# Patent

## Title
Meningococcus adhesins NadA, App and ORF 40

## Abstract

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

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of Ser. No. 12/775,457, filed May 6, 2010, now abandoned, which is a Continuation of Ser. No. 10/484,703, filed Mar. 7, 2005, now abandoned, which is the National Phase of PCT Application PCT/IB02/03396, filed Jul. 26, 2002, which claims the benefit of GB Application 0211025.2, filed May 14, 2002, GB Application 0121591.2, filed Sep. 6, 2001, and GB Application 0118401.9, filed Jul. 27, 2001, all of which are incorporated herein by reference in their entirety.


TECHNICAL FIELD
This invention is in the field of biochemistry and, in particular, the biochemistry of the pathogenic bacteria in the genus Neisseria (e.g. N. meningitidis and N. gonorrhea).
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: 223002100702SUBSEQLISTING2.TXT, date recorded: Apr. 30, 2015, size: 109 KB).
BACKGROUND
International patent applications WO99/24578, WO99/36544, WO99/57280 and WO00/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhea. The complete genome sequence of serogroup B N. meningitidis has been published [Tettelin et al. (2000) Science 287:1809-1815] and has been subjected to analysis in order to identify vaccine antigens [Pizza et al. (2000) Science 287:1816-1820]. Approaches to expression of the proteins are disclosed in WO01/64922. The complete genome sequence of serogroup A N. meningitidis is also known [Parkhill et al. (2000) Nature 404:502-506].
Sequence data alone, however, does not reveal everything about this pathogen. Objects of the present invention include: (a) to provide ways of intervening in Neisseria biochemistry; (b) to provide new uses for known Neisseria proteins; (c) to provide alternative and improved forms of known Neisseria proteins, such as enzymatically inactive forms of known proteins or proteolytic products of known proteins; and (d) to provide materials useful for studying and modulating Neisserial adhesion.
DISCLOSURE OF THE INVENTION
Nomenclature Used Herein
‘ORF40’ is disclosed in example 1 of WO99/36544. Sequences from serogroups A and B of N. meningitidis are disclosed (SEQ IDs 1 to 6 therein). Other forms of the protein are disclosed in WO99/31132 and WO99/58683, and can also be found in GenBank (see gi accession numbers: 11352902, 7228562, 14578015, 12958107, 7228586, 7228572, 7228594, 7228588, 14578013, 7228568, 7228546, 7228548, 7228592, 14578009, 7228558, 7228600, 7228596, 7228542, 7228574, 7228552, 7228554, 14578023, 14578021, 11354080, 7228584 & 7228590). ‘App’ (adhesion and penetration protein) is disclosed as ‘ORF1’ in example 77 of WO99/24578. Sequences from serogroups A and B of N. meningitidis and from N. gonorrhea are disclosed (SEQ IDs 647 to 654 therein). Other forms of the protein are disclosed in WO99/55873, and can also be found in GenBank (see gi accession numbers: 11280386, 7227246, 11071865, 6977941, 11071863, 11280387, 7379205).
‘NadA’ (Neisserial adhesin A) from serogroup B of N. meningitidis is disclosed as protein ‘961’ in WO99/57280 (SEQ IDs 2943 & 2944) and as ‘NMB1994’ by Tettelin et al. (see also GenBank accession numbers: 11352904 & 7227256) and in FIG. 9 herein. These proteins are preferably expressed other than as a fusion protein (e.g. without GST, MBP, his-tag or similar).
Preferred proteins for use according to the invention are those of serogroup B N. meningitidis strain MC58, strain 2996 or strain 394/98 (a New Zealand strain). It will be appreciated, however, that the invention is not in general limited by strain references to a particular protein (e.g. ‘ORF40’, ‘App’ etc.) may be taken to include that protein from any strain. In general, therefore, reference to any particular protein includes proteins which share sequence identity with one of the sequences disclosed above. The degree of ‘sequence identity’ is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants. In the context of the present invention, sequence identity is preferably determined by the Smith-Waterman homology, search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an sane gap search with parameters gap open penalty=12 and gap extension penalty=1. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.
The naming conventions used in WO99/24578, WO99/36544 and WO99/57280 are also used herein (e.g. ‘ORF4’, ‘ORF40’, ‘ORF40-1’ etc. as used in V099/24578 and WO99/36544, ‘m919’, ‘g919’ and ‘a919’ etc. as used in WO99/57280).
Secreted App
It has been found that, when expressed in E. coli without a GST or his-tag fusion partner, App is exported to the outer membrane as a precursor of about 160 kDa, where it is processed and secreted into the culture.
The invention therefore provides a method for purifying processed App protein, comprising the steps of: expressing a gene encoding App protein in a non-Neisserial host cell; and purifying processed App protein from the culture medium.
The invention also provides purified protein obtainable by this process.
The App protein preferably includes its wild-type 42 residue signal peptide at the N-terminus i.e. no N-terminus fusion partner is used. It is also preferred not to include a C-terminus fusion partner.
To purify the protein front the culture medium the culture can be centrifuged and the protein can be recovered from the supernatant.
The non-Neisserial host cell is preferably a bacterium and is most preferably E. coli. 
Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5) to the RNA polymerase binding sequencer example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic, enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistake.” In interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tar promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sri. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of seine genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promote system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).
In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is railed the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34]. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokatyotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual]. 
A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vivo incubation with a bacterial methionine N-terminal peptidase (EP-A-0219237).
Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.
Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200 and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.
Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least ene sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0127328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.
Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter aria, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature, 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].
Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbio. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990), J. Bacteriol. 172949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6121; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Bayer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus]. 
Adherence Proteins
Example 22 of international patent application WO01/64922 discloses that E. coli which expresses protein NadA can adhere to human epithelial cells. This adherence activity has been further studied and it had also been found for App and ORF40.
The invention provides methods for preventing the attachment of Neisserial cells to epithelial cells.
References to a “Neisserial cell” in this section include any species of the bacterial genus Neisseria, including N. gonorrhoeae and N. lactamica. Preferably, however, the species is N. meningitidis. The N. meningitidis may be from any serogroup, including serogroups A, C, W135 and Y. Most preferably, however, it is N. meningitidis serogroup B.
References to an “epithelial cell” in this section include any cell found in or derived from the epithelium of a mammal. The cell may be in vitro (e.g. in cell culture) or in vivo. Preferred epithelial cells are from the nasopharynx. The cells are most preferably human cells.
Blocking the Neisseria-Epithelium Interaction
The invention provides a method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein the ability of one or more App, ORF40 and/or NadA to bind to the epithelial cell is blocked.
The ability to bind may be blocked in various ways but, most conveniently, an antibody specific for App, ORF40 and/or NadA is used. The invention also provides antibody which is specific for App, ORF40 or NadA. This antibody preferably has an affinity for App, ORF40 and/or NadA of at least 10−7 M e.g. 10−8 M, 10−9 M, 10−10 M or tighter.
Antibodies for use in accordance with the invention may be polyclonal, but are preferably monoclonal. It will be appreciated that the term “antibody” includes whole antibodies (e.g. IgG, IgA etc), derivatives of whole antibodies which retain the antigen-binding sites (e.g. Fab, Fab′, F(ab′)2 etc.), single chain antibodies (e.g. sFv), chimeric antibodies, CDR-grafted antibodies, humanised antibodies, univalent antibodies, human monoclonal antibodies [e.g. Green (1999) J. Immunol Methods 231:11-23; Kipriyanov & Little (1999) Mol. Biotechnol 12:173-201 etc.] and the like. Humanised antibodies may be preferable to those which are fully human [e.g. Fletcher (2001) Nature Biotechnology 19:395-96].
As an alternative to using antibodies, antagonists of the interaction between App, ORF40 or NadA and its receptor on the epithelial cell may be used. As a further alternative, a soluble form of the epithelial cell receptor may be used as a decoy. These can be produced by removing the receptor's transmembrane and, optionally, cytoplasmic regions [e.g. EP-B2-0139417, EP-A-0609580 etc.].
The antibodies, antagonists and soluble receptors of the invention may be used as medicaments to prevent the attachment of a Neisserial cell to an epithelial cell.
Inhibiting Expression of the Neisserial Gene
The invention provides a method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein protein expression from one or more of App, ORF40 and/or NadA is inhibited. The inhibition may be at the level of transcription and/or translation.
A preferred technique for inhibiting expression of the gene is antisense [e.g. Piddock (1998) Curr Opin Microbiol 1:502-8; Nielsen (2001) Expert Opin Investig Drugs 10:331-41; Good & Nielsen (1998) Nature Biotechnol 16:355-358; Rabman et al. (1991) Antisense Res Dev 1:319-327; Methods in Enzymology volumes 313 & 314; Manual of Antisense Methodology (eds. Hartmann & Endres); Antisense Therapeutics (ed. Agrawal) etc.]. Antibacterial antisense techniques are disclosed in, for example, international patent applications WO99/02673 and WO99/13893.
The invention also provides nucleic acid comprising a fragment of x or more nucleotides from nucleic acid which encodes App, ORF40 or NadA, wherein x is at least 8 (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30 or more). The nucleic acid will typically be single-stranded.
The nucleic acid is preferably of the formula 5′-(N)a—(X)—(N)b-3′, wherein 0≧a≧15, 0≧b≧15, N is any nucleotide, and X is a fragment of a nucleic acid which encodes App, ORF40 or NadA. X preferably comprises at least 8 nucleotides (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30 or more). The values of a and b may independently be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. Each individual nucleotide N in the —(N)a— and —(N)b— portions of the nucleic acid may be the same or different. The length of the nucleic acid (i.e. a+b+length of X) is preferably less than 100 (e.g. less than 90, 80, 70, 60, 50, 40, 30 etc.).
It will be appreciated that the term “nucleic acid” includes DNA, RNA, DNA/RNA hybrids, DNA and RNA analogues such as those containing modified backbones (with modifications in the sugar and/or phosphates e.g. phosphorothioates, phosphoramidites etc.), and also peptide nucleic acids (PNA) and any other polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases etc. Nucleic acid according to the invention can be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various for (e.g. single stranded, double stranded, vectors, probes etc.).
The antisense nucleic acids of the invention may be used as medicaments to prevent the attachment of a Neisserial cell to an epithelial cell.
Knockout of the Neisserial gene
The invention provides a method far preventing the attachment of a Neisserial cell to an epithelial cell, wherein one or more of App, ORF40 and/or NadA is knocked out.
The invention also provides a Neisseria bacterium in which one or more of App, ORF40 and/or NadA has been knocked out.
Techniques for producing knockout bacteria are well known, and knockout Neisseria have been reported [e.g. Moe et al. (2001) Infect. Immun. 69:3762-3771; Seifert (1997) Gene 188:215-220; Zhu et al. (2000) J. Bacteriol. 182:439-447 etc.].
The knockout mutation may be situated in the coding region of the gene or may lie within its transcriptional control regions (e.g. within its promoter).
The knockout mutation will reduce the level of mRNA encoding App, ORF40 and/or NadA to <1% of that produced by the wild-type bacterium, preferably <0.5%, more preferably <0.1%, and most preferably to 0%.
The knockout mutants of the invention may be used as immunogenic compositions (e.g. as vaccines) to prevent Neisserial infection. Such a vaccine may include the mutant as a live attenuated bacterium.
Mutagenesis of the Neisserial Gene
The invention provides a method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein one or more of App, ORF40 and/or NadA has a mutation which inhibits its activity.
The invention also provides a mutant protein, wherein the mutant protein comprises the amino acid sequence of App, ORF40 and/or NadA, or a fragment thereof, but wherein one or more amino acids of said amino acid sequence is/are mutated (e.g. see below for App).
The amino acids which is/are mutated preferably result in the reduction or removal of an activity of App, ORF40 and/or NadA which is responsible directly or indirectly for adhesion to epithelial cells.
For example, the mutation may inhibit an enzymatic activity or may remove a binding site in the protein.
The invention also provides nucleic acid encoding this mutant protein.
The invention also provides a method for producing this nucleic acid, comprising the steps of: (a) providing source nucleic acid encoding App, ORF40 or NadA, and (b) performing mutagenesis (e.g. site-directed mutagenesis) on said source nucleic acid to provide nucleic acid encoding a mutant protein.
Mutation may involve deletion, substitution, and/or insertion, any of which may be involve one or more amino acids. As an alternative, the mutation may involve truncation.
Mutagenesis of virulence factors is a well-established science for many bacteria [e.g. toxin mutagenesis described in WO93/13202; Rappuoli & Pizza, Chapter 1 of Sourcebook of Bacterial Protein Toxins (ISBN 0-12-053078-3); Pizza et al. (2001) Vaccine 19:2554-41; Alape-Giron et al. (2000) Eur J Biochem 267:5191-5197; Kitten et al. (2000) Infect Immun 68:4441-4451; Gubba et al. (2000) Infect Immun 68:3716-3719; Boulnois et al. (1991) Mol Microbiol 5:2611-2616 etc.] including Neisseria [e.g. Power et al. (2000) Microbiology 146:967-979; Forest et al. (1999) Mol Microbiol 31:743-752; Comelissen et al. (1998) Mol Microbiol 27:611-616; Lee et al. (1995) Infect Immun 63:2508-2515; Robertson et al. (1993) Mol Microbiol 8:891-901 etc.].
Mutagenesis may be specifically targeted to nucleic acid encoding App, ORF40 and/or NadA. Alternatively, mutagenesis may be global or random (e.g. by irradiation, chemical mutagenesis etc.), which will typically be followed by screening bacteria for those in which a mutation has been introduced into App, ORF40 and/or NadA. Such screening may be by hybridisation assays (e.g. Southern or Northern blots etc.), printer-based amplification (e.g. PCR), sequencing, proteomics, aberrant SDS-PAGE gel migration etc.
The mutant proteins and nucleic acids of the invention may be used as immunogenic compositions (e.g., as vaccines) to prevent Neisserial infection.
Screening Methods
The invention also provides methods for screening compounds to identify those (antagonists) which inhibit the binding of a Neisserial cell to an epithelial cell.
Potential antagonists for screening include small organic molecules, peptides, peptoids, polypeptides, lipids, metals, nucleotides, nucleosides, polyamines, antibodies, and derivatives thereof. Small organic molecules have a molecular weight between 50 and about 2,500 daltons, and most preferably in the range 200-800 daltons. Complex mixtures of substances, such as extracts containing natural products, compound libraries or the products of mixed combinatorial syntheses also contain potential antagonists.
Typically, App, ORF40 and/or NadA protein is incubated with an epithelial cell and a test compound, and the mixture is then tested to see if the interaction between the protein and the epithelial cell has been inhibited.
Inhibition will, of course, be determined relative to a standard (e.g. the native protein/cell interaction). Preferably, the standard is a control value measured in the absence of the test compound. It will be appreciated that the standard may have been determined before performing the method, or may be determined during or after the method has been performed. It may also be an absolute standard.
The protein, cell and compound may be mixed in any order.
For preferred high-throughput screening methods, all the biochemical steps for this assay are performed in a single solution in, for instance, a test tube or microtitre plate, and the test compounds are analysed initially at a single compound concentration. For the purposes of high throughput screening, the experimental conditions are adjusted to achieve a proportion of test compounds identified as “positive” compounds from amongst the total compounds screened.
Other methods which may be used include, for example, reverse two hybrid screening [e.g. Vidal & Endoh (1999) TIBTECH 17:374-381] in which the inhibition of the Neisseria:receptor interaction is reported as a failure to activate transcription.
The method may also simply involve incubating one or more test compound(s) with App, ORF40 and/or NadA and determining if they interact. Compounds that interact with the protein can then be tested for their ability to block an interaction between the protein and an epithelial cell.
The invention also provides a compound identified using the method. These can be used to treat or prevent Neisserial infection. The compound preferably has an affinity for App, ORF40 and/or NadA of at least 10−7 M e.g. 10−8 M, 10−9 M, 10−10 M or tighter.
The invention also provides a composition comprising (a) an E. coli bacterium which expresses App and/or ORF40 (and, optionally, NadA) and (b) an epithelial coil (e.g. a human epithelial cell).
Expression in Eater Membrane Vesicles (OMVs)
International patent application WO01/52885 discloses that the addition of further defined components to OMV vaccines significantly broadens their efficacy.
The preparation of OMVs from NmB is well-known in the art. Methods for obtaining suitable preparations are disclosed in, for instance: Claassen et al. [Vaccine (1996) 14:1001-1008]; Cartwright et al. [Vaccine (1999) 17:2612-2619]; Peeters et al. [Vaccine (1996) 14:1009-1015]; Fu et al. [Biotechnology NY (1995) 12:170-74]; Davies et al. [J. Immunol. Meth. (1990) 134:215-225]; Saunders et al. (Infect. Immun. (1999) 67:113-119); Draabick et al. [Vaccine (2000) 18:160-172]; Moreno et al. [Infect. Immun. (1985) 47:527-533]; Milagres et al. [Infect. Immun. (1994) 62:4419-4424]; Naess et al. [Infect. Immun. (1998) 66:959-965]; Rosengvist et al. [Dev. Biol. Stand. (1998) 92:323-333]; Haneberg et al. [Infect. Immun. (1998) 66:1334-41]; Andersen at al. [Vaccine (1997) 15:1225-34]; Bjune et al. [Lancet (1991) 338:1093-96] etc.
It has now been found that OMVs prepared from E. coli which express a heterologous Neisseria gene can give better results in standard immunogenicity tests than the antigens in purified form.
The invention therefore provides a method for preparing an OMV from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding App, ORF40 or NadA protein.
The invention also provides (a) OMVs obtainable by this process, and (b) an outer membrane vesicle from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding App, ORF40 or NadA protein.
The non-Neisserial host cell is preferably a bacterium and is most preferably E. coli. 
More generally, the invention provides a method for preparing an OMV from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding one or more of the following proteins:

    
    
        (A) Even SEQ IDs 2-892 from WO99/24578;
        (B) Even SEQ IDs 2-90 from WO99/36544;
        (C) Even SEQ IDs 2-3020 from WO99/57280;
        (D) Even SEQ IDS 3040-3114 from WO99/57280;
        (E) SEQ IDs 3115-3241 from WO99/57280;
        (F) The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [supra];
        (G) A protein comprising the amino acid sequence of one or more of (A) to (F);
        (H) A protein sharing sequence identity with the amino acid sequence of one or more of (A) to (F); and
        (I) A protein comprising a fragment of one or more of (A) to (F).
    
    


Similarly, the invention also provides (a) OMVs obtainable by this process, and (b) an outer membrane vesicle from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding one or more of proteins (A) to (I) described above.
The degree of ‘sequence identity’ referred to in (H) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more) and this includes mutants and allelic variants
The ‘fragment’ referred to in (I) should comprise at least n consecutive amino acids from one or more of (A) to (F) and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragment comprises an epitope from one or more of (A) to CF). Preferred fragments are those disclosed in WO00/71574 and WO01/04316.
Preferred proteins for (A) to (F) are found in N. meningitidis serogroup B.
Mutants of App
Amino acid 267 of SEQ ID 650 of WO99/24578 (SEQ ID 32 herein) is a serine. App is believed to be a serine protease and this serine is believed to be a catalytic residue at its active site. It will be appreciated that standard sequence alignment techniques will reveal the amino acid corresponding to this Ser-267 for any other App sequence (e.g. Ser-260 in SEQ ID 652 of WO99/24578, Ser-267 in SEQ ID 654 etc.).
The invention provides a protein comprising the amino acid sequence of App, except that one or more of amino acids Ser-267, Asp-158 and His-115 (numbered according to SEQ ID 32) is/are mutated. The mutation may be a deletion, an insertion or, preferably, a substitution. The substitution is preferably with one of the 19 other naturally-occurring amino acids and is more preferably with glycine, alanine, tyrosine or lysine.
App is believed to cleaved at a site between amino acids 1063 and 1171 (numbered according to SEQ ID 32). It will be appreciated that standard sequence alignment techniques will reveal the amino acids corresponding to these two residues for any other App sequence.
The invention provides a protein comprising the amino acid sequence of App, except that one or more amino acid(s) between Ser-1064 and Arg-1171 (numbered according to SEQ ID 32) is mutated. The mutation may be a deletion, an insertion, truncation or, preferably, a substitution. The substitution is preferably with one of the 19 other naturally-occurring amino acids. The residue which is mutated is preferably S-1064, D-1065, K-1066, L-1067, G-1068, K-1069, A-1076, E-1071, A-1072, K-1073, K-1074, Q-1075, A-1076, E-1077, K-1078, D-2079, N-1080, A-1081, Q-1082, S-1083, L-1084, D-1085, A-1086, L-1087, I-1088, A-1089, A-1090, G-1091, R-1092, D-1093, A-1094, V-1095, E-1096, K-1097, T-1098, E-1099, S-100, V-1101, A-1102, E-1103, P-1104, A-1105, R-1106, Q-1107, A-1108, G-1109, G-1110, E-1111, N-1112, V-1113, G-1114, I-1115, M-1116, Q-1117, A-1118, E-1119, E-1120, E-1121, K-1122, K-1123, R-1124, V-1125, Q-1126, A-1127, D-1128, K-1129, D-1130, T-1131, A-1132, L-1133, A-1134, K-1135, Q-1136, R-1137, E-1138, 1139, E-1140, T-1141, R-1142, P-1143, A-1144, T-1145, T-1146, A-1147, F-1148, P-1149, R-1150, A-1151, R-1152, R-1153, A-1154, R-1155, R-1156, D-1157, L-1158, P-1159, Q-1160, L-1161, Q-1162, P-1163, Q-1164, P-1165, Q-1166, P-1167, Q-1168, P-1169, Q-1170 and/or R-1171.
App is alternatively believed to cleaved at amino acid 956 and/or amino acid 1178 (numbered according SEQ ID 32). It will be appreciated that standard sequence alignment techniques will reveal the amino acids corresponding to these residues for any other App sequence.
The invention provides a protein comprising the amino acid sequence of App, except that one or more of amino acids Phe-956, Asn-957, Ala-1178 & Asn-1179 (numbered according to SEQ ID 32) is mutated. The mutation may be a deletion, an insertion, truncation or, preferably, a substitution. The substitution is preferably with one of the 19 other naturally-occurring amino acids.
The invention also provides nucleic acid encoding these mutant proteins.
The invention also provides a method for producing this nucleic acid, comprising the steps of: (a) providing source nucleic acid encoding App, ORF40 or NadA, and (b) performing mutagenesis (e.g. site-directed mutagenesis) on said souses nucleic acid to provide nucleic acid encoding a mutant protein.
The invention provides mature App.
The invention also provides a protein comprising the amino acid sequence of a processed App, wherein said processed App does not comprise the C-terminus domain which is downstream of an autoproteloytic cleavage site in full-length App. Per example, based on SEQ ID 32 as full-length App, the invention provides SEQ IDs 33 to 36. C-terminus domains which may be removed during autoproteolysis are SEQ IDs 38 and 39.
The invention also provides a protein comprising the amino acid sequence of a processed App, wherein the C-terminus of said processed. App is Phe-956 (numbered according to SEQ ID 32). For example, the invention provides SEQ IDs 33 and 35. The amino acid corresponding to Phe-956 in other App sequences can be identified by standard sequence alignment techniques.
The invention also provides a protein comprising the amino acid sequence of a processed App, wherein the C-terminus of said processed App is Ala-1178 (numbered according to SEQ ID 32). For example, the invention provides SEQ IDs 34 and 36. The amino acid corresponding to Ala-1178 in other App sequences can be identified by standard sequence alignment techniques.
The invention also provides a protein comprising the amino acid sequence of a processed App, wherein said processed App does not comprise SEQ ID 37, 38 of 39.
The invention also provides a protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 33, 34, 35, 36, 37, 38 & 39.
The invention also provides a protein comprising an amino acid sequence with at least p % sequence identity to one or more of SEQ IDs 33, 34, 35, 36, 37, 38 & 39. Depending on the particular sequence, the value of p is preferably 50 or more (e.g. 60, 70, 80, 90, 95, 99 or more). These proteins include homologs, orthologs, allelic variants and functional mutants. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affirm gap search with parameters gap open penalty=12 and gap extension penalty=1.
The invention further provides proteins comprising a fragment of one or more of SEQ IDs 33, 34, 35, 36, 37, 38 & 39. The fragments should comprise at least q consecutive amino acids from the sequences and, depending on the particular sequence, q is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragments comprise one or more epitopes from the sequence.
The invention also provides nucleic acid encoding these proteins of the invention.
Alleles of NadA
The invention provides a protein comprising the amino acid sequence of one or more of SEQ IDs 1 to 14.
The invention also provides a protein comprising an amino acid sequence having at least x % sequence identity to one or more of SEQ IDs 1 to 14. The value of x is at least 50% (e.g. 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more). This includes variants e.g. allelic, variants, homologs, orthologs, paralogs, mutants, etc.
A preferred allele of NadA for use with the present invention is SEQ ID 3 (or SEQ ID 6).
The invention also provides a protein comprising a fragment of one or more of SEQ IDs 1 to 14. These should comprise at least n consecutive nucleotides from one or more of SEQ IDs 1 to 14, wherein n is 6 or more (e.g. 7, 8, 9, 10, 11, 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 or more). The fragment may comprise a sequence which is common to SEQ IDs 1 to 14, or may comprise a sequence which is not common to SEQ IDs 1 to 14.
Preferred fragments comprise one or mare epitopes from SEQ IDs 1 to 14. Other preferred fragments are (a) the N-terminal leader peptides of SEQ IDs 1 to 14, (b) SEQ IDs 1 to 14, but without k N-terminal amino acid residue(s), wherein k is 1 or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 etc.), and (c) SEQ IDs 1 to 14, but without l C-terminal amino acid residue(s), wherein l is 1 or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50 etc.). Preferred fragments fall within both (b) and (c) i.e. truncation at both C- and N-termini.
Preferred fragments within category (b) lack the N-terminal leader peptide. For SEQ IDs 1, 2, 3, 7, 9, 11 & 13 the value of k is thus 23; for SEQ IDs 4, 5, 6, 8, 10, 12& 14 the value of k is 2.5. The leader peptide may be replaced with the leader peptide from another protein, by another protein (i.e. to form a fusion protein) or by an alternative N-terminus sequence to allow efficient expression.
Preferred fragments within category (c) lack the C-terminal membrane anchor. The value of l is the 54. Minor variants of this C-terminal deletion may be used (e.g. when l is 45, 46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66).
Proteins with the N-terminus sequence MKH or MQH are preferred to those with N-terminus sequence MSM.
The protein of the invention may include the heptad sequence (AA1AA2AA3AA4AA5AA6AA7)r wherein: AA1 is Leu, Ile, Val or Met; each of AA2AA3AA4AA5AA6 and AA7 may independently be any amino acid; r is an integer of 1 or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.). Where r is 2 or more, the meaning of each AA1AA2AA3AA4AA5AA6 and AA7 may be the same or different in each of the r heptad repeats. The heptad(s) can form a leucine-zipper domain.
Proteins of the invention can be prepared in many ways e.g. by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself (e.g. isolation from prostate tissue), from a cell line source, etc.
Proteins of the invention can be prepared in various forms e.g. native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated etc.
The protein is preferably in the form of an oligomer.
Proteins of the invention may be attached or immobilised to a solid support.
Proteins of the invention may comprise a detectable label e.g. a radioactive label, a fluorescent label, or a biotin label. This is particularly useful in immunoassay techniques.
Proteins of the invention are preferably in isolated or substantially isolated form.
In general, the proteins of the invention are provided in a non-naturally occurring environment e.g. they are separated from their naturally-occurring environment. In certain embodiments, the subject protein is present in a composition that is enriched for the protein as compared to a control. As such, purified protein is provided, whereby purified is meant that the protein is present in a composition that is substantially free of other expressed proteins, where by substantially free is meant that less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed proteins.
The term “protein” refers to amino acid polymers of any length. The polymer may be linear or branched, it comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example proteins containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Proteins can occur as single chains or associated chain.
Mutants can include amino acid substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid substituted. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the polypeptide (e.g. a functional domain and/or, where the polypeptide is a member of a polypeptide family, a region associated with a consensus sequence). Selection of amino acid alterations for production of variants can be based upon the accessibility (interior vs. exterior) of the amino acid, the thermostability of the variant polypeptide, desired disulfide bridges, desired metal binding sites etc.
The invention also provides nucleic acid encoding a protein of the invention as defined above. The invention also provides nucleic acid comprising a fragment of at least n consecutive nucleotides from said nucleic acid, wherein n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 or more).
Furthermore, the invention provides nucleic acid which can hybridise to nucleic acid encoding a protein of the invention, preferably under “high stringency” conditions (e.g. 65° C. in a 0.1×SSC, 0.5% SDS solution).
Nucleic acids of the invention can be used in hybridisation reactions (e.g. Northern or Southern blots, or in nucleic acid microarrays or ‘gene chips’) and amplification reactions (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA, etc.) and other nucleic acid techniques.
Nucleic acids of the invention can be, prepared in many ways e.g. by chemical synthesis in whole or part, by digesting longer polynucleotides using nucleases (e.g. restriction enzymes), from genomic or cDNA libraries, from the bacterium itself, etc.
Nucleic acids of the invention can take various forms e.g. single-stranded, double-stranded, vectors, printers, probes, labelled, unlabelled, etc.
Nucleic acids of the invention are preferably in isolated or substantially isolated form.
The invention includes nucleic acid comprising sequences complementary to those described above e.g. for antisense or probing, or for use as primers.
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.
Nucleic acid according to the invention may be labelled e.g. with a radioactive or fluorescent label. This is particularly useful where the nucleic acid is to be used in nucleic acid detection techniques e.g. Where the nuclei acid is a primer or as a probe for use in techniques such as PCR, LCR TMA, NASBA, etc.
The invention also provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors, such as those suitable for nucleic acid immunisation) and host cells transformed with such vectors.
Immunisation
The invention provides an immunogenic composition comprising (a) a Neisserial NadA protein and/or (b) nucleic acid encoding NadA protein.
The invention also provides a method for raising an antibody response in a mammal, comprising administering an immunogenic composition of the invention to the mammal. The antibody response is preferably a protective antibody response. The protective antibody preferably blocks the attachment of NadA and/or App to epithelial cells.
The invention also provides a method for protecting a mammal against a Neisserial intention, comprising administering to the mammal an immunogenic composition of the invention.
The invention also provides Neisserial NadA protein for use as a medicament.
The invention also provides the use of a NadA protein in the manufacture of a medicament for preventing Neisserial infection in a mammal
The invention also provides the use of nucleic acid encoding a NadA protein in the manufacture of a medicament for preventing Neisserial infection in a mammal.
The Mammal is preferably a human. The human may be an adult or, preferably, a child.
The NadA protein is preferably a N. meningitidis NadA. It preferably comprises the amino acid sequence of one or more of SEQ IDs 1 to 14, or an amino acid sequence having sequence identity thereto or comprising a fragment thereof (see above). The NadA protein is preferably in the form of an oligomer (e.g. a timer, trimer, tetramer or higher). Within SEQ IDs 1 to 14, SEQ IDs 1 to 12 are preferred, as antibodies against these NadA proteins are bactericidal across the various hypervirulent alleles. Where an immune response against a non-hypervirulent NadA+ strain is desired, however, SEQ IDs 13 & 14 are preferred. Of course, NadA mixtures are also possible, particularly mixtures containing more than one NadA allele.
Immunogenic compositions of the invention may be used therapeutically (i.e. to treat an existing infection) or prophylactically (i.e. to prevent future infection).
The uses and methods of the invention are particularly useful for treating/protecting against infections of Neisseria meningitidis, including serogroups A, B, and C. They are particularly useful against strains of N. meningitidis from hypervirulent lineages ET-5, EY-37 and cluster A4.
The uses and methods are particularly useful for preventing/treating diseases including, but not limited to, meningitis (particularly bacterial meningitis) and bacteremia.
Efficacy of therapeutic treatment can be tested by monitoring Neisserial infection after administration of the composition of the invention. Efficacy of prophylactic treatment can be tested by monitoring immune responses against NadA after administration of the composition.
The composition of the invention may additionally comprise an antigen which, when administered to a mammal, elicits an immune response which is protective against a lineage III strain of N. meningitidis. 
Compositions of the invention will generally be administered directly to a patient Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, or pulmonary administration.
The invention may be used to elicit systemic and/or mucosal immunity.
Dosage treatment can be a single dose schedule or a multiple dose schedule.
The immunogenic composition of the invention will generally include a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly-metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Liposomes are suitable carriers. A thorough discussion of pharmaceutical carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.
Neisserial infections affect various areas of the body and so the compositions of die invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a line powder or a spray. Tire composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops.
The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7.
Immunogenic compositions comprise an immunologically effective amount of immunogen, as well as any other of other specified components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses). The composition may be administered in conjunction with other immunoregulatory agents.
The immunogenic composition may include an adjuvant. Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to (A) aluminium compounds (e.g. an aluminium hydroxide such as oxyhydroxide, or an aluminium phosphate such as hydroxyphosphate or orthophosphate, aluminium sulphate etc.), or mixtures of different aluminium compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous etc.), and with adsorption being preferred; (B) MP59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer); (C) liposomes; (D) ISCOMs, which may be devoid of additional detergent; (F) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion; (F) RibiTM adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DetoxTM); (G) saponin adjuvants, such as QuilA or QS21, also known as StimulonTM; (H) chitosan; (I) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, tumor necrosis factor, etc.; (K) microparticles (i.e. a particle of  ̃100 nm to  ̃150 μm in diameter, more preferably  ̃200 nm to  ̃30 μm in diameter, and most preferably  ̃500 nm to  ̃10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone etc.); (L) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL); (M) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions; (N) oligonucleotides comprising CpG motifs i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (O) a polyoxyethylene ether or a polyoxyethylene ester; (P) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional nonionic surfactant arch as an octoxynol; (Q) an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide) and a saponin; (R) an immunostimulant and a particle of metal salt; (S) a saponin and an oil-in-water emulsion; (T) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol); (U) E. coli heat-labile enterotoxin (“LT”), or detoxified mutants thereof, such as the K63 or R72 mutants; (V) cholera toxin (“CT”), or detoxified mutants thereof; (W) microparticles (i.e. a particle of  ̃100 nm to  ̃150 μm in diameter, more preferably  ̃200 nm to  ̃30 μm in diameter, and most preferably  ̃500 nm to  ̃10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid) such as poly(lactide-co-glycolide), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone etc.); and (X) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Aluminium salts (aluminium phosphates and particularly hydroxyphosphates, and/or hydroxides and particularly oxyhydroxide) and MF59 are preferred adjuvants for parenteral immunisation. Toxin mutants are preferred mucosal adjuvants.
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.
Compositions of the invention may comprise antigens (e.g. protective antigens against N. meningitidis or against other organisms) in addition to NadA e.g. DTP antigens, Hib antigen etc.
Immunogenic compositions of the invention may be used therapeutically (i.e. to treat an existing infection) or prophylactically (i.e. to prevent future infection). Therapeutic immunisation is particularly useful for treating Candida infection in immunocompromised subjects.
As an alternative to using proteins antigens in the immunogenic compositions of the invention, nucleic acid (preferably DNA e.g. in the form of a plasmid) encoding the antigen may be used.
Disclaimers
The invention preferably excludes: (a) amino acid and nucleic acid sequences available in public sequence databases (e.g. GenBank or GENESEQ) prior to 26 Jul. 2002 and, more preferably, prior to 27 Jul. 2001; (b) amino acid and nucleic acid sequences disclosed in patent applications having a filing date or, where applicable, a priority date prior to 26 Jul. 2002 and, more preferably, prior to 27 Jul. 2001. In particular, SEQ ID entries in the following patent applications may be excluded WO99/24578; WO99/36544; WO99/57280; WO00/22430; WO00/66741; WO00/66791; WO00/71574; WO00/71725; WO01/04316; WO01/31019; WO01/37863; WO01/38350; WO01/52885; WO01/64920; WO01/64922.
DEFINITIONS
The term “comprising” means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1A-FIG. 1C shows expression data for ORF40. FIG. 1A shows a whole cell Western blot for ORF40. FIG. 1B shows a whole cell immunoblot for ORF40. FIG. 1C shows FACS analysis of ORF40.
FIG. 2A-FIG. 2C shows expression data for App. FIG. 2A shows a Western blot for App. FIG. 2B shows a whole cell immunoblot for App. FIG. 2C shows FACS analysis of App.
FIG. 3A-FIG. 3C shows expression data for NadA. FIG. 3A shows SDS-PAGE analysis of NadA. FIG. 3B shows a whole cell immunoblot for NadA. FIG. 3C shows FACS analysis of NadA.
FIG. 4A-FIG. 4D show FACS analysis data. Adhesion of cells expressing ORF40 (FIG. 4A), App (FIG. 4B), NadA (FIG. 4C), or an HSF positive control (FIG. 4D) to human epithelial cells is shown.
FIG. 5A-FIG. 5D show FACS analysis data. Adhesion of ORF40 protein (FIG. 5A), App protein (FIG. 5B), NadA protein (FIG. 5C), or a GNA2132 negative control protein (FIG. 5D) to human epithelial cells is shown.
FIG. 6A-FIG. 6B shows binding of proteins to human epithelial cells. FIG. 6A shows binding of ORF40 (♦), App (•), NadA (♦) and GNA2132 (▪) to human epithelial cells as a function of protein concentration. FIG. 6B shows binding of NadA (♦) and GNA2132 (▪) to human epithelial cells as a function of protein concentration.
FIG. 7 shows homologies of ORF40.
FIG. 8 shows homologies of App.
FIG. 9A-FIG. 9C show an alignment of NadA alleles 1 to 3. FIG. 9A shows a sequence alignment of NadA alleles 1 to 3 (SEQ ID NOS: 4-6) starting with the first Met residue in the open reading frame. FIG. 9B shows an alignment of NadA alleles 1 to 3 (SEQ ID NOS: 1-3) starting with the second Met residue in the open reading frame. FIG. 9C shows an alignment of allele C (SEQ ID NO: 14) with NadA alleles 1 to 3 (SEQ NOs: 4-6).
FIG. 10 shows the relationship of NadA alleles 1 to 3 (SEQ ID NOs: 4-6).
FIG. 11 shows predicted secondary structure for NadA, (SEQ ID NO: 40) (top) and (SEQ ID NO: 52) (bottom).
FIG. 12A-FIG. 12B show NadA sequence information. FIG. 12A shows analysis of sequences upstream and downstream of NadA (SEQ ID NO: 41). FIG. 12B shows alignment of the NadA locus in the MC58 and Z2491 strains (SEQ ID NO: 42).
FIG. 13 shows PCR analysis of NadA expression in different strains of N. meningitidis. 
FIG. 14 shows immunoblot analysis of NadA expression in different strains of N. meningitidis. 
FIG. 15A-FIG. 15B show variation of NadA expression with culture time. FIG. 15A shows a Western blot of NadA expression over time in the MC58 strain (left panel) with a corresponding analysis of strain growth rate (right panel). FIG. 15B shows a Western blot of NadA expression over time in the 2996 strain (left panel) with a corresponding analysis of strain growth rate (right panel).
FIG. 16 shows NadA FACS of isogenic capsulated (NMB) and non-capsulated (M7) N. meningitidis cells.
FIG. 17A-FIG. 17D show immunofluorescence results obtained using anti-NadA against Chang cells (FIG. 17A, FIG. 17B, and FIG. 17C) or HeLa cells (FIG. 17D).
FIG. 18A-FIG. 18B show immunofluorescence results obtained using anti-NadA against Chang cells after incubation at 37° C. (FIG. 18A) or 4° C. (FIG. 18B).
FIG. 19 shows immunofluorescence results for Chang cells treated with saponin.
FIG. 20A-FIG. 20C show immunofluorescence results obtained using monocytes. FIG. 20A shows NadA binding to monocytes. FIG. 20B shows NadA alone with no staining antibody. FIG. 20C shows NadA stained with pre-immune serum.
FIG. 21 shows immunofluorescence results obtained using macrophages.
FIG. 22 shows IL-α secretion by monocytes in response to NadA treatment.
FIG. 23 shows the effect of anti-CD14 on IL-α secretion by monocytes.
FIG. 24A-FIG. 24B show immunofluorescence results obtained using anti-NadA against E. coli transformed to express NadA (FIG. 24A) or untransformed bacteria (FIG. 24B).
FIG. 25A-FIG. 25C show staining of the transformed E. coli using anti-NadA (FIG. 25A), anti-E. coli (FIG. 25B), or both (FIG. 25C).
FIG. 26 is a schematic representation of App features. The N-terminal leader peptide, the passenger domain and the C-terminal β-domain are indicated. The positions of the serine protease active site, the ATP/GTP binding site, the two Arginine-rich sites (SEQ ID NO: 53) (starting at residue 934) and (SEQ ID NO: 54) (starting at residue 1149) and the Proline-rich region (SEQ ID NO: 55) are shown. In BOX 1, cleavage sites are shown. Sequences shown in BOX 1 correspond to (top to bottom): SEQ ID NO: 43, SEQ ID NO: 56, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47. In BOX 2 a comparison of known proteolytic sites of different autotransporters is shown (SEQ ID NOS: 48-51) (top to bottom) and a consensus signature is derived (SEQ ID NO: 57). Arrows identify the cleavages; X=any amino acid; hyd=hydrophobic residues; (A,S)=Alanine or Serine.
FIG. 27 is a schematic representation of the constructs used for studying App.
FIG. 28 shows a western blot of outer membrane and extracellular proteins in E. coli. 
FIG. 29 shows FACS analysis of outer membrane and extracellular proteins in E. coli. 
FIG. 30A-FIG. 30C show immunofluorescence of outer membrane and extracellular proteins in E. coli harboring pET-App (FIG. 30A), pET-App-His (FIG. 30B), or pET (FIG. 30C).
FIG. 31 shows total E. coli proteins analysed by SDS-PAGE.
FIG. 32 shows an immunoblot of crude precipitated culture supernatants using mouse antiserum against App-his.
FIG. 33 shows FACS adhesion data using rabbit antiserum against E. coli. Percentages of cells positive to adhesion are shown near the fluorescence profiles.
FIG. 34A-FIG. 34G show immunofluorescence microscopy data showing bacterial adherence and aggregation. FIG. 34A and FIG. 34B show pET-App transformants incubated with epithelial cells. FIG. 34C and FIG. 34D show S267A mutants incubated with epithelial cells. FIG. 34E and FIG. 34F show Appβ transformants incubated with epithelial cells. FIG. 34G shows untransformed controls incubated with epithelial cells.
FIG. 35 shows concentration-dependent binding of App-His (♦), Appα-His (▪) and NMB2132 (▴) expressed as net Mean Fluorescence Intensity (MFI).
FIG. 36 shows the effect on binding of App-His (100 μg/ml) of pre-incubation with pronase (left-hand columns) or phospholipase A2 (right-hand columns) with increasing concentration of enzyme. Pronase was tested at 0, 250, 500, 1000 μg/ml; phosholipase A2 was tested at 0, 50, 200, 800 μg/ml.
FIG. 37 is a comparison of cellular binding specificity of App-His protein at 100, 25 or 6.25 μg/ml against various different cells.
FIG. 38 shows association of wild-type or App-knockout N. meningitidis MC58 bacteria.
FIG. 39 shows a western blot analysis of total lysates from N. meningitidis MC58 harvested at 0.5 or 0.8 OD620nm. Lanes 1 & 3 show wild-type MC58 and lanes 2 & 4 show the App knockout.
FIG. 40 shows a western blot analysis of supernatants in parallel to FIG. 39.



MODES FOR CARRYING OUT THE INVENTION
NadA Homology
NadA shows homology to (a) YadA of enteropathogenic Yersinia, a non-pilus associated adhesin implicated in virulence [Cornelis (1998) Microbiol. Mol. Biol. Rev. 62:1315-1352.] and (b) UspA2 of Moraxella catarrhalis, a protein involved in serum resistance and a protective antigen [Chen et al. (1999) Infect. Immun. 67:1310-1316]. Sequence similarity is mainly clustered in the carboxyl terminal region (56-63% identity in the last 70 amino acids). Outside this region the level of identity drops to 23-25%.
YadA and UspA2 have been identified as adhesins [Hoiczyk et al. (2000) EMBO J 19:5989-5999]. Both proteins form very stable and difficult-to-dissociate high molecular weight oligomers (150-200 kDa) anchored to the outer membrane. NadA has also been found to form very stable high molecular weight aggregates on the outer membrane of meningococcus.
The amino acid sequence of NadA was analysed [Nielsen et al. (1997) Protein Engineering 10:1-6; Levin & Garner (1988) Biochim. Biophys. Acta 955:283-295; Berger et al. (1995) PNAS USA 92:8259-8263; Bornberg-Bauer et al. (1998) Nucleic Acids Res. 26:2740-2746]. Secondary structure analysis is shown in FIG. 11. The globular N-terminus and amphipathic C-terminus are indicated, as are the positions of the leader peptide (LP) and a membrane anchor. The carboxyl-terminal region (aa 310-362) has a predicted amphipatic β-structure (β-strands shown in black) and a terminal aromatic amino acid, which are typical features of outer membrane anchoring domains. The amino terminal region (aa 23-90) has no defined secondary structure, but the rest of the protein has mainly α-helix propensity (84.6%). Within this region, residues 90-146 and 183-288 have high probability of forming coiled coils. In addition, residues 122-143 contain four leucine residues in the “a” positions of the heptad repeats (L-x(6)-L-x(6)-L-x(6)-L) that may form a leucine zipper domain (•••). It is known that both coiled coils and leucine zipper sequences are involved in dimerization and may mediate oligomerisation of monomers via association of two or more alpha helices.
Even though primary structure similarity between NadA, YadA and UspA2 is clustered at the C-terminus, therefore, the overall similarity between the three proteins is conserved at secondary structure level. Putative leucine zippers are present in both NadA and UspA2. NadA, YadA and UspA2 have a carboxyl terminal membrane anchor made by four amphipathic β-strands and an internal α-helical region with propensity to form coiled-coils. In YadA and UspA2 these α-helices have been shown to form coiled-coils regions, which mediate oligomerisation of monomers [Hoiczyk et al. (2000) EMBO J 19:5989-5999; Cope et al. (1999) J. Bacteriol. 181:4026-4034].
The absence of cysteine residues in the mature forms of NadA is another feature shared with its homologues.
The Genomic Environment of NadA
The 1086 bp nadA coding region is flanked at the 3′ end by a terminator sequence while at the 5′ end (FIG. 12A) it shows a putative ribosome-binding site (RBS; 5′-AAGG-3′) and a putative promoter region located 8 and 47 base pairs, respectively, upstream the ATG start codon.
130 bp upstream the coding region are nine repeats of the tetranucleotide TAAA (shaded black in FIG. 12A), preceded by a second putative promoter with −10 and −35 regions. Because of the presence of the TAAA repeats, the gene had been listed as one of those that may undergo phase variation, even though the repeats are not in the coding region [Tettelin et al.]. The homologous gene UspA2 has a tetranucleotide repeat (AGAT) located in the same position as in nadA, which varies in different strains [Cope et al. (1999) J. Bacteriol. 181:4026-4034].
The G+C content of the nadA gene and its upstream region is lower than average (45% against an average of the rest of the genome, 51.5%), suggesting acquisition of the gene by horizontal transfer.
The NadA gene and its upstream region are not present in the published sequence of the genome of serogroup A, strain Z2491 [Parkhill et al. (2000) Nature 404:502-506]. In the MenA genome, a short sequence of 16 nucleotides with no homologies in the database, replaces the nadA gene (FIG. 12B), whereas the upstream and downstream genes (nmb1993 and nmb1995) are well conserved (91% and 97% identity). Analysis of the sequences immediately adjacent to the nadA region and absent in the Z2491 serogroup A strain shows that the segment is flanked by the TCAGAC direct repeats. This may indicate a mechanism of recombination. In the A strain the stretch of 16 nucleotides has a disrupted pair of TCAGAC repeats flanking it.
Variation in NadA Genotype
Given the difference in nadA expression between serotypes A and B, 175 different strains of N. meningitidis were chosen for analysis −150 isolates representative of the five disease-associated serogroups (A, B, C, Y and W-135) and 25 strains isolated from healthy carriers. The analysis also included one strain each of N. goriorrhoeae. N. cinerea and N. lactamica. 
Bacteria were grown overnight at 37° C. in a humidified atmosphere of 5% CO2 in air on gonococcus (GC) medium agar (Difco) supplemented with Kellogg's supplement solution (0.22 M D-glucose, 0.03 M L-glutamine, 0.001 M ferric nitrate, and 0.02 M cocarboxylase) (Sigma-Aldrich Chemical Co, St. Louis, Mo.) as previously described [Knapp et al. (1988) Antimicrob. Agents Chemother. 32:765-767; Roberts et al. (1977) J. Bacteriol. 131:557-563]. One loopful of bacteria was dissolved in 500 μl of PBS and chromosomal DNA was prepared as previously described [Tinsley et al. (1996) PNAS USA 93:11109-11114].
The bacteria were screened by PCR and/or dot blot hybridization.
PCR amplification of the nadA genes was performed on 10 ng of chromosomal DNA using primers, mapping 350 nt upstream and downstream from the coding region (forward primer: SEQ ID 16; reverse primer: SEQ ID 17), and Platinum Hifi Taq Polymerase (GIBCO). PCR conditions were: 30 cycles of denaturation at 95° C. for 30 s, annealing at 60° C. for 30 s, and extension at 68° C. for 1 min. PCR products were analysed on 1% agarose gel and the sizes were determined using a molecular weight marker 1 Kb Plus DNA Ladder (GIBCO). The amplified fragments were purified on a Qiaquick column (Qiagen) and then automated cyclo-sequenced (Applied Biosystems model 377) by primer walking on both strands of the amplified fragment.
For dot blotting, the probe used was the whole nadA gene, as amplified from 2996 strain and labelled with digoxigenin using the Roche DIG High-Prime DNA Labelling and Detection Kit. 10 μl aliquot of cell suspension of each strain were boiled for 10 min. and spotted on nylon membrane (Boehringer). The membranes underwent cross-linking of DNA by 2′ exposure to UV light and other standard procedures for preparation and signal detection as reported by the manufacturer.
The nadA gene was absent, in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. In N. meningitidis, however, 47% of isolated were positive for its presence.
PCR generated (FIG. 13) a product of 1800 bp in NadA+ strains MC58 (lane 1), 90/18311 (lane 2) and 2996 (lane 3). It gave a product a 400 bp in NadA− strain Z2491 and NG3/88 (lane 5), Some strains (e.g. 93/4286, C4678, 2022, ISS1113) gave a PCR product of 2500 bp (lane 4: L93/4286).
The presence/absence of NadA in N. meningitidis was correlated with strain lineage. Strains isolated from invasive meningococcal disease have been classified by multilocus enzyme electrophoresis (MLEE) into a small number of hypervirulent lineages: Electrophoretic Types ET37, ET5, cluster A4, lineage III, subgroups I, III and IV-1 [Achtman (1995) Global epidemiology of meningococcal disease. In Meningococcal disease (Cartwight, ed), John Wiley and Sons, Chichester, England. 159-175; Caugant (1998) APMIS 106:505-25]. Recently, a sequence-based classification, multilocus sequence typing (MLST), has been introduced, which classifies the above strains into Sequence Types ST11, ST32, ST8, ST41, ST1, ST5, ST4, respectively [Malden et al. (1998) PNAS USA 95:3140-3145]. Strains isolated from healthy carriers fall into many different ET and ST types.
The nadA gene was present in 51 out of 53 strains (96%) of the hypervirulent lineages ET-5, ET-37 and cluster A4, whereas it was absent in all the tested lineage III strains. Seven of the 25 carrier strains were positive. Most of the serogroup C strains tested were positive even if not belonging to 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 where the other two strains belonging to subgroup IV-1 were negative.
Lineage III has only recently been introduced in Europe and USA and the geographic segregation in New Zealand for many years could have impaired its ability to acquire novel genes. For instance, mutations may have occurred in the surrounding chromosomal regions preventing Lineage III from further recombination events. Mother possible explanation is that ET-5, ET-37 and Cluster A4 strains need nadA to achieve peak fitness whereas Lineage III isolates cannot derive any significant benefit from nadA insertion, thus undergoing a negative selection.
NadA is thus over-represented in three hypervirulent N. meningitidis lineages. It appears to be a foreign gene present in a subset of hypervirulent strains.
NadA Alleles
As PCR products were differently sized (FIG. 13) and most of the NadA+ strains could be grouped in three different sizes, genes were sequenced for 36 strains representative of each sizes: 26 positive strains, 4 strains with a long PCR product, and 6 NadA− strains.
In the negative strains, a 16 bp sequence was found which was identical to the sequence present in the published serogroup A genuine sequence.
Analysis of the sequence of the four long PCR product strains revealed an interruption by a single copy of IS1301, interrupting the protein after 162 amino acids with a stop codon. The insertion site was identical in all four strains, but the orientation of IS1301 differed, indicating independent events. The target consensus for IS1301, 5′-AYTAG-3′ was found within the NadA gene at nucleotide 472, generated by, an A->G mutation, and was accompanied by a TA duplication.
In nadA+ strains gene size ranged from 1086 to 1215 bp, with consequent variation of the amino acid sequences of the encoded proteins flew 362 to 405 amino acids. It was possible to cluster 22 of the 26 NadA genes into three well-defined alleles (FIGS. 9 & 10; Table I). The sequence of the gene within each allele is identical and overall identity between the alleles ranges from 96% to 99%. This level of conservation is surprising and suggests weak selective pressure and/or a very recent acquisition of the nadA gene. The latter possibility is consistent with the low G+C content of the genome in this region (see above).














SEQ


Allele
Found in strains
IDs







1
MC58, BZ83, BZ169, NM066, NM119, CU385, ISS832,
1, 4



ISS1071, ISS1104


2
90/18311, NGP165, PMC8, M986, ISS838 and 961-5945
2, 5


3
C11, 973-1720, ISS759, F6124, 2996, 8047, NMB
3, 6









The sequences shown in FIG. 9A assume that the N-terminus amino acid is the first Met in the open reading frame (SEQ IDs 4 to 6), but the second Met (residue 3 in SEQ IDs 4 to 6) has a better positioned Shine-Dalgarno motif (FIG. 9B). Sequences starting from the second Met codon are thus preferred (SEQ IDs 1 to 3).
Allele 1 codes for a protein of 362-amino acids (SEQ ID 1) and includes strain MC58 and all the ET-5 positive strains sequenced. The other five strains belonging to allele 1 were very recent isolates and they have not been FE-typed yet, although serotype and serosubtype classification (B15:P1.7 and B:4:P1.15) of these strains suggests affiliation of these strains to the ET-5 complex.
Allele 2 codes for a protein of 398 amino acids (SEQ ID 2) resulting from the addition of 2 as after residue 268 (numbering according to SEQ ID 1), addition of 41 as after residue 271, and deletion of 7 an after residue 122, resulting in the deletion of the first heptad repeat of the leucine zipper domain. Leucine residues at a fixed spacing of seven residues commonly identify leucine zippers. One leucine in the repeats has frequently been replaced mostly by Met, Val or Ile. In this case allele 2 could use the Ile upstream or downstream to form the leucine zipper motif.
Allele 3 codes for a protein of 405 amino acids (SEQ ID 3) and, like allele 2, contains 43 extra amino acids at residues 268 and 271 but differs from allele 2 by not having the 7aa deletion after residue 122. Allele 3 is found in serogroup A, B and C strains.
The remaining 4/26 positive strains (ISS1024, ISS759, 973-1720, 95330, marked with in Table 1) contain minor variants of alleles 1 to 3:

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


All insertions and deletions happen in the coiled-coil region and involve 7 or 41 amino acids which, representing 2 or 6 turns of the α-helix, allows for variations in length of the coiled coil region without disturbing the overall structure. Furthermore, the deletion in ISS1024 results in the loss of the first heptad repeat of the leucine zipper domain but does not destroy the domain because leucine residues at a fixed spacing of seven residues eau be replaced mostly by Met, Val or Ile. In this case allele 2 could use the Ile upstream or downstream to form the leucine zipper motif (FIG. 11).
Any of these various NadA sequences and alleles can be used in accordance with the invention.
When sequence analysis was extended to the putative promoter and terminator regions (50 bp upstream, 350 bp downstream), variations were found only in the in the 5′ region. Three Italian strains (ISS1071, ISS832 and ISS1104) differed for a single base mutation while in strain 961-5945 there was a 7 base differences (indicated with * in FIG. 10). Variations were also found in the 5′ regions where the TAAA tetranucleotide was repeated from 4 to 12 times in different strains (Table 1). The number of repeats was variable also within each allele (Table 1).
Further work was performed on carrier strains isolated from healthy individuals by oro-pharyngeal swab. Some strains, even if described as carriers, belong to hypervirulent clusters, and NadA was found in all such carrier strains as described above (i.e. allele 1 in the ET-5 strains and allele 2 in the ET-37 strains).
NadA was also found in five carrier strains (NGE28, 65/96, 149/96, 16269, 16282) which do not belong to a hypervirulent cluster. These five strains shared a sequence (SEQ IDs 13 & 14) which was not found in strains isolated from patients. This allele is referred to as ‘allele C’ (carrier).
An alignment of allele C with alleles 1 to 3 is shown in FIG. 9C. Disruption in the coiled-coil segments of the protein is evident.
Unlike alleles 1 to 3, allele C protein does not readily form a high molecular aggregate when expressed in E. coli. Like alleles 1 to 3, however, allele C is exposed on the surface of N. meningitidis, because it is a target for bactericidal antibody raised against itself. However, these antibodies are not bactericidal against strains carrying alleles 1 to 3; similarly, antibodies raised against alleles 1 to 3 are not bactericidal against allele C strains.
NadA Oligomers on the Cell Surface
WO01/64922 reports that NadA forms oligomeric structures. To study NadA oligomers in more detail, whole cell lysates of N. meningitidis were probed by Western blot.
Bacterial colonies [strains MC58 (allele 1), 90/18311 (allele 2), 2996 (allele 3), L93/4286 (IS1301 insertion) and NG3/88 (nadA−)] were grown to stationary phase in GC broth supplemented with 0.3% glucose. Samples, were taken at different times, pelleted by centrifugation at 3000×g for 10 min, and resuspended in PBS and thawed/frozen up to bacterial lysis. Equal amounts of proteins were subjected to SDS-PAGE on 12.5% Polyaerylamide gels and electrotransferred onto nitrocellulose membranes.
To prepare anti-NadA polyclonal serum, recombinant NadA was expressed and purified. Sequences encoding the three nadA alleles (allele 1: as 24-362; allele 2: as 24-343; allele 3: as 24-350), were amplified by PCR chromosomal DNA and cloned into pET21b+ vector (Novagen). The plasmids were transformed in E. coli BL21 (DE3) to express the proteins as C-terminal histidine fusions. Protein expression was induced at 30° C. by adding 1 mM IPTG at OD600nm 0.3 and growing the bacteria for an additional 3 h; expression was evaluated by SDS-PAGE. Recombinant lesion proteins were purified by affinity chromatography on Ni2+-conjugated chelating fast-flow Sepharose 4B resin, 20 μg of purified protein was used to immunise six-week-old CD1 female mice (4 to 6 per group). Proteins were given intraperitoneally, with complete Freund's adjuvant (CFA) for the first dose and incomplete Freund's adjuvant (IFA) for the second (day 21) and third (day 35) booster doses. Bleed out samples were taken on day 49 and used for the serological analysis.
The blots showed a high molecular weight reactive band in strains MC58 (FIG. 14, lane 1), 90/18311 (lane 2) and 2996 (lane 3). The band was absent in strain NG3/88 (lane 5). Boiling of the sample buffer up to 40 minutes did not change the pattern. The different size of the proteins was consistent with the size of the alleles. Given the expected size ranging from 35 to 40 kDa of monomeric proteins, the high MW of the observed band, could be explained by the presence of an oligomeric form of NadA. This possibility is supported by the fact that in a strain containing the IS1301 insertion, coding for a shorter protein of 162 amino acids and lacking most of the coiled-coil region, the high MW reactive band was absent and replaced by a band of 14.5 kDa (FIG. 14, lane 4), consistent with the predicted molecular weight of the processed monomeric protein.
Although the oligomeric protein was found in all strains containing a functional gene, expression levels varied from strain to strain (Table I). Moreover, the amount of NadA protein varied within the same strain during growth.
Four different strains (MC58, 2996, C11, F6124), chosen as representative of diverse overall NadA expression level, were followed during growth up to stationary phase. FIG. 15 shows growth of two of the tested strains (15A: MC58, with low NadA expression; 15B: 2996, with high NadA expression), with the curve showing OD600. Western blots of samples taken at each point of the OD600 growth curve showed that the NadA band was barely visible at the beginning of the growth and became more intense during growth, up to its maximum, at stationary phase. Alt strains analysed showed the same growth-phase dependent behaviour.
High MW NadA was also seen in western blots of outer membrane vesicles, consistent with NadA being anchored to the outer membrane.
Similarly, FACS analysis on live bacteria during log-phase growth showed that NadA was available for antibody binding on the surface of the FACS intensity in a strain with a polysaccharide capsule (strain NMB) was reduced 1 log in comparison to an isogenic non-encapsulated mutant strain (M7), but the protein was surface-exposed and available for binding in both strains (FIG. 16).
NadA forms surface-exposed oligomers, which are stable to heat, SDS and reduction with β-mercaptoethanol. As the mature form of the lacks cysteine residues, disulphide bond formation cannot be involved in this phenomenon; rather this is consistent with the predicted coiled-coil structure and the presence of leucine zipper motifs that might mediate intermolecular interactions between monomers [Lupas (1996) Trends Biochm. Sci. 21:375-382; O'Shea et al. (1991) Science 254:539-544]. The size of the oligomers is approximately 170 kDa, suggesting a tetrameric structure [WO01/64922]. However, a rigid coiled-coil structure is likely to have an anomalous migration is SDS PAGE and therefore the 170 kDa form may be a trimer.
Protective Immunogenicity
Polyclonal anti-NadA serum was tested for bactericidal activity as previously described [Pizza et al. (2000); Peeters et al. (1999) Vaccine 17:2702-2712], with pooled baby rabbit serum (CedarLane) used as complement source. Serum bactericidal titer, was defined as the serum dilution resulting in a 50% decrease in colony forming units (CFU) per ml after 60 minutes incubation of bacteria in the reaction mixture, compared to control CPU per ml at time 0. Typically, bacteria incubated with the negative control antibody in the presence of complement showed a 150 to 200% increase in CPU/ml during the 60 min. of incubation.
Results were as follows:
















Strain
NadA expression
Allele
Bactericidal titre





















2996
+++
3
32768



C11
+++
3
16384



F6124
+
3
4096



MC58
+
1
8192



BZ232
−
−
<4



NGH38
−
−
<4










As shown, the serum induced complement-mediated killing of all strains that have the nadA gene, and was inactive against the strains that do not have the gene. However, bactericidal titres varied between strains. Titres were higher against strains expressing higher amounts of protein. This result was confirmed when titres were determined in the early and late phase of growth (FIG. 15).
To check whether the differences in the bactericidal activity were due to different allele sequences, immune sera, raised, against the three NadA types, were produced and used in a cross bactericidal assay. The results obtained with the antisera were similar to those shown above, suggesting that the bactericidal activity is not influenced by the allele diversity but rather to the antigen expression level.
The ability of immune sera to protect animals from bacteremia during infection was also tested, using the infant rat model. The sera used were obtained by immunising guinea pigs with 50 μg purified rNadA (allele 3). Immunisation of outbred Wistar rats (5 to 7 days old) was performed subcutaneously together CFA for the first dose and IFA for the further three doses (days 28, 56, 84). Bleed out samples were taken on day 105 and used for the animal protection assay.
Two experiments were performed using two different MenB strains (8047 and 2996). Each strain has been serially passaged three times in infant rats. In experiment 1, groups of four rats were challenged intraperitoneally with 100 μl of a mix of (a) bacteria from strain 8047 (7×103 CPU per rat) and (b) heat inactivated guinea pig antiserum or anti-capsule control mAb (SEAM 3 [Van Der Ley et al. (1992) Infect. Immun. 60:3156]). In experiment 2, group of six rats were treated with the control mAb or with different dilutions of guinea pig antiserum at time 0. Two hours later, they were challenged with the 2996 bacteria (5.6×103 CPU per rat). In both experiments, blood cultures were obtained 18 h after the challenge by puncturing the heart with a syringe and needle containing approximately 25 U of heparin without preservative. Bacteria numbers in the blood cultures were obtained by plating out 1, 10, and 100 μl of blood onto chocolate agar overnight. For calculation of geometric mean CFU/ml, animals with sterile cultures were assigned a value of 1 CPU/ml.
Results were as follows:













Blood culture at 18 hours













CFU/ml


Exp1
Treatment
Positive/Total
(103)













1
Anti-capsular mAb (2 μg/rat)
0/4
<0.001



Anti-NadA antiserum (1:5 dilution)
0/4
<0.001



PBS + 1% BSA
5/5
40.17


2
Anti-capsular mAb (20 μg/rat)
1/6
0.003



Anti-NadA antiserum (1:5 dilution)
1/6
0.002



Anti-NadA antiserum (1:25 dilution)
3/6
0.035



Pre-immune NadA serum
6/6
1.683









Thus anti-NadA antiserum is highly protective in this assay.
Overall, therefore, NadA has several attributes of being a good vaccine antigen: (i) it is a surface-exposed molecule, potentially involved in bacterial adhesion, (ii) it is present in at least 50% of the disease-associated strains and in almost 100% of three hypervirulent lineages; (iii) it elicits protective and bactericidal antibodies in laboratory ordinals; and (iv) each allele induces cross-bactericidal antibodies.
ORF40
ORF40 shows homology to Hsf and its allelic variant Hia, both adhesins of Haemophilus influenzae. The different size among Hia, Hsf and ORF40 is in part explained by the presence of three copies of a large repeated domain in Hsf, which is present in single copy in His and only partially in ORF40 (FIG. 7). In MenB, ORF40 is found on the outer membrane as a protein of about 200 kDa (cf. predicted MW of 59 kDa for mature protein).
App
App shows homology (FIG. 8) to the adhesion and penetration protein Hap of H. influenzae, which is an adhesin with a serine-protease activity that undergoes autoproteolytic cleavage and extracellular release [Hendrixson et al. (1997) Mol Microbiol 26:505-518]. Uncleaved surface-associated Hap mediates adherence to epithelial cells and promotes bacterial aggregation and colonisation.
In N. meningitidis, App is exported to the outer membrane, processed and secreted. Both Hap and App belong to the autotransporter family which comprises proteins from gram-negative bacteria characterized by a distinct mechanism of secretion. This system was first described for IgA1 protease of N. gonorrhoeae, which is considered the prototype of this family. Proteins of the autotransporter family have been implicated in the virulence of many gram-negative pathogens [Henderson & Nataro (2001) Infect Immun 69:1231-1243]. They are synthesized as large precursor proteins comprising at least three functional domains: a typical N-terminal leader sequence, an internal domain (passenger domain) and a C-terminal domain (translocator domain or β-domain). The leader sequence mediates the export (sec-dependent) of the protein to the periplasm. Subsequently the translocator domain inserts into the outer membrane forming a β-barrel pore to allow the export of the passenger domain. Once at the bacterial surface, the passenger domain can be cleaved and released into the environment. Cleavage can occur by an autoproteolytic event directed by protease activity in the passenger domain itself. Passenger domains of autotransporters are widely divergent, reflecting their remarkably disparate roles. On the contrary the β-domains display high degree of conservation consistent with their conserved function.
App possesses the prevailing domains of the autotransporter proteins as well as the conserved serine protease motif (GDSGSP). It has been shown that this motif is responsible for cleavage of human IgA by the Neisseria IgA1 proteases and for autoproteolytic cleavage of Hap protein of H. influenzae, App has been shown to be a conserved antigen among meningococci, to be expressed during infection and carriage, to stimulate B cells and T cells, and to induces a bactericidal antibody response [Hadi et al. (2001) Mol. Microbiol. 41:611-623; Van Ulsen et al. (2001) FEMS Immunol Med Microbiol 324.53-64].
In serogroup B strain 2996, App has 1454 amino acids and a predicted MW of 19,965 Da. FIG. 26 shows the protein's predicted structural features. Three domains can be seen: domain 1 (amino acids 1-42) is the signal peptide; domain 2 is the passenger domain, which is the functionally active protein; domain 3 is the C-terminal translocator domain with β barrel structure.
At the N-terminus of the passenger domain, His-115, Asp-158 and Ser-267 correspond to the serine protease catalytic triad His-98, Asp-140 and Ser-243 from Hap [Fink et al. (2001) J Biol Chem 276:39492-39500]. Residues 285-302 are a putative ATP/GTP-binding site (P loop), which suggests a mechanism of energy coupling for outer membrane translocation. Towards the C-terminus of the passenger domain, two Arg-rich regions are present. The first (RRSRR) is residues 934-938 and the second (RRARR) begins at residue 1149. These motifs are reminiscent of known targets for trypsin-like proteolytic cleavage sites such as the one in diphtheria toxin and those upstream of the auto-cleavage sites of H. influenzae Hap, N. gonorrhoeae IgA-protease and B. pertussis FhaB (FIG. 26, box 1). Downstream of the Arg-rich regions are motifs 934NTL956 and 1176NSG1178, which are identical or similar to the cleavage sites in autotransporters Sap (Serratia marcescens), Pni (Bordetella brinchiseptica), Brka (Bordetella pertussis) [Jose et al., (1995) Mol. Microbiol. 380] and Hap (H. influenzae) (FIG. 26, box 2). Together, these sequence motifs suggest that the two motifs 954NTL956 and 1176NSG1178 and the RR(A,S,R)2RR pattern could act as signals for correct localisation of downstream processing sites.
Further analysis of the App sequence shows a proline-rich region, where the dipeptide motif PQ is repeated four times beginning at residue 1156. A search for homology to known protein sequences reveals some similarity to the surface proteins of S. pneumonie PspA and PspC and to a proline-rich region of the B. pertussis outer membrane protein p69 pertactin, where the (PQP)5 motif is located in a loop containing the major immunoprotective epitope.
Finally, the last three amino acids of App (YRW) are identical to those of Hap where they have been described as crucial for outer membrane localisation and protein stability [Hendrixson et al., 1997].
Expression in E. coli without Fusion Partners
ORF40, App and NadA full-length genes were cloned in pET21b+ vector and the plasmids were transformed in E. coli BL21(DE3) in order to express the genes under control of T7 promoter.
Expression was achieved activating the promoter with IPTG or under non-induced conditions. Localisation and surface-exposure of the proteins were assayed by cell-fractionation experiments (SDS-PAGE and Western blot), FACS analysis and whole-cell immunoblot. As shown in FIGS. 1 to 3, all the three proteins are translocated to the surface of E. coli: 

    
    
        ORF40 is expressed as monomeric form and possibly forms also multimers (FIG. 1).
        App is exported to E. coli outer membrane as a precursor of about 160 kDa, that is processed and secreted in the culture supernatant (FIG. 2).
        NadA is found to the be present in the outer membrane fraction as a single high molecular weight band of approximately 180 kDa. This probably corresponds to an oligomeric form of the protein. Such a band is absent in E. coli expressing intracellular NadA (FIG. 3).
    
    


App expression was studied in more detail.
N. meningitidis strain 2996 genomic DNA was prepared as previously described [Tinsley & Nassif (1996) PNAS USA 93:11109-11114]. DNA devoid of the sequence coding for the signal peptide (amino acids 1 to 42) and of the STOP codon was amplified using PCR primers SEQ IDs 18 & 19 followed by digestion with NheI and XhoI and insertion into the NheI/XhoI sites of the pET-21b expression vector, to give ‘pET-App-His’ (FIG. 27). This plasmid was introduced into E. coli BL21(DE3) and used for the expression of C-terminal His-tagged fusion protein which was purified and used to raise antibodies. The full-length app gene was amplified and cloned in a similar way, using PCR primers SEQ IDs 20 & 21, to give plasmid ‘pET-App’.
Plasmids were introduced into E. coli BL21(DE3) and expression induced by addition of 1 nM IPTG. The expressed protein was detected by western blotting (FIG. 28, lane 1). To verify that the protein was exported to the E. coli surface, FACS (FIG. 29) and immunofluorescence microscopy (FIG. 30) were used. The FACS analysis showed positive surface expression on the pET-App transformants (full-length gene) but no surface expression with App-His (no signal peptide) or with the empty vector. The immunofluorescence results agreed with FACS. Therefore expression of the full-length app gene resulted in the export of App to the surface of E. coli, but deletion of the first 42 amino acids abolished surface-localisation.
Western blot analysis of outer membrane proteins from pET-App transformants revealed a specific reactive band of  ̃0.160 kDa (FIG. 28, lane 1), corresponding to the predicted molecular weight of the full-length protein. A corresponding band was missing in the outer membrane fraction from transformed controls (lane 3). Western blot analysis of culture supernatants revealed a secreted protein of  ̃100 kDa with pET-App (lane 2) that was absent with the untransformed controls (lane 4). Sometimes a very weak band was also detected at  ̃140 kDa in pET-App transformants.
Therefore the full length app gene when introduced into E. coli induces expression of an App protein which is exported to the outer membrane, cleaved and released into the culture supernatant.
Native Expression can Influence the Quality of the Immune Response
To evaluate the role of protein conformation on induction of an immune response, outer membrane vesicles from E. coli expressing ORF40, App or NadA were isolated and used to immunise mice. Sera were tested for bactericidal activity and results compared with those obtained with the fusion proteins. The bactericidal response (strain 2996) was improved 5-10 fold when the proteins are produced in their “native” form in OMVs:














Bactericidal titres*












Antigen
Fusion protein
E. coli OMV















ORF40
256
2048



App
64
1024



NadA
32768
>65536







*Titres expressed as the reciprocal of the serum dilution yielding-50% bacteria killing






App Autoproteolytic Cleavage

E. colipET-App transformants secrete a 100 kDa product into culture supernatant and show a 160 kDa surface product. To test whether the secreted App product derives from an autoproteolytic process, one of the putative catalytic residues (Ser-267) was replaced with Ala.
The pET-AppS267A mutant was obtained by site-directed mutagenesis using the QuikChange kit (Stratagem) and primers SEQ IDs 22 & 23.
SDS-PAGE analysis of total proteins from pET-AppS267A transformants (FIG. 31) lane 2) showed protein similar in size to pET-App transformants (lane 1). The protein was shown to be surface exposed by FACS analysis (FIG. 29). Western blot analysis of culture supernatants showed App in pET-App transformants (FIG. 32, lane 1) but not in pET-AppS267A transformants (lane 2).
Mutation of Ser-267 to Ala thus abolishes processing and secretion of the App precursor, which remains cell-associated. These data suggest that App has a maim protease activity that is responsible for autoproteolytic preening and release in the supernatant of the secreted App domain.
Cleavage at 954NTL956 would leave a fragment with predicted molecular weight of 104190 Da. Cleavage at 1176NSG1378 would give a 128798 Da fragment. These two predicted fragments may match the two bands of  ̃140 and  ̃100 kDa observed in culture supernatants. Cleavage may occur first to give the  ̃140 kDa fragment and then second to give the 100 kDa fragment. The β domain of App would thus begin at residue 1177.
NadA, ORF40 and App Function as Adhesins
Example 22 of international patent application WO01/64922 discloses that NadA expression in E. coli makes the transformed bacterium adhere to human epithelial cells. The adherent phenotype has been further studied for NadA and also for App and ORF40.
E. coli BL21(DE3) bacteria (108 CFU), grown under non-induced or induced conditions, were inoculated onto Chang human epithelial monolayers (105 cells) and incubated at 37° C. for 1 or 2 hours. Cells were then incubated with rabbit anti-E. coli and PE-conjugate secondary antibody. Adhesion was detected by FACS as specific fluorescence intensity associated to Chang cells. Positive controls were E. coli DH5 expressing hsf (DH5/pDC601)); negative controls were BL21(DE3)/pET21b and DH5a/pT7-7. The results in FIG. 4 show that the ability of the recombinant E. coli strains to adhere to cultured epithelial cells is associated with expression of these three proteins.
To confirm that these three proteins are able to promote interaction with host cells, the recombinant proteins themselves were investigated for binding to epithelial cells. 105 Chang human epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, human conjunctiva) were incubated at 4° C. for 30 minutes with medium alone or with different concentration of ORF40 (150 μg/ml), App (150 μg/ml) or NadA (300 μg/ml), or with GNA2132 (300 μg/ml) as negative control [see Pizza (2000)]. Binding was detected by FACS using polyclonal antisera against the single recombinant proteins and a secondary PE-conjugate antibody. The FACS signal shifts (FIG. 5) show that the three proteins are able to bind to human epithelial cells, whereas purified GNA2132 (negative control) does not.
FIG. 6A shows that binding increases in a dose-dependent manner. Binding of NadA reaches a plateau at around 200 μg/ml. GNA2132 fails to bind even at 400 μg/ml (FIG. 6B). Data in FIG. 6 are mean fluorescent intensity (MFI) values plotted against protein concentration (μg/ml).
Using FACS, binding of NadA to cells was also seen with Hep-2 and MOLT-4 cells, but not with HeLa, A549, Hec-1B, Hep-G2, CHO or HUVBC cells. Adhesion to Chang cells could be abolished by treating the cells with pronase, indicating that the human receptor for NadA is a protein.
Adhesion of purified NadA protein to Chang conjunctiva cells was also observed using immunofluorescence microscopy. The protein (lacking its C-terminal anchor domain) was incubated with Chang cells at 37° C. in complete culture medium for 3 hours at various concentrations. Cells were then washed, fixed, and analysed by laser confocal microscopy after staining with anti-NadA mouse polyclonal antibodies and secondary Texas-red coupled anti-Mouse IgG antibodies. No binding was seen at 0 nM (FIG. 1A), but binding was evident at 170 nM (17B) and 280nM (17C), with clustering evident at higher concentrations. In contest, no binding of NadA was seen with HeLa cells, even at 280nM protein (17D).
Binding was much more evident at 37° C. (FIG. 18A) than at 4° C. (FIG. 18B). The dot-like structures seen at 4° C., compared to clusters at 37° C., suggest that lateral interactions between NadA monomers are temperature-dependent (influenced by membrane fluidity).
To distinguish surface and endocytosed protein, saponin detergent was added during the staining procedure. Intracellular clusters having the size of endosomes were more evident (arrow) when saponin was used, but a high proportion of protein remained on the cell surface (FIG. 19).
Immunofluorescence also revealed that NadA binds to monocytes (FIG. 20A). NadA alone (no staining antibody; 20B) and NadA stained with pre-immune serum (20C) were not visible. At high magnification, evidence of uptake into vesicles (either endosomes or phagosomes) was seen.
FIG. 21 shows that murine macrophages (raw 264.1) bind and endocytose NadA (125nM, 3 hours, 37° C.; cells cultured in DMEM).
Beating NadA at 95° C. for 15 minutes prior to incubation removed its ability to bind to monocytes as measured by secretion of IL-α by the cells (FIG. 22). The stimulatory activity of NadA preparations is thus heat-labile. Stimulatory activity was also blocked by the use of anti-CD14 (FIG. 23) or by the removal of NadA from the preparations using bead-immobilised anti-NadA.
Immunofluorescence microscopy was also used to detect binding of E. coli expressing NadA. Transformed E. coli bound strongly (FIG. 24A) whereas untransformed bacteria did not (24B). IL-α release by monocytes was over 1.5× higher using the transformed E. coli than the untransformed bacteria at a bacteria/monocyte ratio of 40:1.
Transformed E. coli were bound to glass cover slips, fixed and double-stained with anti-NadA (FIG. 25A) and anti-E. coli antibodies (25B). When both were used patches of anti-NadA were visible, suggesting that NadA tends to form aggregates on the bacterial surface, which hamper the interaction of antibodies with other surface antigens.
Looking at App, recombinant E. coli strains were incubated with monolayers of Chang conjunctiva epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4 [human conjunctiva], ATCC CCL 20.2) and adhesion was analysed using FACS. Cells obtained from confluent monolayers were seeded at 105 cells per well in 12-well tissue culture plates and incubated for 24 hours. Cultures of bacteria after IPTG induction were washed twice in PBS and resuspended in DMEM+1% PBS to a concentration of 5×108 bacteria per ml. Aliquots of 1 ml of each strain were added to monolayer cultures of Chang cells and incubated for 3 hours at 37° C. in 5% CO2. Non-adherent bacteria were removed by washing three times with PBS, and 300 μl of cell dissociation solution (Sigma) were added to each microtitre well. Incubation was continued at 37° C. for 10 minutes. Cells were harvested and then incubated for 1 hour at 4° C. with rabbit polyclonal anti-E. coli antiserum (DAKO). Cells were washed twice in PBS+5% FBS and incubated for 30 minutes at 4° C. with R-Phycoerythrin-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Cells were then washed in PBS+5% PBS and resuspended in 100 μl PBS. Fluorescence was, measured With FACSCalibur flow cytometer (Becton Dickinson). For each of fluorescence profile, 10000 cells were analysed.
The results reported in FIG. 33 show pET-App transformants were able to adhere to Chang cells, giving a fluorescence shift of 90.3%. S267A transformants were also able to adhere (91.0%). Untransformed E. coli were unable to adhere to Chang cells (bottom FACS plot).
As for NadA, FACS results were in agreement with immunofluorescence microscopy data. As shown in FIGS. 34A & 34B, pET-App transformants incubated with monolayers demonstrated high levels of adhesion to epithelial cells and visible bacteria-bacteria aggregation. For the S267A mutant, adhesion and bacterial aggregation were increased (34C & 34D). Untransformed controls showed no adhesion (34G). Deletion of the first 42 amino acids also abolished adhesion.
In contrast to Chang epithelial cells, no adhesion was seen when HUVBC endothelial cells were tested with pET-App transformants. To cause sepsis and meningitis, N. meningitidis has to interact with human endothelial cells. App may thus be involved in the first step of colonisation at the level of human respiratory epithelial mucosa, rather than in pathological endothelial colonisation.
Localization and Specificity of App Binding Activity.
To identify the binding region of App, a chimeric protein named Appβ was used. This protein consists of the C-terminal domain of App (amino acids 1077 to 1454) fused to the leader peptide of IgA1 protease of N. gonorrhoeae. The gonococcal leader sequence was chosen because it has been well characterized and is functional in E. coli. Plasmid pET-Appβ contains a 1.1 kbp DNA fragment amplified by PCR using SEQ IDs 26 & 27.
The pET-Appβ construct was introduced into E. coli BL21(DE3). FACS localisation studies confirmed that Appβ was localized on the E. coli surface. The in vitro adhesion assay using Chang epithelial cells showed adhesion by immunofluorescence (FIGS. 34E & 34F). FACS analysis showed that the pET-Appβ transformants were still able to adhere to epithelial cells but at lower levels (74.2% shift) than pET-App transformants.
These results indicate that the App binding domain is located in its C-terminal region, in the 100mer fragment between residues 1077 and 1176.
Purified recombinant proteins were also studied. App-α-His consists of the N-terminal portion of App (amino acids 43-1084) fused to a poly-His tag Plasmid pET-Appα-His contains a NheI/XhoI 3.1 kbp fragment amplified by PCR with SEQ IDs 24 & 25. The binding activity of the purified recombinant App-α-His was compared to that of App-His by FACS binding assays. Chang cells were incubated with increased concentrations of recombinant App proteins or lipoprotein NMB12132-His (negative control). Binding of App-His (♦) increased in a close-dependent manner and reached a plateau at a concentration of  ̃50 μg/ml whereas the binding of Appα-his (▪) was very low (FIG. 35). The control NMB32132-His (▴) failed to bind Chang cells.
To explore the biochemical nature of the molecule involved in interaction with App, the Chang cells were treated with pronase or phospholipase A2 before the binding experiments. 105 cells per well were placed in microplates and incubated in FCS-free DMEM at 37° C. in 5% CO2 for 30 minutes with (a) pronase at 250, 500, or 1000 μg/ml or (b) phospholipase A2 at 50,200, or 800 μg/ml. After enzymatic incubation, an equal volume of complete medium was added to each well to stop the reaction. Cells were subsequently mixed with 100 μg/ml App-His or medium alone and incubated for 1 hours at 4° C. As shown in FIG. 36, pronase treatment (left-hand columns) markedly reduced the binding of App-His protein to Chang cells, while treatment with phospholipase A2 (right-hand columns) did not reduce the binding. The receptor for App on Chang cells is thus proteinaceous.
Adhesion to different cell lines were also tested (FIG. 37). After incubation of cultured cells with three different concentrations of App-His (100, 25 & 6.25 μg/ml) high level binding to Chang cells and HepG2 cells was seen, a moderate level of binding to A-549 cells, and minimal binding to HeLa cells. No binding was observed to Hec-1-B, Hep-2, 16HBE14o epithelial cell lines or to HUVBC endothelial cells.
App Knockout
After the work on E. coli suggesting an adhesin role for App, isogenic mutant strain of N. meningitidis was constructed. The starling strain was MC58. Its app gene was truncated and replaced with an antibiotic cassette by transforming the parent strain with the plasmid pBSUDAppERM, which contains a truncated app gene and the ermC gene (erythromycin resistance) for allelic exchange. Briefly, 600 bp of the upstream flailing region including the start codon and 760 bp downstream flanking region including the stop codon were amplified from MC58 using primers SEQ IDs 28 to 31. Fragments were cloned into pBluescript and transformed into E. coli DH5 rising standard techniques. Once all subcloning was complete, naturally competent N. meningitidis strain. MC58 was transformed by selecting a few colonies lawn overnight on GC agar plates, and mixing them with 20 μl of 10 mM TrisHCl pH8.5 containing 1 μg of plasmid DNA. The mixture was spotted onto a GC agar plate, incubated for 6 hrs at 37° C., 5% CO2 then diluted in PBS and spread on GC agar plates containing 5 μg/ml erythromycin. The deletion app gene in the genome of MC58 was confirmed by PCR. Lack of App expression was confirmed by Western blot analysis.
Adhesion of wildtype MC58 and the isogenic MC58Δapp mutant strain was evaluated on Chang cells. There was a  ̃10 fold reduction (ranging from 3- to 27-fold in different experiments) of the association of the knockout mutant compared with the wild type strain (FIG. 38). No difference was observed between the app mutant and the parental strain with Hep2 and 16HBE14o cell lines and with HUVEC endothelial cells, confirming that App does not mediate adhesion to these cells.
No non-pilus adhesins which contribute to adhesion of N. meningitidis in a capsulated background have previously been reported.
App expression was studied in N. meningitidis MC58. Colonies from plates grown overnight were diluted in GC broth and incubated at 37° C. with 5% CO2. Samples were taken when OD620nm=0.5 (mid log phase) and 0.8 (stationary phase) and analysed by western blot. Two bands with apparent molecular weights  ̃160 and  ̃140 kDa were detected in whole cells lysates of log phase bacteria (FIG. 39, lane 1), while stationary phase bacteria showed only a faint band at  ̃140 kDa (lane 3). As expected, no App was observed in the ΔApp mutant (lanes 2 & 4).
In marked contrast, supernatant samples of wild-type MC58 showed a band at  ̃140 kDa and its amount was higher in stationary phase than in log phase (FIG. 40, lanes 3 & 1). The stationary phase sample also showed a reactive band at  ̃100 kDa.
It will be understood that the invention is described above by way of example only modifications may be made whilst remaining within the scope and spirit of the invention.







TABLE I







Characteristics of 26 N. meningitidis strains and their nadA gene allele













Serogroup

nadA
(TAAA)
NadA


Strain
type:subtype
Clonal group
allele
repeats
expression















64/69
NG:15:P1.7,16
ET-5
1
4
+


BZ83
B:15
ET-5
1
5
+++


CU385
B:4:P1.15
ET-5
1
6
++


MC58
B:15:P1.7,16b
ET-5
1
9
+


BZ169
B:15:P1.16
ET-5
1
12
++


95330*
B:4:P1.15
ET-5
1
9
nd


ISS1104
B:15:P1.7,16
nd
1
4
+


ISS1071
B:15:P1.7,16
nd
1
5
+++


ISS832
B:15:P1.7
nd
1
5
++


NM119
B,4.P1.15
nd
1
6
nd


NM066
B:15:P1.7,16
nd
1
12
nd


90/18311
C:NT:P1.5
ET-37
2
9
++


NGP165
B:NT:P1.2
ET-37
2
9
++


FAM18
C:2a:P1.5,2
ET-37
2
9
nd


M986
B:2a:P1.5,2
ET-37
2
12
++


ISS1024*
C:2b:P1.5
nd
2
9
++


ISS838
C:2a:P1.5,2
nd
2
6
++


PMC8
C:
nd
2
10
++++


961-5945
B:2b:P1.21,16
A4
2
12
+++


ISS759*
C:2b:P1.2
nd
3
8
++++


F6124
A
Subgroup III
3
9
+


NMB
B:2b:P1.5,2
nd
3
12
++


8047
B:2b:P1.2
nd
3
12
+++


2996
B:2b:P1.5-1,2
nd
3
12
+++


C11
C:NT:P1.1
nd
3
12
+++


973-1720*
C:2b:P1.2
A4
3
12
+++





*indicates that the strain carriers a minor variant of the relevant allele


nd = not done













TABLE II







Characteristics of N. meningitidis strains analysed for NadA expression





















NadA 


ST
ET
Strain
Year
Serogroup:type:subtype
Country
Disease
gene

















 74
ET5
MC58
1985
B:15:P1.7,16b
UK
case
+


 32
ET5
H44/76
1976
B:15:P1.7,16
Norway
case
+


 32
ET5
BZ169
1985
B:15:P1.16
Netherlands
case
+


 32
ET5
30/00
2000
B:15:P1.7,16
Norway
case
+


 33
ET5
N44/89
1989
B:4,7:P1.19,15
Brazil
case
+


 34
ET5
BZ83
1984
B:15
Netherlands
case
+


—
ET5
72/00
2000
B:15:P1.7,13
Norway
case
+


—
ET5
39/99
1999
C:15:P1.7,16
Norway
case
+


—
ET5
M4102
1996
B:ND
USA
case
+


—
ET5
95330
1995
B:4:P1.15
Canada
case
+


—
ET5
2201731
1993
NG:4:P1.15
Iceland
carrier
+


—
ET5
64/96
1996
NG:15:P1.7,16
Norway
carrier
+


—
ET5
CU385
1980
B:4:P1.15
Cuba
case
+


—
ET5
8680
1987
B
Chile
case
+


—
ET5
204/92
1992
B
Cuba
case
+


—
ET5
EG329
1985
B
Germany
case
+


—
ET5
NG080
1981
B
Norway
case
+


—
ET5
NG144/82
1982
B
Norway
case
+


—
ET5
NG PB24
1985
B
Norway
case
+


—
ET5
196/87
1987
C
Norway
case
+


—
ET5
Mk521/99
1999
B
Ivory Coast
case
+


—
ET5
GR 4/00
2000
—
Greece
case
+


 11
ET37
FAM18
1983
C:2a:P1.5,2
USA
case
+


 11
ET37
L93/4286
1993
C
UK
case
+


—
ET37
NGP165
1974
B:NT:P1.2
Norway
—
+


—
ET37
M986
1963
B:2a:P1.5,2
USA
case
+


—
ET37
C4678
1998
C:2a:P1.5,2
Germany
case
+


—
ET37
95N477
1995
B:2a:P1.2
Australia
case
−


—
ET37
BRAZ10
1976
C
Brazil
case
+


—
ET37
F1576
1984
C
Ghana
case
+


—
ET37
M597
1988
C
Israel
case
+


—
ET37
500
1984
C
Italy
case
+


—
ET37
D1
1989
C
Mali
case
+


—
ET37
NG P20
1969
B
Norway
case
+


—
ET37
MA-5756
1985
C
Spain
case
+


—
ET37
38VI
1964
B
USA
carrier
+


—
ET37
N1/99
1999
C:2a
Norway
case
+


—
ET37
N28/00
2000
W-135:2a
Norway
case
+


 66
A4
973-1720
1997
C:2b:P1.2
Australia
case
+


 153
A4
961-5945
1996
B:2b:P1.21,16
Australia
case
+


—
A4
5/99
1999
B:2b:P1.5,2
Norway
case
+


—
A4
312294
1995
C:2b:P1.5,2
UK
case
+


—
A4 
96217
1996
B:2b:P1.5,10
Canada
case
+


—
A4
G2136
1986
B
UK
case
+


—
A4
312 901
1996
C
UK
case
+


—
A4
AK22
1992
B
Greece
case
+


—
A4
BZ10
1967
B
Holland
case
+


—
A4
BZ163
1979
B
Holland
case
+


—
A4
B6116/77
1977
B
Iceland
case
+


—
A4
94/155
1994
C
New Zealand
case
+


—
A4
SB25
1990
C
South Africa
case
+


—
A4
N53/00
2000
C:2b:P1.5,2
Norway
case
+


—
A4
N62/00
2000
C:2b:P1.5,2
Norway
case
+


 41
Lin.III
BZ198
1986
B:NT
Netherlands
case
−


 42
Lin.III
M198/254
1998
B:4:P1.4
New Zealand
case
−


 158
Lin.III
972-0319
1997 
B:NT:P1.4
Australia
case
−


 159
Lin.III
980-2543
1998
B:NT:P1.4
Australia
case
−


1127
Lin.III
67/00
2000
B:4,7
Norway
case
−


—
Lin.III
93/114
1993
C:4:P1.4
Belgium
case
−


—
Lin.III
M198/172 
1998
B:4:P1.4
New Zealand
case
−


—
Lin.III
347/97
1997
B:4:P1.4
New Zealand
case
−


—
Lin.III
386/98
1998
B:4:P1.4
New Zealand
case
−


—
Lin.III
389/98
1998
B:4:P1.4
New Zealand
case
−


—
Lin.III
392/98
1998
B:4:P1.4
New Zealand
case
−


—
Lin.III
394/98 
1998
B:4:P1.4
New Zealand
case
−


—
Lin.III
400
1991
B
Austria
case
−


—
Lin.III
M40/94
1994
B
Chile
case
−


—
Lin.III
AK50
1992
B
Greece
case
−


—
Lin.III
M-101/93
1993
B
Iceland
case
−


—
Lin.III
931905
1993
B
Netherlands
case
−


—
Lin.III
91/40
1991
B
New Zealand
case
−


—
Lin.III
50/94
1994
B
Norway
case
−


—
Lin.III
N45/96
1996
B
Norway
case
−


—
Lin.III
88/03415
1988
B
Scotland
case
−


  1
s I
BZ133
1977
B:NT
Netherlands
case
−


  5
s III
F6124
1988
A
Chad
case
+


  4
s IV-1
205900
1990
A4,21:P1.7:1
Mali
case
−


  4
s IV-1
Z2491
1983
A
Gambia
case
−


 12
other
NG3/88
1988
B:8(2):P1.1
Norway
case
−


 13
other
NG6/88
1988
B:NT:P1.1
Norway
case
−


 14
other
NGF26
1988
B:NT:P1.16
Norway
carrier
−


 15
other
NGE31
1988
B:NT
Norway
carrier
−


 18
other
528
1989
B:nd
Russia
case
−


 20
other
1000
1988
B:NT:P1.5
Russia
case
−


 22
other
A22
1986
W-135
Norway
carrier
−


 26
other
NGE28
1988
B:4
Norway
carrier
+


 29
other
860800
1986
Y
Netherlands
case
−


 31
other
E32
1988
Z
Norway
carrier
−


 35
other
SWZ107
1986
B:4:P1.2
Switzerland
case
−


 36
other
NGH38
1988
B:NT:P1.3
Norway
carrier
−


 38
other
BZ232
1964
B:NT:P.2
Netherlands
case
−


 39
other
E26
1988
X
Norway
carrier
−


 43
other
NGH15
1988
B:8:P1.15
Norway
carrier
−


 47
other
NGH36
1988
B:8:P1.2
Norway
carrier
−


 48
other
BZ147
1963
B:NT
Netherlands
case
−


 49
other
297-0
1987
B:4:P1.15
Chile
carrier
−


 540
other
2996
1975
B:2b:P1.5-1,2
UK
case
+


1034
other
96/1101
1996
C:14:P1.1,7
Belgium
case
−


—
other
15
1990
B:14,19:P1.9,15
Slovenia
case
−


—
other
M1090
1996
B:4
Israel
case
−


—
other
M1096
1996
C:NT:P1.5
Israel
case
−


—
other
B3937
1995 
B.22P1.16
Germany
case
+


—
other
31
1993
B:4
Finland
case
−


—
other
95074
1995
B:NT:P1.13
Canada
case
+


—
other
660/94
1994
B:4:P1.6
Algeria
case
−


—
other
30/93
1993
B:14:P1.14
Argentina
case
−


—
other
24370
1996
B:ND
South Africa
case
−


—
other
241175I
1993
NG:21:P1.16
Iceland
carrier
−


—
other
171274I
1993
NQ:15:-
Iceland
carrier
−


—
other
65/96
1996
B:4:P1.14
Norway
carrier
+


—
other
66/96
1996
B:17:P1.15
Norway
carrier
−


—
other
149/96
1996
B:1,19:P1.5,2
Belgium
carrier
+


—
other
16060
1991
B:4:P1.14
Belgium
carrier
−


—
other
16489
1991
NG21:P.1.1
Norway
carrier
−


—
other
16990
1991
NG:14:P1.5,2,6
Norway
carrier
−


—
other
2022
1991
NG:4:P1.10
Norway
carrier
+


—
other
M136
1968
B:11:P1.15
USA
case
−


—
other
860060
1988
X
Holland
case
−


—
other
NG H41
1986
B
Norway
carrier
−


—
other
NG G40
1988
B
Norway
carrier
−


—
other
NG4/88
1988
B
Norway
case
−


—
other
EG 327 
1985
B
DDR
case
−


—
other
EG 328
1985
B
DDR
case
−


—
other
3906
1977
B
China
case
−


—
other
NG E30
1988
B
Norway
carrier
−


—
other
71/94
1994
Y
Norway
case
−


—
other
DK24
1940
B
Denmark
case
−


—
—
C11
1965
C:16:P1.7a,1
Germany
—
+


—
—
pmc8
—
C
—
—
+


—
—
NMB
1968
B:2b:P1.5,2
USA
case
+


—
—
8047
1978
B:2b:P1.2
USA
case
+


—
—
S3446
1972
B:14:P1.23,14
USA
case
−


—
—
ISS 749
1996
B.14:P1.13
Italy
case
−


—
—
ISS 759
1996
C:2b:P1.2
Italy
case
+


—
—
ISS 832
1997
B:15:P1.7
Italy
case
+


—
—
ISS 838
1997
C:2a:P1.5,2
Italy
case
+


—
—
ISS1001
1999
B:14:P1.13
Italy
case
−


—
—
ISS1024 
2000
C:2b:P1.5
Italy
case
+


—
—
ISS1026
2000
B:4:P1.13
Italy
case
−


—
—
ISS1071
2000 
B:15:P1.7,16
Italy
case
+


—
—
ISS1102 
2000
B:15:P1.4
Italy
case
−


—
—
ISS1104
2000
B:15:P1.7,16
Italy
case
+


—
—
ISS1106
2000
B:4:P1.4
Italy
case
−


—
—
ISS1113
2000
C:2a:P1.5
Italy
case
+


—
—
NI002/90
—
—
Brazil
—
+


—
—
IMC2135
—
—
Brazil
—
+


—
—
NM001
—
B:4:P1.4
UK
case
−


—
—
NM002
—
B:NT:P1.16
UK
case
−


—
—
NM004
—
B:NT:P1.14
UK
case
−


—
—
NM008
—
B:4:P1.4
UK
case
−


—
—
NM009/10
—
B:4:P1.3,6
UK
case
−


—
—
NM021
—
B:4:P1.16
UK
case
−


—
—
NM036
—
C:2a:P1.10
UK
case
+


—
—
NM037
—
B:2b:P1.10
UK
case
+


—
—
NM050
—
B:NT:P1.9
UK
case
−


—
—
NM058
—
B:NT:NST
UK
case
−


—
—
NM066
—
B:15:P1.7,16
UK
case
+


—
—
NM067
—
C:2a:NST
UK
case
+


—
—
NM069
—
B:15:P1.7,16
UK
case
+


—
—
NM081 
—
C:2a:P1.5,2
UK
case
+


—
—
NM088
—
C:2a:P1.5,2
UK
case
+


—
—
NM092
—
B:4:P1.4
UK
case
−


—
—
NM106
—
B:NT:P1.4
UK
case
−


—
—
NM107/8
—
B:4:P1.4
UK
case
−


—
—
NM117
—
B:21:P1.9
UK
case
−


—
—
NM119
—
B:4:P1.15
UK
case
+


—
—
NM131
—
B
UK
case
−


—
—
NM145
—
C
UK
case
+


—
—
NM154
—
C:NT:P1.5,2
UK
case
+


—
—
NM156
—
B:15:P1.16
UK
case
+


—
—
NM167
—
B
UK
case
−


—
—
NM184
—
B:NT:P1.5,2
UK
case
−


—
—
NM186
—
B
UK
case
−


—
—
NM188
—
B
UK
case
+


—
—
NM200
—
B:4:P1.4
UK
case
−
















TABLE III







SEQUENCE LISTING








SEQ ID NO:
Description











1
allele 1 of 961


2
allele 2 of 961


3
allele 3 of 961


4
allele 1 of 961 (first-ATG start)


5
allele 2 of 961 (first-ATG start)


6
allele 3 of 961 (first-ATG start)


7
variant allele 2 of 961 in strain ISS1024


8
variant allele 2 of 961 (first-ATG start) in strain ISS1024


9
variant allele 3 of 961 in strains 973-1720 and ISS759


10
variant allele 3 of 961 (first-ATG start) in strains



973-1720 and ISS759


11
961 allele 1/2 chimera (strain 95330)


12
961 allele 1/2 chimera (strain 95330) (first-ATG start)


13
961 allele C


14
961 allele C (first-ATG start)


15
coding sequence for SEQ ID 13


16-31
PCR primers


32
SEQ ID 650 from WO99/24578


33-39
Domain derivatives of SEQ ID 32

## Claims

1. An isolated protein comprising:
(a) an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2; or
(b) a fragment of 170 or more amino acids of SEQ ID NO: 2, wherein the isolated protein lacks the C-terminal membrane anchor corresponding to amino acids 310-362 of SEQ ID NO: 1.

2. The isolated protein of claim 1 which comprises the amino acid sequence of (a) and also lacks 1 or more N-terminal amino acid residues of SEQ ID NO: 2.

3. The isolated protein of claim 1 which comprises the fragment of (b) and also lacks 1 or more N-terminal amino acid residues of SEQ ID NO: 2.

4. The isolated protein of claim 1 which comprises the fragment of (b) and also lacks the N-terminal leader peptide anchor corresponding to amino acids 1-23 of SEQ ID NO: 1.

5. The isolated protein of claim 1 which includes the heptad sequence of SEQ ID NO: 2 (AA1AA2AA3AA4AA5AA6AA7)r wherein: AA1 is Leu, Ile, Val or Met; each of AA2, AA3, AA4, AA5, AA6 and AA7 may independently be any amino acid; and r is an integer of 1 or more.

6. An immunogenic composition comprising the protein of claim 1 and an adjuvant.

7. An isolated protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 lacking the C-terminal membrane anchor corresponding to amino acids 310-362 of SEQ ID NO: 1 and the N-terminal leader peptide corresponding to amino acids 1-23 of SEQ ID NO: 1.