# Patent

## Title
Hybrid and tandem expression of Neisserial proteins

## Abstract

Two or more Neisserial proteins are joined such that they are translated as a single polypeptide chain. Hybrid proteins are represented by the formula 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 I. Proteins where each of the n-X— moieties shares sequence identity to each other —X— moiety, the protein is a ‘tandem protein’.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of U.S. patent application Ser. No. 10/488,786, filed Feb. 25, 2005, which is the National Stage of International Patent Application of PCT/IB02/03904, filed Sep. 6, 2002, which claims the benefit of United Kingdom Patent Application Serial No. 0121591.2, filed Sep. 6, 2001, each of which are hereby incorporated by reference in their entirety.


SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
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: 223002100601SubSeqList.txt, date recorded: Aug. 8, 2013, size: 83 KB).
TECHNICAL FIELD
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
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.
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
Hybrid Proteins
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.

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.
The —X— Moieties
There are two main groups of hybrid proteins according to the invention. These two groups are not mutually exclusive.
In the first group, each —X— moiety is:

    
    
        (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;
        (b) an amino acid sequence having sequence identity to an amino acid sequence from (a); or
        (c) an amino acid sequence comprising a fragment of an amino acid sequence from (a).
    
    


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.
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:

    
    
        (d) the 446 even SEQ IDs (i.e. 2, 4, 6, . . . , 890, 892) disclosed in reference 3.
        (e) the 45 even SEQ IDs (i.e. 2, 4, 6, . . . , 88, 90) disclosed in reference 4;
        (f) the 1674 even SEQ IDs 2-3020, even SEQ IDs 3040-3114, and all SEQ IDs 3115-3241, disclosed in reference 5;
        (g) the 2160 amino acid sequences NMB0001 to NMB2160 from reference 7; or
        (h) an amino acid sequence disclosed in reference 1 or reference 2.
    
    


The —Xb— moiety is related to —Xa— such that: (i) —Xb— has sequence identity to —Xa—, and/or (j) —Xb— comprises a fragment of —Xa—.
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—Xc— etc.
The —Xa— and —Xb— moieties may be in either order from N-terminus to C-terminus.
The —Xa— moiety is preferably an orf1, orf4, orf25, orf40, orf46.1, orf83, NMB 1343, 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.
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.
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.
The ‘fragment’ referred to in (c) and (j) should consist of least m consecutive amino acids from an amino acid sequence from (a), (d), (e), (f), (g) or (h) and, depending on the particular sequence, m is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).
Preferably the fragment comprises an epitope from an amino acid sequence from (a), (d), (e), (f), (g) or (h). Preferred fragments are those disclosed in references 9 and 10.
Preferred (c) and (j) fragments are C- and/or N-terminal truncations (e.g. Δ1-287, Δ2-287 etc.).
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).
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).
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.
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.
961 protein has been notionally split into several domains (FIG. 4).
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-.
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:
















X1
X2
X1
X2









ΔG287
230
230
ΔG287



ΔG287
936
936
ΔG287



ΔG287
741
741
ΔG287



ΔG287
961
961
ΔG287



ΔG287
ORF46.1
ORF46.1
ΔG287



ΔG287
919
919
ΔG287



ΔG287
953
953
ΔG287



961c
287
287
961c



961c
230
230
961c



961c
936
936
961c



961c
741
741
961c



961c
983
983
961c



961c
ΔG983
ΔG983
961c



961c
ORF46.1
ORF46.1
961c



961
ORF46.1
ORF46.1
961



961cL
287
287
961cL



961cL
230
230
961cL



961cL
936
936
961cL



ORF46.1
936
936
ORF46.1



ORF46.1
230
230
ORF46.1



ORF46.1
741
741
ORF46.1



ORF46.1
ΔG741
ΔG741 
ORF46.1



ORF46.1
983
983
ORF46.1



ORF46.1
ΔG983
ΔG983
ORF46.1



230
961
961
230



230
741
741
230



230
ΔG741
ΔG741
230



936
961
961
936



936
741
741
936



936
ΔG741
ΔG741
936



ΔG741
741
ΔG287
287



ORF46.1
983
983
ORF46.1



ΔG741
ORF46.1
ORF46.1
ΔG741



ΔG741
983
983
ΔG741



ΔG741
961
961
ΔG741



ΔG741
961c
961c
ΔG741



ΔG983
ORF46.1
ORF46.1
ΔG983



ΔG983
961
961
ΔG983



ΔG983
961c
961c
ΔG983










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.
The -L- Moieties
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.
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.
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.
The -A- Moiety
-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.
The —B— Moiety
—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.
Polymorphic Forms of Proteins
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).
Reference sequences from N. meningitidis serogroup B include:













Protein
Reference
Protein
Reference







orf1
Ref. 3, SEQ ID 650
orf4
Ref. 3, SEQ ID 218


orf25
Ref. 3, SEQ ID 684
orf40
Ref. 4, SEQ ID 4


orf46
Ref. 6, SEQ ID 1049
orf83
Ref. 3, SEQ ID 314


NMB1343
Ref. 7, NMB1343
230
Ref. 5, SEQ ID 830


233
Ref. 5, SEQ ID 860 
287
Ref. 5, SEQ ID 3104


292
Ref. 5, SEQ ID 1220
594
Ref. 5, SEQ ID 1862


687
Ref. 5, SEQ ID 2282
736
Ref. 5, SEQ ID 2506


741
Ref. 5, SEQ ID 2536
907
Ref. 5, SEQ ID 2732


919
Ref. 5, SEQ ID 3070
936
Ref. 5, SEQ ID 2884


953
Ref. 5, SEQ ID 2918
961
Ref. 5, SEQ ID 940


983
Ref. 7, NMB1969









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.
The sequence listing herein includes polymorphic forms of proteins 741 (SEQ IDs 1-22) and NMB1343 (SEQ IDs 23-24) which have been identified. The sequence listing herein includes the amino acid sequence set forth as SEQ ID NO: 1202 in WO99/57280, which corresponds to SEQ ID NO: 41 of the present application. The sequence listing herein includes the amino acid sequence set forth as SEQ ID NO: 2918 in WO99/57280, which corresponds to SEQ ID NO: 42 of the present application. The sequence listing herein includes the amino acid sequence set forth as SEQ ID NO: 2884 in WO99/57280, which corresponds to SEQ ID NO: 43 of the present application. The sequence listing herein includes the amino acid sequence set forth as SEQ ID NO: 2536 in WO99/57280 which corresponds to SEQ ID NO: 44 of the present application.
Serogroups and Strains
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.
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.
Where a form of 287 is used, this is preferably from strain 2996 or from strain 394/98.
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.
Where a form of 961 is used, this is preferably from strain 2996.
Strains are indicated as a subscript e.g. 741MC58 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).
Domain-Based Expression of Protein 961
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]).
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):














Domain name
Amino acids









961-1 ‘L’
 1-23



961-2
 24-87



961-3
 88-143



961-4
144-180



961-5
181-268



961-6
269-286



961-7
287-330



961-8
331-350



961-9
351-405










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

    
    
        961-2 to 961-5 (‘961a’)
        961-6 to 961-9 (‘961b’)
        961-1 to 961-8 (‘961cL’)
        961-2 to 961-8 (‘961c’)
        961-2 to 961-6 and amino acids 287-325 from domain 961-7 (‘961d’)
        961-2 to 961-8 and amino acids 351-383 from domain 961-9 (‘961Δ1’)
        961-1 to 961-8 and amino acids 351-383 from domain 961-9 (‘961Δ1L’)
        961-1 to 961-7 and amino acids 331-343 from domain 961-8 (‘961cL-Δaro’)
        961-1 to 961-6 and amino acids 287-315 from domain 961-7 (‘961cL-Δcc’)
        961-1 to 961-5 (‘961aL’)
        961-1 to 961-4 (‘961aL-Δ1’)
        961-1 to 961-3 (‘961aL-Δ2’)
        961-1 to 961-2 (‘961aL-Δ3’)
    
    


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).
Sequences
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).
The invention also provides nucleic acid encoding such proteins.
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).
The invention also provides nucleic acid encoding proteins according to the invention.
It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).
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.).
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.
Mixtures
The invention also provides a composition comprising two or more (i.e. 2, 3, 4, 5, 6 or 7) of the following proteins:

    
    
        (1) 287
        (2) 741
        (3) ORF46.1
        (4) 961
        (5) NH2-A-[-X-L-]n-B—COOH, wherein n=2, X1=287, X2=953
        (6) NH2-A-[-X-L-]n-B—COOH, wherein n=2, X1=287, X2=919
        (7) NH2-A-[-X-L-]n-B—COOH, wherein n=2, X1=287, X2=961
    
    


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):

    
    
        (8) NH2-A-[-X-L-]n-B—COOH, wherein n=2, X1=287, X2=741
        (9) NH2-A-[-X-L-]n-B—COOH, wherein n=2, X1=936, X2=741
    
    


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].
A preferred mixture comprises the following three proteins:

    
    
        (1) 961c, preferably 961c2996 (e.g. SEQ ID 31 herein);
        (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
        (3) NH2-A-[-X-L-]n-B—COOH, wherein n=2, X1=936 (preferably 9362996), X2=ΔG741 (preferably ΔG741MC58), L1=GSGGGG (e.g. SEQ ID 30 herein).
    
    


The mixtures may also comprise N. meningitidis outer membrane vesicles.
Heterologous Host
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.
Vectors Etc.
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.
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.
To improve solubility, purification of hybrid proteins may involve the refolding techniques disclosed herein.
Immunogenic Compositions and Medicaments
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.
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.
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.
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.
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.
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.
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.
Further Components of the Composition
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. 
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.
The vaccine may be administered in conjunction with other immunoregulatory agents.
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) MF59TM (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) RibiTM 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 (DetoxTM); (2) saponin adjuvants, such as QS21 or StimulonTM (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J. Immunol., 1996, 156, 4570-4575; Halpern et al., Cell. Immunol., 1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messina et al., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998, 160, 5898-5906; International patent applications WO96/02555, WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581] i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (8) a polyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (e.g. WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (e.g. WO01/21152); (10) an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin e.g. WO00/62800; (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) e.g. WO98/57659; (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition.
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-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.
Further Antigens
Further antigens which can be included in the composition of the invention include:

    
    
        an outer-membrane vesicle (OMV) preparation from N. meningitidis serogroup B, such as those disclosed in refs. 14, 15, 16, 17 etc.
        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.
        a saccharide antigen from Streptococcus pneumoniae [e.g. refs. 21, 22, 23].
        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.
        an antigen from hepatitis A virus, such as inactivated virus [e.g. 27, 28].
        an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. 28, 29].
        an antigen from hepatitis C virus [e.g. 30].
        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].
        a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref. 33] e.g. the CRM197 mutant [e.g. 34].
        a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 33].
        a saccharide antigen from Haemophilus influenzae B [e.g. 19].
        an antigen from N. gonorrhoeae [e.g. 3, 4, 5].
        an antigen from Chlamydia pneumoniae [e.g. 35, 36, 37, 38, 39, 40, 41].
        an antigen from Chlamydia trachomatis [e.g. 42].
        an antigen from Porphyromonas gingivalis [e.g. 43].
        polio antigen(s) [e.g. 44, 45] such as IPV or OPV.
        rabies antigen(s) [e.g. 46] such as lyophilised inactivated virus [e.g. 47, RabAvertTM].
        measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref. 33].
        influenza antigen(s) [e.g. chapter 19 of ref. 33], such as the haemagglutinin and/or neuraminidase surface proteins.
        an antigen from Moraxella catarrhalis [e.g. 48].
        a protein antigen from Streptococcus agalactiae (group B streptococcus) [e.g. 49, 50].
        a saccharide antigen from Streptococcus agalactiae 
        an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 50, 51, 52].
        an antigen from Staphylococcus aureus [e.g. 53].
    
    


The composition may comprise one or more of these further antigens.
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.
Any suitable conjugation reaction can be used, with any suitable linker where necessary.
Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means [32]).
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.
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.).
Antigens in the composition will typically be present at a concentration of at least 1 μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
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.
DEFINITIONS
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.
The term “about” in relation to a numerical value x means, for example, x±10%.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows an alignment of twenty-three sequences for protein 741. These are SEQ IDs 1 to 22 plus the sequence from MC58.
FIG. 2 shows an alignment of the NMB1343 sequence from gonococcus (top; SEQ ID 25) and meningococcus (bottom; SEQ ID 26).
FIG. 3 shows hybrid and tandem proteins of the invention.
FIG. 4 shows 9 domains within 9612996, and FIG. 5 shows how these have been manipulated.



MODES FOR CARRYING OUT THE INVENTION
Hybrid Proteins—X1=ΔG287
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).


















#
n
X1
L1
X2
L2









1
2
ΔG287
—
230
(His)6



2
2

—
936
(His)6



3
2

—
741MC58
(His)6



4
2

—
741ET37
(His)6



5
2

—
74190/18311
(His)6



6
2

—
74195N477
(His)6



7
2
ΔG287nz
—
741MC58
(His)6










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:
















Strain(serogroup)
2996(B)
MC58(B)
NGH38(B)
394/98(B)
44/76(B)
F6124(A)





















Al hydroxide
8192
32768
8192
>2048
16384
8192


FCA
16384
262144
8192
>2048
>32768
8192









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:
















2996(B)
MC58(B)
NGH38(B)
394/98(B)
44/76(B)
F6124(A)
BZ133(C)





















8000
65000
4000
4000
32000
8000
16000









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





















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
6932





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









Hybrid Proteins—X1=961c or 961cL

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:


















#
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










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:















Strain(serogroup)
2996(B)
MC58(B)
394/98(B)
44/76(B)
F6124(A)




















Al hydroxide
8192
8192
512
1024
<16


FCA
65536
16384
>2048
>2048
8192









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
















Strain(serogroup)
2996(B)
MC58(B)
394/98(B)
44/76(B)
F6124(A)
BZ133(C)





















Al hydroxide
65536
32768
4096
>32768
16384
>2048


FCA
>16384
262144
4096
>16384
—
>2048









These results could be improved by mixing 961c-741 with ORF46.1 or with ΔG287-919.
Results obtained after immunisation with proteins disclosed in refs. 1 & 2, tested against the homologous strain, were as follows:





















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









Hybrid proteins —X1=ORF46.1
In addition to those disclosed in references 1 & 2, two hybrid proteins with ORF46.1 at the N-terminus were constructed:


















#
n
X1
L1
X2
L2









1
2
ORF46.1
—
936
(His)6



2
2

—
230
(His)6










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





















Bactericidal








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









Hybrid Proteins—X1=230

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


















#
n
X1
L1
X2
L2









1
2
230
—
ORF46.1
(His)6



2
2

—
961
(His)6



3
2

—
961c
(His)6



4
2

—
741MC58
(His)6










Hybrid Proteins—X1=936

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


















#
n
X1
L1
X2
L2









1
2
936
—
ORF46.1
(His)6



2
2

—
961
(His)6



3
2

—
741ET37
(His)6



4
2

—
741MC58
(His)6



5
2

—
74190/18311
(His)6



6
2

—
74195N477
(His)6



7
2

—
741
(His)6










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:















Strain(serogroup)
2996(B)
MC58(B)
394/98(B)
44/76(B)
F6124(A)




















Al hydroxide
16384
32768
1024
2048
<16  


FCA
65536
65536
>2048
8192
2048(36%)









Titres using protein #4 were as follows:















Strain(serogroup)
2996(B)
MC58(B)
394/98(B)
44/76(B)
F6124(A)




















Al hydroxide
256
>262144
>2048
32768
8192


FCA
1024
>262144
>2048
>32768
>32768









Titres using protein #7 were as follows:
















Strain (serogroup)
2996 (B)
MC58 (B)
394/98 (B)
44/76 (B)
F6124 (A)
BZ133 (C)





















Al hydroxide
256
130000
16000
32000
8000
16000









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





















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









Mixtures of Hybrid Proteins

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:



















2996 (B)
MC58 (B)
NGH38
394/98 (B)
H44/76 (B)
F6124 (A)
BZ133 (C)
C11 (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






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




















#
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









Tandem Proteins

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.
Twelve specific tandem proteins are:















#
n
X1
L1
X2
L2




















1
2
ΔG741MC58
—
741MC58
(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
ΔG741MC58
—
741394/98
(His)6


11
2
ΔG741MC58
—
74190/18311
(His)6


12
2
ΔG741MC58
—
74195N477
(His)6









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 liter 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.
Allelic Variants—741
Twenty-two polymorphic sequences of 741 were found (SEQ IDs 1 to 22). These and the MC58 sequence are aligned in FIG. 1.
Allelic Variants—NMB1343
Using PCR on 42 strains of meningococcus of various serogroups, the gene encoding NMB1343 protein was found in 24/42 and was absent in 18/42 strains (Table 1). The NMB1343 gene was sequenced for 10 of the NMB1343+ 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.
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:




An alignment of the corresponding nucleotide sequences is shown in FIG. 2. This shows that the gonococcal sequence has a 4mer insertion in the 5′ region of the NMB 1343 gene which causes a frameshift and consequent loss of the 5′ methionine residue.
Domain Deletion—961
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. 
FIGS. 4 and 5 show domains in protein 961.
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.
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.
ΔG287—Cross-Strain Bactericidal Activity
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:











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






Refolding

To improve the levels of soluble protein for some hybrid proteins, alternative refolding protocols to those disclosed in reference 2 were adopted.
Inclusion bodies (IBs) were isolated as follows:

    
    
        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)
        2. Add 1.5 mg lysozyme per gram cells, mix shortly with an ultraturrax, and incubate at 4° C. for 30 min.
        3. Use sonication or high-pressure homogenization (French press) to disrupt the cells.
        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.
        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.
        6. Spin down inclusion bodies by centrifugation at 31000 g (20 000 rpm) for 10 min, 4° C.
        7. Resuspend pellet in 40 ml 0.1 M tris-HCl pH 7, 20 mM EDTA, using an ultraturrax
        8. Repeat centrifugation step 6.
        9. The inclusion body pellet may be used, or stored frozen at −20° C.
    
    


Hybrid proteins were expressed in E. coli as follows:
















Culture
Flask


Inclusion



volume
volume
Temp
Final
body yield


Protein
(litres)
(litres)
(° 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









The pellets were solubilised, refolded, ultrafiltered, dialysed, and protein was then purified:
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.
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.
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 0.1M 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.
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.
Compared with proteins purified as described in ref. 2, bactericidal assay titres were as follows:














Reference 2
Refolded














Alumin-
Alumin-

Alumin-




ium hy-
ium hy-

ium phos-


Protein
CFA
droxide
droxide
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
—









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
















Culture
Flask


Inclusion



volume
volume
Temp
Final
body yield


Protein
(litres)
(litres)
(° C.)
OD600
(w/w)







orf46.1K
5
5
37
13.7
29.4









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 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 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.
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:


















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










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 1





Strain
1343
Sequence
Strain classification







72/00
+

ET5 B:15:P1.7,13,13a


30/00
+

ET5 B:15:P1.7,16


39/99
+

ET5 C:15:P1.7,16


95330
+

ET5 B:4:P1.15


M4102
+

ET5 nd


MC58(21)
+
+
ET5 B:15:P1.7,16b


BZ169(7)
+
+
ET5 B:NT:P1.16


BZ83(19)
+

ET5 B:15:—.—


CU385
+
+
ET5 B:4:P1.15


220173I
+

ET5 NG:4:P1.15


64/96
+
+
ET5 NG:15:P1.7,16 (carrier)


220173I
+

ET5 B:4:P1.15 (carrier)


ISS1071
+

nd B:15:P1.7,16 (ET5?)


BZ198(2)
+
+
lin.3 B:8:P1.1


980-2543
+
+
lin.3 B:NT:P1.4


16060
+
+
other B:4:P1.14 (carrier)


394-98
+

nd B:4:P1.4 (lin 3?)


ISS1106
+

nd B:4:P1.4 (lin.3?)


BZ133(10)
+
+
sub I B:NT:—.—


S3446
+
+
nd B:14:P1.23,14


ISS1001
+
+
nd B:14:P1.13


241175I
+

other NG:21:P1.16 (carrier)


171274I
+

other NG:15:— (carrier)


66/96
+

other B:17:P1.15 (carrier)


961-5945
−

A4


96217
−

A4


312294
−

A4


90/18311(24)
−

ET37


93/4286(25)
−

ET37


M986
−

ET37


1000(5)
−

other


NGE28(13)
−

other carrier


NGH38(14)
−

other carrier


BZ232(18)
−

other


F6124(23)
−

sub III A:—.—


C11
−

C:—


NMB
−

nd


8047
−

nd


ISS759
−

nd C:2b:P1.2


ISS1113
−

nd C:2:P1.5


65/96
−

nd 4:P1.14


2996(96)
−

nd B:2b:P1.5,2









REFERENCES
The Contents of which are Hereby Incorporated by Reference


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## Claims

1. A composition comprising a first hybrid 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 an integer greater than 1 and
(a) an X1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 41; and
(b) an X2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 42, mixed with a second hybrid protein having the formula:


NH2-A-Y1-L-Y2—B—COOH



wherein
(c) Y1 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 43; and
(d) Y2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 44.

2. The composition of claim 1 wherein n=2.

3. The composition of claim 1, wherein L has 20 or fewer amino acids.

4. The composition of claim 1, wherein L is a poly-glycine linker.

5. The composition of claim 4, wherein the poly-glycine linker is GSGGGG (SEQ ID NO: 27).

6. The composition of claim 1, wherein A has 40 or fewer amino acids.

7. The composition of claim 1, wherein B has 40 or fewer amino acids.

8. The composition of claim 1 further comprising a pharmaceutically acceptable carrier.

9. A method of raising an immune response against Neisseria meningitidis serogroup B bacteria in a subject comprising administering to the subject an effective amount of the composition of claim 1.