Patent application title: METHODS OF INDUCING AN IMMUNE RESPONSE WITH COMPOSITIONS COMPRISING A NEISSERIA MENINGITIDIS 741 PROTEIN
Inventors:
Mariagrazia Pizza (Siena, IT)
Assignees:
GlaxoSmithKline Biologicals, s.a.
IPC8 Class: AA61K39095FI
USPC Class:
Class name:
Publication date: 2015-10-01
Patent application number: 20150273044
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 NH2-A-[--X-L-]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`.Claims:
1. (canceled)
2. A purified polypeptide comprising (i) an amino acid sequence having at least 80% identity to SEQ ID NO: 3, or (ii) at least 50 consecutive amino acids from SEQ ID NO: 3.
3. The purified polypeptide of claim 2, wherein the amino acid sequence has at least 90% identity to SEQ ID NO: 3.
4. The purified polypeptide of claim 2, wherein the polypeptide comprises at least 60 consecutive amino acids from SEQ ID NO: 3.
5. A method of inducing an immune response in a subject comprising administering to the subject an effective amount of a composition comprising the purified polypeptide of claim 2.
6. The method of claim 5, wherein the composition comprises an aluminum salt adjuvant.
7. The method of claim 6, wherein the purified polypeptide is adsorbed to the aluminum salt adjuvant.
8. The method of claim 6, wherein the aluminum salt adjuvant comprises aluminum phosphate.
9. The method of claim 7, wherein the aluminum salt adjuvant comprises aluminum phosphate.
10. The method of claim 6, wherein the aluminum salt adjuvant comprises aluminum phosphate.
11. The method of claim 7, wherein the aluminum salt adjuvant comprises aluminum phosphate.
12. A purified protein having formula: NH2-A-[--X-L-]n-B--COOH wherein 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 2 and X is an amino acid sequence comprising at least 40 consecutive amino acids from a 741 protein, wherein the first 741 protein is SEQ ID NO: 3 and the second 741 protein is SEQ ID NO: 7.
13. A composition comprising the purified protein of claim 12 adsorbed to an aluminum salt adjuvant.
14. A composition comprising the purified protein of claim 12 and an aluminum hydroxyphosphate adjuvant.
15. A method of inducing an immune response in a subject comprising administering to the subject an effective amount of a composition comprising the purified polypeptide of claim 12.
16. The method of claim 15, wherein the composition comprises an aluminum salt adjuvant.
17. The method of claim 16, wherein the purified polypeptide is adsorbed to the aluminum salt adjuvant.
18. The method of claim 16, wherein the aluminum salt adjuvant comprises aluminum phosphate.
19. The method of claim 17, wherein the aluminum salt adjuvant comprises aluminum phosphate.
20. The method of claim 16, wherein the aluminum salt adjuvant comprises aluminum phosphate.
21. The method of claim 17, wherein the aluminum salt adjuvant comprises aluminum phosphate.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application 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, which claims 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; the disclosures of which are herein incorporated by reference in their entirety.
[0002] All documents cited herein are 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: 223002100602SeqList.txt, date recorded: Jun. 12, 2015, size: 70 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 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: NH2-A-[--X-L-]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 (--Xa--) and a second --X-- moiety (--Xb--). The --Xa-- 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 --Xb-- moiety is related to --Xa-- such that: (i) --Xb-- has sequence identity to 13 Xa--, and/or (j) --Xb-- comprises a fragment of --Xa--.
[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 --Xa--Xb--, and three polymorphic forms may be expressed as --Xa--Xb--Xa-- etc.
[0025] The --Xa-- and --Xb-- moieties may be in either order from N-terminus to C-terminus.
[0026] The --Xa-- 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 --Xa-- moiety is more preferably an orf46.1, 230, 287, 741, 919, 936, 953, 961 or 983 amino acid sequence. The --Xa-- 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).
[0030] Preferably the fragment comprises an epitope from an amino acid sequence from (a), (d), (e), (t), (g) or (h). Preferred fragments are those disclosed in references 9 and 10.
[0031] Preferred (c) and (j) fragments are C- and/or N-terminal truncations (e.g. Δ1-287, A2-287 etc.).
[0032] 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)g, where g>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)g--by deletion (e.g. CGGGGS→CGGGS, CGGS, CGS or CS), by substitution (e.g. CGGGGS→CGXGGS, CGXXGS, CGXGXS etc.), and/or by insertion (e.g. CGGGGS→CGGXGGS, CGXGGGS, etc.). Deletion of (Gly)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 `ΔG`. Poly-glycine omission is particularly useful for proteins 287, 741, 983 and Tbp2 (ΔG287, ΔG741, ΔG983 and ΔGTbp2--references 1 & 2).
[0033] 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, ORF461-433, ORF46434-608, 961c-- reference 2; FIGS. 4 and 5 herein).
[0034] 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.
[0035] 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.
[0036] 961 protein has been notionally split into several domains (FIG. 4).
[0037] 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 X1 will be retained, but the leader peptides of X2 . . . Xn will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of X1 as moiety -A-.
[0038] When n=2, preferred pairs of --X-- moieties are: ΔG287 and 230; ΔG287 and 936; ΔG287 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: ΔG741 and 741; ΔG287 and 287. More specifically, the following combinations of X1 and X2 are preferred when n=2:
TABLE-US-00001 X1 X2 ΔG287 230 ΔG287 936 ΔG287 741 ΔG287 961 ΔG287 ORF46.1 ΔG287 919 ΔG287 953 961c 287 961c 230 961c 936 961c 741 961c 983 961c ΔG983 961c ORF46.1 961 ORF46.1 961cL 287 961cL 230 961cL 936 ORF46.1 936 ORF46.1 230 ORF46.1 741 ORF46.1 ΔG741 ORF46.1 983 ORF46.1 ΔG983 230 961 230 741 230 ΔG741 936 961 936 741 936 ΔG741 ΔG741 741 ORF46.1 983 ΔG741 ORF46.1 ΔG741 983 ΔG741 961 ΔG741 961c ΔG983 ORF46.1 ΔG983 961 ΔG983 961c 230 ΔG287 936 ΔG287 741 ΔG287 961 ΔG287 ORF46.1 ΔG287 919 ΔG287 953 ΔG287 287 961c 230 961c 936 961c 741 961c 983 961c ΔG983 961c ORF46.1 961c ORF46.1 961 287 961cL 230 961cL 936 961cL 936 ORF46.1 230 ORF46.1 741 ORF46.1 ΔG741 ORF46.1 983 ORF46.1 ΔG983 ORF46.1 961 230 741 230 ΔG741 230 961 936 741 936 ΔG741 936 ΔG287 287 983 ORF46.1 ORF46.1 ΔG741 983 ΔG741 961 ΔG741 961c ΔG741 ORF46.1 ΔG983 961 ΔG983 961c ΔG983
[0039] 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 ΔG 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 ΔG form of 741.
[0040] The -L- Moieties
[0041] 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 NH2--X1-L1-X2-L2-COOH, NH2--X1--X2--COOH, NH2--X1-L1-X2--COOH, NH2--X1--X2-L2-COOH, etc.
[0042] 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. Glyn where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. Hisn 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 Gly4 tetrapeptide being a typical poly-glycine linker.
[0043] If Xn+1 is a ΔG protein and Ln is a glycine linker, this may be equivalent to Xn+1 not being a ΔG protein and Ln being absent.
[0044] The -A- Moiety
[0045] -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. Hisn 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 X1 lacks its own N-terminus methionine, -A- may be a methionine residue.
[0046] The --B-- Moiety
[0047] --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. Hisn 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.
[0048] Polymorphic Forms of Proteins
[0049] 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).
[0050] 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
[0051] 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.
[0052] The sequence listing herein includes polymorphic forms of proteins 741 (SEQ IDs 1-22) and NMB 1343 (SEQ IDs 23-24) which have been identified.
[0053] Serogroups and Strains
[0054] 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, X2 may be from the same strain as X1 or from a different strain. Where n=3, the strains might be (i) X1═X2═X3 (ii) X1═X2≠X3 (iii) X1≠X2═X3 (iv) X1≠X2≠X3 or (v) X1═X3≠X2, etc.
[0055] 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.
[0056] Where a form of 287 is used, this is preferably from strain 2996 or from strain 394/98.
[0057] 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.
[0058] Where a form of 961 is used, this is preferably from strain 2996.
[0059] 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).
[0060] Domain-Based Expression of Protein 961
[0061] 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]).
[0062] 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 Domain name Amino acids 961-1 `L` 1-23 961-6 269-286 961-2 24-87 961-7 287-330 961-3 88-143 961-8 331-350 961-4 144-180 961-9 351-405 961-5 181-268
[0063] This information can be used to locate the same domains in other forms of 961.
[0064] 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:
[0065] 961-2 to 961-5 (`961a`)
[0066] 961-6 to 961-9 (`961b`)
[0067] 961-1 to 961-8 (`961 cL`)
[0068] 961-2 to 961-8 (`961c`)
[0069] 961-2 to 961-6 and amino acids 287-325 from domain 961-7 (`961d`)
[0070] 961-2 to 961-8 and amino acids 351-383 from domain 961-9 (`961Δ1`)
[0071] 961-1 to 961-8 and amino acids 351-383 from domain 961-9 (`961Δ1L`)
[0072] 961-1 to 961-7 and amino acids 331-343 from domain 961-8 (`961cL-Δaro`)
[0073] 961-1 to 961-6 and amino acids 287-315 from domain 961-7 (`961cL-Δcc`)
[0074] 961-1 to 961-5 (`961aL`)
[0075] 961-1 to 961-4 (`961aL-Δ1`)
[0076] 961-1 to 961-3 (`961aL-Δ2`)
[0077] 961-1 to 961-2 (`961aL-Δ3`)
[0078] 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).
[0079] Sequences
[0080] 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).
[0081] The invention also provides nucleic acid encoding such proteins.
[0082] Furthermore, the invention provides nucleic acid which can hybridise to this nucleic acid, preferably under "high stringency" conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).
[0083] The invention also provides nucleic acid encoding proteins according to the invention.
[0084] It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).
[0085] 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.).
[0086] 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.
[0087] Mixtures
[0088] The invention also provides a composition comprising two or more (i.e. 2, 3, 4, 5, 6 or 7) of the following proteins:
[0089] (1) 287
[0090] (2) 741
[0091] (3) ORF46.1
[0092] (4) 961
[0093] (5) NH2-A-[--X-L-]n-B--COOH, wherein n=2, X1=287, X2=953
[0094] (6) NH2-A-[--X-L-]n-B--COOH, wherein n=2, X1=287, X2=919
[0095] (7) NH2-A-[--X-L-]n-B--COOH, wherein n=2, X1=287, X2=961
[0096] 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):
[0097] (8) NH2-A-[--X-L-]n-B--COOH, wherein n=2, X1=287, X2=741
[0098] (9) NH2-A-[--X-L-]n-B--COOH, wherein n=2, X1=936, X2=741
[0099] 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 `ΔG` 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].
[0100] A preferred mixture comprises the following three proteins:
[0101] (1) 961c, preferably 961c2996 (e.g. SEQ ID 31 herein);
[0102] (2) NH2-A-[--X-L-]n-B--COOH, wherein n is 2, --X1-- is ΔG287 (preferably ΔG287NZ), --X2-- is 953 (preferably 9532996) lacking its leader peptide, -L1- is GSGGGG, and -A- comprises a N-terminus methionine (e.g. -A- is M or MA) (e.g. SEQ IDs 28 & 29 herein); and
[0103] (3) NH2-A-[--X-L-]n-B--COOH, wherein n=2, X1=936 (preferably 9362996), X2=ΔG741 (preferably ΔG741.sub.MC58), L1=GSGGGG (e.g. SEQ ID 30 herein).
[0104] The mixtures may also comprise N. meningitidis outer membrane vesicles.
[0105] Heterologous Host
[0106] 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.
[0107] Vectors Etc.
[0108] 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.
[0109] 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.
[0110] To improve solubility, purification of hybrid proteins may involve the refolding techniques disclosed herein.
[0111] Immunogenic Compositions and Medicaments
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] Further Components of the Composition
[0120] 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.
[0121] 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.
[0122] The vaccine may be administered in conjunction with other immunoregulatory agents.
[0123] 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® (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® 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®); (2) saponin adjuvants, such as QS21 or Stimulon® (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 3-0-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;
[0124] (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.
[0125] 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.
[0126] Further Antigens
[0127] Further antigens which can be included in the composition of the invention include:
[0128] an outer-membrane vesicle (OMV) preparation from N. meningitidis serogroup B, such as those disclosed in refs. 14, 15, 16, 17 etc.
[0129] 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.
[0130] a saccharide antigen from Streptococcus pneumoniae [e.g. refs. 21, 22, 23].
[0131] 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.
[0132] an antigen from hepatitis A virus, such as inactivated virus [e.g. 27, 28].
[0133] an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. 28, 29].
[0134] an antigen from hepatitis C virus [e.g. 30].
[0135] 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].
[0136] a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref. 33] e.g. the CRM197 mutant [e.g. 34].
[0137] a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 33].
[0138] a saccharide antigen from Haemophilus influenzae B [e.g. 19].
[0139] an antigen from N. gonorrhoeae [e.g. 3, 4, 5].
[0140] an antigen from Chlamydia pneumoniae [e.g. 35, 36, 37, 38, 39, 40, 41].
[0141] an antigen from Chlamydia trachomatis [e.g. 42].
[0142] an antigen from Porphyromonas gingivalis [e.g. 43].
[0143] polio antigen(s) [e.g. 44, 45] such as IPV or OPV.
[0144] rabies antigen(s) [e.g. 46] such as lyophilised inactivated virus [e.g.47, RabAvert®]
[0145] measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref. 33].
[0146] influenza antigen(s) [e.g. chapter 19 of ref. 33], such as the haemagglutinin and/or neuraminidase surface proteins.
[0147] an antigen from Moraxella catarrhalis [e.g. 48].
[0148] a protein antigen from Streptococcus agalactiae (group B streptococcus) [e.g. 49, 50].
[0149] a saccharide antigen from Streptococcus agalactiae
[0150] an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 50, 51, 52].
[0151] an antigen from Staphylococcus aureus [e.g. 53].
[0152] The composition may comprise one or more of these further antigens.
[0153] 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 63]. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRM197 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.
[0154] Any suitable conjugation reaction can be used, with any suitable linker where necessary.
[0155] Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [32]).
[0156] 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.
[0157] 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.).
[0158] Antigens in the composition will typically be present at a concentration of at least 1μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
[0159] 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 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.
[0160] Definitions
[0161] 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.
[0162] The term "about" in relation to a numerical value x means, for example, x±10%.
BRIEF DESCRIPTION OF DRAWINGS
[0163] FIG. 1 shows an alignment of twenty-three sequences for protein 741. These are SEQ IDs 1 to 22 plus the sequence from MC58.
[0164] FIG. 2 shows an alignment of the NMB 1343 sequence from gonococcus (top; SEQ ID 25) and meningococcus (bottom; SEQ ID 26).
[0165] FIG. 3 shows hybrid and tandem proteins of the invention.
[0166] FIG. 4 shows 9 domains within 9612996, and FIG. 5 shows how these have been manipulated.
MODES FOR CARRYING OUT THE INVENTION
[0167] Hybrid Proteins--X1=ΔG287
[0168] In addition to those disclosed in references 1 & 2, seven hybrid proteins with ΔG287 from strain 2996 at the N-terminus were constructed. Eight 287 tandem proteins were also made (see below).
TABLE-US-00004 # n X1 L1 X2 L2 1 2 ΔG287 -- 230 (His)6 2 2 -- 936 (His)6 3 2 -- 741.sub.MC58 (His)6 4 2 -- 741.sub.ET37 (His)6 5 2 -- 74190/18311 (His)6 6 2 -- 74195N477 (His)6 7 2 ΔG287nz -- 741.sub.MC58 (His)6
[0169] 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
[0170] 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
[0171] 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 X1 L1 X2 L2 FCA Alum FCA Alum 2 ΔG287394/98 -- 961 (His)6 -- 32768 -- >109350 919 32768 4096 4718 3678 953 >32768 >16384 1900 6936 741 16384 2048 232 862 2 ΔG2872996 -- 961 (His)6 65536 32768 108627 >109350 919 128000 32000 11851 2581 953 65536 -- 3834 -- 741 16384 8192 315 4645
[0172] Hybrid Proteins--X1=961c or 961cL
[0173] 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 X1 L1 X2 L2 1 2 961c -- 287 -- 2 2 -- 287 (His)6 3 2 -- 230 (His)6 4 2 -- 936 (His)6 5 2 961cL -- 287 -- 6 2 -- 287 (His)6 7 2 -- 230 (His)6 8 2 -- 936 (His)6
[0174] 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
[0175] 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
[0176] These results could be improved by mixing 961c-741 with ORF46.1 or with ΔG287-919.
[0177] 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 X1 L1 X2 L2 FCA Alum FCA Alum 2 961c -- ORF46.1 (His)6 32768 1024 >109350 >109350 741 >16384 8192 >109350 >109350 936 >32768 8192 >109350 >109350
[0178] Hybrid Proteins--X1=ORF46.1
[0179] 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 X1 L1 X2 L2 1 2 ORF46.1 -- 936 (His)6 2 2 -- 230 (His)6
[0180] 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.
[0181] Results obtained after immunisation with proteins disclosed in refs. 1 & 2 were as follows:
TABLE-US-00013 Bacteri- cidal titre ELISA n X1 L1 X2 L2 FCA Alum FCA Alum 2 ORF46.1 -- 961 (His)6 8192 8192 21558 >109350 -- 961c (His)6 8192 128 9020 76545
[0182] Hybrid Proteins--X1=230
[0183] In addition to those disclosed in references 1 & 2, four hybrid proteins with 230 at the N-terminus were constructed:
TABLE-US-00014 # n X1 L1 X2 L2 1 2 230 -- ORF46.1 (His)6 2 2 -- 961 (His)6 3 2 -- 961c (His)6 4 2 -- 741.sub.MC58 (His)6
[0184] Hybrid Proteins--Xj=936
[0185] In addition to those disclosed in references 1 & 2, seven hybrid proteins with 936 at the N-terminus were constructed:
TABLE-US-00015 # n X1 L1 X2 L2 1 2 936 -- ORF46.1 (His)6 2 2 -- 961 (His)6 3 2 -- 741.sub.ET37 (His)6 4 2 -- 741.sub.MC58 (His)6 5 2 -- 74190/18311 (His)6 6 2 -- 74195N477 (His)6 7 2 -- 741 (His)6
[0186] 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 (36%)
[0187] 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
[0188] 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
[0189] 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 X1 L1 X2 L2 FCA Alum FCA Alum 2 936 -- 741 (His)6 1024 256 1466 5715 936 >32768 >32768 >109350 >109350
[0190] Mixtures of Hybrid Proteins
[0191] Mice were immunised with of three proteins adjuvanted with aluminium hydroxide, either single or in a triple combination: (1) 287NZ-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
[0192] 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
[0193] Tandem Proteins
[0194] Hybrid proteins of the invention can be represented by formula NH2--[--X-L-]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.
[0195] Twelve specific tandem proteins are:
TABLE-US-00022 # n X1 L1 X2 L2 1 2 ΔG741.sub.MC58 -- 741.sub.MC58 (His)6 2 2 ΔG2872996 (Gly)6 ΔG287394/98 (His)6 3 2 ΔG2872996 (Gly)6 ΔG2872996 (His)6 4 2 ΔG287394/98 (Gly)6 ΔG287394/98 (His)6 5 2 ΔG287394/98 (Gly)6 ΔG2872996 (His)6 6 2 ΔG2872996 (Gly)6 ΔG287394/98 -- 7 2 ΔG2872996 (Gly)6 ΔG2872996 -- 8 2 ΔG287394/98 (Gly)6 ΔG287394/98 -- 9 2 ΔG287394/98 (Gly)6 ΔG2872996 -- 10 2 ΔG741.sub.MC58 -- 741394/98 (His)6 11 2 ΔG741.sub.MC58 -- 74190/18311 (His)6 12 2 ΔG741.sub.MC58 -- 74195N477 (His)6
[0196] 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.
[0197] Allelic Variants--741
[0198] Twenty-two polymorphic sequences of 741 were found (SEQ IDs 1 to 22). These and the MC58 sequence are aligned in FIG. 1.
[0199] Allelic Variants--NMB1343
[0200] 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 NMB 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.
[0201] 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 ##STR00001##
[0202] An alignment of the corresponding nucleotide sequences is shown in FIG. 2. This shows that the gonococcal sequence has a 4 mer insertion in the 5' region of the NMB1343 gene which causes a frameshift and consequent loss of the 5' methionine residue.
[0203] Domain Deletion--961
[0204] 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 A4, 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.
[0205] FIGS. 4 and 5 show domains in protein 961.
[0206] 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.
[0207] To investigate this further, deletion mutants in the C-terminal region of 961 were constructed (961cL-Δaro, 961cLΔcc, 961aL, 961aL-Δ1, 961aL-Δ2, 961aL-Δ3) on the basis of structural features (deletions of aromatic residues in the cases of 961cΔaro 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.
[0208] ΔG287--Cross-Strain Bactericidal Activity
[0209] 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 ΔG287 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
[0210] Refolding
[0211] To improve the levels of soluble protein for some hybrid proteins, alternative refolding protocols to those disclosed in reference 2 were adopted.
[0212] Inclusion Bodies (IBs) Were Isolated as Follows:
[0213] 1. Homogenize cells (5 g wet weight) in 25 ml 0.1 M Tris-HCl pH 7, 1 mM EDTA, at 4° C. using an ultraturrax (10 000 rpm)
[0214] 2. Add 1.5 mg lysozyme per gram cells, mix shortly with an ultraturrax, and incubate at 4° C. for 30 min.
[0215] 3. Use sonication or high-pressure homogenization (French press) to disrupt the cells.
[0216] 4. To digest DNA, add MgCl2 to a final concentration of 3 mM and DNase to a final concentration of 10 μg/ml, and incubate for 30 min at 25° C.
[0217] 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° C.
[0218] 6. Spin down inclusion bodies by centrifugation at 31000 g (20 000 rpm) for 10 min, 4° C.
[0219] 7. Resuspend pellet in 40 ml 0.1 M tris-HCl pH 7, 20 mM EDTA, using an ultraturrax
[0220] 8. Repeat centrifugation step 6.
[0221] 9. The inclusion body pellet may be used, or stored frozen at -20° C.
[0222] Hybrid proteins were expressed in E. coli as follows:
TABLE-US-00025 Culture Flask Inclusion volume volume Temp Final body yield Protein (liters) (liters) (° C.) OD600 (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
[0223] The pellets were solubilised, refolded, ultrafiltered, dialysed, and protein was then purified:
[0224] 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° C., resulting in a protein concentration of 5 μg/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 μg/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° C. in a Beckman centrifuge rotor JA25.5 The supernatant isolated after centrifugation was used for His-tag purification.
[0225] 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° C., resulting in a protein concentration of 5 μg/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 μg/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° C. in a Beckman centrifuge rotor JA25.5. The supernatant isolated after centrifugation was used for His-tag purification.
[0226] 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° C., resulting in a protein concentration of 5 μg/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 μg/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 0.1 M 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° C. in a Beckman centrifuge rotor JA25.5. The supernatant isolated after centrifugation was used for His-tag purification.
[0227] 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° C., resulting in a protein concentration of 50 μg/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 μg/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° C. in a Beckman centrifuge rotor JA25.5 The supernatant isolated after centrifugation was used for His-tag purification.
[0228] Compared with proteins purified as described in ref. 2, bactericidal assay titres were as follows:
TABLE-US-00026 Reference 2 Refolded Alumin- Alumin- Alumin- ium ium ium hydrox- hydrox- phos- Protein CFA ide ide MF59 phate ORF46.1-961-His 8192 8192 32768 -- -- ORF46.1-961c-His 8192 128 <64 8192 -- 961c-ORF46.1His 32768 1024 16384 -- -- orf46.1-741 His <4 16 <4 256 --
[0229] 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 (liters) (liters) (° C.) OD600 (w/w) orf46.1K 5 5 37 13.7 29.4
[0230] 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° C., resulting in a protein concentration of 50μg/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 m/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° 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.
[0231] 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
[0232] 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
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TABLE-US-00030
[0313] SEQUENCE LISTING 741 from strain 1000 SEQ ID 1 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TTPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQTITLASGEFQIYKQNHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ 741 from strain 2201731 (premature stop codon, though reliable sequence) SEQ ID 2 MTRSKPVNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQ TEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGT AFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKP DGKRHAVISGSVLYNQAEKGSYSLGIFGGKA 741 from strain 90/18311 (incomplete) SEQ ID 3 GLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGN GDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHS AVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKA EYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELAS AELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATV KIREKVHET 741 from strain L93/4286 (incomplete) SEQ ID 4 VAADIGAGLADALTAPLDHKDKGLQSLMLDQSVRKNEKLKLAAQG AEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQTITLASGEFQ IYKQNHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFN QLPDGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPE QNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQE IAGSATVKIREKVHEIGIAGKQ 741 from strain 2996 SEQ ID 5 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKAD EKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQ 741 from strain 30/00 SEQ ID 6 KDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKND KVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQD SEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDD AGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHA VISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGL AAKQ 741 from strain 312294 SEQ ID 7 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKAD EKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQ 741 from strain 39/99 (incomplete) SEQ ID 8 DKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDK VSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDS EHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDA GGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAV ISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLA AKQ 741 from strain 5/99 SEQ ID 9 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKA FSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ 741 from strain 67/00 SEQ ID 10 MTRSKPVNRTAFCCFSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQ TEQEQDPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGT AFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKP DEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANG IHHIGLAAKQ 741 from strain BZ169 SEQ ID 11 LQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHS GKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGK LTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISG SVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ 741 from strain 72/00 SEQ ID 12 LQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHS GKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGK LTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISG SVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ 741 from strain 93/114 SEQ ID 13 MTRSKPVNRTAFCCFSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQ TEQEQDPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGT AFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKP DEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANG IHHIGLAAKQ 741 from strain 95N477 SEQ ID 14 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKA FSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ 741 from strain 96217 SEQ ID 15 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKAD EKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQ 741 from strain BZ133 SEQ ID 16 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQ TEQVQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGT AFGSDDASGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKP DKKRHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANG IRHIGLAAKQ 741 from strain BZ232 SEQ ID 17 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQTITLASGEFQIYKQNHSAVVALQ
IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ 741 from strain C11 SEQ ID 18 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKA FSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ 741 from strain M1090 SEQ ID 19 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKA FSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ 741 from strain M1096 SEQ ID 20 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TTPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQTITLASGEFQIYKQNHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ 741 from strain M198/172 SEQ ID 21 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQ TEQVQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGT AFGSDDASGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKP DKKRHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANG IRHIGLAAKQ 741 from strain NGH38 SEQ ID 22 MTRSKPVNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADAL TAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQTITLASGEFQIYKQNHSAVVALQ IEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKA FSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKAD EKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ NMB1343 from ten meningococcal strains SEQ ID 23 MGNFLYRGISCQQDEQNNGQLKPKGNKAEVAIRYDGKFKYDGKAT HGPSVKNAVYAHQIETGLYDGCYISTTTDKEIAKKFATSSGIENG YIYVLNRDLFGQYSIFEYEVEHPENPNEKEVTIRAEDCGCIPEEV IIAKELIEIN NMB1343 from gonococcus SEQ ID 24 INNLWEISYLYRGISCQQDEQNNGQLKPKGNKAEVAIRYDGKFKY DGKATHGPSVKNAVYAHQIETDLYDGCYISTTTDKEIAKKFATSS GIENGYIYVLNRDLFGQYSIFEYEVEHPENPDEKEVTIRAEDCGC IPEEVIIAKELIEIN NMB1343 nucleic acid sequence (gonococcus) SEQ ID 25 TCCGCCGCATTACCTTATAAAATAAAACATCCCTCTCAAGCAGTC TGATAATGTTTGGATTGCTTGAGATTGATGAGTGATGGTGTTAAA TTCAAACTTTAAATTAATAACTTATGGGAAATTTCTTATTTATAT AGAGGCATTAGTTGCCAACAAGATGAGCAAAATAATGGACAGTTA AAACCTAAAGGTAATAAAGCTGAAGTTGCAATTCGTTATGATGGT AAGTTTAAATATGATGGTAAAGCTACACATGGTCCAAGTGTGAAG AATGCAGTTTACGCCCATCAAATTGAAACAGATCTATATGACGGA TGTTATATATCTACGACAACAGACAAGGAAATTGCCAAGAAATTT GCAACAAGCTCCGGCATCGAAAATGGCTATATATATGTTTTAAAT AGAGATTTGTTTGGTCAATATTCTATTTTTGAATATGAGGTTGAA CATCCAGAAAACCCAGATGAGAAGGAAGTAACAATCAGAGCTGAA GATTGTGGCTGTATTCCTGAAGAAGTGATTATTGCTAAAGAGTTG ATAGAAATTAACTAAGTTGAAAGGTCAATATAATGGCTTTAGTTG AATTGAAAGTGCCCGACATTGGCGGACACGAAAATGTAGATATTA TCGC NMB1343 nucleic acid sequence (meningococcus) SEQ ID 26 TCCGCCGCATTACCTTATAAAATAAAACATCCCTCTCAAGCAGTC TGATAATGTTTGGATTGCTTGAGATTGATGAGTAATGGTGTTAAA TTCAACCTTTAAATTAATAACTTATGGGAAATTTCTTATATAGAG GCATTAGTTGCCAACAAGATGAGCAAAATAATGGACAGTTAAAAC CTAAAGGTAATAAAGCTGAAGTTGCAATTCGTTATGATGGTAAGT TTAAATATGATGGTAAAGCTACACATGGTCCAAGTGTGAAGAATG CAGTTTACGCCCATCAAATTGAAACAGGTCTATATGACGGATGTT ATATATCTACGACAACAGACAAGGAAATTGCCAAGAAATTTGCAA CAAGTTCCGGCATCGAAAATGGCTATATATATGTTTTAAATAGGG ATTTGTTTGGTCAATATTCTATTTTTGAATATGAGGTTGAACATC CAGAAAACCCAAATGAGAAGGAAGTAACAATCAGAGCTGAAGATT GTGGCTGTATTCCTGAAGAAGTGATTATTGCTAAAGAGTTGATAG AAATTAACTAAGTTGAAAGGTCAATATAATGGCTTTAGTTGAATT GAAAGTGCCCGACATTGGCGGACACGAAAATGTAGATATTATCGC linker SEQ ID 27 GSGGGG preferred ΔG287NZ-953 hybrid SEQ ID 28 MASPDVKSADTLSKPAAPVVAEKETEVKEDAPQAGSQGQGAPSTQ GSQDMAAVSAENTGNGGAATTDKPKNEDEGPQNDMPQNSAESANQ TGNNQPADSSDSAPASNPAPANGGSNFGRVDLANGVLIDGPSQNI TLTHCKGDSCNGDNLLDEEAPSKSEFENLNESERIEKYKKDGKSD KFTNLVATAVQANGTNKYVIIYKDKSASSSSARFRRSARSRRSLP AEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAE KLPGGSYALRVQGEPAKGEMLAGTAVYNGEVLHFHTENGRPYPTR GRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWT ENGGGDVSGRFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQD GSGGGGATYKVDEYHANARFAIDHFNTSTNVGGFYGLTGSVEFDQ AKRDGKIDITIPVANLQSGSQHFTDHLKSADIFDAAQYPDIRFVS TKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPMAKTEV CGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDIQIEAAKQ ΔG287NZ-953 hybrid SEQ ID 29 MASPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQ GGQDMAAVSEENTGNGGAAATDKPKNEDEGAQNDMPQNAADTDSL TPNHTPASNMPAGNMENQAPDAGESEQPANQPDMANTADGMQGDD PSAGGENAGNTAAQGTNQAENNQTAGSQNPASSTNPSATNSGGDF GRTNVGNSVVIDGPSQNITLTHCKGDSCSGNNFLDEEVQLKSEFE KLSDADKISNYKKDGKNDGKNDKFVGLVADSVQMKGINQYIIFYK PKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTG HSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEPSKGEMLAGTA VYNGEVLHFHTENGRPSPSRGRFAAKVDFGSKSVDGIIDSGDGLH MGTQKFKAAIDGNGFKGTWTENGGGDVSGKFYGPAGEEVAGKYSY RPTDAEKGGFGVFAGKKEQDGSGGGGATYKVDEYHANARFAIDHF NTSTNVGGFYGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTD HLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAP VKLKAEKFNCYQSPMAKTEVCGGDFSTTIDRTKWGVDYLVNVGMT KSVRIDIQIEAAKQ 936-ΔG741 hybrid SEQ ID 30 MKPKPHTVRTLIAAIFSLALSGCVSAVIGSAAVGAKSAVDRRTTG AQTDDNVMALRIETTARSYLRQNNQTKGYTPQISVVGYNRHLLLL GQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDT WNTSKVRATLLGISPATQARVKIVTYGNVTYVMGILTPEEQAQIT QKVSTTVGVQKVITLYQNYVQRGSGGGGVAADIGAGLADALTAPL DHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLK NDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQI
QDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGS DDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKR HAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHI GLAAKQ 961c SEQ ID 31 MATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTIT KKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAE SEIEKLTTKLADTDAALADTDAALDATTNALNKLGENITTFAEET KTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKT ANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEA VAAKVTDIKADIATNKDNIAKKANSADVYTREESDSKFVRIDGLN ATTEKLDTRLASAEKSIADHDTRLNGLDKTVSDLRKETRQGLAEQ AALSGLFQPYNVG
Sequence CWU
1
1
311279PRTNeisseria meningitidis 1Met Thr Arg Ser Lys Pro Val Asn Arg Thr
Ala Phe Cys Cys Leu Ser1 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 Ser1
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
Ser1 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
Pro1 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 Ser1 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 Lys1 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 Ser1 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 Asn1 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 Ser1 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 Ser1 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 Leu1 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 Leu1 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 Ser1 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 Ser1 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 Ser1
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 Ser1 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 Ser1 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 Ser1 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 Ser1
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 Ser1 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 Ser1 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 Ser1 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 Gln1
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
Cys1 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 630276DNANeisseria
meningitidisSynthetic Construct 27gsgggg
628579PRTArtificial SequenceSynthetic
Construct 28Met Ala Ser Pro Asp Val Lys Ser Ala Asp Thr Leu Ser Lys Pro
Ala1 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 Ala1 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 Phe1
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 Ala1 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
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