Patent Publication Number: US-2019183999-A1

Title: Developments in meningococcal outer membrane vesicles

Description:
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE 
     The content of the following submission on ASCII text file is incorporated herein by reference in entirety: a computer readable form (CRF) of the Sequence Listing (file name: 303822003801SeqList.txt, date recorded: Dec. 20, 2015, size: 102 KB). 
     FIELD OF THE INVENTION 
     This invention is in the field of meningococcal vaccines based on membrane vesicles. 
     BACKGROUND ART 
     Various vaccines against serogroup B of  Neisseria meningitidis  (“MenB”) are currently being investigated. Some of these are based on outer membrane vehicles (OMVs), such as the Novartis MENZB™ product, the Finlay Institute VA-MENGOC-BC™ product, and the Norwegian Institute of Public Health MENBVAC™ product. Reference 1 discloses the construction of vesicles from strains modified to express six different PorA subtypes. References 2-4 report pre-clinical studies of an OMV vaccine in which fHbp (also known as GN1870) is over-expressed (and this over-expression can be combined with knockout of LpxL1 [5]). Reference 6 recently reported a clinical study of five formulations of an OMV vaccine in which PorA &amp; FrpB are knocked-out and Hsf &amp; TbpA are over-expressed. Reference 7 reports a native outer membrane vesicle vaccine prepared from bacteria having inactivated synX, lpxL1, and 1gtA genes. 
     It is an object of the invention to provide further and improved meningococcal OMVs, and also to provide further and improved meningococci for use in vaccine production. 
     DISCLOSURE OF THE INVENTION 
     A first aspect of the invention provides meningococcal outer membrane vesicles in which NHBA is over-expressed. A second aspect of the invention provides meningococcal outer membrane vesicles in which NadA is over-expressed. A third aspect of the invention provides a panel of bacterial strains, each member of which is isogenic except for a single gene which in each strain encodes a different variant of an antigen of interest. 
     Over-Expression 
     The first and second aspects of the invention provide meningococcal outer membrane vesicles in which certain antigens are over-expressed. In the first aspect, at least NHBA is over-expressed. In the second aspect, at least NadA is over-expressed. 
     As discussed below, these vesicles are obtained from bacteria which over-express the relevant antigen(s). The bacterium may express the antigen(s) already, but include a genetic modification which, compared to a bacterium without that modification, increases expression of the antigen. This modification will usually be introduced using recombinant techniques, such as site-directed mutagenesis or targeted homologous recombination, so vesicles of the invention are usually obtained from recombinant bacteria. Typically a bacterium will include (i) a gene under the control of a promoter with which it is not found in nature and/or (ii) a knockout of a gene which is found in the bacterium in nature. 
     As a result of the over-expression, outer membrane vesicles prepared from the modified meningococcus contain higher levels of the over-expressed antigen(s). The increase in expression in the OMVs is usefully at least 10%, measured in mass of the relevant antigen per unit mass of OMV, and is more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more. 
     Suitable recombinant modifications which can be used to cause over-expression of an antigen include, but are not limited to: (i) promoter replacement; (ii) gene addition; (iii) gene replacement; or (iv) repressor knockout. 
     In promoter replacement, the promoter which controls expression of the antigen&#39;s gene in a bacterium is replaced with a promoter which provides higher levels of expression. For instance, the gene might be placed under the control of a promoter from a housekeeping metabolic gene. In other embodiments, the antigen&#39;s gene is placed under the control of a constitutive or inducible promoter. Similarly, the gene can be modified to ensure that its expression is not subject to phase variation. Methods for reducing or eliminating phase variability of gene expression in meningococcus are disclosed in reference 8. These methods include promoter replacement, or the removal or replacement of a DNA motif which is responsible for a gene&#39;s phase variability. 
     In gene addition, a bacterium which already expresses the antigen receives a second copy of the relevant gene. This second copy can be integrated into the bacterial chromosome or can be on an episomal element such as a plasmid. The second copy can have a stronger promoter than the existing copy. The gene can be placed under the control of a constitutive or inducible promoter. The effect of the gene addition is to increase the amount of expressed antigen. Where a plasmid is used, it is ideally a plasmid with a high copy number e.g. above 10, or even above 100. 
     In gene replacement, gene addition occurs but is accompanied by deletion of the existing copy of the gene. For instance, this approach was used in reference 4, where a bacterium&#39;s endogenous chromosomal fHbp gene was deleted and replaced by a plasmid-encoded copy (see also reference 9). Expression from the replacement copy is higher than from the previous copy, thus leading to over-expression. 
     In repressor knockout, a protein which represses expression of an antigen of interest is knocked out. Thus the repression does not occur and the antigen of interest can be expressed at a higher level. 
     Promoters for over-expressed genes can advantageously include a CREN [10]. 
     A over-expressing modified strain will generally be isogenic with its parent strain, except for a genetic modification. As a result of the modification, expression of the antigen of interest in the modified strain is higher (under the same conditions) than in the parent strain. A typical modification will be to place a gene under the control of a promoter with which it is not found in nature and/or to knockout a gene which encodes a repressor. 
     In embodiments where NHBA is over-expressed, various approaches can be used. For convenience, the approach already reported in reference 11 can be used i.e. introduction of a NHBA gene under the control of an IPTG-inducible promoter. By this approach the level of expression of NHBA can be proportional to the concentration of IPTG added to a culture. The promoter may include a CREN. 
     In embodiments where NadA is over-expressed, various approaches can be used. One useful approach involves deletion of the gene encoding NadR (NMB1843), which is a transcriptional repressor protein [12] which down-regulates or represses the NadA-encoding gene in all strains tested. Knockout of NadR results in high-level constitutive expression of NadA. An alternative approach to achieve NadA over-expression is to add 4-hydroxyphenylacetic to the culture medium. A further approach is to introduce a NadA gene under the control of an IPTG-inducible promoter. 
     In some embodiments a bacterium over-expresses both NHBA and NadA. 
     In addition to over-expressing NHBA and/or NadA, a bacterium may over-express one or more further antigens. For instance, a bacterium may over-express one or more of: (a) NhhA; (b) TbpA; (c) HmbR; (d) TbpB; (e) NspA; (f) Cu,Zn-superoxide dismutase; (g) Omp85; (h) App; and/or (i) fHbp. Over-expression of NhhA is already reported in references 6 and 13. Over-expression of TbpA is already reported in references 6, 13 and 14. Over-expression of HmbR is already reported in reference 15. Over-expression of TbpB is already reported in reference 14. Over-expression of NspA is already reported in reference 16, in combination with porA and cps knockout. Over-expression of Cu,Zn-superoxide dismutase is already reported in reference 14. Over-expression of fHbp is already reported in references 2-4 &amp; 9, and by a different approach (expressing a constitutively-active mutant FNR) in references 17 &amp; 18. 
     In some embodiments a bacterium over-expresses NHBA, NadA and fHbp. These three antigens are components of the “universal vaccine” disclosed in reference 19 or “4CMenB” [20, 21]. In one embodiment, expression of NHBA is controlled by a strong promoter, NadR is knocked out, and the strain expresses a constitutively active mutant FNR. In another embodiment, expression of NHBA is controlled by a strong promoter, expression of fHbp is controlled by a strong promoter, and NadR is knocked out. The bacterium can also be a bacterium which does not express an active MltA (GNA33), such that it spontaneously releases vesicles which contain NHBA, NadA and fHbp. Ideally, the bacterium does not express a native LPS e.g. it has a mutant or knockout of LpxL1. 
     Vesicles 
     The first and second aspects of the invention provide meningococcal outer membrane vesicles. These outer membrane vesicles include any proteoliposomic vesicle obtained by disruption of or blebbling from a meningococcal outer membrane to form vesicles therefrom that retain antigens from the outer membrane. Thus the term includes, for instance, OMVs (sometimes referred to as ‘blebs’), microvesicles (MVs [22]) and ‘native OMVs’ (‘NOMVs’ [23]). 
     MVs and NOMVs are naturally-occurring membrane vesicles that form spontaneously during bacterial growth and are released into culture medium. MVs can be obtained by culturing  Neisseria  in broth culture medium, separating whole cells from the smaller MVs in the broth culture medium (e.g. by filtration or by low-speed centrifugation to pellet only the cells and not the smaller vesicles), and then collecting the MVs from the cell-depleted medium (e.g. by filtration, by differential precipitation or aggregation of MVs, by high-speed centrifugation to pellet the MVs). Strains for use in production of MVs can generally be selected on the basis of the amount of MVs produced in culture e.g. refs. 24 &amp; 25 describe  Neisseria  with high MV production. 
     OMVs are prepared artificially from bacteria, and may be prepared using detergent treatment (e.g. with deoxycholate), or by non-detergent means (e.g. see reference 26). Techniques for forming OMVs include treating bacteria with a bile acid salt detergent (e.g. salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., with sodium deoxycholate [27 &amp; 28] being preferred for treating  Neisseria ) at a pH sufficiently high not to precipitate the detergent [29]. Other techniques may be performed substantially in the absence of detergent [26] using techniques such as sonication, homogenisation, microfluidisation, cavitation, osmotic shock, grinding, French press, blending, etc. Methods using no or low detergent can retain useful antigens such as NspA [26]. Thus a method may use an OMV extraction buffer with about 0.5% deoxycholate or lower e.g. about 0.2%, about 0.1%, &lt;0.05% or zero. 
     A useful process for OMV preparation is described in reference 30 and involves ultrafiltration on crude OMVs, rather than instead of high speed centrifugation. The process may involve a step of ultracentrifugation after the ultrafiltration takes place. 
     Another useful process for outer membrane vesicle production is to inactivate the mltA gene in a meningococcus, as disclosed in reference 31. These mutant bacteria spontaneously release vesicles into their culture medium. 
     If lipo-oligosaccharide (LOS) is present in a vesicle it is possible to treat the vesicle so as to link its LOS and protein components (“intra-bleb” conjugation [43]). 
     The vesicles may lack LOS altogether, or they may lack hexa-acylated LOS e.g. LOS in the vesciles may have a reduced number of secondary acyl chains per LOS molecule [32]. For example, the vesicles may from a strain which has a lpxL1 deletion or mutation which results in production of a penta-acylated LOS [3, 7]. LOS in a strain may lack a lacto-N-neotetraose epitope e.g. it may be a 1st and/or 1gtB knockout strain [6]. LOS may lack at least one wild-type primary O-linked fatty acid [33]. LOS having. The LOS may have no α chain. The LOS may comprise GlcNAc-Hep 2 phosphoethanolamine-KDO 2 -Lipid A [34]. 
     The vesicles may include one, more than one, or (preferably) zero PorA serosubtypes. Modification of meningococcus to provide multi-PorA OMVs is known e.g. from references 1 and 35. Conversely, modification to remove PorA is also known e.g. from reference 6. 
     The vesicles may be free from one of both of PorA and FrpB. Preferred vesicles are PorA-free. 
     The invention may be used with mixtures of vesicles from different strains. For instance, reference 36 discloses vaccine comprising multivalent meningococcal vesicle compositions, comprising a first vesicle derived from a meningococcal strain with a serosubtype prevalent in a country of use, and a second vesicle derived from a strain that need not have a serosubtype prevent in a country of use. Reference 37 also discloses useful combinations of different vesicles. A combination of vesicles from strains in each of the L2 and L3 immunotypes may be used in some embodiments. 
     Bacteria 
     As mentioned above, OMVs of the invention are prepared from meningococci which over-express the relevant antigen(s) due to genetic modification. The invention also provides these bacteria. The bacteria can be used for preparing OMVs of the invention. 
     In addition to genetic modification(s) which cause over-expression of the antigen(s) of interest, the bacteria may include one or more further modifications. For instance, the bacterium may have a knockout of one or more of lpxL1, 1gtB, porA, frpB, synX, 1gtA, mltA and/or lst. 
     The bacterium, may have low endotoxin levels, achieved by knockout of enzymes involved in LPS biosynthesis [38, 39]. 
     The bacterium may be from any serogroup e.g. A, B, C, W135, Y. It is preferably serogronp B. 
     The bacterium may be of any serotype (e.g., 1, 2a, 2b, 4, 14, 15, 16, etc.), any serosubtype, and any immunotype (e.g. L1; L2; L3; L3,3,7; L10; etc.). Vesicles can usefully be prepared from strains having one of the following subtypes: P1.2; P1.2,5; P1.4; P1.5; P1.5,2; P1.5,c; P1.5c,10; P1.7,16; P1.7,16b; P1.7h,4; P1.9; P1.15; P1.9,15; P1.12,13; P1.13; P1.14; P1.21,16; P1.22,14. 
     The bacterium may be from any suitable lineage, including hyperinvasive and hypervirulent lineages e.g. any of the following seven hypervirulent lineages: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37 complex; A4 cluster; lineage 3. These lineages have been defined by multilocus enzyme electrophoresis (MLEE), but multilocus sequence typing (MLST) has also been used to classify meningococci [ref. 40] e.g. the ET-37 complex is the ST-11 complex by MLST, the ET-5 complex is ST-32 (ET-5), lineage 3 is ST-41/44, etc. 
     In some embodiments a bacterium may include one or more of the knockout and/or hyper-expression mutations disclosed in references 16 and 41-43. Suitable genes for modification include: (a) Cps, CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [41]; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB; and (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, P or B, SiaD, SynA, SynB, and/or SynC. 
     A bacterium may have one or more, or all, of the following characteristics: (i) down-regulated or knocked-out LgtB and/or GalE to truncate the meningococcal LOS; (ii) up-regulated TbpA; (iii) up-regulated NhhA; (iv) up-regulated Omp85; (v) up-regulated LbpA; (vi) up-regulated NspA; (vii) knocked-out PorA; (viii) down-regulated or knocked-out FrpB; (ix) down-regulated or knocked-out Opa; (x) down-regulated or knocked-out Opc; (xii) deleted cps gene complex. A truncated LOS can be one that does not include a sialyl-lacto-N-neotetraose epitope e.g. it might be a galactose-deficient LOS. The LOS may have no α chain. 
     Strain Production 
     The invention provides a process for preparing a meningococcal strain suitable for OMV preparation, comprising steps of (i) choosing a starting strain which expresses a first amount of an antigen when grown in specific culture conditions, then (ii) modifying the starting strain to provide a modified strain, wherein the modified strain expresses a second amount of the antigen when grown in the same specific culture conditions, wherein the second amount is higher than the first amount; wherein the antigen is either NHBA or NadA. The second amount of NHBA or NadA is usefully at least 10%, higher than the first amount, measured in mass of the relevant antigen per unit mass of bacteria, and is more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more. 
     The invention provides a process for preparing a meningococcal strain suitable for OMV preparation, comprising steps of (i) choosing a starting strain which expresses NHBA and/or NadA; and (ii) modifying the starting strain to increase the amount of NHBA and/or NadA which it expresses. The increased amount after modification in step (ii) is usefully at least 10%, higher than the first amount, measured in mass of the relevant antigen per unit mass of bacteria, and is more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more. 
     Either of these processes can be followed by a step of (iii) culturing the modified bacteria obtained in step (ii) to provide a bacterial culture. 
     In step (ii), the modification to increase expression of NHBA and/or NadA can be any of the modifications discussed above. For instance, the strain can be modified by knocking out expression of NadR, thereby increasing expression of NadA. The strain can also be modified to increase or decrease expression of other polypeptides, as described elsewhere herein e.g. to increase its fHbp expression, such as by introducing a gene which encodes a constitutively-active mutant FNR. 
     The invention also provides a process for preparing a meningococcal vesicle, comprising a step of treating a bacterial culture obtained by a process of the invention (as described above) such that its outer membrane forms vesicles. This treatment step can use any of the techniques discussed above. 
     The invention also provides a process for preparing a meningococcal vesicle, comprising a step of treating a meningococcus of the invention such that its outer membrane forms vesicles. This treatment step can use any of the techniques discussed above. 
     Useful starting strains are in meningococcus serogroup B. Four useful starting meningococcal strains for preparing bacteria which over-express an antigen of interest are MC58, NZ05/33, H44/76 and GB013. MC58 has PorA serosubtype 1.7,16; NZ05/33 has serosubtype 1.7-2.4; H44/76 has serosubtype 1.7,16; and GB013 has serosubtype 1.22,9. 
     Isogenic Panels 
     A third aspect of the invention provides a panel of bacterial strains (e.g. meningococci), each member of which is isogenic except for a single gene which in each strain encodes a different variant of an antigen of interest. Thus the only genetic difference between each member of the panel is the coding sequence for this antigen. This panel can be used to study the immunological effect of polymorphic forms of a gene of interest found in different wild-type strains, without having to worry about variability due to differences in those strains which are unrelated to the antigen of interest. For instance, these panels can be used as test strains in a serum bactericidal antibody assay to provide a constant genetic background for assessing the cross-population killing of bacteria by antibodies which were raised against a specific sequence variant. 
     A useful panel for an antigen of interest can be made be selecting a starting strain of meningococcus. A useful starting strain does not express the antigen of interest; if the starting strain does express the antigen of interest then expression of the endogenous gene can be knocked out e.g. by insertion of a marker gene. To create a panel, a site in the bacterial genome is chosen for insertion of a gene encoding the antigen of interest. This site can be under the control of a promoter, such that different coding sequences can be introduced for expression from this promoter, or it can lack a promoter, in which case the introduced sequences should include a promoter. An important feature of the panel is that each member has the same promoter for expression of the antigen of interest, in the same location in the genome, such that the only genetic difference between them is the coding sequence for the antigen of interest. 
     The antigen of interest, which differs between panel members, can be any useful antigen which exists in polymorphic forms across a bacterial population. Thus, for meningococcus, the antigen of interest could be e.g. fHbp, NadA, NHBA, Omp85, HmbR, NhhA, App, NspA, TbpA, etc. 
     The general approach of creating an isogenic panel for testing the effect of sequence variability is not restricted to meningococcus and can be used for any other bacterium. 
     Antigens 
     NHBA (Neisserial Heparin Binding Antigen) 
     NHBA [11] was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] gene NMB2132 (GenBank accession number GI:7227388; SEQ ID NO: 9 herein). Sequences of NHBA from many strains have been published since then. For example, allelic forms of NHBA (referred to as protein ‘287’) can be seen in FIGS. 5 and 15 of reference 44, and in example 13 and FIG. 21 of reference 45 (SEQ IDs 3179 to 3184 therein). Various immunogenic fragments of NHBA have also been reported. 
     Preferred NHBA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 9; and or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 9, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 9. 
     The most useful NHBA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 9. Advantageous NHBA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     NadA (Neisserial adhesin A) 
     The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC 58 [68] as gene NMB1994 (GenBank accession numbers GI:7227256; SEQ ID NO: 10 herein). The sequences of NadA antigen from many strains have been published since then, and the protein&#39;s activity as a Neisserial adhesin has been well documented. Various immunogenic fragments of NadA have also been reported. 
     Preferred NadA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 10; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 10, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 10. 
     The most useful NadA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 10. Advantageous NadA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. SEQ ID NO: 6 is one such fragment. 
     HmbR 
     The full-length HmbR sequence was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB1668 (SEQ ID NO: 7 herein). Reference 46 reports a HmbR sequence from a different strain (SEQ ID NO: 8 herein), and reference 15 reports a further sequence (SEQ ID NO: 19 herein). SEQ ID NOs: 7 and 8 differ in length by 1 amino acid and have 94.2% identity. SEQ ID NO: 19 is one amino acid shorter than SEQ ID NO: 7 and they have 99% identity (one insertion, seven differences) by CLUSTALW. The invention can use any such HmbR polypeptide. 
     The invention can use a polypeptide that comprises a full-length HmbR sequence, but it will often use a polypeptide that comprises a partial HmbR sequence. Thus in some embodiments a HmbR sequence used according to the invention may comprise an amino acid sequence having at least i % sequence identity to SEQ ID NO: 7, where the value of i is 50, 60, 70, 80, 90, 95, 99 or more. In other embodiments a HmbR sequence used according to the invention may comprise a fragment of at least j consecutive amino acids from SEQ ID NO: 7, where the value of j is 7, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more. In other embodiments a HmbR sequence used according to the invention may comprise an amino acid sequence (i) having at least i % sequence identity to SEQ ID NO: 7 and/or (ii) comprising a fragment of at least j consecutive amino acids from SEQ ID NO: 7. 
     Preferred fragments of j amino acids comprise an epitope from SEQ ID NO: 7. Such epitopes will usually comprise amino acids that are located on the surface of HmbR. Useful epitopes include those with amino acids involved in HmbR&#39;s binding to haemoglobin, as antibodies that bind to these epitopes can block the ability of a bacterium to bind to host haemoglobin. The topology of HmbR, and its critical functional residues, were investigated in reference 47. Fragments that retain a transmembrane sequence are useful, because they can be displayed on the bacterial surface e.g. in vesicles. Examples of long fragments of HmbR correspond to SEQ ID NOs: 21 and 22. If soluble HmbR is used, however, sequences omitting the transmembrane sequence, but typically retaining epitope(s) from the extracellular portion, can be used. 
     The most useful HmbR antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 7. Advantageous HmbR antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     fHbp (factor H binding protein) 
     The fHbp antigen has been characterised in detail. It has also been known as protein ‘741’ [SEQ IDs 2535 &amp; 2536 in ref 45], ‘NMB1870’, ‘GNA1870’ [refs. 48-50], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [51-53]. It is naturally a lipoprotein and is expressed across all meningococcal serogronps. The structure of fHbp&#39;s C-terminal immunodominant domain (‘fHbpC’) has been determined by NMR [54]. This part of the protein forms an eight-stranded β-barrel, whose strands are connected by loops of variable lengths. The barrel is preceded by a short α-helix and by a flexible N-terminal tail. 
     The fHbp antigen falls into three distinct variants [55] and it has been found that serum raised against a given family is bactericidal within the same family, but is not active against strains which express one of the other two families i.e. there is intra-family cross-protection, but not inter-family cross-protection. The invention can use a single fHbp variant, but is will usefully include a fHbp from two or three of the variants. Thus it may use a combination of two or three different fHbps, selected from: (a) a first protein, comprising an amino acid sequence having at least a% sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; (b) a second protein, comprising an amino acid sequence having at least b% sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2; and/or (c) a third protein, comprising an amino acid sequence having at least c% sequence identity to SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3. 
     The value of a is at least 85 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more. The value of b is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more. The value of c is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more. The values of a, b and c are not intrinsically related to each other. 
     The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The values of x, y and z are not intrinsically related to each other. 
     Where the invention uses a single fHbp variant, a composition may include a polypeptide comprising (a) an amino acid sequence having at least a% sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; or (b) an amino acid sequence having at least b% sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2; or (c) an amino acid sequence having at least c% sequence identity to SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3. 
     Where the invention uses a fHbp from two or three of the variants, a composition may include a combination of two or three different fHbps selected from: (a) a first polypeptide, comprising an amino acid sequence having at least a% sequence identity to SEQ ID NO: 1 and/or comprising an amine acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; (b) a second polypeptide, comprising an amino acid sequence having at least b% sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2; and/or (c) a third polypeptide, comprising an amino acid sequence having at least c% sequence identity to SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3. The first, second and third polypeptides have different amino acid sequences. 
     Where the invention uses a fHbp from two of the variants, a composition can include both: (a) a first polypeptide, comprising an amino acid sequence having at least a% sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; and (b) a second polypeptide, comprising an amino acid sequence having at least b% sequence identity to SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2. The first and second polypeptides have different amino acid sequences. 
     Where the invention uses a fHbp from two of the variants, a composition can include both: (a) a first polypeptide, comprising an amino acid sequence having at least a% sequence identity to SEQ ID NO: 1 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; (b) a second polypeptide, comprising an amino acid sequence having at least c% sequence identity to SEQ ID NO: 3 and/or comprising an. amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3. The first and second polypeptides have different amino acid sequences. 
     Another useful fHbp which can be used according to the invention is one of the modified forms disclosed, for example, in reference 56 e.g. comprising SEQ ID NO: 20 or 23 therefrom. These modified forms can elicit antibody responses which are broadly bactericidal against meningococci. 
     fHbp protein(s) in a OMV will usually be lipidated e.g. at a N-terminus cysteine. In other embodiments they will not be lipidated. 
     NspA (Neisserial surface protein A) 
     The NspA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB0663 (GenBank accession number GI:7225888; SEQ ID NO: 11 herein). The antigen was previously known from references 57 &amp; 58. The sequences of NspA antigen from many strains have been published since then. Various immunogenic fragments of NspA have also been reported. 
     Preferred NspA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 11; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO:  11 , wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 11. 
     The most useful NspA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 11. Advantageous NspA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     NhhA (Neisseria hia homologue) 
     The NhhA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB0992 (GenBank accession number GI:7226232; SEQ ID NO: 12 herein). The sequences of NhhA antigen from many strains have been published since e.g. refs 44 &amp; 59, and various immunogenic fragments of NhhA have been reported. It is also known as Hsf. 
     Preferred NhhA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 12; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 12, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 12. 
     The most useful NhhA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 12. Advantageous NhhA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     App (Adhesion and penetration protein) 
     The App antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB1985 (GenBank accession number GI:7227246; SEQ ID NO: 13 herein). The sequences of App antigen from many strains have been published since then. It has also been known as ‘ORF1’ and ‘Hap’. Various immunogenic fragments of App have also been reported. 
     Preferred App antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 13; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 13, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 13. 
     The most useful App antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 13. Advantageous App antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     Omp85 (85 kDa outer membrane protein) 
     The Omp85 antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB0182 (GenBank accession number GI:7225401; SEQ ID NO: 14 herein). The sequences of Omp85 antigen from many strains have been published since then. Further information on Omp85 can be found in references 60 and 61. Various immunogenic fragments of Omp85 have also been reported. 
     Preferred Omp85 antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 14; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 14, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 14. 
     The most useful Omp85 antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 14. Advantageous Omp85 antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     TbpA 
     The TbpA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB0461 (GenBank accession number GI:7225687; SEQ ID NO: 23 herein). The sequences of TbpA from many strains have been published since then. Various immunogenic fragments of TbpA have also been reported. 
     Preferred TbpA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 23; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 23, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 23. 
     The most useful TbpA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 23. Advantageous TbpA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     TbpB 
     The TbpB antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB1398 (GenBank accession number GI:7225686; SEQ ID NO: 24 herein). The sequences of TbpB from many strains have been published since then. Various immunogenic fragments of TbpB have also been reported. 
     Preferred TbpB antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 24; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 24, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 24. 
     The most useful TbpB antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 24. Advantageous TbpB antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     Cu,Zn-superoxide Dismutase 
     The Cu,Zn-superoxide dismutase antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [68] as gene NMB1398 (GenBank accession number GI:7226637; SEQ ID NO: 25 herein). The sequences of Cu,Zn-superoxide dismutase from many strains have been published since then. Various immunogenic fragments of Cu,Zn-superoxide dismutase have also been reported. 
     Preferred Cu,Zn-superoxide dismutase antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 25; and/or (b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 25, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 25. 
     The most useful Cu,Zn-superoxide dismutase antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 25. Advantageous Cu,Zn-superoxide dismutase antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. 
     Pharmaceutical Compositions 
     Vesicles of the invention are useful as active ingredients in immunogenic pharmaceutical compositions for administration to a patient. These will typically include a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in reference 62. 
     Effective dosage volumes can be routinely established, but a typical human dose of the composition has a volume of about 0.5 ml e.g. for intramuscular injection. The RIVM OMV-based vaccine was administered in a 0.5 ml volume [63] by intramuscular injection to the thigh or upper arm. MeNZB™ is administered in a 0.5 ml by intramuscular injection to the anterolateral thigh or the deltoid region of the arm. Similar doses may be used for other delivery routes e.g, an intranasal OMV-based vaccine for atomisation may have a volume of about 100 μl or about 130 μl per spray, with four sprays administered to give a total dose of about 0.5 ml. 
     The pH of a composition of the invention is usually between 6 and 8, and more preferably between 6.5 and 7.5 (e.g. about 7). The pH of the RIVM OMV-based vaccine is 7.4 [64], and a pH&lt;7.5 is preferred for compositions of the invention. The RIVM OMV-based vaccine maintains pH by using a 10 mM Tris/HCl buffer, and stable pH in compositions of the invention may be maintained by the use of a buffer e.g. a Tris buffer, a citrate buffer, phosphate buffer, or a histidine buffer. Thus compositions of the invention will generally include a buffer. 
     The composition may be sterile and/or pyrogen-free. Compositions of the invention may be isotonic with respect to humans. 
     Compositions of the invention for administration to patients are immunogenic, and are more preferably vaccine compositions. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other 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&#39;s immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor&#39;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. The antigen content of compositions of the invention will generally be expressed in terms of the amount of protein per dose. A dose of about 0.9 mg protein per ml is typical for OMV-based intranasal vaccines. 
     Compositions of the invention may include an immunological adjuvant. Thus, for example, they may include an aluminium salt adjuvant or an oil-in-water emulsion (e.g. a squalene-in-water emulsion). Suitable aluminium salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), (e.g. see chapters 8 &amp; 9 of ref. 65), or mixtures thereof. The salts can take any suitable form (e.g. gel, crystalline, amorphous, etc.), with adsorption of antigen to the salt being preferred. The concentration of Al +++  in a composition for administration to a patient is preferably less than 5 mg/ml e.g. ≤4 mg/ml, ≤3 mg/ml, ≤2 mg/ml, ≤1 mg/ml, etc. A preferred range is between 0.3 and 1 mg/ml. A maximum of 0.85 mg/dose is preferred. Aluminium hydroxide adjuvants are particularly suitable for use with meningococcal vaccines. 
     Meningococci affect various areas of the body and so the compositions of the invention may be prepared in various liquid forms. For example, the compositions may be prepared as injectable, either as solutions or suspensions. The composition may be prepared for pulmonary administration e.g, by an inhaler, using a fine spray. The composition may be prepared for nasal, aural or ocular administration e.g. as spray or drops. Injectables for intramuscular administration are typical. 
     Compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose format. Antimicrobials such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, but it is preferred to use either a mercury-free preservative or no preservative at all. 
     Compositions of the invention may comprise detergent e.g. a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g. &lt;0.01%. 
     Compositions of the invention may include residual detergent (e.g. deoxycholate) from OMV preparation. The amount of residual detergent is preferably less than 0.4 μg (more preferably less than 0.2 μg) for every μg of MenB protein. 
     If a composition of the invention includes LOS, the amount of LOS is preferably less than 0.12 μg (more preferably less than 0.05 μg) for every μg of protein. 
     Compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 9 mg/ml. 
     Methods of Treatment 
     The invention also provides a method for raising an immune response in a mammal, comprising administering a composition of the invention to the mammal. The immune response is preferably protective and preferably involves antibodies. The method may raise a booster response in a patient that has already been primed against  N. meningitidis.    
     The mammal is preferably a human. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. 
     The invention also provides vesicles of the invention for use as a medicament. The medicament is preferably used to raise an immune response in a mammal (i.e. it is an immunogenic composition) and is more preferably a vaccine. 
     The invention also provides the use of vesicles of the invention in the manufacture of a medicament for raising an immune response in a mammal. 
     These uses and methods are preferably for the prevention and/or treatment of a disease caused by  N. meningitidis  e.g. bacterial (or, more specifically, meningococcal) meningitis, or septicemia. 
     One way of checking efficacy of therapeutic treatment involves monitoring Neisserial infection after administration of the composition of the invention. One way of checking efficacy of prophylactic treatment involves monitoring immune responses against antigens after administration of the composition. Immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age, or animal models [66]) and then determining standard parameters including serum bactericidal antibodies (SBA) and ELISA titres (GMT). These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. A SBA increase of at least 4-fold or 8-fold is preferred. Where more than one dose of the composition is administered, more than one post-administration determination may be made. 
     In general, compositions of the invention are able to induce serum bactericidal antibody responses after being administered to a subject. These responses are conveniently measured in mice and are a standard indicator of vaccine efficacy. Serum bactericidal activity (SBA) measures bacterial killing mediated by complement, and can be assayed using human or baby rabbit complement. WHO standards require a vaccine to induce at least a 4-fold rise in SBA in more than 90% of recipients. MeNZB™ elicits a 4-fold rise in SBA 4-6 weeks after administration of the third dose. 
     Preferred compositions can confer an antibody titre in a human subject patient that is superior to the criterion for seroprotection for an acceptable percentage of subjects. Antigens with an associated antibody titre above which a host is considered to be seroconverted against the antigen are well known, and such titres are published by organisations such as WHO. Preferably more than 80% of a statistically significant sample of subjects is seroconverted, more preferably more than 90%, still more preferably more than 93% and most preferably 96-100%. 
     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 any other suitable route. The invention may be used to elicit systemic and/or mucosal immunity. Intramuscular administration to the thigh or the upper arm is preferred. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 ml. 
     Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined. The OMV-based RIVM vaccine was tested using a 3- or 4-dose primary schedule, with vaccination at 0, 2 &amp; 8 or 0, 1, 2 &amp; 8 months. MeNZB™ is administered as three doses at six week intervals. 
     Compositions of the invention may be used to induce bactericidal antibody responses against more than one hypervirulent lineage of meningococcus. In particular, they can preferably induce bactericidal responses against two or three of the following three hypervirulent lineages: (i) cluster A4; (ii) ET5 complex; and (iii) lineage 3. They may additionally induce bactericidal antibody responses against one or more of hypervirulent lineages subgroup I, subgroup III, subgroup IV-1 or ET-37 complex, and against other lineages e.g. hyperinvasive lineages. This does not necessarily mean that the composition can induce bactericidal antibodies against each and every strain of meningococcus within these hypervirulent lineages e.g. rather, for any given group of four of more strains of meningococcus within a particular hypervirulent lineage, the antibodies induced by the composition are bactericidal against at least 50% (e.g. 60%, 70%, 80%, 90% or more) of the group. Preferred groups of strains will include strains isolated in at least four of the following countries: GB, AU, CA, NO, IT, US, NZ, NL, BR, and CU. The serum preferably has a bactericidal titre of at least 1024 (e.g. 2 10 , 2 11 , 2 12 , 2 13 , 2 14 , 2 15 , 2 16 , 2 17 , 2 18  or higher, preferably at least 2 14 ) e.g. the serum is able to kill at least 50% of test bacteria of a particular strain when diluted 1/1024. 
     Useful compositions can induce bactericidal responses against the following strains of serogroup B meningococcus: (i) from cluster A4, strain 961-5945 (B:2b:P1.21,16) and/or strain G2136 (B:-); (ii) from ET-5 complex, strain MC58 (B:15:P1.7,16b) and/or strain 44/76 (B:15:P1.7,16); (iii) from lineage 3, strain 394/98 (B:4;P1.4) and/or strain BZ198 (B:NT:-). More preferred compositions can induce bactericidal responses against strains 961-5945, 44/76 and 394/98. 
     Strains 961-5945 and G2136 are both  Neisseria  MLST reference strains [ids 638 &amp; 1002 in ref. 67]. Strain MC58 is widely available (e.g. ATCC BAA-335) and was the strain sequenced in reference 68. Strain 44/76 has been widely used and characterised (e.g. ref 69) and is one of the  Neisseria  MLST reference strains [id 237 in ref. 67; row 32 of Table 2 in ref 40]. Strain 394/98 was originally isolated in New Zealand in 1998, and there have been several published studies using this strain, (e.g. refs. 70 &amp; 71). Strain BZ198 is another MLST reference strain (id 409 in ref 67; row 41 of Table 2 in ref. 40). 
     Further Antigenic Components 
     In addition to vesicles of the invention, an immunogenic composition can include further antigens. 
     In some embodiments, a composition includes one or more capsular saccharides from meningococci e.g. from serogroups A, C, W135 and/or Y. These saccharides will usually be conjugated to a protein carrier. A composition of the invention may include one or more conjugates of capsular saccharides from 1, 2, 3, or 4 of meningococcal serogroup A, C, W135 and Y e.g. A+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, A+W135+Y, A+C+W135+Y, etc. Components including saccharides from all four of serogroups A, C, W135 and Y are ideal. 
     As well as containing antigens from  N. meningitidis , compositions may include antigens from further pathogens. For example, the composition may comprise one or more of the following further antigens:
         an antigen from  Streptococcus pneumoniae , such as a saccharide (typically conjugated)   an antigen from hepatitis B virus, such as the surface antigen HBsAg.   an antigen from  Bordetella pertussis , such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from  B. pertussis , optionally also in combination with pertactin and/or agglutinogens 2 and 3.   a diphtheria antigen, such as a diphtheria toxoid.   a tetanus antigen, such as a tetanus toxoid.   a saccharide antigen from  Haemophilus influenzae  B (Hib), typically conjugated.   inactivated poliovirus antigens.       

     Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred. 
     If a Hib saccharide is included (typically as a conjugate), the saccharide moiety may be a polysaccharide (e.g. full-length polyribosylribitol phosphate (PRP) as purified from bacteria), but it is also possible to fragment the purified saccharide to make oligosaccharides (e.g: MW from ˜1 to ˜5 kDa) e.g. by hydrolysis. The concentration of Hib conjugate in a composition will usually be in the range of 0.5 μg to 50 μg e.g. from 1-20 μg, from 10-15 μg, from 12-16 μg, etc. The amount may be about 15 g, or about 12.5 μg in some embodiments. A mass of less than 5 μg may be suitable [72] e.g. in the range 1-5 μg, 2-4 μg, or about 2.5 μg. As described above, in combinations that include Hib saccharide and meningococcal saccharides, the dose of the former may be selected based on the dose of the latter (in particular, with multiple meningococcal serogroups, their mean mass). Further characteristics of Hib conjugates are as disclosed above for meningococcal conjugates, including choice of carrier protein (e.g. CRM197 or tetanus toxoid), linkages, ratios, etc. 
     If a  S. pneumoniae  antigen is included, this may be a polypeptide or a saccharide. Conjugates capsular saccharides are particularly useful for immunising against pneumococcal. The saccharide may be a polysaccharide having the size that arises during purification of the saccharide from bacteria, or it may be an oligosaccharide achieved by fragmentation of such a polysaccharide. In the 7-valent PREVNAR™ product, for instance, 6 of the saccharides are presented as intact polysaccharides while one (the 18C serotype) is presented as an oligosaccharide. A composition may include a capsular saccharide from one or more of the following pneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. A composition may include multiple serotypes e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes, 7-valent, 9-valent, 10-valent, 11-valent and 13-valent conjugate combinations are already known in the art, as is a 23-valent unconjugated combination. For example, an 10-valent combination may include saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent combination may further include saccharide from serotype 3. A 12-valent combination may add to the 10-valent mixture: serotypes 6A and 19A; 6A and 22F; 19A and 22F; 6A and 15B; 19A and 15B; r 22F and 15B; A 13-valent combination may add to the 11-valent mixture: serotypes 19A and 22F; 8 and 12F; 8 and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12F and 22F; 15B and 19A; 15B and 22F. etc. Further characteristics of pneumococcal conjugates are as disclosed above for meningococcal conjugates, including choice of carrier protein (e.g. CRM197 or tetanus toxoid), linkages, ratios, etc. Where a composition includes more than one conjugate, each conjugate may use the same carrier protein or a different carrier protein. Reference 73 describes potential advantages when using different carrier proteins in multivalent pneumococcal conjugate vaccines. 
     General 
     The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 74-80, etc. 
     The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g; X+Y. 
     The term “about” in relation to a numerical value x is optional and means, for example, x±10%. 
     Where the invention concerns an “epitope”, this epitope may be a B-cell epitope and/or a T-cell epitope, but will usually be a B-cell epitope. Such epitopes can be identified empirically (e.g. using PEPSCAN [81, 82] or similar methods), or they can be predicted (e.g. using the Jameson-Wolf antigenic index [83], matrix-based approaches [84], MAPITOPE [85], TEPITOPE [86, 87], neural networks [88], OptiMer &amp; EpiMer [89, 90], ADEPT [91], Tsites [92], hydrophilicity [93], antigenic index [94] or the methods disclosed in references 95-99, etc.). Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as “antigenic determinants”. 
     References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. 100. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. 101. 
     The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  illustrates the approach for constructing an isogenic panel by knocking out nadA and nhba (GNA2132) to create a background strain. 
         FIG. 2  shows the insertion of fHbp genes into the background strain to make a panel of isogenic strains expressing different fHbp genes under the control of a Ptac promoter. 
         FIG. 3A - FIG. 3B  shows expression levels of fHbp in the isogenic panel strains described in  FIG. 2 . 
         FIG. 4A - FIG. 4B  shows expression of NadA (upper panel) and NadR (lower panel) in eight wild-type strains ( FIG. 4A ) or their NadR knockout forms ( FIG. 4B ). The numbers in  FIG. 4A  show the number of TAAA tetranucleotide repeats in the strain.  FIG. 4C - FIG. 4D  shows expression of NadA and NadR in 7 strains, in the presence of absence of 4HPA. 
         FIG. 5A - FIG. 5C  shows ( FIG. 5A ) starting strain MC58 ( FIG. 5B ) MC58Δnhba and ( FIG. 5C ) MC58Δnhba transformed with a complementing nhba gene with an upstream CREN and IPTG-inducible promoter. 
         FIG. 6  shows NHBA expression by MC58 and derivative strains. The left two lanes show expression in MC58 and MC58Δnhba. The next 8 lanes show expression in complemented strains at four concentrations of IPTG. The lanes are arranged in pairs, with the right-hand lane being a strain complemented with nhba having an upstream CREN. 
         FIG. 7  shows NHBA expression by 95N477 and derivative strains. The left two lanes show expression in 95N477 and 95N477Δnhba. The next 5 lanes show expression in complemented strains at the indicated concentrations of IPTG. 
         FIG. 8  shows NHBA expression for five strains in an isogenic panel. From top to bottom the expressed NHBA is from strain NZ98/254, UK013, UK355, 2996 and NM117. 
     
    
    
     MODES FOR CARRYING OUT THE INVENTION 
     NHBA 
     The endogenous nhba gene is knocked out in various serogroup B strains to create strains MC58Δnhba, 95N477Δnhba, NGH38Δnhba and UK013Δnhba. These strains are then transformed with pCOMPpind-287 vector containing a gene encoding nhba from strain 394/98, with or without an upstream CREN (contact regulatory element of  Neisseria ), under the control of an IPTG-inducible promoter. The vectors insert the nhba gene (±CREN) between the endogenous nmb1428 and nmb1429 genes by homologous recombination. 
       FIG. 5  shows the starting MC58 strain, the MC58Δnhba strain, and the complemented MC58 strain (+CREN).  FIG. 6  shows expression of NHBA by the various MC58 strains with increasing IPTG concentration. The complemented strains show high levels of inducible NHBA expression, with the highest levels seen with the inserted gene has an upstream CREN. 
       FIG. 7  shows expression in the 95N477 strains. The endogenous nhba gene in this strain encodes a 427aa protein, whereas the inserted complementing gene has 492aa. Increased expression levels of the larger NHBA protein are clearly visible, and this expression increases with IPTG concentration. 
     Although in some strains (e.g. M4407) it was not possible to obtain a Δnhba knockout using the transformation protocols, for strains which could be transformed these results show that strains which over-express NHBA can readily be obtained. 
     NadR (NMB1843) 
     The nadA gene is present in approximately 50% of meningococcal isolates. NadA exhibits growth-phase dependent expression, with maximal levels in the stationary growth phase of all strains tested. Expression is controlled by a tetranucleotide repeat (TAAA) located upstream of the nadA promoter. The number of repeats can be modified during replication through slipped strand mispairing, and consequently can influence the expression of the nadA gene by creating variants where changes in the repeat number result in promoters with low, medium or high activity. 
     An area of the nadA promoterm upstream of the TAAA repeat, is responsible for repression of nadA expression during logarithmic phase of growth. This area is called the ‘GPR region’. DNA-affinity fractionation identified a protein present in meningococcus crude extracts which binds to the GPR region. This protein is NadR (NMB1843) and is a member of the MarR family of repressors. NadR binds to three operators (binding sites) in the nadA promoter and results in repression of NadA expression. Knockout of NadR in strains expressing high, medium or low levels of NadA results in almost comparable high level expression in each strain. Thus NadR is the repressor that contributes to the differential expression levels exhibited by meningococcal strains, or phase variants in the same strain, with different numbers of repeats in their promoter. NadR is expressed to similar levels in different strains but can repress more or less efficiently the nadA promoter depending on the number of repeats present in the variant promoter. 
     Knockout of NadR in various meningococcus backgrounds results in almost comparable high levels of expression of NadA across the panel. Strains are transformed with the knockout construct for the allelic replacement of nmb1843 with a chloramphenicol cassette. Expression levels in eight different strains are shown in  FIG. 4 . 
     A small molecule ligand 4-hydroxyphenylacetic acid (4HPA) can induce NadA expression in vitro due to derepression of NadR ( FIG. 4C ). Addition of the molecule to the purified NadR protein in vitro can inhibit the binding activity of the protein for the nadA promoter. 4HPA is a metabolite of the catabolic pathway of the aromatic amino acids and is secreted in human saliva and urine, and so in vivo expression of NadA may be higher than is seen during in vitro growth. 
     Thus strains which over-express NadA can readily be obtained by inactivation of NadR and/or by addition of a small molecule inducer to the growth medium. 
     Isogenic Panel—NHBA 
     NHBA is an antigen in the 4CMenB product. An isogenic panel was used to study the potential cross protection of NHBA-induced bactericidal antibodies. 
     The nhba genes from six different meningococcal strains were amplified to provide the mature form of the polypeptide with a C-terminus histidine tag. These were cloned into the pET-21b+ plasmid vector and expressed in  E. coli . The purified NHBA peptides were then used to immunize mice (20 μg dose) and obtain mouse antisera. ELISA assays were performed in order to confirm the presence of antibodies in all the mouse sera obtained. 
     To evaluate the immunogenicity and the contribution of amino acid sequence variability to vaccine coverage, a starting strain was engineered to be susceptible to bactericidal killing only by anti-NHBA antibodies (rather than the other antigens in 4CMenB).  N. meningitidis  strain 5/99 naturally expresses high levels of NadA, but very low levels of NHBA and fHbp. Its nadA and nhba genes were respectively replaced by ery and kan resistance cassettes (5/99ΔΔ). The nhba gene to be complemented was then inserted in the intergenic region between the open reading frames nmb1428 and nmb1429. Thus the final strain panel was isogenic except for the chosen nhba gene, and this gene should be inducable for expression at equal levels in all members of the panel. 
     FACS showed that the panel members showed a comparable amount of the different NHBA polypeptides in each strain ( FIG. 8 ). Several mouse antisera raised against the different NHBA polypeptides were tested in western blot and the detection appeared to be variant-specific, showing a stronger recognition for the homologous variant. 
     The panel was also used for testing the bactericidal effect of the mouse antisera. As the strains were isogenic then any difference in bactericidal effect should arise only from the different expressed NHBA polypeptides. In parallel the sera were tested against wild-type strains which express the relevant NHBA polypeptide sequence, to see if the common genetic background of the isogenic panel did enable the detection of differences which would be concealed by natural variation if wild-type strains were used. Results were as follows: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                 Antiserum NHBA 
                 5/99ΔΔ 
                 Wild-type 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 NZ98/254 
                 &gt;8192 
                 8192 
               
               
                   
                 WC58 
                 8192 
                 512 
               
               
                   
                 UK013 
                 256 
                 128 
               
               
                   
                 UK355 
                 128 
                 256 
               
               
                   
                 2996 
                 128 
                 256 
               
               
                   
                 NM117 
                 2048 
                 4096 
               
               
                   
                   
               
            
           
         
       
     
     Thus the panel does seem to compensate for variability which is unrelated to the NHBA antigen itself. For instance, serum raised against the MC58 sequence is much more effective against the MC58 polypeptide in the isogenic panel than against the wild-type MC58 strain. 
     Isogenic Panel—fHbp 
     Sequencing of the fHbp gene in a large collection of meningococcal isolates revealed three variants with low levels of cross-protective bactericidal response. A serum bactericidal assay was used to evaluate the cross-protective capabilities of human antibodies raised against different fHbp variants, but the killing mediated by bactericidal antibodies in this assay is dependent by several factors. Thus the potential coverage of a single antigen may be difficult to estimate. 
     A genetic approach was used to overcome variability due to strain-specific serum susceptibility, limitations of compatible complement sources, and variable expression of fHbp and other surface-exposed factors affecting resistance to serum (e.g. the capsule). A well-characterized meningococcal isolate (5/99) was engineered to generate isogenic strains expressing ten different fHbp sub-variants from a constitutive heterologous promoter. The fHbp genes were inserted between endogenous nmb1428 and nmb1429 genes. This panel was then used as the test strain in a serum bactericidal antibody (SBA) assay to assess the ability of a single fHbp variant to elicit a broadly-protective immune response. 
     In order to have a genetic background to express different fHbp sub-variants without the interfering action of the other antigens, the nadA and nhba genes in the starting 5/99 strain were inactivated by insertion of erm and kan resistance cassettes, respectively ( FIG. 1 ). The resulting double mutant strain (named 5/99ΔΔ) was manipulated to express different fHbp subvariants under the control of a Ptac promoter to standardize the amount of fHbp expressed ( FIGS. 1 &amp; 2 ). In total, ten different fHbp coding sequences were cloned in the pComp-RBS vector and transferred to the 5/99ΔΔ genetic background. 
     To evaluate the expression of fHbp in the recombinant strains, we performed FACS analysis using a mouse polyclonal serum against a single fHbp variant. The analysis showed a comparable amount of the different fHbp sub-variants on the surface of the recombinant strains generated ( FIG. 3 ). 
     The recombinant strains were analyzed for their susceptibility to killing by bactericidal antibodies from mice in a SBA using rabbit complement. Pooled sera from mice immunized with the “universal vaccine” of reference 19 or with its GNA2091-fHbp component were tested for their ability to kill the 5/99 wild-type, the intermediate 5/99ΔΔ strain expressing neither NHBA nor NadA antigens, and the ten recombinant strains. The 5/99 strain was killed by sera raised against the universal vaccine, but not by sera raised against the single antigen GNA2091-fHbp. The 5/99ΔΔ strain was resistant to killing by all sera. All of the complemented strains except one showed significant susceptibility to sera derived from mice immunized with the universal vaccine or with GNA2091-fHbp antigen. The single surviving strain expressed a fHbp in family III, confirming the absence of cross-reactivity between the fHbp families. The nine susceptible strains confirm that the specific fHbp sequence in the universal vaccine can raise antibodies which are broadly protective across the whole of fHbp family 1. 
     The panel was also tested using sera obtained from human adults who were immunised with 4CMenB. The results were comparable to those seen using mice. 
     It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope end spirit of the invention. 
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