HYBRID AND TANDEM EXPRESSION OF NEISSERIAL PROTEINS

Abstract

Two or more Neisserial proteins are joined such that they are translated as a single polypeptide chain. Hybrid proteins are represented by the formula NH.sub.2-A-[--X-L-].sub.n--B--COOH where X is an amino acid sequence, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is an integer greater than 1. Proteins where each of the n --X-- moieties shares sequence identity to each other --X-- moiety, the protein is a `tandem protein`.

Description

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

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application is a Continuation of U.S. patent application Ser. No. 14/739,985, filed Jun. 15, 2015, which is a Continuation of U.S. patent application Ser. No. 14/305,979, filed Jun. 16, 2014, now U.S. Pat. No. 9,056,075, which is a Divisional of U.S. patent application Ser. No. 13/366,252, filed Feb. 3, 2012, now U.S. Pat. No. 8,840,907, which is a Divisional of U.S. patent application Ser. No. 10/488,786, with an international filing date of Sep. 6, 2002, now U.S. Pat. No. 8,980,277, which is the National Stage of International Patent Application No. PCT/IB2002/003904, filed Sep. 6, 2002, which claims the benefit of United Kingdom Patent Application No. 0121591.2, filed Sep. 6, 2001, each of which are hereby incorporated by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

[0003] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 303822001703SubSeqList.txt, date recorded: Feb. 15, 2018, size: 72 KB).

TECHNICAL FIELD

[0004] This invention is in the field of protein expression. In particular, it relates to the expression of proteins from Neisseria (e.g. N. gonorrhoeae or, preferably, N. meningitidis).

BACKGROUND ART

[0005] References 1 and 2 disclose alternative and improved approaches for the expression of the Neisserial proteins disclosed in references 3 to 4 5 6. One such method is to produce `hybrid` proteins in which two or more Neisserial proteins are expressed as a single polypeptide chain. This approach offers two advantages. First, a protein that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem. Second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two separately-useful proteins.

[0006] It is an object of the present invention to provide further alternative and improved approaches for the expression of Neisserial proteins.

DISCLOSURE OF THE INVENTION

[0007] Hybrid Proteins

[0008] Thus the invention provides a method for the simultaneous expression of two or more (e.g. 3, 4, 5, 6 or more) Neisserial proteins, in which said two or more proteins are joined such that they are translated as a single polypeptide chain. In general, the hybrid proteins of the invention can be represented by the formula: NH.sub.2-A-[--X-L-].sub.n--B--COOH

wherein X is an amino acid sequence, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is an integer greater than 1.

[0009] The value of n is between 2 and x, and the value of x is typically 3, 4, 5, 6, 7, 8, 9 or 10. Preferably n is 2, 3 or 4; it is more preferably 2 or 3; most preferably, n=2.

[0010] The --X-- Moieties

[0011] There are two main groups of hybrid proteins according to the invention. These two groups are not mutually exclusive.

[0012] In the first group, each --X-- moiety is:

[0013] (a) an orf1, orf4, orf25, orf40, orf46.1, orf83, NMB1343, 230, 233, 287, 292, 594, 687, 736, 741, 907, 919, 936, 953, 961 or 983 amino acid sequence;

[0014] (b) an amino acid sequence having sequence identity to an amino acid sequence from (a); or

[0015] (c) an amino acid sequence comprising a fragment of an amino acid sequence from (a).

[0016] A preferred subset of (a) is: orf46.1, 230, 287, 741, 919, 936, 953, 961 and 983. A more preferred subset of (a) is: orf46.1, 287, 741 and 961. FIG. 3 shows preferred hybrid proteins.

[0017] In the second group, the hybrid protein comprises a first --X-- moiety (--X.sub.a--) and a second --X-- moiety (--X.sub.b--). The --X.sub.a-- moiety has one of the following amino acid sequences:

[0018] (d) the 446 even SEQ IDs (i.e. 2, 4, 6, . . . , 890, 892) disclosed in reference 3.

[0019] (e) the 45 even SEQ IDs (i.e. 2, 4, 6, . . . , 88, 90) disclosed in reference 4;

[0020] (f) the 1674 even SEQ IDs 2-3020, even SEQ IDs 3040-3114, and all SEQ IDs 3115-3241, disclosed in reference 5;

[0021] (g) the 2160 amino acid sequences NMB0001 to NMB2160 from reference 7; or

[0022] (h) an amino acid sequence disclosed in reference 1 or reference 2.

[0023] The --X.sub.b-- moiety is related to --X.sub.a-- such that: (i) --X.sub.b-- has sequence identity to --X.sub.a--, and/or (j) --X.sub.b-comprises a fragment of --X.sub.a--.

[0024] Examples of this second type of hybrid protein include proteins in which two or more --X-- moieties are identical, or in which they are variants of the same protein e.g. two polymorphic forms of the same protein may be expressed as --X.sub.a--X.sub.b--, and three polymorphic forms may be expressed as --X.sub.a--X.sub.b--X.sub.c-- etc.

[0025] The --X.sub.a-- and --X.sub.b-- moieties may be in either order from N-terminus to C-terminus.

[0026] The --X.sub.a-- moiety is preferably an orf1, orf4, orf25, orf40, orf46.1, orf83, NMB1343, 230, 233, 287, 292, 594, 687, 736, 741, 907, 919, 936, 953, 961 or 983 amino acid sequence. The --X.sub.a-- moiety is more preferably an orf46.1, 230, 287, 741, 919, 936, 953, 961 or 983 amino acid sequence. The --X.sub.a-moiety is most preferably an orf46.1, 287, 741 or 961 amino acid sequence.

[0027] In proteins where each of the n --X-- moieties shares sequence identity to each other --X-- moiety, the protein is referred to as a `tandem protein`. Tandem proteins in which n=2 are preferred.

[0028] The degree of `sequence identity` referred to in (b) and (i) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, up to 100%). This includes mutants, homologs, orthologs, allelic variants etc. [e.g. see ref. 8]. 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 as an indication of functional equivalence.

[0029] The `fragment` referred to in (c) and (j) should consist of least m consecutive amino acids from an amino acid sequence from (a), (d), (e), (f), (g) or (h) and, depending on the particular sequence, m is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). Preferably the fragment comprises an epitope from an amino acid sequence from (a), (d), (e), (f), (g) or (h). Preferred fragments are those disclosed in references 9 and 10.

[0030] Preferred (c) and (j) fragments are C- and/or N-terminal truncations (e.g. .DELTA.1-287, .DELTA.2-287 etc.).

[0031] Preferred (b), (c), (i) and (j) sequences omit poly-glycine sequences. This has been found to aid expression [ref. 2]. Poly-glycine sequences can be represented as (Gly).sub.g, where g.gtoreq.3 (e.g. 4, 5, 6, 7, 8, 9 or more). If a --X-- moiety includes a poly-glycine sequence in its wild-type form, it is preferred to omit this sequence in the hybrid proteins of the invention. This may be by disrupting or removing the (Gly).sub.g--by deletion (e.g. CGGGGS.fwdarw.CGGGS, CGGS, CGS or CS), by substitution (e.g. CGGGGS.fwdarw.CGXGGS, CGXXGS, CGXGXS etc.), and/or by insertion (e.g. CGGGGS.fwdarw.CGGXGGS, CGXGGGS, etc.). Deletion of (Gly).sub.g is preferred, and deletion of the N-terminus portion of a protein up to and including the poly-glycine sequence (e.g. deletion of residues 1-32 in SEQ ID 1) is referred to herein as `.DELTA.G`. Poly-glycine omission is particularly useful for proteins 287, 741, 983 and Tbp2 (.DELTA.G287, .DELTA.G741, .DELTA.G983 and .DELTA.GTbp2--references 1 & 2).

[0032] Preferred (c) and (j) fragments omit complete protein domains. This is particularly useful for protein 961, 287, and ORF46. Once a protein has been notional divided into domains, (c) and (j) fragments can omit one or more of these domains (e.g. 287B, 287C, 287BC, ORF46.sub.1-433, ORF46.sub.434-608, 961c--reference 2; FIGS. 4 and 5 herein).

[0033] 287 protein has been notionally split into three domains, referred to as A, B & C (see FIG. 5 of reference 2). Domain B aligns with IgA proteases, domain C aligns with transferrin-binding proteins, and domain A shows no strong alignment with database sequences. An alignment of polymorphic forms of 287 is disclosed in reference 8.

[0034] ORF46 has been notionally split into two domains--a first domain (amino acids 1-433; ORF46.1) which is well-conserved between species and serogroups, and a second domain (amino acids 434-608) which is not well-conserved. The second domain is preferably deleted, leaving ORF46.1. An alignment of polymorphic forms of ORF46 is disclosed in reference 8.

[0035] 961 protein has been notionally split into several domains (FIG. 4).

[0036] If a --X-- moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the hybrid proteins of the invention. Where the leader peptide is omitted, this is a preferred example of an amino acid sequence within (c) and (j). In one embodiment, the leader peptides will be deleted except for that of the --X-- moiety located at the N-terminus of the hybrid protein i.e. the leader peptide of X.sub.1 will be retained, but the leader peptides of X.sub.2 . . . X.sub.n will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of X.sub.1 as moiety -A-.

[0037] When n=2, preferred pairs of --X-- moieties are: .DELTA.G287 and 230; .DELTA.G287 and 936; .DELTA.G287 and 741; 961c and 287; 961c and 230; 961c and 936; 961cL and 287; 961cL and 230; 961cL and 936; ORF46.1 and 936; ORF46.1 and 230; 230 and 961; 230 and 741; 936 and 961; 936 and 741. When n=2, preferred pairs of --X-- moieties for tandem proteins are: .DELTA.G741 and 741; .DELTA.G287 and 287. More specifically, the following combinations of X.sub.1 and X.sub.2 are preferred when n=2:

TABLE-US-00001 X.sub.1 X.sub.2 .DELTA.G287 230 .DELTA.G287 936 .DELTA.G287 741 .DELTA.G287 961 .DELTA.G287 ORF46.1 .DELTA.G287 919 .DELTA.G287 953 961c 287 961c 230 961c 936 961c 741 961c 983 961c .DELTA.G983 961c ORF46.1 961 ORF46.1 961cL 287 961cL 230 961cL 936 ORF46.1 936 ORF46.1 230 ORF46.1 741 ORF46.1 .DELTA.G741 ORF46.1 983 ORF46.1 .DELTA.G983 230 961 230 741 230 .DELTA.G741 936 961 936 741 936 .DELTA.G741 .DELTA.G741 741 ORF46.1 983 .DELTA.G741 ORF46.1 .DELTA.G741 983 .DELTA.G741 961 .DELTA.G741 961c .DELTA.G983 ORF46.1 .DELTA.G983 961 .DELTA.G983 961c 230 .DELTA.G287 936 .DELTA.G287 741 .DELTA.G287 961 .DELTA.G287 ORF46.1 .DELTA.G287 919 .DELTA.G287 953 .DELTA.G287 287 961c 230 961c 936 961c 741 961c 983 961c .DELTA.G983 961c ORF46.1 961c ORF46.1 961 287 961cL 230 961cL 936 961cL 936 ORF46.1 230 ORF46.1 741 ORF46.1 .DELTA.G741 ORF46.1 983 ORF46.1 .DELTA.G983 ORF46.1 961 230 741 230 .DELTA.G741 230 961 936 741 936 .DELTA.G741 936 .DELTA.G287 287 983 ORF46.1 ORF46.1 .DELTA.G741 983 .DELTA.G741 961 .DELTA.G741 961c .DELTA.G741 ORF46.1 .DELTA.G983 961 .DELTA.G983 961c .DELTA.G983

[0038] Where 287 is used in full-length form, it is preferably at the C-terminal end of a hybrid protein; if it is to be used at the N-terminus, if is preferred to use a .DELTA.G form of 287. Similarly, Where 741 is used in full-length form, it is preferably at the C-terminal end of a hybrid protein; if it is to be used at the N-terminus, if is preferred to use a .DELTA.G form of 741.

[0039] The -L- Moieties

[0040] For each n instances of [--X-L-], linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH.sub.2--X.sub.1-L.sub.1-X.sub.2-L.sub.2-COOH, NH.sub.2--X.sub.1--X.sub.2--COOH, NH.sub.2--X.sub.1-L.sub.1-X.sub.2--COOH, NH.sub.2--X.sub.1--X.sub.2-L.sub.2-COOH, etc.

[0041] Linker amino acid sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences which facilitate cloning, poly-glycine linkers (i.e. Gly, where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. A useful linker is GSGGGG (SEQ ID 27), with the Gly-Ser dipeptide being formed from a BamHI restriction site, thus aiding cloning and manipulation, and the Gly.sub.4 tetrapeptide being a typical poly-glycine linker.

[0042] If X.sub.n+1 is a .DELTA.G protein and L.sub.n is a glycine linker, this may be equivalent to X.sub.n+1 not being a .DELTA.G protein and L.sub.n being absent.

[0043] The -A- Moiety

[0044] -A- is an optional N-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e.g. histidine tags i.e. His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art. If X.sub.1 lacks its own N-terminus methionine, -A- may be a methionine residue.

[0045] The --B-- Moiety

[0046] --B-- is an optional C-terminal amino acid sequence. This will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences which facilitate cloning or purification (e.g. comprising histidine tags i.e. His.sub.n where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences which enhance protein stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

[0047] Polymorphic Forms of Proteins

[0048] The invention can use amino acid sequences from any strains of N. meningitidis. References to a particular protein (e.g. `287`, or `ORF46.1`) therefore include that protein from any strain. Sequence variations between strains are included within (b), (c), (i) and (j).

[0049] Reference sequences from N. meningitidis serogroup B include:

TABLE-US-00002 Protein Reference orf1 Ref. 3, SEQ ID 650 orf25 Ref. 3, SEQ ID 684 orf46 Ref. 6, SEQ ID 1049 NMB1343 Ref. 7, NMB1343 233 Ref. 5, SEQ ID 860 292 Ref. 5, SEQ ID 1220 687 Ref. 5, SEQ ID 2282 741 Ref. 5, SEQ ID 2536 919 Ref. 5, SEQ ID 3070 953 Ref. 5, SEQ ID 2918 983 Ref. 7, NMB1969 orf4 Ref. 3, SEQ ID 218 orf40 Ref. 4, SEQ ID 4 orf83 Ref. 3, SEQ ID 314 230 Ref. 5, SEQ ID 830 287 Ref. 5, SEQ ID 3104 594 Ref. 5, SEQ ID 1862 736 Ref. 5, SEQ ID 2506 907 Ref. 5, SEQ ID 2732 936 Ref. 5, SEQ ID 2884 961 Ref. 5, SEQ ID 940

[0050] Reference 8 discloses polymorphic forms of proteins ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953. Polymorphic forms of 961 are disclosed in references 11 & 12. Any of these polymorphic forms may be used in accordance with the present invention.

[0051] The sequence listing herein includes polymorphic forms of proteins 741 (SEQ IDs 1-22) and NMB1343 (SEQ IDs 23-24) which have been identified.

[0052] Serogroups and Strains

[0053] Preferred proteins of the invention comprise --X-- moieties having an amino acid sequence found in N. meningitidis serogroup B. Within a single protein of the invention, individual --X-- moieties may be from one or more strains. Where n=2, for instance, X.sub.2 may be from the same strain as X.sub.1 or from a different strain. Where n=3, the strains might be (i) X.sub.1=X.sub.2=X.sub.3 (ii) X.sub.1=X.sub.2X.sub.3 (iii) X.sub.1X.sub.2=X.sub.3 (iv) X.sub.1X.sub.2X.sub.3 or (v) X.sub.1=X.sub.3X.sub.2, etc.

[0054] Within serogroup B, preferred --X-- moieties are from strains 2996, MC58, 95N477, or 394/98. Strain 95N477 is sometimes referred to herein as `ET37`, this being its electrophoretic type. Strain 394/98 is sometimes referred to herein as `nz`, as it is a New Zealand strain.

[0055] Where a form of 287 is used, this is preferably from strain 2996 or from strain 394/98.

[0056] Where a form of 741 is used, this is preferably from serogroup B strains MC58, 2996, 394/98, or 95N477, or from serogroup C strain 90/18311.

[0057] Where a form of 961 is used, this is preferably from strain 2996.

[0058] Strains are indicated as a subscript e.g. 741.sub.MC58 is protein 741 from strain MC58. Unless otherwise stated, proteins mentioned herein (e.g. with no subscript) are from N. meningitidis strain 2996, which can be taken as a `reference` strain. It will be appreciated, however, that the invention is not in general limited by strain. As mentioned above, general references to a protein (e.g. `287`, `919` etc.) may be taken to include that protein from any strain. This will typically have sequence identity to 2996 of 90% or more (eg. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

[0059] Domain-Based Expression of Protein 961

[0060] References 1 and 2 disclose how a protein can be notionally divided into domains and how the protein can be manipulated based on these domains. The present invention extends the application of this approach to protein 961 (also known as `NadA` [11,12]).

[0061] In N. meningitidis serogroup B strain 2996, NadA has 405 amino acids. This protein has notionally been divided into the following nine domains (FIG. 4):

TABLE-US-00003 Domain name Amino acids 961-1 `L` 1-23 961-2 24-87 961-3 88-143 961-4 144-180 961-5 181-268 961-6 269-286 961-7 287-330 961-8 331-350 961-9 351-405

[0062] This information can be used to locate the same domains in other forms of 961.

[0063] These domains have been deleted from 961 in strain 2996 in various ways (FIG. 5). Preferred fragments of 961 omit one or more of these nine domains e.g. the following:

[0064] 961-2 to 961-5 (`961a`)

[0065] 961-6 to 961-9 (`961b`)

[0066] 961-1 to 961-8 (`961cL`)

[0067] 961-2 to 961-8 (`961c`)

[0068] 961-2 to 961-6 and amino acids 287-325 from domain 961-7 (`961d`)

[0069] 961-2 to 961-8 and amino acids 351-383 from domain 961-9 (`961.DELTA.1`)

[0070] 961-1 to 961-8 and amino acids 351-383 from domain 961-9 (`961.DELTA.1L`)

[0071] 961-1 to 961-7 and amino acids 331-343 from domain 961-8 (`961cL-.DELTA.aro`)

[0072] 961-1 to 961-6 and amino acids 287-315 from domain 961-7 (`961cL-.DELTA.cc`)

[0073] 961-1 to 961-5 (`961aL`)

[0074] 961-1 to 961-4 (`961aL-.DELTA.1`)

[0075] 961-1 to 961-3 (`961aL-.DELTA.2`)

[0076] 961-1 to 961-2 (`961aL-.DELTA.3`)

[0077] These thirteen fragments (and sub-fragments thereof missing 1, 2, 3, 4 or 5 amino acids at either or both ends) are preferred (c) and (j) fragments, but they may also be expressed in their own right i.e. not in the form of a hybrid protein of the invention. Thus the invention provides a protein comprising one of these fragments, providing that the protein is not full-length 961 and is not a protein specifically disclosed in reference 1 or 2. This protein may be a fusion protein (e.g. a GST-fusion or a His-tag fusion).

[0078] Sequences

[0079] The invention also provides a protein having an amino acid sequence from SEQ IDs 1 to 24. It also provides proteins and nucleic acid having sequence identity to these. As described above, the degree of `sequence identity` is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).

[0080] The invention also provides nucleic acid encoding such proteins.

[0081] Furthermore, the invention provides nucleic acid which can hybridise to this nucleic acid, preferably under "high stringency" conditions (eg. 65.degree. C. in a 0.1.times.SSC, 0.5% SDS solution).

[0082] The invention also provides nucleic acid encoding proteins according to the invention.

[0083] It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).

[0084] Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).

[0085] In addition, 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.

[0086] Mixtures

[0087] The invention also provides a composition comprising two or more (i.e. 2, 3, 4, 5, 6 or 7) of the following proteins:

[0088] (1) 287

[0089] (2) 741

[0090] (3) ORF46.1

[0091] (4) 961

[0092] (5) NH.sub.2-A-[--X-L-].sub.n--B--COOH, wherein n 2, X.sub.1=287, X.sub.2=953

[0093] (6) NH.sub.2-A-[--X-L-].sub.n--B--COOH, wherein n 2, X.sub.1=287, X.sub.2=919

[0094] (7) NH.sub.2-A-[--X-L-].sub.n--B--COOH, wherein n 2, X.sub.1=287, X.sub.2=961

[0095] The mixture may include one or both of the following proteins, either in combination with two or more of (1) to (7), or in combination with only one of (1) to (7):

[0096] (8) NH.sub.2-A-[--X-L-].sub.n--B--COOH, wherein n 2, X.sub.1=287, X.sub.2=741

[0097] (9) NH.sub.2-A-[--X-L-].sub.n--B--COOH, wherein n 2, X.sub.1=936, X.sub.2=741

[0098] Where proteins 287 and 741 are included in the mixture (i.e. in protein 1, 2, 5, 6, 7 or 8), they may be in the `.DELTA.G` form. Where protein 961 is included, it is preferably in the form of `961c` in which the N-terminus leader and C-terminus membrane anchor are absent [e.g. see refs. 1, 2 & 11].

[0099] A preferred mixture comprises the following three proteins:

[0100] (1) 961c, preferably 961c.sub.2996 (e.g. SEQ ID 31 herein);

[0101] (2) NH.sub.2-A-[--X-L-].sub.n--B--COOH, wherein n is 2, --X.sub.1-- is .DELTA.G287 (preferably .DELTA.G287.sub.NZ), --X.sub.2-- is 953 (preferably 953.sub.2996) lacking its leader peptide, -L.sub.1- is GSGGGG, and -A- comprises a N-terminus methionine (e.g. -A- is M or MA) (e.g. SEQ IDs 28 & 29 herein); and

[0102] (3) NH.sub.2-A-[--X-L-].sub.n--B--COOH, wherein n 2, X.sub.1=936 (preferably 936.sub.2996), X.sub.2=.DELTA.G741 (preferably .DELTA.G741.sub.MC58), L.sub.1=GSGGGG (e.g. SEQ ID 30 herein).

[0103] The mixtures may also comprise N. meningitidis outer membrane vesicles.

[0104] Heterologous Host

[0105] Whilst expression of the proteins of the invention may take place in Neisseria, the present invention preferably utilises a heterologous host. The heterologous host may be prokaryotic (e.g. a bacterium) or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonenna typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g. M. tuberculosis), yeast etc.

[0106] Vectors etc.

[0107] The invention provides (a) nucleic acid encoding the proteins described above (b) vectors comprising these nucleic acid sequences (c) host cells containing said vectors (d) compositions comprising the proteins or nucleic acids of the invention, which may be suitable as immunogenic compositions (e.g. vaccines) or as diagnostic reagents (e) these compositions for use as medicaments (e.g. as vaccines) or as diagnostic reagents (f) the use of these compositions in the manufacture of (1) a medicament for treating or preventing infection due to Neisserial bacteria (2) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisseria bacteria, and/or (3) a reagent which can raise antibodies against Neisseria bacteria and (g) a method of treating a patient, comprising administering to the patient a therapeutically effective amount of these compositions.

[0108] Implementing the invention will typically involve the basic steps of: obtaining a first nucleic acid encoding a first protein; obtaining a second nucleic acid encoding a second protein; and ligating the first and second nucleic acids. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.

[0109] To improve solubility, purification of hybrid proteins may involve the refolding techniques disclosed herein.

[0110] Immunogenic Compositions and Medicaments

[0111] The compositions of the invention are preferably immunogenic composition, and are more preferably vaccine compositions. The pH of the composition is preferably between 6 and 7. The pH may be maintained by the use of a buffer. The composition may be sterile.

[0112] Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic.

[0113] The invention also provides a composition of the invention for use as a medicament. The medicament is preferably able to raise an immune response in a mammal (i.e. it is an immunogenic composition) and is more preferably a vaccine.

[0114] The invention also provides the use of a composition of the invention in the manufacture of a medicament for raising an immune response in a mammal. The medicament is preferably a vaccine.

[0115] The invention also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the invention. The immune response is preferably protective. The method may raise a booster response.

[0116] The mammal is preferably a human. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant); where the vaccine is for prophylactic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.

[0117] These uses and methods are preferably for the prevention and/or treatment of a disease caused by a Neisseria (e.g. meningitis, septicaemia, gonorrhoea etc.). The prevention and/or treatment of bacterial meningitis is preferred.

[0118] Further Components of the Composition

[0119] The composition of the invention will typically, in addition to the components mentioned above, comprise one or more `pharmaceutically acceptable carriers`, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose (WO00/56365) and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences.

[0120] Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen, as well as any other of the above-mentioned 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 vaccine may be administered in conjunction with other immunoregulatory agents.

[0121] The vaccine may be administered in conjunction with other immunoregulatory agents.

[0122] The composition may include other adjuvants in addition to (or in place of) the aluminium salt. Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59.TM. (WO90/14837; Chapter 10 in ref. 13), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) 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.); (2) saponin adjuvants, such as QS21 or Stimulon.TM. (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J. Immunol., 1996, 156, 4570-4575; Halpern et al., Cell. Immunol., 1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messina et al., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998, 160, 5898-5906; International patent applications WO96/02555, WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581] i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (8) a polyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (e.g. WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (e.g. WO01/21152); (10) an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin e.g. WO00/62800; (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) e.g. WO98/57659; (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition.

[0123] 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.

[0124] Further Antigens

[0125] Further antigens which can be included in the composition of the invention include:

[0126] an outer-membrane vesicle (OMV) preparation from N. meningitidis serogroup B, such as those disclosed in refs. 14, 15, 16, 17 etc.

[0127] a saccharide antigen from N. meningitidis serogroup A, C, W135 and/or Y, such as the oligosaccharide disclosed in ref. 18 from serogroup C [see also ref. 19] or the oligosaccharides of ref. 20.

[0128] a saccharide antigen from Streptococcus pneumoniae [e.g. refs. 21, 22, 23].

[0129] a protein antigen from Helicobacter pylori such as CagA [e.g. 24], VacA [e.g. 24], NAP [e.g. 25], HopX [e.g. 26], HopY [e.g. 26] and/or urease.

[0130] an antigen from hepatitis A virus, such as inactivated virus [e.g. 27, 28].

[0131] an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. 28, 29].

[0132] an antigen from hepatitis C virus [e.g. 30].

[0133] an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 31 & 32].

[0134] a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref. 33] e.g. the CRM.sub.197 mutant [e.g. 34].

[0135] a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 33].

[0136] a saccharide antigen from Haemophilus influenzae B [e.g. 19].

[0137] an antigen from N. gonorrhoeae [e.g. 3, 4, 5].

[0138] an antigen from Chlamydia pneumoniae [e.g. 35, 36, 37, 38, 39, 40, 41].

[0139] an antigen from Chlamydia trachomatis [e.g. 42].

[0140] an antigen from Porphyromonas gingivalis [e.g. 43].

[0141] polio antigen(s) [e.g. 44, 45] such as IPV or OPV.

[0142] rabies antigen(s) [e.g. 46] such as lyophilised inactivated virus [e.g. 47, RabAvert.TM.].

[0143] measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref. 33].

[0144] influenza antigen(s) [e.g. chapter 19 of ref. 33], such as the haemagglutinin and/or neuraminidase surface proteins.

[0145] an antigen from Moraxella catarrhalis [e.g. 48].

[0146] a protein antigen from Streptococcus agalactiae (group B streptococcus) [e.g. 49, 50].

[0147] a saccharide antigen from Streptococcus agalactiae

[0148] an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 50, 51, 52].

[0149] an antigen from Staphylococcus aureus [e.g. 53].

[0150] The composition may comprise one or more of these further antigens.

[0151] Where a saccharide or carbohydrate antigen is used, it is preferably conjugated to a carrier protein in order to enhance immunogenicity [e.g. refs. 54 to, 55, 56, 57, 58, 59, 60, 61, 62, 63]. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRM.sub.197 diphtheria toxoid is particularly preferred. Other suitable carrier proteins include the N. meningitidis outer membrane protein [e.g. ref. 64], synthetic peptides [e.g. 65, 66], heat shock proteins [e.g. 67], pertussis proteins [e.g. 68, 69], protein D from H. influenzae [e.g. 70], toxin A or B from C. difficile [e.g. 71], etc. Where a mixture comprises capsular saccharides from both serogroups A and C, it is preferred that the ratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Saccharides from different serogroups of N. meningitidis may be conjugated to the same or different carrier proteins.

[0152] Any suitable conjugation reaction can be used, with any suitable linker where necessary.

[0153] Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [32]).

[0154] Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens.

[0155] Antigens are preferably mixed with (and more preferably adsorbed to) an aluminium salt (e.g. phosphate, hydroxide, hydroxyphosphate, oxyhydroxide, orthophosphate, sulphate). The salt may take any suitable form (e.g. gel, crystalline, amorphous etc.).

[0156] Antigens in the composition will typically be present at a concentration of at least 1 .mu.g/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.

[0157] As an alternative to using proteins antigens in the composition of the invention, nucleic acid encoding the antigen may be used [e.g. refs. 72 to, 73, 74, 75, 76, 77, 78, 79, 80]. Protein components of the compositions of the invention may thus be replaced by nucleic acid (preferably DNA e.g. in the form of a plasmid) that encodes the protein.

Definitions

[0158] 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.

[0159] The term "about" in relation to a numerical value x means, for example, x+10%.

BRIEF DESCRIPTION OF DRAWINGS

[0160] FIGS. 1A-C shows an alignment of twenty-three sequences for protein 741. FIG. 1A shows the first 100 amino acids of the alignment. FIG. 1B shows the second 100 amino acids of the alignment. FIG. 1C shows the remaining 80 amino acids of the alignment. These are SEQ IDs 1 to 22 plus the sequence from MC58.

[0161] FIG. 2 shows an alignment of the NMB1343 sequence from gonococcus (top; SEQ ID 25) and meningococcus (bottom; SEQ ID 26).

[0162] FIG. 3 shows hybrid and tandem proteins of the invention.

[0163] FIG. 4 shows 9 domains within 961.sub.2996 and highlights the coiled coil domain (SEQ ID NO: 40) in 961.

[0164] FIG. 5 shows how these have been manipulated by making various constructs with one or more domains of 961 deleted.

MODES FOR CARRYING OUT THE INVENTION

[0165] Hybrid Proteins --X.sub.1=a G287

[0166] In addition to those disclosed in references 1 & 2, seven hybrid proteins with .DELTA.G287 from strain 2996 at the N-terminus were constructed. Eight 287 tandem proteins were also made (see below).

TABLE-US-00004 # n X.sub.1 L.sub.1 X.sub.2 L.sub.2 1 2 .DELTA.G287 -- 230 (His).sub.6 2 2 -- 936 (His).sub.6 3 2 -- 741.sub.MC58 (His).sub.6 4 2 -- 741.sub.ET37 (His).sub.6 5 2 -- 741.sub.90/18311 (His).sub.6 6 2 -- 741.sub.95N477 (His).sub.6 7 2 .DELTA.G287.sub.nz -- 741.sub.MC58 (His).sub.6

[0167] These proteins were adjuvanted with either Freund's complete adjuvant (FCA) or 3 mg/ml alum and used to immunise mice. The resulting sera were tested against various Neisserial strains using the bactericidal assay. Titres using protein #3 were as follows:

TABLE-US-00005 Strain.sup.(serogroup) 2996.sup.(B) MC58.sup.(B) NGH38.sup.(B) 394/98.sup.(B) 44/76.sup.(B) F6124.sup.(A) Al hydroxide 8192 32768 8192 >2048 16384 8192 FCA 16384 262144 8192 >2048 >32768 8192

[0168] In further experiments using protein #3 adjuvanted with aluminium hydroxide, anti-287 and anti-741 ELISA titres each exceeded 984150 and BCA titres were as follows:

TABLE-US-00006 2996.sup.(B) MC58.sup.(B) NGH38.sup.(B) 394/98.sup.(B) 44/76.sup.(B) F6124.sup.(A) BZ133.sup.(C) 8000 65000 4000 4000 32000 8000 16000

[0169] Results obtained after immunisation with proteins disclosed in refs. 1 & 2, tested against the homologous strain, were as follows:

TABLE-US-00007 Bactericidal titre ELISA n X.sub.1 L.sub.1 X.sub.2 L.sub.2 FCA Alum FCA Alum 2 .DELTA.G287.sub.394/98 -- 961 (His).sub.6 -- 32768 -- >109350 919 32768 4096 4718 3678 953 >32768 >16384 1900 6936 741 16384 2048 232 862 2 .DELTA.G287.sub.2996 -- 961 (His).sub.6 65536 32768 108627 >109350 919 128000 32000 11851 2581 953 65536 -- 3834 -- 741 16384 8192 315 4645

[0170] Hybrid Proteins--X.sub.1=961c or 961cL

[0171] In addition to those disclosed in references 1 & 2, eight hybrid proteins with either 961c or 961cL (i.e. 961c+leader peptide) at the N-terminus were constructed:

TABLE-US-00008 # n X.sub.1 L.sub.1 X.sub.2 L.sub.2 1 2 961c -- 287 -- 2 2 -- 287 (His).sub.6 3 2 -- 230 (His).sub.6 4 2 -- 936 (His).sub.6 5 2 961cL -- 287 -- 6 2 -- 287 (His).sub.6 7 2 -- 230 (His).sub.6 8 2 -- 936 (His).sub.6

[0172] These proteins were adjuvanted with either Freund's complete adjuvant (FCA) or 3.3 mg/ml alum and used to immunise mice. The resulting sera were tested against various Neisserial strains using the bactericidal assay. Titres using protein #8 were as follows:

TABLE-US-00009 Strain.sup.(serogroup) 2996.sup.(B) MC58.sup.(B) 394/98.sup.(B) 44/76.sup.(B) F6124.sup.(A) Al hydroxide 8192 8192 512 1024 <16 FCA 65536 16384 >2048 >2048 8192

[0173] Titres obtained after immunisation with 961c-741 [refs. 1 & 2] were as follows:

TABLE-US-00010 Strain.sup.(serogroup) 2996.sup.(B) MC58.sup.(B) 394/98.sup.(B) 44/76.sup.(B) F6124.sup.(A) BZ133.sup.(C) Al hydroxide 65536 32768 4096 >32768 16384 >2048 FCA >16384 262144 4096 >16384 -- >2048

[0174] These results could be improved by mixing 961c-741 with ORF46.1 or with .DELTA.G287-919.

[0175] Results obtained after immunisation with proteins disclosed in refs. 1 & 2, tested against the homologous strain, were as follows:

TABLE-US-00011 Bactericidal titre ELISA n X.sub.1 L.sub.1 X.sub.2 L.sub.2 FCA Alum FCA Alum 2 961c -- ORF46.1 (His).sub.6 32768 1024 >109350 >109350 741 >16384 8192 >109350 >109350 936 >32768 8192 >109350 >109350

[0176] Hybrid proteins --X.sub.1=ORF46.1

[0177] In addition to those disclosed in references 1 & 2, two hybrid proteins with ORF46.1 at the N-terminus were constructed:

TABLE-US-00012 # n X.sub.1 L.sub.1 X.sub.2 L.sub.2 1 2 ORF46.1 -- 936 (His).sub.6 2 2 -- 230 (His).sub.6

[0178] These proteins were adjuvanted with either Freund's complete adjuvant (FCA) or 3 mg/ml alum and used to immunise mice. The resulting sera were tested against the homologous strain using the bactericidal assay and by ELISA.

[0179] Results obtained after immunisation with proteins disclosed in refs. 1 & 2 were as follows:

TABLE-US-00013 Bactericidal titre ELISA n X.sub.1 L.sub.1 X.sub.2 L.sub.2 FCA Alum FCA Alum 2 ORF46.1 -- 961 (His).sub.6 8192 8192 21558 >109350 -- 961c (His).sub.6 8192 128 9020 76545

[0180] Hybrid Proteins--X.sub.1=230

[0181] In addition to those disclosed in references 1 & 2, four hybrid proteins with 230 at the N-terminus were constructed:

TABLE-US-00014 # n X.sub.1 L.sub.1 X.sub.2 L.sub.2 1 2 230 -- ORF46.1 (His).sub.6 2 2 -- 961 (His).sub.6 3 2 -- 961c (His).sub.6 4 2 -- 741.sub.MC58 (His).sub.6

[0182] Hybrid Proteins--X.sub.1=936

[0183] In addition to those disclosed in references 1 & 2, seven hybrid proteins with 936 at the N-terminus were constructed:

TABLE-US-00015 # n X.sub.1 L.sub.1 X.sub.2 L.sub.2 1 2 936 -- ORF46.1 (His).sub.6 2 2 -- 961 (His).sub.6 3 2 -- 741.sub.ET37 (His).sub.6 4 2 -- 741.sub.MC58 (His).sub.6 5 2 -- 741.sub.90/18311 (His).sub.6 6 2 -- 741.sub.95N477 (His).sub.6 7 2 -- 741 (His).sub.6

[0184] These proteins were adjuvanted with either Freund's complete adjuvant (FCA) or 3 mg/ml alum and used to immunise mice. The resulting sera were tested against various Neisserial strains using the bactericidal assay. Titres using protein #2 were as follows:

TABLE-US-00016 Strain.sup.(serogroup) 2996.sup.(B) MC58.sup.(B) 394/98.sup.(B) 44/76.sup.(B) F6124.sup.(A) Al hydroxide 16384 32768 1024 2048 <16 FCA 65536 65536 >2048 8192 2048.sub.(36%)

[0185] Titres using protein #4 were as follows:

TABLE-US-00017 Strain.sup.(serogroup) 2996.sup.(B) MC58.sup.(B) 394/98.sup.(B) 44/76.sup.(B) F6124.sup.(A) Al hydroxide 256 >262144 >2048 32768 8192 FCA 1024 >262144 >2048 >32768 >32768

[0186] Titres using protein #7 were as follows:

TABLE-US-00018 Strain.sup.(serogroup) 2996.sup.(B) MC58.sup.(B) 394/98.sup.(B) 44/76.sup.(B) F6124.sup.(A) BZ133.sup.(C) Al hydroxide 256 130000 16000 32000 8000 16000

[0187] Results obtained after immunisation with proteins disclosed in refs. 1 & 2, tested against the homologous strain, were as follows:

TABLE-US-00019 Bactericidal titre ELISA n X.sub.1 L.sub.1 X.sub.2 L.sub.2 FCA Alum FCA Alum 2 936 -- 741 (His).sub.6 1024 256 1466 5715 936 >32768 >32768 >109350 >109350

[0188] Mixtures of Hybrid Proteins

[0189] Mice were immunised with of three proteins adjuvanted with aluminium hydroxide, either single or in a triple combination: (1) 287.sub.NZ-953; (2) 936-741; and (3) 961c. The mixture was able to induce high bactericidal titres against various strains:

TABLE-US-00020 2996.sup.(B) MC58.sup.(B) NGH38 394/98.sup.(B) H44/76.sup.(B) F6124.sup.(A) BZ133.sup.(C) C11.sup.(C) (1) 32000 16000 130000 16000 32000 8000 16000 8000 (2) 256 131000 128 16000 32000 8000 16000 <4 (3) 32000 8000 -- -- -- 8000 -- 32000 mix 32000 32000 65000 16000 260000 65000 >65000 8000 (X) 4000 4000 1000 1000 >4000 1000 4000 n.d. `--` indicates that this strain contains no NadA gene (X) was a combination of protein 287 with outer membrane vesicles, for comparison

[0190] Looking at individual mice, the mixture induced high and consistent bactericidal titres:

TABLE-US-00021 # 1 2 3 4 5 6 7 8 9 10 2996 32768 16384 65536 32768 32768 65536 65536 32768 65536 8192 MC58 65536 32768 65536 65536 65536 8192 65536 32768 32768 65536 394/98 65536 4096 16384 4096 8192 4096 32768 16384 8192 16384

[0191] Tandem Proteins

[0192] Hybrid proteins of the invention can be represented by formula NH.sub.2--[--X-L-].sub.n--COOH. Where all n instances of --X-- are the same basic protein (either identical, or the same protein from different strains or species), the protein is referred to as a `tandem` protein.

[0193] Twelve specific tandem proteins are:

TABLE-US-00022 # n X.sub.1 L.sub.1 X.sub.2 L.sub.2 1 2 .DELTA.G741.sub.MC58 -- 741.sub.MC58 (His).sub.6 2 2 .DELTA.G287.sub.2996 (Gly).sub.6 .DELTA.G287.sub.394/98 (His).sub.6 3 2 .DELTA.G287.sub.2996 (Gly).sub.6 .DELTA.G287.sub.2996 (His).sub.6 4 2 .DELTA.G287.sub.394/98 (Gly).sub.6 .DELTA.G287.sub.394/98 (His).sub.6 5 2 .DELTA.G287.sub.394/98 (Gly).sub.6 .DELTA.G287.sub.2996 (His).sub.6 6 2 .DELTA.G287.sub.2996 (Gly).sub.6 .DELTA.G287.sub.394/98 -- 7 2 .DELTA.G287.sub.2996 (Gly).sub.6 .DELTA.G287.sub.2996 -- 8 2 .DELTA.G287.sub.394/98 (Gly).sub.6 .DELTA.G287.sub.394/98 -- 9 2 .DELTA.G287.sub.394/98 (Gly).sub.6 .DELTA.G287.sub.2996 -- 10 2 .DELTA.G741.sub.MC58 -- 741.sub.394/98 (His).sub.6 11 2 .DELTA.G741.sub.MC58 -- 741.sub.90/18311 (His).sub.6 12 2 .DELTA.G741.sub.MC58 -- 741.sub.95N477 (His).sub.6

[0194] Proteins #1 to #5 have all been expressed in soluble form in E. coli. Expression levels were between 0.24 and 0.50 mg protein per litre of culture. The tandem proteins were purified and mixed with aluminium phosphate as an adjuvant. Tandem proteins #2, #4 and #5 adsorbed readily to aluminium phosphate; adsorption was less complete for tandem proteins #1 and #3.

[0195] Allelic Variants--741

[0196] Twenty-two polymorphic sequences of 741 were found (SEQ IDs 1 to 22). These and the MC58 sequence are aligned in FIG. 1.

[0197] Allelic Variants--NMB1343

[0198] Using PCR on 42 strains of meningococcus of various serogroups, the gene encoding NMB1343 protein was found in 24/42 and was absent in 18/42 strains (Table 1). The NMB1343 gene was sequenced for 10 of the NMB1343.sup.+ strains (Table 1, column 3). The nucleic acid sequence (and thus amino acid sequence SEQ ID 23; GenBank AAF41718) was identical in all 10 strains.

[0199] NMB1343 was also detected in two strains of N. gonorrhoeae (F62 and SN4). The amino acid sequence from gonococcus is SEQ ID 24. An alignment with the meningococcal sequence is:

TABLE-US-00023 . . . .10 . . . .20 . . . .30 . . . .40 . . . .50 Ng 1 :INNLWEISYLYRGISCQQDEQNNGQLKPKGNKAEVAIRYDGKFKYDGKAT: 50 Nm 1 :~~~~~MGNFLYRGISCQQDEQNNGQLKPKGNKAEVAIRYDGKFKYDGKAT: 45 ***************************************** . . . .60 . . . .70 . . . .80 . . . .90 . . . 100 Ng 51 :HGPSVKNAVYAHQIETDLYDGCYISTTTDKEIAKKFATSSGIENGYIYVL: 100 Nm 46 :HGPSVKNAVYAHQIETGLYDGCYISTTTDKEIAKKFATSSGIENGYIYVL: 95 **************** ********************************* . . . 110 . . . 120 . . . 130 . . . 140 . . . 150 Ng 101 :NRDLFGQYSIFEYEVEHPENPDEKEVTIRAEDCGCIPEEVIIAKELIEIN: 150 Nm 96 :NRDLFGQYSIFEYEVEHPENPNEKEVTIRAEDCGCIPEEVIIAKELIEIN: 145 ********************* ****************************

[0200] An alignment of the corresponding nucleotide sequences is shown in FIG. 2. This shows that the gonococcal sequence has a 4mer insertion in the 5' region of the NMB1343 gene which causes a frameshift and consequent loss of the 5' methionine residue.

[0201] Domain Deletion--961

[0202] 961 is not present in the N. meningitidis serogroup A genome sequence [81], even though the surrounding regions are conserved (>90%) between serogroups A and B. References 11 and 12 disclose polymorphic forms of 961. The gene was found to be present in 91% of serogroup B strains belonging to hypervirulent lineages ET-5, ET-37 and cluster .DELTA.4, but was absent in all strains of lineage 3 tested. Most of the serogroup C strains tested were positive even if not belonging to hypervirulent 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. 961 was absent in N. gonorrhoeae and in commensal species N. lactamica and N. cinerea.

[0203] FIGS. 4 and 5 show domains in protein 961.

[0204] When the anchor region (domain 9) of protein 961 is deleted (`961cL`) and expressed in E. coli, the protein is exported in the periplasm and secreted in the supernatant of the culture.

[0205] To investigate this further, deletion mutants in the C-terminal region of 961 were constructed (961cL-.DELTA.aro, 961cL.DELTA.cc, 961aL, 961aL-.DELTA.1, 961aL-.DELTA.2, 961aL-.DELTA.3) on the basis of structural features (deletions of aromatic residues in the cases of 961c.DELTA.aro mutant, and of coiled-coil regions for the others). These were analysed for expression and secretion into the periplasm and the supernatant of the culture. In all of these deletion mutants, the protein is produced in large amount, is present in periplasmic fraction, and is released in the supernatant of the culture.

[0206] .DELTA.G287--Cross-Strain Bactericidal Activity

[0207] 287 was cloned for five different N. meningitidis serogroup B strains and was manipulated to delete the N-terminus up to the end of the poly-glycine region and to introduce a C-terminal his-tag. This gave five .DELTA.G287 proteins. These were adjuvanted with FCA and used to raise immune sera in mice, which were then tested for bactericidal activity against all five serogroup B strains and also against serogroup A and C strains. Bactericidal titres were as follows:

TABLE-US-00024 Protein Sera tested for bactericidal activity against strain* strain 2996 BZ232 MC58 1000 394/98 F6124 BZ133 2996 16000 128 4096 4096 1024 8000 16000 BZ232 >8000 256 2048 8000 2048 16000 8000 MC58 >8000 64 >8000 8000 2048 8000 8000 1000 >8000 64 4096 8000 1024 16000 16000 394/98 >16000 128 16000 >2048 >16000 -- -- *titres against homologous strain shown in bold

[0208] Refolding

[0209] To improve the levels of soluble protein for some hybrid proteins, alternative refolding protocols to those disclosed in reference 2 were adopted.

[0210] Inclusion bodies (IBs) were isolated as follows:

[0211] 1. Homogenize cells (5 g wet weight) in 25 ml 0.1 M Tris-HCl pH 7, 1 mM EDTA, at 4.degree. C. using an ultraturrax (10 000 rpm)

[0212] 2. Add 1.5 mg lysozyme per gram cells, mix shortly with an ultraturrax, and incubate at 4.degree. C. for 30 min.

[0213] 3. Use sonication or high-pressure homogenization (French press) to disrupt the cells.

[0214] 4. To digest DNA, add MgCl.sub.2 to a final concentration of 3 mM and DNase to a final concentration of 10 .mu.g/ml, and incubate for 30 min at 25.degree. C.

[0215] 5. Add 0.5 vol. 60 mM EDTA, 6% Triton X-100, 1,5M NaCl pH7, to the solution, and incubate for 30 min at 4.degree. C.

[0216] 6. Spin down inclusion bodies by centrifugation at 31000 g (20 000 rpm) for 10 min, 4.degree. C.

[0217] 7. Resuspend pellet in 40 ml 0.1 M tris-HCl pH 7, 20 mM EDTA, using an ultraturrax

[0218] 8. Repeat centrifugation step 6.

[0219] 9. The inclusion body pellet may be used, or stored frozen at -20.degree. C.

[0220] Hybrid proteins were expressed in E. coli as follows:

TABLE-US-00025 Culture Flask Inclusion volume volume Temp Final body yield Protein (litres) (litres) (.degree. C.) OD.sub.600 (w/w) ORF46.1-961-His 1 2 37 1.51 33.2% ORF46.1-961c-His 1 2 37 1.6 28.3% 961c-ORF46.1His 1 2 37 1.18 23.5% orf46.1-741 His 5 5 37 12.42 35.2

[0221] The pellets were solubilised, refolded, ultrafiltered, dialysed, and protein was then purified:

[0222] ORF46.1-961-His IBs were solubilised as follows: IB proteins were resuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to a final protein concentration of 1 mg/ml. To refold the protein, 2 ml of solubilised protein was diluted in 400 ml of refolding buffer (0.1M Tris HCl, 1M L-arginine, 2 mM EDTA pH 8.2) and incubated for 1 hour at 15.degree. C., resulting in a protein concentration of 5 .mu.g/ml. Subsequently, another 2 ml of the solubilised protein was added and incubated for an additional hour at the same temperature resulting in a final protein concentration of 10 .mu.g/ml. The material was ultrafiltered using a 300 ml Amicon ultrafiltration cell (8400), applying a 3 bar pressure on an Amicon membrane with a 30 kDa cut-off (YM30) resulting in 130 ml final volume. The ultrafiltered material was dialysed using a regenerated cellulose tubular membrane with a 12-14 kDa cutoff (Cellusep--Step bio) for 24 hours against 10 L of 0.1M Tris HCl pH 8.2 buffer. A second dialysis of 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH 8.0 buffer was performed. The dialysed material was centrifuged at 22000 rpm for 45 minutes at 4.degree. C. in a Beckman centrifuge rotor JA25.5 The supernatant isolated after centrifugation was used for His-tag purification.

[0223] orf 46.1-961c-His IBs were solubilised as follows: IB proteins were resuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to a final protein concentration of 1 mg/ml. To refold the protein, 2 ml of the solubilised protein was diluted in 400 ml refolding buffer (0.5M Tris HCl, 1M L-arginine, 2 mM EDTA pH 8.2) and incubated for 1 h at 15.degree. C., resulting in a protein concentration of 5 .mu.g/ml. Subsequently another 2 ml of the solubilised protein was added and incubated for an additional hour at the same temperature resulting in a final protein concentration of 10 .mu.g/ml. The material was ultrafiltered using a 300 ml Amicon ultrafiltration cell (8400), applying a 3 bar pressure on an Amicon membrane with a 30 kDa cut-off (YM30) resulting in 150 ml final volume. The ultrafiltered material was dialysed using a regenerated cellulose tubular membrane with a 12-14 kDa cutoff (Cellusep--Step bio) for 24 h against 10 L of 0.1M Tris HCl pH 8.2 buffer. A second dialysis of 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH 8.0 buffer was performed. The dialysed material was centrifuged at 22000 rpm for 45 minutes at 4.degree. C. in a Beckman centrifuge rotor JA25.5. The supernatant isolated after centrifugation was used for His-tag purification.

[0224] 961c-orf46.1-His IBs were solubilised as follows: IB proteins were resuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to a final protein concentration of 1 mg/ml. To refold the protein, 2 ml of the solubilised protein was diluted in 400 ml refolding buffer (0.1M Tris HCl, 0.5 M L-arginine, 2 mM EDTA pH 8.2) and incubated for 1 h at 15.degree. C., resulting in a protein concentration of 5 .mu.g/ml. Subsequently another 2 ml of the solubilized protein was added and incubated for an additional hour at the same temperature resulting in a final protein concentration of 10 .mu.g/ml. The material was ultrafiltered using a 300 ml Amicon ultrafiltration cell (8400), applying a 3 bar pressure on an Amicon membrane with a 30 kDa cut-off (YM30) resulting in 150 ml final volume. The ultrafiltered material was dialysed using a regenerated cellulose tubular membrane with a 12-14 kDa cutoff (Cellusep--Step bio) for 24 h against 10 L of 0.1M Tris HCl pH 8.2 buffer. A second dialysis of 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH 8.0 buffer was performed. The dialysed material was centrifuged at 22000 rpm for 45 minutes at 4.degree. C. in a Beckman centrifuge rotor JA25.5. The supernatant isolated after centrifugation was used for His-tag purification.

[0225] orf46.1-741-His IBs were solubilised as follows: IB proteins were resuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to a final protein concentration of 10 mg/ml. To refold, 2 ml of the solubilised protein was diluted in 400 ml of the refolding buffer (0.5M Tris HCl, 0.7 M L-arginine, 2 mM EDTA pH 7.2) and incubated for 1 h at 15.degree. C., resulting in a protein concentration of 50 .mu.g/ml. Subsequently another 2 ml of the solubilised protein was added and incubated for an additional hour at the same temperature resulting in a final protein concentration of 100 g/ml. The material was ultrafiltered using a 300 ml Amicon ultrafiltration cell (8400), applying a 3 bar pressure on an Amicon membrane with a 30 kDa cut-off (YM30) resulting in 120 ml final volume. The ultrafiltered material was dialysed using a regenerated cellulose tubular membrane with a 12-14 kDa cutoff (Cellusep--Step bio) for 24 h against 10 L of 0.1M Tris HCl pH 8.2 buffer. A second dialysis of 24 h against 10 L of 300 mM NaCl, 50 mM sodium phosphate pH 8.0 buffer was performed. The dialysed material was centrifuged at 22000 rpm for 45 minutes at 4.degree. C. in a Beckman centrifuge rotor JA25.5 The supernatant isolated after centrifugation was used for His-tag purification.

[0226] Compared with proteins purified as described in ref. 2, bactericidal assay titres were as follows:

TABLE-US-00026 Reference 2 Alu- Refolded minium Aluminium Aluminium Protein CFA hydroxide hydroxide MF59 phosphate ORF46.1- 8192 8192 32768 -- -- 961-His ORF46.1- 8192 128 <64 8192 -- 961c-His 961c- 32768 1024 16384 -- -- ORF46.1His orf46.1- <4 16 <4 256 -- 741 His

[0227] Similar procedures were used for ORF46.1 to purify the protein from IBs when expressed with no His-tag (`ORF46.1K`):

TABLE-US-00027 Culture Flask Inclusion volume volume Temp Final body yield Protein (litres) (litres) (.degree. C.) OD.sub.600 (w/w) orf46.1K 5 5 37 13.7 29.4

[0228] IB proteins were resuspended in 4 ml of 6M guanidine HCl, 1 mM EDTA pH 8.5 buffer, to a final protein concentration of 10 mg/ml. To refold, 2 ml of the solubilised protein was diluted in 400 ml of the refolding buffer (0.5M Tris HCl, 0.7 M L-arginine, 2 mM EDTA pH 7.2) and incubated for 1 hours at 15.degree. C., resulting in a protein concentration of 50 .mu.g/ml. Subsequently another 2 ml of the solubilised protein was added and incubated for an additional hour at the same temperature resulting in a final protein concentration of 100 g/ml. The material was ultrafiltered using a 300 ml Amicon ultrafiltration cell (8400), applying a 3 bar pressure on an Amicon membrane with a 30 kDa cut-off (YM30) resulting in 120 ml final volume. The ultrafiltered material was dialysed using a regenerated cellulose tubular membrane with a 12-14 kDa cutoff (Cellusep--Step bio) for 12 h against 10 L of 50 mM sodium phosphate, 2 mM EDTA, pH 7,2 buffer. A second dialysis of 24 h against 10 L of the same buffer was performed. The dialysed material was centrifuged at 22000 rpm for 45 minutes at 4.degree. C. in a Beckman centrifuge rotor JA25.5. The supernatant isolated after centrifugation was used for cationic exchange chromatography. The purification was done on a AKTA explorer chromatography system (Amersham-Pharmacia Biotech) using a 5 ml HiTrap SP sepharose HP column (Amersham-Pharmacia Biotech). The flow rate applied was of 1.5 ml per minute. The column was washed with 35 ml of 50 mM sodium phosphate buffer pH 7.2. A linear gradient (0-1 M NaCl) was performed using a 50 mM sodium phosphate buffer pH 7.2. The protein eluted in two peaks at 92 mM and 380 mM NaCl. The fractions constituting each peak were pooled and respectively named pool 1 and pool 2.

[0229] Compared with proteins purified as described in ref. 2, bactericidal assay titres when adjuvanted with aluminium hydroxide were improved from <4 to 1024. The titre using aluminium phosphate adjuvant with the refolded protein was 2048. ELISA titres were as follows:

TABLE-US-00028 Elisa SBA Protein Aluminium adjuvant (M7) (2996) Orf46.1k (pool 1) Hydroxide 3.3 mg/ml 1212 512 Phosphate 0.6 mg/ml 154 1024 Orf46.1k (pool 2) Hydroxide 3.3 mg/ml 1085 1024 Phosphate 0.6 mg/ml 250 1024

[0230] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

TABLE-US-00029 TABLE 1 Strain 1343 Sequence Strain classification 72/00 + ET5 B:15:P1.7,13,13a 30/00 + ET5 B:15:P1.7,16 39/99 + ET5 C:15:P1.7,16 95330 + ET5 B:4:P1.15 M4102 + ET5 nd MC58(21) + + ET5 B:15:P1.7,16b BZ169(7) + + ET5 B:NT:P1.16 BZ83(19) + ET5 B:15:--.-- CU385 + + ET5 B:4:P1.15 220173I + ET5 NG:4:P1.15 64/96 + + ET5 NG:15:P1.7,16 (carrier) 220173I + ET5 B:4:P1.15 (carrier) ISS1071 + nd B:15:P1.7,16 (ET5?) BZ198(2) + + lin.3 B:8:P1.1 980-2543 + + lin.3 B:NT:P1.4 16060 + + other B:4:P1.14 (carrier) 394-98 + nd B:4:P1.4 (lin 3?) ISS1106 + nd B:4:P1.4 (lin.3?) BZ133(10) + + sub I B:NT:--.-- S3446 + + nd B:14:P1.23,14 ISS1001 + + nd B:14:P1.13 241175I + other NG:21:P1.16 (carrier) 171274I + other NG:15:--(carrier) 66/96 + other B:17:P1.15 (carrier) 961-5945 - A4 96217 - A4 312294 - A4 90/18311(24) - ET37 93/4286(25) - ET37 M986 - ET37 1000(5) - other NGE28(13) - other carrier NGH38(14) - other carrier BZ232(18) - other F6124(23) - sub III A:--.-- C11 - C:-- NMB - nd 8047 - nd ISS759 - nd C:2b:P1.2 ISS1113 - nd C:2:P1.5 65/96 - nd 4:P1.14 2996(96) - nd B:2b:P1.5,2

REFERENCES (THE CONTENTS OF WHICH ARE HEREBY INCORPORATED BY REFERENCE)



[0231] 1--International patent application WO01/64920.

[0232] 2--International patent application WO01/64922.

[0233] 3--International patent application WO99/24578.

[0234] 4--International patent application WO99/36544.

[0235] 5--International patent application WO99/57280.

[0236] 6--International patent application WO00/22430.

[0237] 7--Tettelin et al. (2000) Science 287:1809-1815.

[0238] 8--International patent application WO00/66741.

[0239] 9--International patent application WO00/71574.

[0240] 10--International patent application WO01/04316

[0241] 11--International patent application PCT/IB02/03396.

[0242] 12--Comanducci et al. (2002) J Exp Med 195:1445-1454.

[0243] 13--Vaccine Design: subunit & adjuvant approach (1995) Powell & Newman (ISBN: 030644867X).

[0244] 14--International patent application WO01/52885.

[0245] 15--Bjune et al. (1991) Lancet 338(8775): 1093-1096.

[0246] 16--Fukasawa et al. (1999) Vaccine 17:2951-2958.

[0247] 17--Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333.

[0248] 18--Costantino et al. (1992) Vaccine 10:691-698.

[0249] 19--Costantino et al. (1999) Vaccine 17:1251-1263.

[0250] 20--International patent application PCT/IB02/03191.

[0251] 21--Watson (2000) Pediatr Infect Dis J 19:331-332.

[0252] 22--Rubin (2000) Pediatr Clin North Am 47:269-285, v.

[0253] 23--Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.

[0254] 24--International patent application WO93/18150.

[0255] 25--International patent application WO99/53310.

[0256] 26--International patent application WO98/04702.

[0257] 27--Bell (2000) Pediatr Infect Dis J 19:1187-1188.

[0258] 28--Iwarson (1995) APMIS 103:321-326.

[0259] 29--Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.

[0260] 30--Hsu et al. (1999) Clin Liver Dis 3:901-915.

[0261] 31--Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355.

[0262] 32--Rappuoli et al. (1991) TIBTECH 9:232-238.

[0263] 33--Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.

[0264] 34--Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.

[0265] 35--International patent application WO02/02606.

[0266] 36--Kalman et al. (1999) Nature Genetics 21:385-389.

[0267] 37--Read et al. (2000) Nucleic Acids Res 28:1397-406.

[0268] 38--Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527.

[0269] 39--International patent application WO99/27105.

[0270] 40--International patent application WO00/27994.

[0271] 41--International patent application WO00/37494.

[0272] 42--International patent application WO99/28475.

[0273] 43--Ross et al. (2001) Vaccine 19:4135-4142.

[0274] 44--Sutter et al. (2000) Pediatr Clin North Am 47:287-308.

[0275] 45--Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126.

[0276] 46--Dreesen (1997) Vaccine 15 Suppl:S2-6.

[0277] 47--MIWR Morb Mortal Wkly Rep 1998 Jan. 16; 47(1):12, 19.

[0278] 48--McMichael (2000) Vaccine 19 Suppl 1:S101-107.

[0279] 49--Schuchat (1999) Lancet 353(9146):51-6.

[0280] 50--WO02/34771.

[0281] 51--Dale (1999) Infect Dis Clin North Am 13:227-43, viii.

[0282] 52--Ferretti et al. (2001) PNAS USA 98: 4658-4663.

[0283] 53--Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages 1218-1219.

[0284] 54--Ramsay et al. (2001) Lancet 357(9251):195-196.

[0285] 55--Lindberg (1999) Vaccine 17 Suppl 2: S28-36.

[0286] 56--Buttery & Moxon (2000) JR Coil Physicians Lond 34:163-168.

[0287] 57--Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii.

[0288] 58--Goldblatt (1998) J. Med. Microbiol. 47:563-567.

[0289] 59--European patent 0 477 508.

[0290] 60--U.S. Pat. No. 5,306,492.

[0291] 61--International patent application WO98/42721.

[0292] 62--Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114.

[0293] 63--Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X.

[0294] 64--European patent application 0372501.

[0295] 65--European patent application 0378881.

[0296] 66--European patent application 0427347.

[0297] 67--International patent application WO93/17712.

[0298] 68--International patent application WO98/58668.

[0299] 69--European patent application 0471177.

[0300] 70--International patent application WO00/56360.

[0301] 71--International patent application WO00/61761.

[0302] 72--Robinson & Torres (1997) Seminars in Immunology 9:271-283.

[0303] 73--Donnelly et al. (1997) Annu Rev Immunol 15:617-648.

[0304] 74--Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs 9:471-480.

[0305] 75--Apostolopoulos & Plebanski (2000) Curr Opin Mol Ther 2:441-447.

[0306] 76--Ilan (1999) Curr Opin Mol Ther 1:116-120.

[0307] 77--Dubensky et al. (2000) Mol Med 6:723-732.

[0308] 78--Robinson & Pertmer (2000) Adv Virus Res 55:1-74.

[0309] 79--Donnelly et al. (2000) Am J Respir Crit Care Med 162(4 Pt 2):S190-193.

[0310] 80--Davis (1999) Mt. Sinai J. Med. 66:84-90.

[0311] 81--Parkhill et al. (2000) Nature 404:502-506.

TABLE-US-00030

[0311] SEQUENCE LISTING SEQ ID 1-741 from strain 1000 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTTPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQTITLASGEFQTYKQNHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDPNGRLHYSIDETKKQGYGRIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 2-741 from strain 2201731 (premature stop codon, though reliable sequence) MTRSKPVNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQ DSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLK SPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKA SEQ ID 3-741 from strain 90/18311 (incomplete) GLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEV DGQLITLESGEFQTYKQDHSAVVALQIEKINNPDKIDSLINQRSELVSGLGGEHTAFNQLPSGKAEYHGK AFSSDDPNGRLHYSIDETKKQGYGRIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLA LFGDRAQEIAGSATVKIREKVHET SEQ ID 4-741 from strain L93/4286 (incomplete) VAADIGAGLADALTAPLDHKDKGLQSLMLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRED FIRQIEVDGQTITLASGEFQTYKQNHSAVVALQIEKINNPDKIDSLINQRSELVSGLGGEHTAFNQLPDG KAEYHGKAFSSDDPNGRLHYSIDETKKQGYGRIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEE KGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 5-741 from strain 2996 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQTYKQDHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ SEQ ID 6-741 from strain 30/00 KDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEF QVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKL TYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIEGGKAQEVAG SAEVKTVNGIRHIGLAAKQ SEQ ID 7-741 from strain 312294 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQTYKQDHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ SEQ ID 8-741 from strain 39/99 (incomplete) DKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQ VYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLT YTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIEGGKAQEVAGS AEVKTVNGIRHIGLAAKQ SEQ ID 9-741 from strain 5/99 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQTYKQDHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDETKKQGYGRIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 10-741 from strain 67/00 MTRSKPVNRTAFCCFSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQ DPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLK SPELNVELATAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIEGGQAQEVAGSAEVETANGTHHIGLAAKQ SEQ ID 11-741 from strain BZ169 LQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQVYK QSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTI DFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEV KTVNGIRHIGLAAKQ SEQ ID 12-741 from strain 72/00 LQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQVYK QSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTI DFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEV KTVNGIRHIGLAAKQ SEQ ID 13-741 from strain 93/114 MTRSKPVNRTAFCCFSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQ DPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLK SPELNVELATAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIEGGQAQEVAGSAEVETANGTHHIGLAAKQ SEQ ID 14-741 from strain 95N477 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQTYKQDHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDETKKQGYGRIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 15-741 from strain 96217 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQTYKQDHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ SEQ ID 16-741 from strain BZ133 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQ DSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDASGKLTYTIDFAAKQGHGKIEHLK SPELNVDLAASDIKPDKKRHAVISGSVLYNQAEKGSYSLGIEGGQAQEVAGSAEVETANGIRHIGLAAKQ SEQ ID 17-741 from strain BZ232 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQTITLASGEFQTYKQNHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDPNGRLHYSIDETKKQGYGRIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 18-741 from strain C11 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQTYKQDHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDETKKQGYGRIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 19-741 from strain M1090 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQTYKQDHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 20-741 from strain M1096 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTTPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQTITLASGEFQTYKQNHSAVVALQIEKIN NPDKIDSLINQRSELVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDPNGRLHYSIDETKKQGYGRIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 21-741 from strain M198/172 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQ DSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDASGKLTYTIDFAAKQGHGKIEHLK SPELNVDLAASDIKPDKKRHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ SEQ ID 22-741 from strain NGH38 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEK LKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQTITLASGEFQIYKQNHSAVVALQIEKIN NPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKT PEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ SEQ ID 23-NMB1343 from ten meningococcal strains MGNFLYRGISCQQDEQNNGQLKPKGNKAEVAIRYDGKFKYDGKATHGPSVKNAVYAHQIETGLYDGCYIS TTTDKEIAKKFATSSGIENGYIYVLNRDLFGQYSIFEYEVEHPENPNEKEVTIRAEDCGCIPEEVITAKE LIEIN SEQ ID 24-NMB1343 from gonococcus INNLWEISYLYRGISCQQDEQNNGQLKPKGNKAEVAIRYDGKFKYDGKATHGPSVKNAVYAHQIETDLYD GCYISTTTDKEIAKKFATSSGIENGYIYVLNRDLFGQYSIFEYEVEHPENPDEKEVTIRAEDCGCIPEEV IIAKELIEIN SEQ ID 25-NMB1343 nucleic acid sequence (gonococcus) TCCGCCGCATTACCTTATAAAATAAAACATCCCTCTCAAGCAGTCTGATAATGTTTGGATTGCTTGAGAT TGATGAGTGATGGTGTTAAATTCAAACTTTAAATTAATAACTTATGGGAAATTTCTTATTTATATAGAGG CATTAGTTGCCAACAAGATGAGCAAAATAATGGACAGTTAAAACCTAAAGGTAATAAAGCTGAAGTTGCA ATTCGTTATGATGGTAAGTTTAAATATGATGGTAAAGCTACACATGGTCCAAGTGTGAAGAATGCAGTTT ACGCCCATCAAATTGAAACAGATCTATATGACGGATGTTATATATCTACGACAACAGACAAGGAAATTGC CAAGAAATTTGCAACAAGCTCCGGCATCGAAAATGGCTATATATATGTTTTAAATAGAGATTTGTTTGGT CAATATTCTATTTTTGAATATGAGGTTGAACATCCAGAAAACCCAGATGAGAAGGAAGTAACAATCAGAG CTGAAGATTGTGGCTGTATTCCTGAAGAAGTGATTATTGCTAAAGAGTTGATAGAAATTAACTAAGTTGA AAGGTCAATATAATGGCTTTAGTTGAATTGAAAGTGCCCGACATTGGCGGACACGAAAATGTAGATATTA TCGC SEQ ID 26-NMB1343 nucleic acid sequence (meningococcus) TCCGCCGCATTACCTTATAAAATAAAACATCCCTCTCAAGCAGTCTGATAATGTTTGGATTGCTTGAGAT TGATGAGTAATGGTGTTAAATTCAACCTTTAAATTAATAACTTATGGGAAATTTCTTATATAGAGGCATT AGTTGCCAACAAGATGAGCAAAATAATGGACAGTTAAAACCTAAAGGTAATAAAGCTGAAGTTGCAATTC GTTATGATGGTAAGTTTAAATATGATGGTAAAGCTACACATGGTCCAAGTGTGAAGAATGCAGTTTACGC CCATCAAATTGAAACAGGTCTATATGACGGATGTTATATATCTACGACAACAGACAAGGAAATTGCCAAG AAATTTGCAACAAGTTCCGGCATCGAAAATGGCTATATATATGTTTTAAATAGGGATTTGTTTGGTCAAT ATTCTATTTTTGAATATGAGGTTGAACATCCAGAAAACCCAAATGAGAAGGAAGTAACAATCAGAGCTGA AGATTGTGGCTGTATTCCTGAAGAAGTGATTATTGCTAAAGAGTTGATAGAAATTAACTAAGTTGAAAGG TCAATATAATGGCTTTAGTTGAATTGAAAGTGCCCGACATTGGCGGACACGAAAATGTAGATATTATCGC SEQ ID 27-linker GSGGGG SEQ ID 28-preferred A G287-953 hybrid MASPDVKSADTLSKPAAPVVAEKETEVKEDAPQAGSQGQGAPSTQGSQDMAAVSAENTGNGGAATTDKPK NEDEGPQNDMPQNSAESANQTGNNQPADSSDSAPASNPAPANGGSNEGRVDLANGVLIDGPSQNITLTHC KGDSCNGDNLLDEEAPSKSEFENLNESERIEKYKKDGKSDKFTNLVATAVQANGTNKYVIIYKDKSASSS SARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALR VQGEPAKGEMLAGTAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAI DGNGFKGTWTENGGGDVSGRFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGSGGGGATYKVDEYH ANARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTDHLKSADIFDAAQYPD IREVSTKENENGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTEVCGGDFSTTIDRTKWGVDYLV NVGMTKSVRIDIQIEAAKQ SEQ ID 29-A G287Nz-953 hybrid MASPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGGQDMAAVSEENTGNGGAAATDKPK NEDEGAQNDMPQNAADTDSLTPNHTPASNMPAGNMENQAPDAGESEQPANQPDMANTADGMQGDDPSAGG ENAGNTAAQGTNQAENNQTAGSQNPASSTNPSATNSGGDFGRTNVGNSVVIDGPSQNITLTHCKGDSCSG NNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDGKNDKFVGLVADSVQMKGINQYIIFYKPKPTSFARFR RSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEP SKGEMLAGTAVYNGEVLHFHTENGRPSPSRGRFAAKVDFGSKSVDGIIDSGDGLHMGTQKFKAAIDGNGF KGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGSGGGGATYKVDEYHANARF AIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTDHLKSADIFDAAQYPDIREVS TKENENGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTEVCGGDFSTTIDRTKWGVDYLVNVGMT KSVRIDIQIEAAKQ SEQ ID 30-936-A G741 hybrid MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQ TKGYTPQISVVGYNRHLLLLGQVATEGEKQFVGQTARSEQAAEGVYNYITVASLPRTAGDIAGDTWNTSK VRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQRGSGGGGVA ADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSREDFI RQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGR ATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEK GSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ SEQ ID 31-961c MATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDATAADVEADDFKGLGLKKVVTN LTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDATTNALNKLGENITTFAEETKTNIV KIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAE AAAGTANTAADKAEAVAAKVTDIKADIATNKDNIAKKANSADVYTREESDSKFVRIDGLNATTEKLDTRL ASAEKSIADHDTRLNGLDKTVSDLRKETRQGLAEQAALSGLFQPYNVG

Sequence CWU 1

1

391279PRTNeisseria meningitidis 1Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Thr Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asn His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr 180 185 190 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 2256PRTNeisseria meningitidis 2Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Thr Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 115 120 125 Ser Ala Leu Thr Ala Phe Gln Thr Glu Gln Ile Gln Asp Ser Glu His 130 135 140 Ser Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala145 150 155 160 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr 165 170 175 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr 180 185 190 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly Asn Gly Lys Ile Glu His 195 200 205 Leu Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Ala Ala Asp Ile Lys 210 215 220 Pro Asp Gly Lys Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn225 230 235 240 Gln Ala Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala 245 250 255 3234PRTNeisseria meningitidis 3Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Ser 1 5 10 15 Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu 20 25 30 Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser 35 40 45 Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe 50 55 60 Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly65 70 75 80 Glu Phe Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln 85 90 95 Ile Glu Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln 100 105 110 Arg Ser Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn 115 120 125 Gln Leu Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser Ser 130 135 140 Asp Asp Pro Asn Gly Arg Leu His Tyr Ser Ile Asp Phe Thr Lys Lys145 150 155 160 Gln Gly Tyr Gly Arg Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val 165 170 175 Glu Leu Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val 180 185 190 Ile Leu Gly Asp Thr Arg Tyr Gly Gly Glu Glu Lys Gly Thr Tyr His 195 200 205 Leu Ala Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr 210 215 220 Val Lys Ile Arg Glu Lys Val His Glu Thr225 230 4247PRTNeisseria meningitidis 4Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Met Leu Asp Gln Ser 20 25 30 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln65 70 75 80 Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asn His 85 90 95 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 100 105 110 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly 115 120 125 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 130 135 140 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr145 150 155 160 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 165 170 175 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 180 185 190 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly 195 200 205 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 210 215 220 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu225 230 235 240 Ile Gly Ile Ala Gly Lys Gln 245 5279PRTNeisseria meningitidis 5Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr 180 185 190 Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ala Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 6229PRTNeisseria meningitidis 6Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys 1 5 10 15 Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly 20 25 30 Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser 35 40 45 Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr 50 55 60 Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu65 70 75 80 Thr Ala Phe Gln Thr Glu Gln Ile Gln Asp Ser Glu His Ser Gly Lys 85 90 95 Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala Gly Glu His 100 105 110 Thr Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly 115 120 125 Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile 130 135 140 Asp Phe Ala Ala Lys Gln Gly Asn Gly Lys Ile Glu His Leu Lys Ser145 150 155 160 Pro Glu Leu Asn Val Asp Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly 165 170 175 Lys Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu 180 185 190 Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala Gln Glu Val 195 200 205 Ala Gly Ser Ala Glu Val Lys Thr Val Asn Gly Ile Arg His Ile Gly 210 215 220 Leu Ala Ala Lys Gln225 7279PRTNeisseria meningitidis 7Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr 180 185 190 Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ala Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 8228PRTNeisseria meningitidis 8Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn 1 5 10 15 Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn 20 25 30 Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg 35 40 45 Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu 50 55 60 Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr65 70 75 80 Ala Phe Gln Thr Glu Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met 85 90 95 Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr 100 105 110 Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr 115 120 125 Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp 130 135 140 Phe Ala Ala Lys Gln Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro145 150 155 160 Glu Leu Asn Val Asp Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys 165 170 175 Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys 180 185 190 Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala 195 200 205 Gly Ser Ala Glu Val Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu 210 215 220 Ala Ala Lys Gln225 9279PRTNeisseria meningitidis 9Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Ser Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr 180 185 190 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr

Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 10280PRTNeisseria meningitidis 10Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Phe Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 115 120 125 Ser Ala Leu Thr Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His 130 135 140 Ser Gly Lys Met Val Ala Lys Arg Arg Phe Lys Ile Gly Asp Ile Ala145 150 155 160 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr 165 170 175 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr 180 185 190 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His 195 200 205 Leu Lys Ser Pro Glu Leu Asn Val Glu Leu Ala Thr Ala Tyr Ile Lys 210 215 220 Pro Asp Glu Lys His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn225 230 235 240 Gln Asp Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Gln Ala 245 250 255 Gln Glu Val Ala Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His 260 265 270 His Ile Gly Leu Ala Ala Lys Gln 275 280 11225PRTNeisseria meningitidis 11Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu 1 5 10 15 Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser 20 25 30 Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe 35 40 45 Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly 50 55 60 Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln65 70 75 80 Thr Glu Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys 85 90 95 Arg Gln Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp 100 105 110 Lys Leu Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly 115 120 125 Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala 130 135 140 Lys Gln Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn145 150 155 160 Val Asp Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala 165 170 175 Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr 180 185 190 Ser Leu Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala 195 200 205 Glu Val Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys 210 215 220 Gln225 12225PRTNeisseria meningitidis 12Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu 1 5 10 15 Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser 20 25 30 Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe 35 40 45 Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly 50 55 60 Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln65 70 75 80 Thr Glu Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys 85 90 95 Arg Gln Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp 100 105 110 Lys Leu Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe Gly 115 120 125 Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala 130 135 140 Lys Gln Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn145 150 155 160 Val Asp Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His Ala 165 170 175 Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser Tyr 180 185 190 Ser Leu Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser Ala 195 200 205 Glu Val Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys 210 215 220 Gln225 13280PRTNeisseria meningitidis 13Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Phe Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 115 120 125 Ser Ala Leu Thr Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His 130 135 140 Ser Gly Lys Met Val Ala Lys Arg Arg Phe Lys Ile Gly Asp Ile Ala145 150 155 160 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr 165 170 175 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr 180 185 190 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His 195 200 205 Leu Lys Ser Pro Glu Leu Asn Val Glu Leu Ala Thr Ala Tyr Ile Lys 210 215 220 Pro Asp Glu Lys His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn225 230 235 240 Gln Asp Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Gln Ala 245 250 255 Gln Glu Val Ala Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His 260 265 270 His Ile Gly Leu Ala Ala Lys Gln 275 280 14279PRTNeisseria meningitidis 14Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Ser Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr 180 185 190 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 15279PRTNeisseria meningitidis 15Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr 180 185 190 Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ala Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 16280PRTNeisseria meningitidis 16Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 115 120 125 Ser Ala Leu Thr Ala Leu Gln Thr Glu Gln Val Gln Asp Ser Glu His 130 135 140 Ser Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala145 150 155 160 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr 165 170 175 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Ser Gly Lys Leu Thr 180 185 190 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His 195 200 205 Leu Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Ala Ser Asp Ile Lys 210 215 220 Pro Asp Lys Lys Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn225 230 235 240 Gln Ala Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Gln Ala 245 250 255 Gln Glu Val Ala Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile Arg 260 265 270 His Ile Gly Leu Ala Ala Lys Gln 275 280 17279PRTNeisseria meningitidis 17Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asn His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr 180 185 190 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 18279PRTNeisseria meningitidis 18Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100

105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Ser Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr 180 185 190 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 19279PRTNeisseria meningitidis 19Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Ser Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr 180 185 190 Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 20279PRTNeisseria meningitidis 20Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Thr Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asn His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr 180 185 190 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 21280PRTNeisseria meningitidis 21Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 115 120 125 Ser Ala Leu Thr Ala Leu Gln Thr Glu Gln Val Gln Asp Ser Glu His 130 135 140 Ser Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala145 150 155 160 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr 165 170 175 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Ser Gly Lys Leu Thr 180 185 190 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His 195 200 205 Leu Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Ala Ser Asp Ile Lys 210 215 220 Pro Asp Lys Lys Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn225 230 235 240 Gln Ala Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Gln Ala 245 250 255 Gln Glu Val Ala Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile Arg 260 265 270 His Ile Gly Leu Ala Ala Lys Gln 275 280 22279PRTNeisseria meningitidis 22Met Thr Arg Ser Lys Pro Val Asn Arg Thr Ala Phe Cys Cys Leu Ser 1 5 10 15 Leu Thr Ala Ala Leu Ile Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly 20 25 30 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 35 40 45 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 50 55 60 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys65 70 75 80 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 85 90 95 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 100 105 110 Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asn His 115 120 125 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 130 135 140 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly145 150 155 160 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 165 170 175 His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu His Tyr 180 185 190 Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu His Leu 195 200 205 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala 210 215 220 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Gly225 230 235 240 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 245 250 255 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu 260 265 270 Ile Gly Ile Ala Gly Lys Gln 275 23145PRTNeisseria meningitidis 23Met Gly Asn Phe Leu Tyr Arg Gly Ile Ser Cys Gln Gln Asp Glu Gln 1 5 10 15 Asn Asn Gly Gln Leu Lys Pro Lys Gly Asn Lys Ala Glu Val Ala Ile 20 25 30 Arg Tyr Asp Gly Lys Phe Lys Tyr Asp Gly Lys Ala Thr His Gly Pro 35 40 45 Ser Val Lys Asn Ala Val Tyr Ala His Gln Ile Glu Thr Gly Leu Tyr 50 55 60 Asp Gly Cys Tyr Ile Ser Thr Thr Thr Asp Lys Glu Ile Ala Lys Lys65 70 75 80 Phe Ala Thr Ser Ser Gly Ile Glu Asn Gly Tyr Ile Tyr Val Leu Asn 85 90 95 Arg Asp Leu Phe Gly Gln Tyr Ser Ile Phe Glu Tyr Glu Val Glu His 100 105 110 Pro Glu Asn Pro Asn Glu Lys Glu Val Thr Leu Arg Ala Glu Asp Cys 115 120 125 Gly Cys Ile Pro Glu Glu Val Ile Ile Ala Lys Glu Leu Ile Glu Ile 130 135 140 Asn145 24150PRTNeisseria gonorrhoeae 24Ile Asn Asn Leu Trp Glu Ile Ser Tyr Leu Tyr Arg Gly Ile Ser Cys 1 5 10 15 Gln Gln Asp Glu Gln Asn Asn Gly Gln Leu Lys Pro Lys Gly Asn Lys 20 25 30 Ala Glu Val Ala Ile Arg Tyr Asp Gly Lys Phe Lys Tyr Asp Gly Lys 35 40 45 Ala Thr His Gly Pro Ser Val Lys Asn Ala Val Tyr Ala His Gln Ile 50 55 60 Glu Thr Asp Leu Tyr Asp Gly Cys Tyr Ile Ser Thr Thr Thr Asp Lys65 70 75 80 Glu Ile Ala Lys Lys Phe Ala Thr Ser Ser Gly Ile Glu Asn Gly Tyr 85 90 95 Ile Tyr Val Leu Asn Arg Asp Leu Phe Gly Gln Tyr Ser Ile Phe Glu 100 105 110 Tyr Glu Val Glu His Pro Glu Asn Pro Asp Glu Lys Glu Val Thr Leu 115 120 125 Arg Ala Glu Asp Cys Gly Cys Ile Pro Glu Glu Val Ile Ile Ala Lys 130 135 140 Glu Leu Ile Glu Ile Asn145 150 25634DNANeisseria gonorrhoeae 25tccgccgcat taccttataa aataaaacat ccctctcaag cagtctgata atgtttggat 60tgcttgagat tgatgagtga tggtgttaaa ttcaaacttt aaattaataa cttatgggaa 120atttcttatt tatatagagg cattagttgc caacaagatg agcaaaataa tggacagtta 180aaacctaaag gtaataaagc tgaagttgca attcgttatg atggtaagtt taaatatgat 240ggtaaagcta cacatggtcc aagtgtgaag aatgcagttt acgcccatca aattgaaaca 300gatctatatg acggatgtta tatatctacg acaacagaca aggaaattgc caagaaattt 360gcaacaagct ccggcatcga aaatggctat atatatgttt taaatagaga tttgtttggt 420caatattcta tttttgaata tgaggttgaa catccagaaa acccagatga gaaggaagta 480acaatcagag ctgaagattg tggctgtatt cctgaagaag tgattattgc taaagagttg 540atagaaatta actaagttga aaggtcaata taatggcttt agttgaattg aaagtgcccg 600acattggcgg acacgaaaat gtagatatta tcgc 63426630DNANeisseria meningitidis 26tccgccgcat taccttataa aataaaacat ccctctcaag cagtctgata atgtttggat 60tgcttgagat tgatgagtaa tggtgttaaa ttcaaccttt aaattaataa cttatgggaa 120atttcttata tagaggcatt agttgccaac aagatgagca aaataatgga cagttaaaac 180ctaaaggtaa taaagctgaa gttgcaattc gttatgatgg taagtttaaa tatgatggta 240aagctacaca tggtccaagt gtgaagaatg cagtttacgc ccatcaaatt gaaacaggtc 300tatatgacgg atgttatata tctacgacaa cagacaagga aattgccaag aaatttgcaa 360caagttccgg catcgaaaat ggctatatat atgttttaaa tagggatttg tttggtcaat 420attctatttt tgaatatgag gttgaacatc cagaaaaccc aaatgagaag gaagtaacaa 480tcagagctga agattgtggc tgtattcctg aagaagtgat tattgctaaa gagttgatag 540aaattaacta agttgaaagg tcaatataat ggctttagtt gaattgaaag tgcccgacat 600tggcggacac gaaaatgtag atattatcgc 630276PRTArtificial SequenceSynthetic Construct 27Gly Ser Gly Gly Gly Gly1 5 28579PRTArtificial SequenceSynthetic Construct 28Met Ala Ser Pro Asp Val Lys Ser Ala Asp Thr Leu Ser Lys Pro Ala 1 5 10 15 Ala Pro Val Val Ala Glu Lys Glu Thr Glu Val Lys Glu Asp Ala Pro 20 25 30 Gln Ala Gly Ser Gln Gly Gln Gly Ala Pro Ser Thr Gln Gly Ser Gln 35 40 45 Asp Met Ala Ala Val Ser Ala Glu Asn Thr Gly Asn Gly Gly Ala Ala 50 55 60 Thr Thr Asp Lys Pro Lys Asn Glu Asp Glu Gly Pro Gln Asn Asp Met65 70 75 80 Pro Gln Asn Ser Ala Glu Ser Ala Asn Gln Thr Gly Asn Asn Gln Pro 85 90 95 Ala Asp Ser Ser Asp Ser Ala Pro Ala Ser Asn Pro Ala Pro Ala Asn 100 105 110 Gly Gly Ser Asn Phe Gly Arg Val Asp Leu Ala Asn Gly Val Leu Ile 115 120 125 Asp Gly Pro Ser Gln Asn Ile Thr Leu Thr His Cys Lys Gly Asp Ser 130 135 140 Cys Asn Gly Asp Asn Leu Leu Asp Glu Glu Ala Pro Ser Lys Ser Glu145 150 155 160 Phe Glu Asn Leu Asn Glu Ser Glu Arg Ile Glu Lys Tyr Lys Lys Asp 165 170 175 Gly Lys Ser Asp Lys Phe Thr Asn Leu Val Ala Thr Ala Val Gln Ala 180 185 190 Asn Gly Thr Asn Lys Tyr Val Ile Ile Tyr Lys Asp Lys Ser Ala Ser 195 200 205 Ser Ser Ser Ala Arg Phe Arg Arg Ser Ala Arg Ser Arg Arg Ser Leu 210 215 220 Pro Ala Glu Met Pro Leu Ile Pro Val Asn Gln Ala Asp Thr Leu Ile225 230 235 240 Val Asp Gly Glu Ala Val Ser Leu Thr Gly His Ser Gly Asn Ile Phe 245 250 255 Ala Pro Glu Gly Asn Tyr Arg Tyr Leu Thr Tyr Gly Ala Glu Lys Leu 260 265 270 Pro Gly Gly Ser Tyr Ala Leu Arg Val Gln Gly Glu Pro Ala Lys Gly 275 280 285 Glu Met Leu Ala Gly Thr Ala Val Tyr Asn Gly Glu Val Leu His Phe 290 295 300 His Thr Glu Asn Gly Arg Pro Tyr Pro Thr Arg Gly Arg Phe Ala Ala305 310 315 320 Lys Val Asp Phe Gly Ser Lys Ser Val Asp Gly Ile Ile Asp Ser Gly 325 330 335 Asp Asp Leu His Met Gly Thr Gln Lys Phe Lys Ala Ala Ile Asp Gly 340 345 350 Asn Gly Phe Lys Gly Thr Trp Thr Glu Asn Gly Gly Gly Asp Val Ser 355 360 365 Gly Arg Phe Tyr Gly Pro Ala Gly Glu Glu Val Ala Gly Lys Tyr Ser 370 375 380 Tyr Arg Pro Thr Asp Ala Glu Lys Gly Gly Phe Gly Val Phe Ala Gly385 390 395 400 Lys Lys Glu Gln Asp Gly Ser Gly Gly Gly Gly Ala Thr Tyr Lys Val 405 410 415 Asp Glu Tyr His Ala Asn Ala Arg Phe Ala Ile Asp His Phe Asn Thr 420 425 430 Ser Thr Asn Val Gly Gly Phe Tyr Gly Leu Thr Gly Ser Val Glu Phe 435 440 445 Asp Gln Ala Lys Arg Asp Gly Lys Ile Asp Ile Thr Ile Pro Val Ala 450 455 460 Asn Leu Gln Ser Gly Ser Gln His Phe Thr Asp His Leu Lys Ser Ala465

470 475 480 Asp Ile Phe Asp Ala Ala Gln Tyr Pro Asp Ile Arg Phe Val Ser Thr 485 490 495 Lys Phe Asn Phe Asn Gly Lys Lys Leu Val Ser Val Asp Gly Asn Leu 500 505 510 Thr Met His Gly Lys Thr Ala Pro Val Lys Leu Lys Ala Glu Lys Phe 515 520 525 Asn Cys Tyr Gln Ser Pro Met Ala Lys Thr Glu Val Cys Gly Gly Asp 530 535 540 Phe Ser Thr Thr Ile Asp Arg Thr Lys Trp Gly Val Asp Tyr Leu Val545 550 555 560 Asn Val Gly Met Thr Lys Ser Val Arg Ile Asp Ile Gln Leu Glu Ala 565 570 575 Ala Lys Gln29644PRTArtificial SequenceSynthetic Construct 29Met Ala Ser Pro Asp Val Lys Ser Ala Asp Thr Leu Ser Lys Pro Ala 1 5 10 15 Ala Pro Val Val Ser Glu Lys Glu Thr Glu Ala Lys Glu Asp Ala Pro 20 25 30 Gln Ala Gly Ser Gln Gly Gln Gly Ala Pro Ser Ala Gln Gly Gly Gln 35 40 45 Asp Met Ala Ala Val Ser Glu Glu Asn Thr Gly Asn Gly Gly Ala Ala 50 55 60 Ala Thr Asp Lys Pro Lys Asn Glu Asp Glu Gly Ala Gln Asn Asp Met65 70 75 80 Pro Gln Asn Ala Ala Asp Thr Asp Ser Leu Thr Pro Asn His Thr Pro 85 90 95 Ala Ser Asn Met Pro Ala Gly Asn Met Glu Asn Gln Ala Pro Asp Ala 100 105 110 Gly Glu Ser Glu Gln Pro Ala Asn Gln Pro Asp Met Ala Asn Thr Ala 115 120 125 Asp Gly Met Gln Gly Asp Asp Pro Ser Ala Gly Gly Glu Asn Ala Gly 130 135 140 Asn Thr Ala Ala Gln Gly Thr Asn Gln Ala Glu Asn Asn Gln Thr Ala145 150 155 160 Gly Ser Gln Asn Pro Ala Ser Ser Thr Asn Pro Ser Ala Thr Asn Ser 165 170 175 Gly Gly Asp Phe Gly Arg Thr Asn Val Gly Asn Ser Val Val Ile Asp 180 185 190 Gly Pro Ser Gln Asn Ile Thr Leu Thr His Cys Lys Gly Asp Ser Cys 195 200 205 Ser Gly Asn Asn Phe Leu Asp Glu Glu Val Gln Leu Lys Ser Glu Phe 210 215 220 Glu Lys Leu Ser Asp Ala Asp Lys Ile Ser Asn Tyr Lys Lys Asp Gly225 230 235 240 Lys Asn Asp Gly Lys Asn Asp Lys Phe Val Gly Leu Val Ala Asp Ser 245 250 255 Val Gln Met Lys Gly Ile Asn Gln Tyr Ile Ile Phe Tyr Lys Pro Lys 260 265 270 Pro Thr Ser Phe Ala Arg Phe Arg Arg Ser Ala Arg Ser Arg Arg Ser 275 280 285 Leu Pro Ala Glu Met Pro Leu Ile Pro Val Asn Gln Ala Asp Thr Leu 290 295 300 Ile Val Asp Gly Glu Ala Val Ser Leu Thr Gly His Ser Gly Asn Ile305 310 315 320 Phe Ala Pro Glu Gly Asn Tyr Arg Tyr Leu Thr Tyr Gly Ala Glu Lys 325 330 335 Leu Pro Gly Gly Ser Tyr Ala Leu Arg Val Gln Gly Glu Pro Ser Lys 340 345 350 Gly Glu Met Leu Ala Gly Thr Ala Val Tyr Asn Gly Glu Val Leu His 355 360 365 Phe His Thr Glu Asn Gly Arg Pro Ser Pro Ser Arg Gly Arg Phe Ala 370 375 380 Ala Lys Val Asp Phe Gly Ser Lys Ser Val Asp Gly Ile Ile Asp Ser385 390 395 400 Gly Asp Gly Leu His Met Gly Thr Gln Lys Phe Lys Ala Ala Ile Asp 405 410 415 Gly Asn Gly Phe Lys Gly Thr Trp Thr Glu Asn Gly Gly Gly Asp Val 420 425 430 Ser Gly Lys Phe Tyr Gly Pro Ala Gly Glu Glu Val Ala Gly Lys Tyr 435 440 445 Ser Tyr Arg Pro Thr Asp Ala Glu Lys Gly Gly Phe Gly Val Phe Ala 450 455 460 Gly Lys Lys Glu Gln Asp Gly Ser Gly Gly Gly Gly Ala Thr Tyr Lys465 470 475 480 Val Asp Glu Tyr His Ala Asn Ala Arg Phe Ala Ile Asp His Phe Asn 485 490 495 Thr Ser Thr Asn Val Gly Gly Phe Tyr Gly Leu Thr Gly Ser Val Glu 500 505 510 Phe Asp Gln Ala Lys Arg Asp Gly Lys Ile Asp Ile Thr Ile Pro Val 515 520 525 Ala Asn Leu Gln Ser Gly Ser Gln His Phe Thr Asp His Leu Lys Ser 530 535 540 Ala Asp Ile Phe Asp Ala Ala Gln Tyr Pro Asp Ile Arg Phe Val Ser545 550 555 560 Thr Lys Phe Asn Phe Asn Gly Lys Lys Leu Val Ser Val Asp Gly Asn 565 570 575 Leu Thr Met His Gly Lys Thr Ala Pro Val Lys Leu Lys Ala Glu Lys 580 585 590 Phe Asn Cys Tyr Gln Ser Pro Met Ala Lys Thr Glu Val Cys Gly Gly 595 600 605 Asp Phe Ser Thr Thr Ile Asp Arg Thr Lys Trp Gly Val Asp Tyr Leu 610 615 620 Val Asn Val Gly Met Thr Lys Ser Val Arg Ile Asp Ile Gln Ile Glu625 630 635 640 Ala Ala Lys Gln30456PRTArtificial SequenceSynthetic Construct 30Met Lys Pro Lys Pro His Thr Val Arg Thr Leu Leu Ala Ala Ile Phe 1 5 10 15 Ser Leu Ala Leu Ser Gly Cys Val Ser Ala Val Ile Gly Ser Ala Ala 20 25 30 Val Gly Ala Lys Ser Ala Val Asp Arg Arg Thr Thr Gly Ala Gln Thr 35 40 45 Asp Asp Asn Val Met Ala Leu Arg Ile Glu Thr Thr Ala Arg Ser Tyr 50 55 60 Leu Arg Gln Asn Asn Gln Thr Lys Gly Tyr Thr Pro Gln Ile Ser Val65 70 75 80 Val Gly Tyr Asn Arg His Leu Leu Leu Leu Gly Gln Val Ala Thr Glu 85 90 95 Gly Glu Lys Gln Phe Val Gly Gln Ile Ala Arg Ser Glu Gln Ala Ala 100 105 110 Glu Gly Val Tyr Asn Tyr Ile Thr Val Ala Ser Leu Pro Arg Thr Ala 115 120 125 Gly Asp Ile Ala Gly Asp Thr Trp Asn Thr Ser Lys Val Arg Ala Thr 130 135 140 Leu Leu Gly Ile Ser Pro Ala Thr Gln Ala Arg Val Lys Ile Val Thr145 150 155 160 Tyr Gly Asn Val Thr Tyr Val Met Gly Ile Leu Thr Pro Glu Glu Gln 165 170 175 Ala Gln Ile Thr Gln Lys Val Ser Thr Thr Val Gly Val Gln Lys Val 180 185 190 Ile Thr Leu Tyr Gln Asn Tyr Val Gln Arg Gly Ser Gly Gly Gly Gly 195 200 205 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 210 215 220 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser225 230 235 240 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 245 250 255 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 260 265 270 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 275 280 285 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 290 295 300 Ser Ala Leu Thr Ala Phe Gln Thr Glu Gln Ile Gln Asp Ser Glu His305 310 315 320 Ser Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala 325 330 335 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr 340 345 350 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr 355 360 365 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly Asn Gly Lys Ile Glu His 370 375 380 Leu Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Ala Ala Asp Ile Lys385 390 395 400 Pro Asp Gly Lys Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn 405 410 415 Gln Ala Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala 420 425 430 Gln Glu Val Ala Gly Ser Ala Glu Val Lys Thr Val Asn Gly Ile Arg 435 440 445 His Ile Gly Leu Ala Ala Lys Gln 450 455 31327PRTNeisseria meningitidis 31Met Thr Asn Asp Asp Asp Val Lys Lys Ala Ala Thr Val Ala Leu Ala 1 5 10 15 Ala Ala Tyr Asn Asn Gly Gln Glu Ile Asn Gly Phe Lys Ala Gly Glu 20 25 30 Thr Ile Tyr Asp Ile Asp Glu Asp Gly Thr Ile Thr Lys Lys Asp Ala 35 40 45 Thr Ala Ala Asp Val Glu Ala Asp Asp Phe Lys Gly Leu Gly Leu Lys 50 55 60 Lys Val Val Thr Asn Leu Thr Lys Thr Val Asn Glu Asn Lys Gln Asn65 70 75 80 Val Asp Ala Lys Val Lys Ala Ala Glu Ser Glu Ile Glu Lys Leu Thr 85 90 95 Thr Lys Leu Ala Asp Thr Asp Ala Ala Leu Ala Asp Thr Asp Ala Ala 100 105 110 Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn Ile Thr 115 120 125 Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp Glu Lys 130 135 140 Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala Phe Asn145 150 155 160 Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp Glu Ala 165 170 175 Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr Lys Gln 180 185 190 Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly Lys Ala 195 200 205 Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala Ala Asp Lys Ala Glu Ala 210 215 220 Val Ala Ala Lys Val Thr Asp Leu Lys Ala Asp Ile Ala Thr Asn Lys225 230 235 240 Asp Asn Ile Ala Lys Lys Ala Asn Ser Ala Asp Val Tyr Thr Arg Glu 245 250 255 Glu Ser Asp Ser Lys Phe Val Arg Ile Asp Gly Leu Asn Ala Thr Thr 260 265 270 Glu Lys Leu Asp Thr Arg Leu Ala Ser Ala Glu Lys Ser Ile Ala Asp 275 280 285 His Asp Thr Arg Leu Asn Gly Leu Asp Lys Thr Val Ser Asp Leu Arg 290 295 300 Lys Glu Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu305 310 315 320 Phe Gln Pro Tyr Asn Val Gly 325 326PRTArtificial SequenceSynthetic Construct 32Cys Gly Gly Gly Gly Ser1 5 335PRTArtificial SequenceSynthetic Construct 33Cys Gly Gly Gly Ser1 5 344PRTArtificial SequenceSynthetic Construct 34Cys Gly Gly Ser1 356PRTArtificial SequenceSynthetic ConstructVARIANT3Xaa = Any Amino Acid 35Cys Gly Xaa Gly Gly Ser1 5 366PRTArtificial SequenceSynthetic ConstructVARIANT3, 4Xaa = Any Amino Acid 36Cys Gly Xaa Xaa Gly Ser1 5 376PRTArtificial SequenceSynthetic ConstructVARIANT3, 5Xaa = Any Amino Acid 37Cys Gly Xaa Gly Xaa Ser1 5 387PRTArtificial SequenceSynthetic ConstructVARIANT4Xaa = Any Amino Acid 38Cys Gly Gly Xaa Gly Gly Ser1 5 397PRTArtificial SequenceSynthetic ConstructVARIANT3Xaa = Any Amino Acid 39Cys Gly Xaa Gly Gly Gly Ser1 5

Claims

1-22. (canceled)

23: An immunogenic composition comprising an aluminum salt adjuvant at a concentration from 0.6 mg/ml to 3.3 mg/ml and a purified polypeptide comprising (i) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 50 consecutive amino acids from SEQ ID NO: 3.

24: The immunogenic composition of claim 23, wherein the polypeptide comprises (i) the amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 60 consecutive amino acids from SEQ ID NO: 3.

25: An immunogenic composition comprising an aluminum salt adjuvant at a concentration from 0.6 mg/ml to 3.3 mg/ml and a purified protein having formula: NH.sub.2-A-X.sub.1-L-X.sub.2--B--COOH wherein A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, L is an optional amino acid sequence between X.sub.1 and X.sub.2, and X.sub.1 is an amino acid sequence comprising at least 40 consecutive amino acids from SEQ ID NO: 3 and X.sub.2 is an amino acid sequence comprising at least 40 consecutive amino acids from SEQ ID NO: 7.

26: The composition of claim 25, wherein the purified polypeptide is adsorbed to the aluminum salt adjuvant.

27: The composition of claim 25, wherein the aluminum salt adjuvant is an aluminum hydroxyphosphate adjuvant.

28: A composition comprising a purified protein comprising (i) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 50 consecutive amino acids from SEQ ID NO: 3, adsorbed to an aluminum salt adjuvant.

29: The composition of claim 28, wherein the polypeptide comprises (i) the amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 60 consecutive amino acids from SEQ ID NO: 3.

30: A composition comprising a purified protein comprising (i) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 50 consecutive amino acids from SEQ ID NO: 3, and an aluminum hydroxyphosphate adjuvant.

31: The composition of claim 30, wherein the polypeptide comprises (i) the amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 60 consecutive amino acids from SEQ ID NO: 3.

32: A method of inducing an immune response in a subject comprising administering to the subject the immunogenic composition of claim 23.

33: The method of claim 32, wherein the polypeptide comprises (i) the amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 60 consecutive amino acids from SEQ ID NO: 3.

34: A method of inducing an immune response in a subject comprising administering to the subject the immunogenic composition of claim 25.

35: The method of claim 34, wherein the purified polypeptide is adsorbed to the aluminum salt adjuvant.

36: The method of claim 34, wherein the aluminum salt adjuvant is an aluminum hydroxyphosphate adjuvant.

37: A method of inducing an immune response in a subject comprising administering to the subject the immunogenic composition of claim 28.

38: The method of claim 37, wherein the polypeptide comprises (i) the amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 60 consecutive amino acids from SEQ ID NO: 3.

39: A method of inducing an immune response in a subject comprising administering to the subject the immunogenic composition of claim 30.

40: The method of claim 39, wherein the polypeptide comprises (i) the amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3, and (ii) at least 60 consecutive amino acids from SEQ ID NO: 3.