Glu Gly Lys Asp Asn Lys Pro Leu Ser Glu1010 1015 1020Asn Leu Asn Phe Thr Leu Gln Asn Glu His Val Asp Ala Gly Ala1025 1030 1035Trp Arg Tyr Gln Leu Ile Arg Lys Asp Gly Glu Phe Arg Leu His1040 1045 1050Asn Pro Val Lys Glu Gln Glu Leu Ser Asp Lys Leu Gly Lys Ala1055 1060 1065Glu Ala Lys Lys Gln Ala Glu Lys Asp Asn Ala Gln Ser Leu Asp1070 1075 1080Ala Leu Ile Ala Ala Gly Arg Asp Ala Val Glu Lys Thr Glu Ser1085 1090 1095Val Ala Glu Pro Ala Arg Gln Ala Gly Gly Glu Asn Val Gly Ile1100 1105 1110Met Gln Ala Glu Glu Glu Lys Lys Arg Val Gln Ala Asp Lys Asp1115 1120 1125Thr Ala Leu Ala Lys Gln Arg Glu Ala Glu Thr Arg Pro Ala Thr1130 1135 1140Thr Ala Phe Pro Arg Ala Arg Arg Ala Arg Arg Asp Leu Pro Gln1145 1150 1155Leu Gln Pro Gln Pro Gln Pro Gln Pro Gln Arg Asp Leu Ile Ser1160 1165 1170Arg Tyr Ala Asn Ser Gly Leu Ser Glu Phe Ser Ala Thr Leu Asn1175 1180 1185Ser Val Phe Ala Val Gln Asp Glu Leu Asp Arg Val Phe Ala Glu1190 1195 1200Asp Arg Arg Asn Ala Val Trp Thr Ser Gly Ile Arg Asp Thr Lys1205 1210 1215His Tyr Arg Ser Gln Asp Phe Arg Ala Tyr Arg Gln Gln Thr Asp1220 1225 1230Leu Arg Gln Ile Gly Met Gln Lys Asn Leu Gly Ser Gly Arg Val1235 1240 1245Gly Ile Leu Phe Ser His Asn Arg Thr Glu Asn Thr Phe Asp Asp1250 1255 1260Gly Ile Gly Asn Ser Ala Arg Leu Ala His Gly Ala Val Phe Gly1265 1270 1275Gln Tyr Gly Ile Asp Arg Phe Tyr Ile Gly Ile Ser Ala Gly Ala1280 1285 1290Gly Phe Ser Ser Gly Ser Leu Ser Asp Gly Ile Gly Gly Lys Ile1295 1300 1305Arg Arg Arg Val Leu His Tyr Gly Ile Gln Ala Arg Tyr Arg Ala1310 1315 1320Gly Phe Gly Gly Phe Gly Ile Glu Pro His Ile Gly Ala Thr Arg1325 1330 1335Tyr Phe Val Gln Lys Ala Asp Tyr Arg Tyr Glu Asn Val Asn Ile1340 1345 1350Ala Thr Pro Gly Leu Ala Phe Asn Arg Tyr Arg Ala Gly Ile Lys1355 1360 1365Ala Asp Tyr Ser Phe Lys Pro Ala Gln His Ile Ser Ile Thr Pro1370 1375 1380Tyr Leu Ser Leu Ser Tyr Thr Asp Ala Ala Ser Gly Lys Val Arg1385 1390 1395Thr Arg Val Asn Thr Ala Val Leu Ala Gln Asp Phe Gly Lys Thr1400 1405 1410Arg Ser Ala Glu Trp Gly Val Asn Ala Glu Ile Lys Gly Phe Thr1415 1420 1425Leu Ser Leu His Ala Ala Ala Ala Lys Gly Pro Gln Leu Glu Ala1430 1435 1440Gln His Ser Ala Gly Ile Lys Leu Gly Tyr Arg Trp1445 1450 145533956PRTNeisseria speciesMISC_FEATUREApp domain derivative 33Met Lys Thr Thr Asp Lys Arg Thr Thr Glu Thr His Arg Lys Ala Pro1 5 10 15Lys Thr Gly Arg Ile Arg Phe Ser Pro Ala Tyr Leu Ala Ile Cys Leu20 25 30Ser Phe Gly Ile Leu Pro Gln Ala Trp Ala Gly His Thr Tyr Phe Gly35 40 45Ile Asn Tyr Gln Tyr Tyr Arg Asp Phe Ala Glu Asn Lys Gly Lys Phe50 55 60Ala Val Gly Ala Lys Asp Ile Glu Val Tyr Asn Lys Lys Gly Glu Leu65 70 75 80Val Gly Lys Ser Met Thr Lys Ala Pro Met Ile Asp Phe Ser Val Val85 90 95Ser Arg Asn Gly Val Ala Ala Leu Val Gly Asp Gln Tyr Ile Val Ser100 105 110Val Ala His Asn Gly Gly Tyr Asn Asn Val Asp Phe Gly Ala Glu Gly115 120 125Arg Asn Pro Asp Gln His Arg Phe Thr Tyr Lys Ile Val Lys Arg Asn130 135 140Asn Tyr Lys Ala Gly Thr Lys Gly His Pro Tyr Gly Gly Asp Tyr His145 150 155 160Met Pro Arg Leu His Lys Phe Val Thr Asp Ala Glu Pro Val Glu Met165 170 175Thr Ser Tyr Met Asp Gly Arg Lys Tyr Ile Asp Gln Asn Asn Tyr Pro180 185 190Asp Arg Val Arg Ile Gly Ala Gly Arg Gln Tyr Trp Arg Ser Asp Glu195 200 205Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr His Ile Ala Ser Ala Tyr210 215 220Ser Trp Leu Val Gly Gly Asn Thr Phe Ala Gln Asn Gly Ser Gly Gly225 230 235 240Gly Thr Val Asn Leu Gly Ser Glu Lys Ile Lys His Ser Pro Tyr Gly245 250 255Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp Ser Gly Ser Pro Met Phe260 265 270Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu Ile Asn Gly Val Leu Gln275 280 285Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn Gly Phe Gln Leu Val Arg290 295 300Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala Gly Asp Thr His Ser Val305 310 315 320Phe Tyr Glu Pro Arg Gln Asn Gly Lys Tyr Ser Phe Asn Asp Asp Asn325 330 335Asn Gly Thr Gly Lys Ile Asn Ala Lys His Glu His Asn Ser Leu Pro340 345 350Asn Arg Leu Lys Thr Arg Thr Val Gln Leu Phe Asn Val Ser Leu Ser355 360 365Glu Thr Ala Arg Glu Pro Val Tyr His Ala Ala Gly Gly Val Asn Ser370 375 380Tyr Arg Pro Arg Leu Asn Asn Gly Glu Asn Ile Ser Phe Ile Asp Glu385 390 395 400Gly Lys Gly Glu Leu Ile Leu Thr Ser Asn Ile Asn Gln Gly Ala Gly405 410 415Gly Leu Tyr Phe Gln Gly Asp Phe Thr Val Ser Pro Glu Asn Asn Glu420 425 430Thr Trp Gln Gly Ala Gly Val His Ile Ser Glu Asp Ser Thr Val Thr435 440 445Trp Lys Val Asn Gly Val Ala Asn Asp Arg Leu Ser Lys Ile Gly Lys450 455 460Gly Thr Leu His Val Gln Ala Lys Gly Glu Asn Gln

Gly Ser Ile Ser465 470 475 480Val Gly Asp Gly Thr Val Ile Leu Asp Gln Gln Ala Asp Asp Lys Gly485 490 495Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu Val Ser Gly Arg Gly Thr500 505 510Val Gln Leu Asn Ala Asp Asn Gln Phe Asn Pro Asp Lys Leu Tyr Phe515 520 525Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn Gly His Ser Leu Ser Phe530 535 540His Arg Ile Gln Asn Thr Asp Glu Gly Ala Met Ile Val Asn His Asn545 550 555 560Gln Asp Lys Glu Ser Thr Val Thr Ile Thr Gly Asn Lys Asp Ile Ala565 570 575Thr Thr Gly Asn Asn Asn Ser Leu Asp Ser Lys Lys Glu Ile Ala Tyr580 585 590Asn Gly Trp Phe Gly Glu Lys Asp Thr Thr Lys Thr Asn Gly Arg Leu595 600 605Asn Leu Val Tyr Gln Pro Ala Ala Glu Asp Arg Thr Leu Leu Leu Ser610 615 620Gly Gly Thr Asn Leu Asn Gly Asn Ile Thr Gln Thr Asn Gly Lys Leu625 630 635 640Phe Phe Ser Gly Arg Pro Thr Pro His Ala Tyr Asn His Leu Asn Asp645 650 655His Trp Ser Gln Lys Glu Gly Ile Pro Arg Gly Glu Ile Val Trp Asp660 665 670Asn Asp Trp Ile Asn Arg Thr Phe Lys Ala Glu Asn Phe Gln Ile Lys675 680 685Gly Gly Gln Ala Val Val Ser Arg Asn Val Ala Lys Val Lys Gly Asp690 695 700Trp His Leu Ser Asn His Ala Gln Ala Val Phe Gly Val Ala Pro His705 710 715 720Gln Ser His Thr Ile Cys Thr Arg Ser Asp Trp Thr Gly Leu Thr Asn725 730 735Cys Val Glu Lys Thr Ile Thr Asp Asp Lys Val Ile Ala Ser Leu Thr740 745 750Lys Thr Asp Ile Ser Gly Asn Val Asp Leu Ala Asp His Ala His Leu755 760 765Asn Leu Thr Gly Leu Ala Thr Leu Asn Gly Asn Leu Ser Ala Asn Gly770 775 780Asp Thr Arg Tyr Thr Val Ser His Asn Ala Thr Gln Asn Gly Asn Leu785 790 795 800Ser Leu Val Gly Asn Ala Gln Ala Thr Phe Asn Gln Ala Thr Leu Asn805 810 815Gly Asn Thr Ser Ala Ser Gly Asn Ala Ser Phe Asn Leu Ser Asp His820 825 830Ala Val Gln Asn Gly Ser Leu Thr Leu Ser Gly Asn Ala Lys Ala Asn835 840 845Val Ser His Ser Ala Leu Asn Gly Asn Val Ser Leu Ala Asp Lys Ala850 855 860Val Phe His Phe Glu Ser Ser Arg Phe Thr Gly Gln Ile Ser Gly Gly865 870 875 880Lys Asp Thr Ala Leu His Leu Lys Asp Ser Glu Trp Thr Leu Pro Ser885 890 895Gly Thr Glu Leu Gly Asn Leu Asn Leu Asp Asn Ala Thr Ile Thr Leu900 905 910Asn Ser Ala Tyr Arg His Asp Ala Ala Gly Ala Gln Thr Gly Ser Ala915 920 925Thr Asp Ala Pro Arg Arg Arg Ser Arg Arg Ser Arg Arg Ser Leu Leu930 935 940Ser Val Thr Pro Pro Thr Ser Val Glu Ser Arg Phe945 950 955341178PRTNeisseria speciesMISC_FEATUREApp domain derivative 34Met Lys Thr Thr Asp Lys Arg Thr Thr Glu Thr His Arg Lys Ala Pro1 5 10 15Lys Thr Gly Arg Ile Arg Phe Ser Pro Ala Tyr Leu Ala Ile Cys Leu20 25 30Ser Phe Gly Ile Leu Pro Gln Ala Trp Ala Gly His Thr Tyr Phe Gly35 40 45Ile Asn Tyr Gln Tyr Tyr Arg Asp Phe Ala Glu Asn Lys Gly Lys Phe50 55 60Ala Val Gly Ala Lys Asp Ile Glu Val Tyr Asn Lys Lys Gly Glu Leu65 70 75 80Val Gly Lys Ser Met Thr Lys Ala Pro Met Ile Asp Phe Ser Val Val85 90 95Ser Arg Asn Gly Val Ala Ala Leu Val Gly Asp Gln Tyr Ile Val Ser100 105 110Val Ala His Asn Gly Gly Tyr Asn Asn Val Asp Phe Gly Ala Glu Gly115 120 125Arg Asn Pro Asp Gln His Arg Phe Thr Tyr Lys Ile Val Lys Arg Asn130 135 140Asn Tyr Lys Ala Gly Thr Lys Gly His Pro Tyr Gly Gly Asp Tyr His145 150 155 160Met Pro Arg Leu His Lys Phe Val Thr Asp Ala Glu Pro Val Glu Met165 170 175Thr Ser Tyr Met Asp Gly Arg Lys Tyr Ile Asp Gln Asn Asn Tyr Pro180 185 190Asp Arg Val Arg Ile Gly Ala Gly Arg Gln Tyr Trp Arg Ser Asp Glu195 200 205Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr His Ile Ala Ser Ala Tyr210 215 220Ser Trp Leu Val Gly Gly Asn Thr Phe Ala Gln Asn Gly Ser Gly Gly225 230 235 240Gly Thr Val Asn Leu Gly Ser Glu Lys Ile Lys His Ser Pro Tyr Gly245 250 255Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp Ser Gly Ser Pro Met Phe260 265 270Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu Ile Asn Gly Val Leu Gln275 280 285Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn Gly Phe Gln Leu Val Arg290 295 300Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala Gly Asp Thr His Ser Val305 310 315 320Phe Tyr Glu Pro Arg Gln Asn Gly Lys Tyr Ser Phe Asn Asp Asp Asn325 330 335Asn Gly Thr Gly Lys Ile Asn Ala Lys His Glu His Asn Ser Leu Pro340 345 350Asn Arg Leu Lys Thr Arg Thr Val Gln Leu Phe Asn Val Ser Leu Ser355 360 365Glu Thr Ala Arg Glu Pro Val Tyr His Ala Ala Gly Gly Val Asn Ser370 375 380Tyr Arg Pro Arg Leu Asn Asn Gly Glu Asn Ile Ser Phe Ile Asp Glu385 390 395 400Gly Lys Gly Glu Leu Ile Leu Thr Ser Asn Ile Asn Gln Gly Ala Gly405 410 415Gly Leu Tyr Phe Gln Gly Asp Phe Thr Val Ser Pro Glu Asn Asn Glu420 425 430Thr Trp Gln Gly Ala Gly Val His Ile Ser Glu Asp Ser Thr Val Thr435 440 445Trp Lys Val Asn Gly Val Ala Asn Asp Arg Leu Ser Lys Ile Gly Lys450 455 460Gly Thr Leu His Val Gln Ala Lys Gly Glu Asn Gln Gly Ser Ile Ser465 470 475 480Val Gly Asp Gly Thr Val Ile Leu Asp Gln Gln Ala Asp Asp Lys Gly485 490 495Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu Val Ser Gly Arg Gly Thr500 505 510Val Gln Leu Asn Ala Asp Asn Gln Phe Asn Pro Asp Lys Leu Tyr Phe515 520 525Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn Gly His Ser Leu Ser Phe530 535 540His Arg Ile Gln Asn Thr Asp Glu Gly Ala Met Ile Val Asn His Asn545 550 555 560Gln Asp Lys Glu Ser Thr Val Thr Ile Thr Gly Asn Lys Asp Ile Ala565 570 575Thr Thr Gly Asn Asn Asn Ser Leu Asp Ser Lys Lys Glu Ile Ala Tyr580 585 590Asn Gly Trp Phe Gly Glu Lys Asp Thr Thr Lys Thr Asn Gly Arg Leu595 600 605Asn Leu Val Tyr Gln Pro Ala Ala Glu Asp Arg Thr Leu Leu Leu Ser610 615 620Gly Gly Thr Asn Leu Asn Gly Asn Ile Thr Gln Thr Asn Gly Lys Leu625 630 635 640Phe Phe Ser Gly Arg Pro Thr Pro His Ala Tyr Asn His Leu Asn Asp645 650 655His Trp Ser Gln Lys Glu Gly Ile Pro Arg Gly Glu Ile Val Trp Asp660 665 670Asn Asp Trp Ile Asn Arg Thr Phe Lys Ala Glu Asn Phe Gln Ile Lys675 680 685Gly Gly Gln Ala Val Val Ser Arg Asn Val Ala Lys Val Lys Gly Asp690 695 700Trp His Leu Ser Asn His Ala Gln Ala Val Phe Gly Val Ala Pro His705 710 715 720Gln Ser His Thr Ile Cys Thr Arg Ser Asp Trp Thr Gly Leu Thr Asn725 730 735Cys Val Glu Lys Thr Ile Thr Asp Asp Lys Val Ile Ala Ser Leu Thr740 745 750Lys Thr Asp Ile Ser Gly Asn Val Asp Leu Ala Asp His Ala His Leu755 760 765Asn Leu Thr Gly Leu Ala Thr Leu Asn Gly Asn Leu Ser Ala Asn Gly770 775 780Asp Thr Arg Tyr Thr Val Ser His Asn Ala Thr Gln Asn Gly Asn Leu785 790 795 800Ser Leu Val Gly Asn Ala Gln Ala Thr Phe Asn Gln Ala Thr Leu Asn805 810 815Gly Asn Thr Ser Ala Ser Gly Asn Ala Ser Phe Asn Leu Ser Asp His820 825 830Ala Val Gln Asn Gly Ser Leu Thr Leu Ser Gly Asn Ala Lys Ala Asn835 840 845Val Ser His Ser Ala Leu Asn Gly Asn Val Ser Leu Ala Asp Lys Ala850 855 860Val Phe His Phe Glu Ser Ser Arg Phe Thr Gly Gln Ile Ser Gly Gly865 870 875 880Lys Asp Thr Ala Leu His Leu Lys Asp Ser Glu Trp Thr Leu Pro Ser885 890 895Gly Thr Glu Leu Gly Asn Leu Asn Leu Asp Asn Ala Thr Ile Thr Leu900 905 910Asn Ser Ala Tyr Arg His Asp Ala Ala Gly Ala Gln Thr Gly Ser Ala915 920 925Thr Asp Ala Pro Arg Arg Arg Ser Arg Arg Ser Arg Arg Ser Leu Leu930 935 940Ser Val Thr Pro Pro Thr Ser Val Glu Ser Arg Phe Asn Thr Leu Thr945 950 955 960Val Asn Gly Lys Leu Asn Gly Gln Gly Thr Phe Arg Phe Met Ser Glu965 970 975Leu Phe Gly Tyr Arg Ser Asp Lys Leu Lys Leu Ala Glu Ser Ser Glu980 985 990Gly Thr Tyr Thr Leu Ala Val Asn Asn Thr Gly Asn Glu Pro Ala Ser995 1000 1005Leu Glu Gln Leu Thr Val Val Glu Gly Lys Asp Asn Lys Pro Leu1010 1015 1020Ser Glu Asn Leu Asn Phe Thr Leu Gln Asn Glu His Val Asp Ala1025 1030 1035Gly Ala Trp Arg Tyr Gln Leu Ile Arg Lys Asp Gly Glu Phe Arg1040 1045 1050Leu His Asn Pro Val Lys Glu Gln Glu Leu Ser Asp Lys Leu Gly1055 1060 1065Lys Ala Glu Ala Lys Lys Gln Ala Glu Lys Asp Asn Ala Gln Ser1070 1075 1080Leu Asp Ala Leu Ile Ala Ala Gly Arg Asp Ala Val Glu Lys Thr1085 1090 1095Glu Ser Val Ala Glu Pro Ala Arg Gln Ala Gly Gly Glu Asn Val1100 1105 1110Gly Ile Met Gln Ala Glu Glu Glu Lys Lys Arg Val Gln Ala Asp1115 1120 1125Lys Asp Thr Ala Leu Ala Lys Gln Arg Glu Ala Glu Thr Arg Pro1130 1135 1140Ala Thr Thr Ala Phe Pro Arg Ala Arg Arg Ala Arg Arg Asp Leu1145 1150 1155Pro Gln Leu Gln Pro Gln Pro Gln Pro Gln Pro Gln Arg Asp Leu1160 1165 1170Ile Ser Arg Tyr Ala117535914PRTNeisseria speciesMISC_FEATUREApp domain derivative 35Gly His Thr Tyr Phe Gly Ile Asn Tyr Gln Tyr Tyr Arg Asp Phe Ala1 5 10 15Glu Asn Lys Gly Lys Phe Ala Val Gly Ala Lys Asp Ile Glu Val Tyr20 25 30Asn Lys Lys Gly Glu Leu Val Gly Lys Ser Met Thr Lys Ala Pro Met35 40 45Ile Asp Phe Ser Val Val Ser Arg Asn Gly Val Ala Ala Leu Val Gly50 55 60Asp Gln Tyr Ile Val Ser Val Ala His Asn Gly Gly Tyr Asn Asn Val65 70 75 80Asp Phe Gly Ala Glu Gly Arg Asn Pro Asp Gln His Arg Phe Thr Tyr85 90 95Lys Ile Val Lys Arg Asn Asn Tyr Lys Ala Gly Thr Lys Gly His Pro100 105 110Tyr Gly Gly Asp Tyr His Met Pro Arg Leu His Lys Phe Val Thr Asp115 120 125Ala Glu Pro Val Glu Met Thr Ser Tyr Met Asp Gly Arg Lys Tyr Ile130 135 140Asp Gln Asn Asn Tyr Pro Asp Arg Val Arg Ile Gly Ala Gly Arg Gln145 150 155 160Tyr Trp Arg Ser Asp Glu Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr165 170 175His Ile Ala Ser Ala Tyr Ser Trp Leu Val Gly Gly Asn Thr Phe Ala180 185 190Gln Asn Gly Ser Gly Gly Gly Thr Val Asn Leu Gly Ser Glu Lys Ile195 200 205Lys His Ser Pro Tyr Gly Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp210 215 220Ser Gly Ser Pro Met Phe Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu225 230 235 240Ile Asn Gly Val Leu Gln Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn245 250 255Gly Phe Gln Leu Val Arg Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala260 265 270Gly Asp Thr His Ser Val Phe Tyr Glu Pro Arg Gln Asn Gly Lys Tyr275 280 285Ser Phe Asn Asp Asp Asn Asn Gly Thr Gly Lys Ile Asn Ala Lys His290 295 300Glu His Asn Ser Leu Pro Asn Arg Leu Lys Thr Arg Thr Val Gln Leu305 310 315 320Phe Asn Val Ser Leu Ser Glu Thr Ala Arg Glu Pro Val Tyr His Ala325 330 335Ala Gly Gly Val Asn Ser Tyr Arg Pro Arg Leu Asn Asn Gly Glu Asn340 345 350Ile Ser Phe Ile Asp Glu Gly Lys Gly Glu Leu Ile Leu Thr Ser Asn355 360 365Ile Asn Gln Gly Ala Gly Gly Leu Tyr Phe Gln Gly Asp Phe Thr Val370 375 380Ser Pro Glu Asn Asn Glu Thr Trp Gln Gly Ala Gly Val His Ile Ser385 390 395 400Glu Asp Ser Thr Val Thr Trp Lys Val Asn Gly Val Ala Asn Asp Arg405 410 415Leu Ser Lys Ile Gly Lys Gly Thr Leu His Val Gln Ala Lys Gly Glu420 425 430Asn Gln Gly Ser Ile Ser Val Gly Asp Gly Thr Val Ile Leu Asp Gln435 440 445Gln Ala Asp Asp Lys Gly Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu450 455 460Val Ser Gly Arg Gly Thr Val Gln Leu Asn Ala Asp Asn Gln Phe Asn465 470 475 480Pro Asp Lys Leu Tyr Phe Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn485 490 495Gly His Ser Leu Ser Phe His Arg Ile Gln Asn Thr Asp Glu Gly Ala500 505 510Met Ile Val Asn His Asn Gln Asp Lys Glu Ser Thr Val Thr Ile Thr515 520 525Gly Asn Lys Asp Ile Ala Thr Thr Gly Asn Asn Asn Ser Leu Asp Ser530 535 540Lys Lys Glu Ile Ala Tyr Asn Gly Trp Phe Gly Glu Lys Asp Thr Thr545 550 555 560Lys Thr Asn Gly Arg Leu Asn Leu Val Tyr Gln Pro Ala Ala Glu Asp565 570 575Arg Thr Leu Leu Leu Ser Gly Gly Thr Asn Leu Asn Gly Asn Ile Thr580 585 590Gln Thr Asn Gly Lys Leu Phe Phe Ser Gly Arg Pro Thr Pro His Ala595 600 605Tyr Asn His Leu Asn Asp His Trp Ser Gln Lys Glu Gly Ile Pro Arg610 615 620Gly Glu Ile Val Trp Asp Asn Asp Trp Ile Asn Arg Thr Phe Lys Ala625 630 635 640Glu Asn Phe Gln Ile Lys Gly Gly Gln Ala Val Val Ser Arg Asn Val645 650 655Ala Lys Val Lys Gly Asp Trp His Leu Ser Asn His Ala Gln Ala Val660 665 670Phe Gly Val Ala Pro His Gln Ser His Thr Ile Cys Thr Arg Ser Asp675 680 685Trp Thr Gly Leu Thr Asn Cys Val Glu Lys Thr Ile Thr Asp Asp Lys690 695 700Val Ile Ala Ser Leu Thr Lys Thr Asp Ile Ser Gly Asn Val Asp Leu705 710 715 720Ala Asp His Ala His Leu Asn Leu Thr Gly Leu Ala Thr Leu Asn Gly725 730 735Asn Leu Ser Ala Asn Gly Asp Thr Arg Tyr Thr Val Ser His Asn Ala740 745 750Thr Gln Asn Gly Asn Leu Ser Leu Val Gly Asn Ala Gln Ala Thr Phe755 760 765Asn Gln Ala Thr Leu Asn Gly Asn Thr Ser Ala Ser Gly Asn Ala Ser770 775 780Phe Asn Leu Ser Asp His Ala Val Gln Asn Gly Ser Leu Thr Leu Ser785 790 795 800Gly Asn Ala Lys Ala Asn Val Ser His Ser Ala Leu Asn Gly Asn Val805 810 815Ser Leu Ala Asp Lys Ala Val Phe His Phe Glu Ser Ser Arg Phe Thr820 825 830Gly Gln Ile Ser Gly Gly Lys Asp Thr Ala Leu His Leu Lys Asp Ser835 840 845Glu Trp Thr Leu Pro Ser Gly Thr Glu Leu Gly Asn Leu Asn Leu Asp850 855 860Asn Ala Thr Ile Thr Leu Asn Ser Ala Tyr Arg His Asp Ala Ala Gly865 870 875 880Ala Gln Thr Gly Ser Ala Thr Asp Ala Pro Arg Arg Arg Ser Arg Arg885 890 895Ser Arg Arg Ser Leu Leu Ser Val Thr Pro Pro Thr Ser Val Glu Ser900 905 910Arg Phe361136PRTNeisseria speciesMISC_FEATUREApp domain derivative 36Gly His Thr Tyr Phe Gly Ile Asn Tyr Gln Tyr Tyr Arg Asp Phe Ala1 5 10 15Glu Asn Lys Gly Lys Phe Ala Val Gly Ala Lys Asp Ile Glu Val Tyr20 25 30Asn Lys Lys Gly Glu Leu Val Gly Lys Ser Met Thr Lys Ala Pro Met35

40 45Ile Asp Phe Ser Val Val Ser Arg Asn Gly Val Ala Ala Leu Val Gly50 55 60Asp Gln Tyr Ile Val Ser Val Ala His Asn Gly Gly Tyr Asn Asn Val65 70 75 80Asp Phe Gly Ala Glu Gly Arg Asn Pro Asp Gln His Arg Phe Thr Tyr85 90 95Lys Ile Val Lys Arg Asn Asn Tyr Lys Ala Gly Thr Lys Gly His Pro100 105 110Tyr Gly Gly Asp Tyr His Met Pro Arg Leu His Lys Phe Val Thr Asp115 120 125Ala Glu Pro Val Glu Met Thr Ser Tyr Met Asp Gly Arg Lys Tyr Ile130 135 140Asp Gln Asn Asn Tyr Pro Asp Arg Val Arg Ile Gly Ala Gly Arg Gln145 150 155 160Tyr Trp Arg Ser Asp Glu Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr165 170 175His Ile Ala Ser Ala Tyr Ser Trp Leu Val Gly Gly Asn Thr Phe Ala180 185 190Gln Asn Gly Ser Gly Gly Gly Thr Val Asn Leu Gly Ser Glu Lys Ile195 200 205Lys His Ser Pro Tyr Gly Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp210 215 220Ser Gly Ser Pro Met Phe Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu225 230 235 240Ile Asn Gly Val Leu Gln Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn245 250 255Gly Phe Gln Leu Val Arg Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala260 265 270Gly Asp Thr His Ser Val Phe Tyr Glu Pro Arg Gln Asn Gly Lys Tyr275 280 285Ser Phe Asn Asp Asp Asn Asn Gly Thr Gly Lys Ile Asn Ala Lys His290 295 300Glu His Asn Ser Leu Pro Asn Arg Leu Lys Thr Arg Thr Val Gln Leu305 310 315 320Phe Asn Val Ser Leu Ser Glu Thr Ala Arg Glu Pro Val Tyr His Ala325 330 335Ala Gly Gly Val Asn Ser Tyr Arg Pro Arg Leu Asn Asn Gly Glu Asn340 345 350Ile Ser Phe Ile Asp Glu Gly Lys Gly Glu Leu Ile Leu Thr Ser Asn355 360 365Ile Asn Gln Gly Ala Gly Gly Leu Tyr Phe Gln Gly Asp Phe Thr Val370 375 380Ser Pro Glu Asn Asn Glu Thr Trp Gln Gly Ala Gly Val His Ile Ser385 390 395 400Glu Asp Ser Thr Val Thr Trp Lys Val Asn Gly Val Ala Asn Asp Arg405 410 415Leu Ser Lys Ile Gly Lys Gly Thr Leu His Val Gln Ala Lys Gly Glu420 425 430Asn Gln Gly Ser Ile Ser Val Gly Asp Gly Thr Val Ile Leu Asp Gln435 440 445Gln Ala Asp Asp Lys Gly Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu450 455 460Val Ser Gly Arg Gly Thr Val Gln Leu Asn Ala Asp Asn Gln Phe Asn465 470 475 480Pro Asp Lys Leu Tyr Phe Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn485 490 495Gly His Ser Leu Ser Phe His Arg Ile Gln Asn Thr Asp Glu Gly Ala500 505 510Met Ile Val Asn His Asn Gln Asp Lys Glu Ser Thr Val Thr Ile Thr515 520 525Gly Asn Lys Asp Ile Ala Thr Thr Gly Asn Asn Asn Ser Leu Asp Ser530 535 540Lys Lys Glu Ile Ala Tyr Asn Gly Trp Phe Gly Glu Lys Asp Thr Thr545 550 555 560Lys Thr Asn Gly Arg Leu Asn Leu Val Tyr Gln Pro Ala Ala Glu Asp565 570 575Arg Thr Leu Leu Leu Ser Gly Gly Thr Asn Leu Asn Gly Asn Ile Thr580 585 590Gln Thr Asn Gly Lys Leu Phe Phe Ser Gly Arg Pro Thr Pro His Ala595 600 605Tyr Asn His Leu Asn Asp His Trp Ser Gln Lys Glu Gly Ile Pro Arg610 615 620Gly Glu Ile Val Trp Asp Asn Asp Trp Ile Asn Arg Thr Phe Lys Ala625 630 635 640Glu Asn Phe Gln Ile Lys Gly Gly Gln Ala Val Val Ser Arg Asn Val645 650 655Ala Lys Val Lys Gly Asp Trp His Leu Ser Asn His Ala Gln Ala Val660 665 670Phe Gly Val Ala Pro His Gln Ser His Thr Ile Cys Thr Arg Ser Asp675 680 685Trp Thr Gly Leu Thr Asn Cys Val Glu Lys Thr Ile Thr Asp Asp Lys690 695 700Val Ile Ala Ser Leu Thr Lys Thr Asp Ile Ser Gly Asn Val Asp Leu705 710 715 720Ala Asp His Ala His Leu Asn Leu Thr Gly Leu Ala Thr Leu Asn Gly725 730 735Asn Leu Ser Ala Asn Gly Asp Thr Arg Tyr Thr Val Ser His Asn Ala740 745 750Thr Gln Asn Gly Asn Leu Ser Leu Val Gly Asn Ala Gln Ala Thr Phe755 760 765Asn Gln Ala Thr Leu Asn Gly Asn Thr Ser Ala Ser Gly Asn Ala Ser770 775 780Phe Asn Leu Ser Asp His Ala Val Gln Asn Gly Ser Leu Thr Leu Ser785 790 795 800Gly Asn Ala Lys Ala Asn Val Ser His Ser Ala Leu Asn Gly Asn Val805 810 815Ser Leu Ala Asp Lys Ala Val Phe His Phe Glu Ser Ser Arg Phe Thr820 825 830Gly Gln Ile Ser Gly Gly Lys Asp Thr Ala Leu His Leu Lys Asp Ser835 840 845Glu Trp Thr Leu Pro Ser Gly Thr Glu Leu Gly Asn Leu Asn Leu Asp850 855 860Asn Ala Thr Ile Thr Leu Asn Ser Ala Tyr Arg His Asp Ala Ala Gly865 870 875 880Ala Gln Arg Gly Ser Ala Thr Asp Ala Pro Arg Arg Arg Ser Arg Arg885 890 895Ser Arg Arg Ser Leu Leu Ser Val Thr Pro Pro Thr Ser Val Glu Ser900 905 910Arg Phe Asn Thr Leu Thr Val Asn Gly Lys Leu Asn Gly Gln Gly Thr915 920 925Phe Arg Phe Met Ser Glu Leu Phe Gly Tyr Arg Ser Asp Lys Leu Lys930 935 940Leu Ala Glu Ser Ser Glu Gly Thr Tyr Thr Leu Ala Val Asn Asn Thr945 950 955 960Gly Asn Glu Pro Ala Ser Leu Glu Gln Leu Thr Val Val Glu Gly Lys965 970 975Asp Asn Lys Pro Leu Ser Glu Asn Leu Asn Phe Thr Leu Gln Asn Glu980 985 990His Val Asp Ala Gly Ala Trp Arg Tyr Gln Leu Ile Arg Lys Asp Gly995 1000 1005Glu Phe Arg Leu His Asn Pro Val Lys Glu Gln Glu Leu Ser Asp1010 1015 1020Lys Leu Gly Lys Ala Glu Ala Lys Lys Gln Ala Glu Lys Asp Asn1025 1030 1035Ala Gln Ser Leu Asp Ala Leu Ile Ala Ala Gly Arg Asp Ala Val1040 1045 1050Glu Lys Thr Glu Ser Val Ala Glu Pro Ala Arg Gln Ala Gly Gly1055 1060 1065Glu Asn Val Gly Ile Met Gln Ala Glu Glu Glu Lys Lys Arg Val1070 1075 1080Gln Ala Asp Lys Asp Thr Ala Leu Ala Lys Gln Arg Glu Ala Glu1085 1090 1095Thr Arg Pro Ala Thr Thr Ala Phe Pro Arg Ala Arg Arg Ala Arg1100 1105 1110Arg Asp Leu Pro Gln Leu Gln Pro Gln Pro Gln Pro Gln Pro Gln1115 1120 1125Arg Asp Leu Ile Ser Arg Tyr Ala1130 113537221PRTNeisseria speciesMISC_FEATUREApp domain derivative 37Asn Thr Leu Thr Val Asn Gly Lys Leu Asn Gly Gln Gly Thr Phe Arg1 5 10 15Phe Met Ser Glu Leu Phe Gly Tyr Arg Ser Asp Lys Leu Lys Leu Ala20 25 30Glu Ser Ser Glu Gly Thr Tyr Thr Leu Ala Val Asn Asn Thr Gly Asn35 40 45Glu Pro Ala Ser Leu Glu Gln Leu Thr Trp Glu Gly Lys Asp Asn Lys50 55 60Pro Leu Ser Glu Asn Leu Asn Phe Thr Leu Gln Asn Glu His Val Asp65 70 75 80Ala Gly Ala Trp Arg Tyr Gln Leu Ile Arg Lys Asp Gly Glu Phe Arg85 90 95Leu His Asn Pro Val Lys Glu Gln Glu Leu Ser Asp Lys Leu Gly Lys100 105 110Ala Glu Ala Lys Lys Gln Ala Glu Lys Asp Asn Ala Gln Ser Leu Asp115 120 125Ala Leu Ile Ala Ala Gly Arg Asp Ala Val Glu Lys Thr Glu Ser Val130 135 140Ala Glu Pro Ala Arg Gln Ala Gly Gly Glu Asn Val Gly Ile Met Gln145 150 155 160Ala Glu Glu Glu Lys Lys Arg Val Gln Ala Asp Lys Asp Thr Ala Leu165 170 175Ala Lys Gln Arg Glu Ala Glu Thr Arg Pro Ala Thr Thr Ala Phe Pro180 185 190Arg Ala Arg Arg Ala Arg Arg Asp Leu Pro Gln Leu Gln Pro Gln Pro195 200 205Gln Pro Gln Pro Gln Arg Asp Leu Ile Ser Arg Tyr Ala210 215 22038279PRTNeisseria speciesMISC_FEATUREApp domain derivative 38Asn Ser Gly Leu Ser Glu Phe Ser Ala Thr Leu Asn Ser Val Phe Ala1 5 10 15Val Gln Asp Glu Leu Asp Arg Val Phe Ala Glu Asp Arg Arg Asn Ala20 25 30Val Trp Thr Ser Gly Ile Arg Asp Thr Lys His Tyr Arg Ser Gln Asp35 40 45Phe Arg Ala Tyr Arg Gln Gln Thr Asp Leu Arg Gln Ile Gly Met Gln50 55 60Lys Asn Leu Gly Ser Gly Arg Val Gly Ile Leu Phe Ser His Asn Arg65 70 75 80Thr Glu Asn Thr Phe Asp Asp Gly Ile Gly Asn Ser Ala Arg Leu Ala85 90 95His Gly Ala Val Phe Gly Gln Tyr Gly Ile Asp Arg Phe Tyr Ile Gly100 105 110Ile Ser Ala Gly Ala Gly Phe Ser Ser Gly Ser Leu Ser Asp Gly Ile115 120 125Gly Gly Lys Ile Arg Arg Arg Val Leu His Tyr Gly Ile Gln Ala Arg130 135 140Tyr Arg Ala Gly Phe Gly Gly Phe Gly Ile Glu Pro His Ile Gly Ala145 150 155 160Thr Arg Tyr Phe Val Gln Lys Ala Asp Tyr Arg Tyr Glu Asn Val Asn165 170 175Ile Ala Thr Pro Gly Leu Ala Phe Asn Arg Tyr Arg Ala Gly Ile Lys180 185 190Ala Asp Tyr Ser Phe Lys Pro Ala Gln His Ile Ser Ile Thr Pro Tyr195 200 205Leu Ser Leu Ser Tyr Thr Asp Ala Ala Ser Gly Lys Val Arg Thr Arg210 215 220Val Asn Thr Ala Val Leu Ala Gln Asp Phe Gly Lys Thr Arg Ser Ala225 230 235 240Glu Trp Gly Val Asn Ala Glu Ile Lys Gly Phe Thr Leu Ser Leu His245 250 255Ala Ala Ala Ala Lys Gly Pro Gln Leu Glu Ala Gln His Ser Ala Gly260 265 270Ile Lys Leu Gly Tyr Arg Trp27539501PRTNeisseria speciesMISC_FEATUREApp domain derivative 39Asn Thr Leu Thr Val Asn Gly Lys Leu Asn Gly Gln Gly Thr Phe Arg1 5 10 15Phe Met Ser Glu Leu Phe Gly Tyr Arg Ser Asp Lys Leu Lys Leu Ala20 25 30Glu Ser Ser Glu Gly Thr Tyr Thr Leu Ala Val Asn Asn Thr Gly Asn35 40 45Glu Pro Ala Ser Leu Glu Gln Leu Thr Val Val Glu Gly Lys Asp Asn50 55 60Lys Pro Leu Ser Glu Asn Leu Asn Phe Thr Leu Gln Asn Glu His Val65 70 75 80Asp Ala Gly Ala Trp Arg Tyr Gln Leu Ile Arg Lys Asp Gly Glu Phe85 90 95Arg Leu His Asn Pro Val Lys Glu Gln Glu Leu Ser Asp Lys Leu Gly100 105 110Lys Ala Glu Ala Lys Lys Gln Ala Glu Lys Asp Asn Ala Gln Ser Leu115 120 125Asp Ala Leu Ile Ala Ala Gly Arg Asp Ala Val Glu Lys Thr Glu Ser130 135 140Val Ala Glu Pro Ala Arg Gln Ala Gly Gly Glu Asn Val Gly Ile Met145 150 155 160Gln Ala Glu Glu Glu Lys Lys Arg Val Gln Ala Asp Lys Asp Thr Ala165 170 175Leu Ala Lys Gln Arg Glu Ala Glu Thr Arg Pro Ala Thr Thr Ala Phe180 185 190Pro Arg Ala Arg Arg Ala Arg Arg Asp Leu Pro Gln Leu Gln Pro Gln195 200 205Pro Gln Pro Gln Pro Gln Arg Asp Leu Ile Ser Arg Tyr Ala Asn Ser210 215 220Gly Leu Ser Glu Phe Ser Ala Thr Leu Asn Ser Val Phe Ala Val Gln225 230 235 240Asp Glu Leu Asp Arg Val Phe Ala Glu Asp Arg Arg Asn Ala Val Trp245 250 255Thr Ser Gly Ile Arg Asp Thr Lys His Tyr Arg Ser Gln Asp Phe Arg260 265 270Ala Tyr Arg Gln Gln Thr Asp Leu Arg Gln Ile Gly Met Gln Lys Asn275 280 285Leu Gly Ser Gly Arg Val Gly Ile Leu Phe Ser His Asn Arg Thr Glu290 295 300Asn Thr Phe Asp Asp Gly Ile Gly Asn Ser Ala Arg Leu Ala His Gly305 310 315 320Ala Val Phe Gly Gln Tyr Gly Ile Asp Arg Phe Tyr Ile Gly Ile Ser325 330 335Ala Gly Ala Gly Phe Ser Ser Gly Ser Leu Ser Asp Gly Ile Gly Gly340 345 350Lys Ile Arg Arg Arg Val Leu His Tyr Gly Ile Gln Ala Arg Tyr Arg355 360 365Ala Gly Phe Gly Gly Phe Gly Ile Glu Pro His Ile Gly Ala Thr Arg370 375 380Tyr Phe Val Gln Lys Ala Asp Tyr Arg Tyr Glu Asn Val Asn Ile Ala385 390 395 400Thr Pro Gly Leu Ala Phe Asn Arg Tyr Arg Ala Gly Ile Lys Ala Asp405 410 415Tyr Ser Phe Lys Pro Ala Gln His Ile Ser Ile Thr Pro Tyr Leu Ser420 425 430Leu Ser Tyr Thr Asp Ala Ala Ser Gly Lys Val Arg Thr Arg Val Asn435 440 445Thr Ala Val Leu Ala Gln Asp Phe Gly Lys Thr Arg Ser Ala Glu Trp450 455 460Gly Val Asn Ala Glu Ile Lys Gly Phe Thr Leu Ser Leu His Ala Ala465 470 475 480Ala Ala Lys Gly Pro Gln Leu Glu Ala Gln His Ser Ala Gly Ile Lys485 490 495Leu Gly Tyr Arg Trp5004036PRTNeisseria speciesMISC_FEATUREResidues of NadA 40Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala Leu Ala1 5 10 15Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala Leu Asn Lys Leu20 25 30Gly Glu Asn Ile3541270DNANeisseria speciesmisc_featurepartial coding sequence for NadA 41acccatatcc tgacaaaatt aagacacgac accggcagaa ttgacatcag cataatatgc 60acatattaac agatattaat gccgaactac ctaactgcaa gaattaaata aataaataaa 120taaataaata aataaataaa ttgcgacaat gtattgtata tatgcctcct ttcatatata 180ctttaatatg taaacaaact tggtggggat aaaatactta caaaagattt ccgccccatt 240ttttatccac tcacaaaggt aatgagcatg 2704216DNANeisseria speciesmisc_featurePartial sequence of MenA genome 42tttccattcc aaacgc 164328PRTNeisseria speciesMISC_FEATUREN. meningitidis 43Arg Arg Ser Arg Arg Ser Leu Leu Ser Val Thr Pro Pro Ala Ser Ala1 5 10 15Glu Ser His Phe Asn Thr Leu Thr Val Asn Gly Lys20 254433PRTNeisseria speciesMISC_FEATUREN. meningitidis 44Arg Arg Ala Arg Arg Asp Leu Pro Gln Pro Gln Pro Gln Pro Gln Pro1 5 10 15Gln Pro Gln Arg Asp Glu Lys Leu Ile Ser Arg Tyr Ala Asn Ser Gly20 25 30Leu4521PRTHaemophilus speciesMISC_FEATUREH. influenzae 45Arg Arg Ala Ala Arg Ala Ala Phe Pro Asp Thr Leu Pro Asp Gln Ser1 5 10 15Leu Leu Asn Ala Leu204626PRTNeisseria speciesMISC_FEATUREN. gonorrhoeae 46Arg Arg Arg Arg Arg Ala Ile Leu Pro Arg Pro Pro Ala Pro Val Phe1 5 10 15Ser Leu Asp Asp Tyr Asp Ala Lys Asp Asn20 254728PRTBordetella speciesMISC_FEATUREB. pertussis 47Arg Arg Ala Arg Arg Ala Leu Arg Gln Asp Phe Phe Thr Pro Gly Ser1 5 10 15Val Val Arg Ala Gln Gly Asn Val Thr Val Gly Arg20 25484PRTBordetella speciesMISC_FEATUREB. bronchiseptica 48Ser Asn Ala Leu1494PRTSerratia speciesMISC_FEATURES. marcescens 49Leu Asn Ser Gly1504PRTBordetella speciesMISC_FEATUREB. pertussis 50Val Asn Ala Ala1514PRTHaemophilus speciesMISC_FEATUREH. influenzae 51Leu Asn Ala Leu1

Claims

1. An isolated antibody that specifically binds to NMB1994, wherein NMB1994 comprises residues 24-343 of SEQ ID NO:2, or residues 24-350 of SEQ ID NO:3.

2. The antibody of claim 1, wherein said antibody is a polyclonal antibody.

3. The antibody of claim 1, wherein said antibody is a monoclonal antibody.

4. The antibody of claim 1, wherein said antibody specifically binds to said NMB1994 comprising residues 24-343 of SEQ ID NO:2.

5. The antibody of claim 1, wherein said antibody specifically binds to said NMB1994 comprising residues 24-350 of SEQ ID NO:3.

6. The antibody of claim 1, wherein said antibody is able to bind to a bacterium of a Neisseria meningitidis strain that expresses NadA.

7. The antibody of claim 1, wherein said antibody is able to induce complement-mediated killing of a Neisseria meningitidis strain that expresses NadA.

8. The antibody of claim 7, wherein said strain is selected from the group consisting of 2996, C11, F6124 and MC58.

9. The antibody of claim 1, wherein said antibody is able to reduce bacteremia in a subject exposed to a Neisseria meningitidis strain that expresses NadA.

10. A method, comprising: administering an isolated antibody that specifically binds to NMB1994 to a subject, wherein NMB1994 comprises residues 24-343 of SEQ ID NO:2, or residues 24-350 of SEQ ID NO:3.

11. The method of claim 10, wherein said subject has been exposed to a Neisseria meningitidis strain that expresses NadA.

12. The method of claim 11, wherein said administering reduces bacteremia.

13. The method of claim 10, wherein said antibody specifically binds to said NMB1994 comprising residues 24-343 of SEQ ID NO:2.

14. The method of claim 10, wherein said antibody specifically binds to said NMB1994 comprising residues 24-350 of SEQ ID NO:3.

15. A method, comprising: contacting a bacterium of a Neisseria meningitidis strain that expresses NadA with an isolated antibody that specifically binds to NMB1994, wherein NMB1994 comprises residues 24-343 of SEQ ID NO:2, or residues 24-350 of SEQ ID NO:3.

16. The method of claim 15, wherein said antibody binds to said bacterium.

17. The method of claim 16, wherein the ability of said bacterium to bind to an epithelial cell is blocked when bound to said antibody

18. A method for preventing the attachment of a Neisseria meningitidis cell to an epithelial cell through NMB1994 mediated adhesion in a human subject by administering to the human subject an antibody that specifically binds to NMB1994, wherein NMB1994 comprises residues 24-343 of SEQ ID NO:2 or residues 24-350 of SEQ ID NO:3, and wherein the antibody inhibits NMB1994's ability to bind to the epithelial cell.

19. The method of claim 18, wherein NMB1994 comprises residues 24-343 of SEQ ID NO:2.

20. The method of claim 18, wherein NMB1994 comprises residues 24-350 of SEQ ID NO:3.