Combination Vaccines With Serogroup B Meningococcus And D/t/p

Abstract

Serogroup B meningococcus antigens can successfully be combined with diphtheria, tetanus and pertussis toxoids ("DTP") to provide effective combination vaccines for protecting against multiple pathogens. These combinations are effective with a range of different adjuvants, and with both pediatric-type and booster-type DTP ratios. The adjuvant can improve the immune response which the composition elicits; alternatively, by including an adjuvant it is possible for the compositions to have a relatively lower amount of antigen while nevertheless having immunogenicity which is comparable to unadjuvanted combination vaccines.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional of U.S. application Ser. No. 14/420,238, with an international filing date of Sep. 6, 2013, now U.S. Pat. No. 9,526,776; which is the National Phase of PCT Application No. PCT/EP2013/068414, filed Sep. 6, 2013; which claims the benefit of U.S. Provisional Application No. 61/697,756, filed Sep. 6, 2012; all of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] 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: 303822012710SeqList.txt, date recorded: Dec. 16, 2016, size: 122 KB).

TECHNICAL FIELD

[0003] This invention is in the field of combination vaccines i.e. vaccines containing mixed immunogens from more than one pathogen, such that administration of the vaccine can simultaneously immunize a subject against more than one pathogen.

BACKGROUND ART

[0004] Vaccines containing antigens from more than one pathogenic organism within a single dose are known as "multivalent" or "combination" vaccines. Various combination vaccines have been approved for human use, including trivalent vaccines for protecting against diphtheria, tetanus and pertussis or against measles, mumps and rubella. These vaccines offer patients the advantage of receiving a reduced number of injections, which can lead to the clinical advantage of increased compliance (e.g. see chapter 29 of ref 1), particularly in pediatric patients.

[0005] One difficulty when providing new combination vaccines is the potential for adverse vaccine-vaccine interactions between the mixed components, which may be due to physical or chemical factors. For instance, reference 2 discusses potential alterations in immunogenicity when antigens are combined, and reference 3 reports that the development of combination vaccines involves much more than the simple mixing of existing antigens. Similarly, reference 4 reviews a variety of clinically-relevant interactions (see also reference 5), and reference 6 reviews the technical challenges faced when making a combination vaccine.

[0006] It is an object of the invention to provide further and improved combination vaccines, and in particular those which can protect against serogroup B meningococcus and other pathogens.

SUMMARY OF THE INVENTION

[0007] The inventors have shown that serogroup B meningococcus antigens can successfully be combined with diphtheria, tetanus and pertussis toxoids ("DTP") to provide effective combination vaccines for protecting against multiple pathogens. These combinations are effective with a range of different adjuvants, and with both pediatric-type and booster-type DTP ratios. The adjuvant can improve the immune response which the composition elicits; alternatively, by including an adjuvant it is possible for the compositions to have a relatively lower amount of antigen while nevertheless having immunogenicity which is comparable to unadjuvanted combination vaccines.

[0008] In general, therefore, the invention provides an immunogenic composition comprising (a) a serogroup B meningococcus immunogen and (b) at least one of a diphtheria toxoid, a tetanus toxoid, and/or a pertussis toxoid. The composition will usually also include an adjuvant, such as an aluminium salt or an oil-in-water emulsion. Preferably component (b) includes all three of a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid. In some embodiments component (b) includes more diphtheria toxoid than tetanus toxoid (measured in Lf units), but in other embodiments it includes more tetanus toxoid than diphtheria toxoid.

[0009] In a first embodiment the invention provides an immunogenic composition comprising: (a) a serogroup B meningococcus immunogen; (b) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid; and (c) an adjuvant. The adjuvant can comprise one or more of an aluminium salt adjuvant, a TLR agonist, or an oil-in-water emulsion.

[0010] In a second embodiment the invention provides an immunogenic composition comprising: (a) a serogroup B meningococcus immunogen; and (b) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, wherein the diphtheria toxoid is present in an excess relative to tetanus toxoid as measured in Lf units. This composition can also include an adjuvant, and this can comprise one or more of an aluminium salt adjuvant, a TLR agonist, or an oil-in-water emulsion.

[0011] In a third embodiment the invention provides an immunogenic composition comprising: (a) a serogroup B meningococcus immunogen; and (b) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, wherein the tetanus toxoid is present in an excess relative to diphtheria toxoid as measured in Lf units. This composition can also include an adjuvant, and this can comprise one or more of an aluminium salt adjuvant, a TLR agonist, or an oil-in-water emulsion.

[0012] Compositions of the invention can include antigens in addition to diphtheria toxoid, tetanus toxoid, and pertussis toxoid e.g. they can include Hib capsular saccharide (ideally conjugated), HBsAg, IPV, meningococcal capsular saccharide (ideally conjugated), etc.

[0013] Serogroup B Meningococcus Immunogens

[0014] Immunogenic compositions of the invention include a serogroup B meningococcus immunogen. When administered to human beings (or to a suitable animal model) the immunogen can elicit a bactericidal immune response. These immunogens can be proteins, liposaccharides, or vesicles.

[0015] Various serogroup B meningococcus protein immunogens are known in the art, including but not limited to NHBA, fHbp and NadA as found in the BEXSERO.TM. product [7,8]. Further protein immunogens which can be included in compositions of the invention are HmbR, NspA, NhhA, App, Omp85, TbpA, TbpB, Cu,Zn-superoxide dismutase, and ZnuD. Further details of these immunogens are discussed below.

[0016] A vaccine may include one or more of these various immunogens e.g. it can include each of NHBA, fHbp and NadA. It can also include variant forms of a single immunogen e.g. it can include more than one variant of meningococcal fHbp (i.e. two fHbp proteins with different sequences [191, 9]).

[0017] The serogroup B meningococcus protein immunogens can be present as fusion proteins. For instance, the BEXSERO.TM. product includes two fusion proteins: SEQ ID NO: 4 is a fusion of NMB2091 and a fHbp; and SEQ ID NO: 5 is a fusion of a NHBA and NMB1030. One useful fusion protein is SEQ ID NO: 19, which includes NMB2091 and two copies of a fHbp.

[0018] Two useful combinations of serogroup B immunogens include: a NHBA e.g. SEQ ID NO: 5; a fHbp e.g. either SEQ ID NO: 4 or SEQ ID NO: 19; and a NadA e.g. SEQ ID NO: 6. Other useful combinations include proteins which differ from SEQ ID NOs: 5, 4, 19 & 6 by up to 5 amino acids each but which retain the ability to elicit antibodies which recognise SEQ ID NOs: 5, 4, 19 & 6.

[0019] Compositions which include at least one fHbp immunogen are preferred e.g. those containing two different fHbp sequences. Details of suitable fHbp combinations are discussed below.

[0020] Thus compositions of the invention can usefully include (a) the mixture of three serogroup B meningococcus protein immunogens disclosed as `5CVMB` in reference 8 or (b) the mixture of serogroup B meningococcus protein immunogens disclosed as `rLP2086` in reference 10.

[0021] Usually, the serogroup B meningococcus immunogens are purified soluble recombinant proteins. In some embodiments, however, they can be present in meningococcal vesicles. Thus the composition can include mcningococcal vesicles i.e. any protcoliposomic vesicle obtained by disruption of or blebbing from a meningococcal outer membrane to form vesicles therefrom that retain antigens from the outer membrane. Thus this term includes, for instance, OMVs (sometimes referred to as `blebs`), microvesicles (MVs) and `native OMVs` (`NOMVs`). Various such vesicles are known in the art (e.g. see references 11 to 25) and any of these can be included within a composition of the invention. Further details of these vesicles are given below. In some embodiments, however, the composition is vesicle-free.

[0022] A composition of the invention can preferably elicit a serum bactericidal assay after being administered. 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. For instance, a composition may induce at least a 4-fold rise in SBA in more than 90% of recipients.

[0023] A composition of the invention can preferably elicit an immune response in human beings which is protective against serogroup B meningococcus. For instance, the vaccine may elicit an immune response which is protective at least against a prototype serogroup B strain such as MC58, which is widely available (e.g. ATCC BAA-335) and was the strain sequenced in reference 26. Other strains can also be used, but a response against MC58 is easily tested.

[0024] Diphtheria Toxoid

[0025] Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positive non-sporing aerobic bacterium. This organism expresses a prophage-encoded ADP-ribosylating exotoxin (`diphtheria toxin`), which can be treated (e.g. using formaldehyde) to give a toxoid that is no longer toxic but that remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. Diphtheria toxoids are disclosed in more detail in chapter 13 of reference 1. Preferred diphtheria toxoids are those prepared by formaldehyde treatment. The diphtheria toxoid can be obtained by growing C. diphtheriae in growth medium (e.g. Fenton medium, or Linggoud & Fenton medium), which may be supplemented with bovine extract, followed by formaldehyde treatment, ultrafiltration and precipitation. The toxoided material may then be treated by a process comprising sterile filtration and/or dialysis.

[0026] A composition should include enough diphtheria toxoid to elicit circulating diphtheria antitoxin levels of at least 0.01 IU/ml. Quantities of diphtheria toxoid are generally measured in the `Lf` unit ("flocculating units", or the "limes flocculating dose", or the "limit of flocculation"), defined as the amount of toxin/toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture [27,28]. For example, the NIBSC supplies `Diphtheria Toxoid, Plain` [29], which contains 300 LF per ampoule, and also supplies `The 1st International Reference Reagent For Diphtheria Toxoid For Flocculation Test` [30] which contains 900 Lf per ampoule. The concentration of diphtheria toxoid in a composition can readily be determined using a flocculation assay by comparison with a reference material calibrated against such reference reagents.

[0027] The immunizing potency of diphtheria toxoid in a composition is generally expressed in international units (IU). The potency can be assessed by comparing the protection afforded by a composition in laboratory animals (typically guinea pigs) with a reference vaccine that has been calibrated in IUs. NIBSC supplies the `Diphtheria Toxoid Adsorbed Third International Standard 1999` [31,32], which contains 160 IU per ampoule, and is suitable for calibrating such assays.

[0028] The conversion between IU and Lf systems depends on the particular toxoid preparation.

[0029] Compositions of the invention typically include, per unit dose, between 1-40 Lf diphtheria toxoid. In a pediatric-type composition, where the diphtheria toxoid is present in an excess relative to tetanus toxoid (in Lf units), the composition will generally include between 10-35 Lf diphtheria toxoid per unit dose e.g. between 15-30 Lf, such as 15, 25 or 30 Lf. In a booster-type composition, where tetanus toxoid is present in an excess relative to the diphtheria toxoid (in Lf units), the composition will generally include between 1-4 Lf diphtheria toxoid per unit dose e.g. between 1.5-3 Lf, such as 2 or 2.5 Lf. If a composition includes saccharide antigen(s) conjugated to diphtheria toxoid then these amounts exclude the amount of carrier protein in those conjugate(s).

[0030] By IU measurements, pediatric-type compositions will generally include .gtoreq.25 IU diphtheria toxoid per unit dose, whereas booster-type compositions will generally include 1-3 IU per unit dose.

[0031] If a composition includes an aluminium salt adjuvant then diphtheria toxoid in the composition is preferably adsorbed (more preferably totally adsorbed) onto it, and preferably onto an aluminium hydroxide adjuvant.

[0032] Tetanus Toxoid

[0033] Tetanus is caused by Clostridium tetani, a Gram-positive, spore-forming bacillus. This organism expresses an endopeptidase (`tetanus toxin`), which can be treated to give a toxoid that is no longer toxic but that remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. Tetanus toxoids are disclosed in more detail in chapter 27 of reference 1. Preferred tetanus toxoids are those prepared by formaldehyde treatment. The tetanus toxoid can be obtained by growing C. tetani in growth medium (e.g. a Latham medium derived from bovine casein), followed by fobrmaldehyde treatment, ultrafiltration and precipitation. The material may then be treated by a process comprising sterile filtration and/or dialysis.

[0034] A composition should include enough tetanus toxoid to elicit circulating tetanus antitoxin levels of at least 0.01 IU/ml. Quantities of tetanus toxoid are generally expressed in `Lf` units (see above), defined as the amount of toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture [27]. The NIBSC supplies `The 1st International Reference Reagent for Tetanus Toxoid For Flocculation Test` [33] which contains 1000 LF per ampoule, by which measurements can be calibrated.

[0035] The immunizing potency of tetanus toxoid is measured in international units (IU), assessed by comparing the protection afforded by a composition in laboratory animals (typically guinea pigs) with a reference vaccine e.g. using NIBSC's `Tetanus Toxoid Adsorbed Third International Standard 2000` [34,35], which contains 469 IU per ampoule.

[0036] The conversion between IU and Lf systems depends on the particular toxoid preparation.

[0037] Compositions of the invention typically include between 2.5-25 Lf of tetanus toxoid per unit dose. In a pediatric-type composition, where diphtheria toxoid is present in an excess relative to the tetanus toxoid (in Lf units), the composition will generally include between 4-15 Lf tetanus toxoid per unit dose e.g. between 5-10 Lf, such as 5 or 10 Lf. In a booster-type composition, where the tetanus toxoid is present in an excess relative to diphtheria toxoid (in Lf units), the composition will generally include between 4-6 Lf tetanus toxoid per unit dose e.g. 5 Lf. If a composition includes saccharide antigen(s) conjugated to tetanus toxoid then these amounts exclude the amount of carrier protein in those conjugate(s).

[0038] By IU measurements, pediatric-type compositions will generally include .gtoreq.40 IU tetanus toxoid per unit dose, whereas booster-type compositions will generally include 15-25 IU per unit dose.

[0039] If a composition includes an aluminium salt adjuvant then tetanus toxoid in the composition is preferably adsorbed (sometimes totally adsorbed) onto an aluminium salt, preferably onto an aluminium hydroxide adjuvant.

[0040] Pertussis Toxoid

[0041] Bordetella pertussis causes whooping cough. Compositions of the invention include pertussis toxoid (`PT`) i.e. a detoxified form of pertussis toxin. The invention can use a PT-containing whole-cell pertussis antigen ("wP") but preferably a composition is free from wP and instead includes an acellular ("aP") PT-containing antigen i.e. a defined mixture of purified pertussis antigens. When using an aP antigen a composition of the invention will typically include, in addition to the PT, filamentous hemagglutinin (FHA) and/or pertactin (also known as the `69 kiloDalton outer membrane protein`). It can also optionally include fimbriae types 2 and 3. Preparation of these various Pa antigens is well known in the art.

[0042] PT can be detoxified by treatment with formaldehyde and/or glutaraldehyde, and FHA and pertactin can also be treated in the same way. As an alternative to chemical detoxification of PT, the invention can use a mutant PT in which wild-type enzymatic activity has been reduced by mutagenesis [36] e.g. the 9K/129G double mutant [37]. The use of such genetically-detoxified PT is preferred,

[0043] Quantities of acellular pertussis antigens are usually expressed in micrograms. Compositions of the invention typically include between 2-30 .mu.g PT per unit dose. In a pediatric-type composition, PT can be present at between 5-30 .mu.g per unit dose (e.g. 5, 7.5, 20 or 25 .mu.g), whereas in a booster-type composition the composition will generally include between 2-0 .mu.g PT per unit dose (e.g. 2.5 .mu.g or 8 .mu.g). Where a composition includes FHA, it is typically present between 2-30 .mu.g per unit dose. In a pediatric-type composition, FHA can be present at between 2.5-25 .mu.g per unit dose (e.g. 2.5, 5, 10, 20 or 25 .mu.g), whereas in a booster-type composition FHA can be present at between 4-10 .mu.g per unit dose (e.g. 5 .mu.g or 8 .mu.g). Where a composition includes pertactin, this is typically present between 2-10 .mu.g per unit dose. In a pediatric-type composition, pertactin can be present at between 2.5-10 .mu.g per unit dose (e.g. 2.5, 3, 8 or 10 .mu.g), whereas in a booster-type composition pertactin can be present at between 2-3 .mu.g per unit dose (e.g. 2.5 .mu.g or 3 .mu.g).

[0044] A composition normally contains .ltoreq.80 .mu.g per unit dose of total acellular pertussis antigens. Each individual antigen will usually be present at .ltoreq.30 .mu.g per unit dose.

[0045] It is usual that each of PT. FHA and pertactin are present in a composition of the invention. These may be present at various ratios (by mass), such as PT:FHA:p69 ratios of 20:20:3, 25:25:8, 16:16:5, 5:10:6, or 10:5:3. It is usual to have a mass excess of FHA relative to pertactin if both are present.

[0046] If a composition includes an aluminium salt adjuvant then PT in the composition is preferably adsorbed (sometimes totally adsorbed) onto an aluminium salt, preferably onto an aluminium hydroxide adjuvant. Any FHA can also be adsorbed to the aluminium salt. Any pertactin can be adsorbed to the aluminium salt adjuvant, but the presence of pertactin normally means that the composition requires the presence of aluminium hydroxide to ensure stable adsorption [38].

[0047] Hib Conjugates

[0048] Haemophilus influenzae type b (`Hib`) causes bacterial meningitis. Hib vaccines are typically based on the capsular saccharide antigen (e.g. chapter 14 of ref. 1), the preparation of which is well documented (e.g. references 39 to 48). The Hib saccharide is conjugated to a carrier protein in order to enhance its immunogenicity, especially in children. Typical carrier proteins are tetanus toxoid, diphtheria toxoid, the CRM197 derivative of diphtheria toxoid, or the outer membrane protein complex from serogroup B meningococcus. Tetanus toxoid is a useful carrier, as used in the product commonly referred to as `PRP-T`. PRP-T can be made by activating a Hib capsular polysaccharide using cyanogen bromide, coupling the activated saccharide to an adipic acid linker (such as (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), typically the hydrochloride salt), and then reacting the linker-saccharide entity with a tetanus toxoid carrier protein. CRM197 is another useful carrier for Hib conjugate in compositions of the invention.

[0049] The saccharide moiety of the conjugate may comprise full-length polyribosylribitol phosphate (PRP) as prepared from Hib bacteria, and/or fragments of full-length PRP. Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e. excess protein) and 5:1 (i.e. excess saccharide) may be used e.g. ratios between 1:2 and 5:1 and ratios between 1:1.25 and 1:2.5. In preferred vaccines, however, the weight ratio of saccharide to carrier protein is between 1:2.5 and 1:3.5. In vaccines where tetanus toxoid is present both as an antigen and as a carrier protein then the weight ratio of saccharide to carrier protein in the conjugate may be between 1:0.3 and 1:2 [49]. Administration of the Hib conjugate preferably results in an anti-PRP antibody concentration of .gtoreq.0.15 .mu.g/ml, and more preferably .gtoreq.1 .mu.g/ml, and these are the standard response thresholds.

[0050] Quantities of Hib antigens are typically expressed in micrograms of saccharide. If a composition of the invention includes a Hib antigen then a normal quantity per unit dose is between 5-15 .mu.g e.g. 10 .mu.g or 12 .mu.g.

[0051] If a composition includes an aluminium salt adjuvant then Hib antigen can be adsorbed onto it or can be unadsorbed.

[0052] Hepatitis B Virus Surface Antigen

[0053] Hepatitis B virus (HBV) is one of the known agents which causes viral hepatitis. The HBV virion consists of an inner core surrounded by an outer protein coat or capsid, and the viral core contains the viral DNA genome. The major component of the capsid is a protein known as HBV surface antigen or, more commonly, `HBsAg`, which is typically a 226-amino acid polypeptide with a molecular weight of .about.24 kDa. All existing hepatitis B vaccines contain HBsAg, and when this antigen is administered to a normal vaccinee it stimulates the production of anti-HBsAg antibodies which protect against HBV infection.

[0054] For vaccine manufacture, HBsAg can be made in two ways. The first method involves purifying the antigen in particulate form from the plasma of chronic hepatitis B carriers, as large quantities of HBsAg are synthesized in the liver and released into the blood stream during an HBV infection. The second way involves expressing the protein by recombinant DNA methods. HBsAg for use with the method of the invention is recombinantly expressed e.g. in yeast or CHO cells. Suitable yeasts include Saccharomyces (such as S. cerevisiae) or Hanensula (such as H. polymorpha) hosts.

[0055] Unlike native HBsAg (i.e. as in the plasma-purified product), yeast-expressed HBsAg is generally non-glycosylated, and this is the most preferred form of HBsAg for use with the invention. Yeast-expressed HBsAg is highly immunogenic and can be prepared without the risk of blood product contamination.

[0056] The HBsAg will generally be in the form of substantially-spherical particles (average diameter of about 20 nm), including a lipid matrix comprising phospholipids. Yeast-expressed HBsAg particles may include phosphatidylinositol, which is not found in natural HBV virions. The particles may also include a non-toxic amount of LPS in order to stimulate the immune system [50]. The particles may retain non-ionic surfactant (e.g. polysorbate 20) if this was used during disruption of yeast [51].

[0057] A preferred method for HBsAg purification involves, after cell disruption: ultrafiltration; size exclusion chromatography; anion exchange chromatography; ultracentrifugation; desalting; and sterile filtration. Lysates may be precipitated after cell disruption (e.g. using a polyethylene glycol), leaving HBsAg in solution, ready for ultrafiltration.

[0058] After purification HBsAg may be subjected to dialysis (e.g. with cysteine), which can be used to remove any mercurial preservatives such as thimerosal that may have been used during HBsAg preparation [52]. Thimerosal-free preparation is preferred.

[0059] The HBsAg is preferably from HBV subtype adw2.

[0060] Quantities of HBsAg are typically expressed in micrograms. If a composition of the invention includes HBsAg then a normal quantity per unit dose is between 5-25 .mu.g e.g. 10 .mu.g or 20 .mu.g.

[0061] If a composition includes an aluminium salt adjuvant then HBsAg can be adsorbed onto it (preferably adsorbed onto an aluminium phosphate adjuvant).

[0062] Inactivated Poliovirus Antigen (IPV)

[0063] Poliomyelitis can be caused by one of three types of poliovirus. The three types are similar and cause identical symptoms, but they are antigenically very different and infection by one type does not protect against infection by others. As explained in chapter 24 of reference 1, it is therefore preferred to use three poliovirus antigens with the invention--poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirus Type 3 (e.g. Saukett strain). As an alternative to these strains ("Salk" strains), Sabin strains of types 1 to 3 can be used e.g. as discussed in references 53 & 54. These strains can be more potent than the normal Salk strains.

[0064] Polioviruses may be grown in cell culture. A preferred culture uses a Vero cell line, which is a continuous cell line derived from monkey kidney. Vero cells can conveniently be cultured microcarriers. Culture of the Vero cells before and during viral infection may involve the use of bovine-derived material, such as calf serum, and of lactalbumin hydrolysate (e.g. obtained by enzymatic degradation of lactalbumin). Such bovine-derived material should be obtained from sources which are free from BSE or other TSEs.

[0065] After growth, virions may be purified using techniques such as ultrafiltration, diafiltration, and chromatography. Prior to administration to patients, polioviruses must be inactivated, and this can be achieved by treatment with formaldehyde before the viruses are used in the process of the invention.

[0066] The viruses are preferably grown, purified and inactivated individually, and are then combined to give a bulk mixture for use with the invention.

[0067] Quantities of IPV are typically expressed in the `DU` unit (the "D-antigen unit" [55]). Where all three of Types 1, 2 and 3 poliovirus are present the three antigens can be present at a DU ratio of 5:1:4 respectively, or at any other suitable ratio e.g. a ratio of 15:32:45 when using Sabin strains [53]. Typical amounts of Salk IPV strains per unit dose are 40 DU type 1, 8 DU type 2 and 32 DU type 3, although lower doses can also be used. A low amount of antigen from Sabin strains is particularly useful, with .ltoreq.15 DU type 1, .ltoreq.5 DU type 2, and .ltoreq.25 DU type 3 (per unit dose).

[0068] If a composition includes an aluminium salt adjuvant then IPV antigens are often not pre-adsorbed to any adjuvant before they are formulated, but after formulation they may become adsorbed onto the aluminium salt(s).

[0069] Further Antigens

[0070] Compositions of the invention include D, T, and P antigens. As mentioned above, they may also include Hib, HBsAg, and/or poliovirus antigens. Immunogenic compositions of the invention may include antigens from further pathogens. For example, these antigens may be from N. meningitidis (one or more of serogroups A, B, C, W135 and/or Y) or S. pneumoniae.

[0071] Meningococcal Saccharides

[0072] Where a composition includes a Neisseria meningitidis capsular saccharide conjugate there may be one or more than one such conjugate. Including 2, 3, or 4 of serogroups A, C, W135 and Y is typical 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 useful, as in the MENACTRA.TM. and MENVEO.TM. products. Where conjugates from more than one serogroup are included then they may be present at substantially equal masses e.g. the mass of each serogroup's saccharide is within +10% of each other. A typical quantity per serogroup is between 1 .mu.g and 20 .mu.g e.g. between 2 and 10 .mu.g per serogroup, or about 4 .mu.g or about 5 .mu.g or about 10 .mu.g. As an alternative to a substantially equal ratio, a double mass of serogroup A saccharide may be used.

[0073] Administration of a conjugate preferably results in an increase in serum bactericidal assay (SBA) titre for the relevant serogroup of at least 4-fold, and preferably at least 8-fold. SBA titres can be measured using baby rabbit complement or human complement [56].

[0074] The capsular saccharide of serogroup A meningococcus is a homopolymer of (.alpha.l.fwdarw.6)-linked N-acetyl-D-mannosamine-1-phosphate, with partial O-acetylation in the C3 and C4 positions. Acetylation at the C-3 position can be 70-95%. Conditions used to purify the saccharide can result in de-O-acetylation (e.g. under basic conditions), but it is useful to retain OAc at this C-3 position. In some embodiments, at least 50% (e.g. at least 60%, 70%, 80%, 90%, 95% or more) of the mannosamine residues in a serogroup A saccharides are O-acetylated at the C-3 position. Acetyl groups can be replaced with blocking groups to prevent hydrolysis [57], and such modified saccharides are still serogroup A saccharides within the meaning of the invention.

[0075] The serogroup C capsular saccharide is a homopolymer of (.alpha.2.fwdarw.9)-linked sialic acid (N-acetyl neuraminic acid, or `NeuNAc`). The saccharide structure is written as .fwdarw.9)-Neu p NAc 7/8 OAc-(.alpha.2.fwdarw.. Most serogroup C strains have O-acetyl groups at C-7 and/or C-8 of the sialic acid residues, but about 15% of clinical isolates lack these O-acetyl groups [58,59]. The presence or absence of OAc groups generates unique epitopes, and the specificity of antibody binding to the saccharide may affect its bactericidal activity against O-acetylated (OAc-) and de-O-acetylated (OAc+) strains [60-62]. Serogroup C saccharides used with the invention may be prepared from either OAc+ or OAc- strains. Licensed MenC conjugate vaccines include both OAc- (NEISVAC-C.TM.) and OAc+ (MENJUGATE.TM. & MENINGITEC.TM.) saccharides. In some embodiments, strains for production of serogroup C conjugates are OAc+ strains, e.g. of serotype 16, serosubtype P1.7a,1, etc. Thus C:16:P1.7a,1 OAc+ strains may be used. OAc+ strains in serosubtype P1.1 are also useful, such as the C11 strain. Preferred MenC saccharides are taken from OAc+ strains, such as strain C11.

[0076] The serogroup W135 saccharide is a polymer of sialic acid-galactose disaccharide units. Like the serogroup C saccharide, it has variable O-acetylation, but at sialic acid 7 and 9 positions [63]. The structure is written as: .fwdarw.4)-D-Neup5Ac(7/9OAc)-.alpha.-(2.fwdarw.6)-D-Gal-.alpha.-(1.fwdarw- ..

[0077] The serogroup Y saccharide is similar to the serogroup W135 saccharide, except that the disaccharide repeating unit includes glucose instead of galactose. Like serogroup W135, it has variable O-acetylation at sialic acid 7 and 9 positions [63]. The serogroup Y structure is written as: .fwdarw.4)-D-Neup5Ac(7/9OAc)-.alpha.-(2.fwdarw.6)-D-Glc-.alpha.-(1.fwdarw- ..

[0078] The saccharides used according to the invention may be O-acetylated as described above (e.g. with the same O-acetylation pattern as seen in native capsular saccharides), or they may be partially or totally de-O-acetylated at one or more positions of the saccharide rings, or they may be hyper-O-acetylated relative to the native capsular saccharides. For example, reference 64 reports the use of serogroup Y saccharides that are more than 80% de-O-acetylated.

[0079] The saccharide moieties in meningococcal conjugates may comprise full-length saccharides as prepared from meningococci, and/or may comprise fragments of full-length saccharides i.e. the saccharides may be shorter than the native capsular saccharides seen in bacteria. The saccharides may thus be depolymerised, with depolymerisation occurring during or after saccharide purification but before conjugation. Depolymerisation reduces the chain length of the saccharides. One depolymerisation method involves the use of hydrogen peroxide [65]. Hydrogen peroxide is added to a saccharide (e.g. to give a final H.sub.2O.sub.2 concentration of 1%), and the mixture is then incubated (e.g. at about 55.degree. C.) until a desired chain length reduction has been achieved. Another depolymerisation method involves acid hydrolysis [66]. Other depolymerisation methods are known in the art. The saccharides used to prepare conjugates for use according to the invention may be obtainable by any of these depolymerisation methods. Depolymerisation can be used in order to provide an optimum chain length for immunogenicity and/or to reduce chain length for physical manageability of the saccharides. In some embodiments, saccharides have the following range of average degrees of polymerisation (Dp): A=10-20; C=12-22; W135=15-25; Y=15-25. In terms of molecular weight, rather than Dp, useful ranges are, for all serogroups: <100 kDa; 5 kDa-75 kDa; 7 kDa-50 kDa; 8 kDa-35 kDa; 12 kDa-25 kDa; 15 kDa-22 kDa. In other embodiments, the average molecular weight for saccharides from each of meningococcal serogroups A, C, W135 and Y may be more than 50 kDa e.g. .gtoreq.75 kDa, .gtoreq.100 kDa, .gtoreq.110 kDa, .gtoreq.120 kDa, .gtoreq.130 kDa, etc. [67], and even up to 1500 kDa, in particular as determined by MALLS. For instance: a MenA saccharide may be in the range 50-500 kDa e.g. 60-80 kDa; a MenC saccharide may be in the range 100-210 kDa; a MenW135 saccharide may be in the range 60-190 kDa e.g. 120-140 kDa; and/or a MenY saccharide may be in the range 60-190 kDa e.g. 150-160 kDa.

[0080] If a component or composition includes both Hib and meningococcal conjugates then, in some embodiments, the mass of Hib saccharide can be substantially the same as the mass of a particular meningococcal serogroup saccharide. In some embodiments, the mass of Hib saccharide will be more than (e.g. at least 1.5.times.) the mass of a particular meningococcal serogroup saccharide. In some embodiments, the mass of Hib saccharide will be less than (e.g. at least 1.5.times. less) the mass of a particular meningococcal serogroup saccharide.

[0081] Where a composition includes saccharide from more than one meningococcal serogroup, there is an mean saccharide mass per serogroup. If substantially equal masses of each serogroup are used then the mean mass will be the same as each individual mass; where non-equal masses are used then the mean will differ e.g. with a 10:5:5:5 .mu.g amount for a MenACWY mixture, the mean mass is 6.25 .mu.g per serogroup. In some embodiments, the mass of Hib saccharide will be substantially the same as the mean mass of meningococcal saccharide per serogroup. In some embodiments, the mass of Hib saccharide will be more than (e.g. at least 1.5.times.) the mean mass of meningococcal saccharide per serogroup. In some embodiments, the mass of Hib saccharide will be less than (e.g. at least 1.5.times.) the mean mass of meningococcal saccharide per serogroup [68].

[0082] Pneumococcal Saccharides

[0083] Streptococcus pneumoniae causes bacterial meningitis and existing vaccines are based on capsular saccharides. Thus compositions of the invention can include at least one pneumococcal capsular saccharide conjugated to a carrier protein.

[0084] The invention can include capsular saccharide from one or more different pneumococcal serotypes. Where a composition includes saccharide antigens from more than one serotype, these are preferably prepared separately, conjugated separately, and then combined. Methods for purifying pneumococcal capsular saccharides are known in the art (e.g. see reference 69) and vaccines based on purified saccharides from 23 different serotypes have been known for many years. Improvements to these methods have also been described e.g. for serotype 3 as described in reference 70, or for serotypes 1, 4, 5, 6A, 6B, 7F and 19A as described in reference 71.

[0085] Pneumococcal capsular saccharide(s) will typically be selected from the following 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. Thus, in total, a composition may include a capsular saccharide from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more different serotypes. Compositions which include at least serotype 6B saccharide are useful.

[0086] A useful combination of serotypes is a 7-valent combination e.g. including capsular saccharide from each of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Another useful combination is a 9-valent combination e.g. including capsular saccharide from each of serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. Another useful combination is a 10-valent combination e.g. including capsular saccharide from each of 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; or 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; 6A and 19A, etc.

[0087] Thus a useful 13-valent combination includes capsular saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19 (or 19A), 19F and 23F e.g. prepared as disclosed in references 72 to 75. One such combination includes serotype 6B saccharide at about 8 .mu.g/ml and the other 12 saccharides at concentrations of about 4 .mu.g/ml each. Another such combination includes serotype 6A and 6B saccharides at about 8 .mu.g/ml each and the other 11 saccharides at about 4.+-.g/ml each.

[0088] Suitable carrier proteins for conjugates include bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. These are commonly used in conjugate vaccines. For example, the CRM197 diphtheria toxin mutant is useful [76]. Other suitable carrier proteins include synthetic peptides [77,78], heat shock proteins [79,80], pertussis proteins [81,82], cytokines [83], lymphokines [83], hormones [83], growth factors [83], artificial proteins comprising multiple human CD4.sup.+ T cell epitopes from various pathogen-derived antigens [84] such as N19 [85], protein D from H. influenzae [86-88], pneumolysin [89] or its non-toxic derivatives [90], pneumococcal surface protein PspA [91], iron-uptake proteins [92], toxin A or B from C. difficile [93], recombinant Pseudomonas aeruginosa exoprotein A (rEPA) [94], etc.

[0089] Particularly useful carrier proteins for pneumococcal conjugate vaccines are CRM197, tetanus toxoid, diphtheria toxoid and H. influenzae protein D. CRM197 is used in PREVNAR.TM.. A 13-valent mixture may use CRM197 as the carrier protein for each of the 13 conjugates, and CRM197 may be present at about 55-60 .mu.g/ml.

[0090] Where a composition includes conjugates from more than one pneumococcal serotype, it is possible to use the same carrier protein for each separate conjugate, or to use different carrier proteins. In both cases, though, a mixture of different conjugates will usually be formed by preparing each serotype conjugate separately, and then mixing them to form a mixture of separate conjugates. Reference 95 describes potential advantages when using different carrier proteins in multivalent pneumococcal conjugate vaccines, but the PREVNAR.TM. product successfully uses the same carrier for each of seven different serotypes.

[0091] A carrier protein may be covalently conjugated to a pneumococcal saccharide directly or via a linker. Various linkers are known. For example, attachment may be via a carbonyl, which may be formed by reaction of a free hydroxyl group of a modified saccharide with CDI [96.97]followed by reaction with a protein to form a carbamate linkage. Carbodiimide condensation can be used [98]. An adipic acid linker can be used, which may be formed by coupling a free --NH.sub.2 group (e.g. introduced to a saccharide by amination) with adipic acid (using, for example, diimide activation), and then coupling a protein to the resulting saccharide-adipic acid intermediate [99,100]. Other linkers include .beta.-propionamido [101], nitrophenyl-ethylamine [102], haloacyl halides [103], glycosidic linkages [104], 6-aminocaproic acid [105], N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [106], adipic acid dihydrazide ADH [107], C.sub.4 to C.sub.12 moieties [108], etc.

[0092] Conjugation via reductive amination can be used. The saccharide may first be oxidised with periodate to introduce an aldehyde group which can then form a direct covalent linkage to a carrier protein by reductive amination e.g. to a lysine's .epsilon.-amino group. If the saccharide includes multiple aldehyde groups per molecule then this linkage technique can lead to a cross-linked product, where multiple aldehydes react with multiple carrier amines. This cross-linking conjugation technique is particularly useful for at least pneumococcal serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

[0093] A pneumococcal saccharide may comprise a full-length intact saccharide as prepared from pneumococcus, and/or may comprise fragments of full-length saccharides i.e. the saccharides may be shorter than the native capsular saccharides seen in bacteria. The saccharides may thus be depolymerised, with depolymerisation occurring during or after saccharide purification but before conjugation. Depolymerisation reduces the chain length of the saccharides.

[0094] Depolymerisation can be used in order to provide an optimum chain length for immunogenicity and/or to reduce chain length for physical manageability of the saccharides. Where more than one pneumococcal serotype is used then it is possible to use intact saccharides for each serotype, fragments for each serotype, or to use intact saccharides for some serotypes and fragments for other serotypes.

[0095] Where a composition includes saccharide from any of serotypes 4, 6B, 9V, 14, 19F and 23F, these saccharides are preferably intact. In contrast, where a composition includes saccharide from serotype 18C, this saccharide is preferably depolymerised.

[0096] A serotype 3 saccharide may also be depolymerised, For instance, a serotype 3 saccharide can be subjected to acid hydrolysis for depolymerisation [72] e.g. using acetic acid. The resulting fragments may then be oxidised for activation (e.g. periodate oxidation, maybe in the presence of bivalent cations e.g. with MgCl.sub.2), conjugated to a carrier (e.g. CRM197) under reducing conditions (e.g. using sodium cyanoborohydride), and then (optionally) any unreacted aldehydes in the saccharide can be capped (e.g. using sodium borohydride) [72]. Conjugation may be performed on lyophilized material e.g. after co-lyophilizing activated saccharide and carrier. A serotype 1 saccharide may be at least partially de-O-acetylated e.g. achieved by alkaline pH buffer treatment [73] such as by using a bicarbonate/carbonate buffer. Such (partially) de-O-acetylated saccharides can be oxidised for activation (e.g. periodate oxidation), conjugated to a carrier (e.g. CRM197) under reducing conditions (e.g. using sodium cyanoborohydride), and then (optionally) any unreacted aldehydes in the saccharide can be capped (e.g. using sodium borohydride) [73]. Conjugation may be performed on lyophilized material e.g. after co-lyophilizing activated saccharide and carrier.

[0097] A serotype 19A saccharide may be oxidised for activation (e.g. periodate oxidation), conjugated to a carrier (e.g. CRM197) in DMSO under reducing conditions, and then (optionally) any unreacted aldehydes in the saccharide can be capped (e.g. using sodium borohydride) [109]. Conjugation may be performed on lyophilized material e.g. after co-lyophilizing activated saccharide and carrier.

[0098] One or more pneumococcal capsular saccharide conjugates may be present in lyophilised form.

[0099] Pneumococcal conjugates can ideally elicit anticapsular antibodies that bind to the relevant saccharide e.g. elicit an anti-saccharide antibody level .gtoreq.0.20 .mu.g/mL [110]. The antibodies may be evaluated by enzyme immunoassay (EIA) and/or measurement of opsonophagocytic activity (OPA). The EIA method has been extensively validated and there is a link between antibody concentration and vaccine efficacy.

[0100] Adjuvants

[0101] Compositions of the invention can include an adjuvant, such as (i) an oil-in-water emulsion (ii) at least one aluminium salt or (iii) at least one TLR agonist.

[0102] In some embodiments a composition includes a mixture of an aluminium salt and a TLR agonist, and the TLR agonist can be adsorbed to the aluminium salt to improve adjuvant effects [142]. This can lead to a better (stronger, or more quickly achieved) immune response and/or can permit a reduction in the amount of aluminium in the composition while maintaining an equivalent adjuvant effect.

[0103] Where a composition includes aluminium salt adjuvant(s) then between one and all of the immunogens in the composition can be adsorbed to the salt(s). Moreover, if the composition includes a TLR adjuvant then this can also be adsorbed to the salt(s), as discussed below.

[0104] Where a composition includes an aluminium salt adjuvant then preferably it does not also include an oil-in-water emulsion adjuvant. Conversely, where a composition includes an oil-in-water emulsion adjuvant then preferably it does not also include an aluminium salt adjuvant.

[0105] Oil-in-Water Emulsion Adjuvants

[0106] According to the invention's second aspect a vaccine is adjuvanted with an oil-in-water emulsion. Various such emulsions are known e.g. MF59 and AS03 are both authorised in Europe.

[0107] Useful emulsion adjuvants they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible. The oil droplets in the emulsion generally have a sub-micron diameter, and these small sizes can readily be achieved with a microfluidiser to provide stable emulsions, or by alternative methods e.g. phase inversion. Emulsions in which at least 80% (by number) of droplets have a diameter of less than 220 nm are preferred, as they can be subjected to filter sterilization.

[0108] The emulsion can include oil(s) from an animal (such as fish) and/or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolisable and may therefore be used with the invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.

[0109] Most fish contain metabolisable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred for use with the invention (see below). Squalane, the saturated analog to squalene, is also a useful oil. Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art. Other preferred oils are the tocopherols (see below). Mixtures of oils can be used.

[0110] Preferred amounts of total oil (% by volume) in an adjuvant emulsion are between 1 and 20% e.g. between 2-10%. A squalane content of 5% by volume is particularly useful.

[0111] Surfactants can be classified by their `HLB` (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10 e.g. about 15. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 or polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX.TM. tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol.TM. NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85) or sorbitan monolaurate.

[0112] Emulsions used with the invention preferably include non-ionic surfactant(s). Preferred surfactants for including in the emulsion are polysorbate 80 (polyoxyethylene sorbitan monooleate; Tween 80), Span 85 (sorbitan trioleate), lecithin or Triton X-100. Mixtures of surfactants can be used e.g. a mixture of polysorbate 80 and sorbitan trioleate. A combination of a polyoxyethylene sorbitan ester such as polysorbate 80 (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also useful. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol. Where a mixture of surfactants is used then the HLB of the mixture is calculated according to their relative weightings (by volume) e.g. the preferred 1:1 mixture by volume of polysorbate 80 and sorbitan trioleate has a HLB of 8.4.

[0113] Preferred amounts of total surfactant (% by volume) in an adjuvant emulsion are between 0.1 and 2% e.g. between 0.25-2%. A total content of 1% by volume is particularly useful e.g. 0.5% by volume of polysorbate 80 and 0.5% by volume of sorbitan trioleate.

[0114] Useful emulsions can be prepared using known techniques e.g. see references 132 and 111-112117

[0115] Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to: [0116] A submicron emulsion of squalene, polysorbate 80, and sorbitan trioleate. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% sorbitan trioleate. This adjuvant is known as `MF59` [118-120], as described in more detail in Chapter 10 of ref. 131 and chapter 12 of ref. 132. The MF59 emulsion advantageously includes citrate ions e.g. 10 mM sodium citrate buffer. [0117] An emulsion of squalene, a tocopherol, and polysorbate 80. The emulsion may include phosphate buffered saline. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% polysorbate 80, and the weight ratio of squalene:tocopherol is preferably .ltoreq.1 (e.g. 0.90) as this can provide a more stable emulsion. Squalene and polysorbate 80 may be present volume ratio of about 5:2, or at a weight ratio of about 11:5. Thus the three components (squalene, tocopherol, polysorbate 80) may be present at a weight ratio of 1068:1186:485 or around 55:61:25. This adjuvant is known as `AS03`. Another useful emulsion of this type may comprise, per human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate 80 [121] e.g. in the ratios discussed above. [0118] An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles [122]. [0119] An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant. As described in reference 123, preferred phospholipid components are phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, phosphatidylinositol phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous. [0120] An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or `Span 80`). The emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [124]. The emulsion may also include one or more of: alditol; a cryoprotective agent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. It may also include a TLR4 agonist, such as one whose chemical structure does not include a sugar ring [125]. Such emulsions may be lyophilized. The `AF03` product is one such emulsion.

[0121] Preferred oil-in-water emulsions used with the invention comprise squalene and polysorbate 80.

[0122] The emulsions may be mixed with TdaP antigens during vaccine manufacture, or they may be mixed extemporaneously at the time of delivery. Thus, in some embodiments, the adjuvant and antigens may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use. At the time of mixing (whether during bulk manufacture, or at the point of use) the antigen will generally be in an aqueous form, such that the final vaccine is prepared by mixing two liquids. The volume ratio of the two liquids for mixing can vary (e.g. between 5:1 and 1:5) but is generally about 1:1. If emulsion and antigen are stored separately in a kit then the product may be presented as a vial containing emulsion and a vial containing aqueous antigen, for mixing to give adjuvanted liquid vaccine (monodose or multi-dose).

[0123] Preferred emulsions of the invention include squalene oil. This is usually prepared from shark oil but alternative sources are known e.g. see references 126 (yeast) and 127 (olive oil). Squalene which contains less than 661 picograms of PCBs per gram of squalene (TEQ) is preferred for use with the invention, as disclosed in reference 128. The emulsions are preferably made from squalene of high purity e.g. prepared by double-distillation as disclosed in reference 129.

[0124] Where a composition includes a tocopherol, any of the a, 03, y, 8, c or 4 tocopherols can be used, but .alpha.-tocopherols are preferred. The tocopherol can take several forms e.g. different salts and/or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. D-.alpha.-tocopherol and DL-.alpha.-tocopherol can both be used. Tocopherols have antioxidant properties that may help to stabilize the emulsions [130]. A preferred .alpha.-tocopherol is DL-.alpha.-tocopherol, and a preferred salt of this tocopherol is the succinate.

[0125] Aluminium Salt Adjuvants

[0126] Compositions of the invention can include an aluminium salt adjuvant. Aluminium salt adjuvants currently in use are typically referred to either as "aluminium hydroxide" or as "aluminium phosphate" adjuvants. These are names of convenience, however, as neither is a precise description of the actual chemical compound which is present (e.g. see chapter 9 of reference 131, and chapter 4 of reference 132). The invention can use any of the "hydroxide" or "phosphate" salts that useful as adjuvants. Aluminium salts which include hydroxide ions are preferred if adsorption of a TLR agonist is desired as these hydroxide ions can readily undergo ligand exchange for adsorption of the TLR agonist. Thus preferred salts for adsorption of TLR agonists are aluminium hydroxide and/or aluminium hydroxyphosphate. These have surface hydroxyl moieties which can readily undergo ligand exchange with phosphorus-containing groups (e.g. phosphates, phosphonates) to provide stable adsorption. An aluminium hydroxide adjuvant is thus most preferred.

[0127] The adjuvants known as "aluminium hydroxide" are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AlO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH).sub.3, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070 cm.sup.-1 and a strong shoulder at 3090-3100 cm.sup.-1 (chapter 9 of ref. 131). The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants e.g. with needle-like particles with diameters about 2 nm. The PZC of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al.sup.+++ at pH 7.4 have been reported for aluminium hydroxide adjuvants.

[0128] The adjuvants known as "aluminium phosphate" are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate. They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PO.sub.4/Al molar ratio between 0.3 and 0.99. Hydroxyphosphates can be distinguished from strict AlPO.sub.4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm.sup.-1 (e.g. when heated to 200.degree. C.) indicates the presence of structural hydroxyls (chapter 9 of ref. 131).

[0129] The PO.sub.4/Al.sup.j molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95.+-.0.1. The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO.sub.4/Al molar ratio between 0.84 and 0.92, included at 0.6 mg Al.sup.3+/ml. The aluminium phosphate will generally be particulate. Typical diameters of the particles are in the range 0.5-20 .mu.m (e.g. about 5-10 .mu.m) after any antigen adsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mg Al.sup.+++ at pH 7.4 have been reported for aluminium phosphate adjuvants.

[0130] The PZC of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate=more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

[0131] In solution both aluminium phosphate and hydroxide adjuvants tend to form stable porous aggregates 1-10 .mu.m in diameter [133].

[0132] A composition can include a mixture of both an aluminium hydroxide and an aluminium phosphate, and components may be adsorbed to one or both of these salts.

[0133] An aluminium phosphate solution used to prepare a composition of the invention may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary. The aluminium phosphate solution is preferably sterile and pyrogen-free. The aluminium phosphate solution may include free aqueous phosphate ions e.g. present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM. The aluminium phosphate solution may also comprise sodium chloride. The concentration of sodium chloride is preferably in the range of 0.1 to 100 mg/ml (e.g. 0.5-50 mg/ml, 1-20 mg/ml, 2-10 mg/ml) and is more preferably about 3.+-.1 mg/ml. The presence of NaCl facilitates the correct measurement of pH prior to adsorption of antigens.

[0134] A composition of the invention ideally includes less than 0.85 mg Al.sup.+++ per unit dose. In some embodiments of the invention a composition includes less than 0.5 mg Al.sup.+++ per unit dose. The amount of Al.sup.+++ can be lower than this e.g. <250 .mu.g, <200 .mu.g, <150 .mu.g, <100 .mu.g, <75 Ag, <50 .mu.g, <25 .mu.g, <10 .mu.g, etc.

[0135] Where compositions of the invention include an aluminium-based adjuvant, settling of components may occur during storage. The composition should therefore be shaken prior to administration to a patient. The shaken composition will be a turbid white suspension.

[0136] If a TLR agonist and an aluminium salt are both present, in general the weight ratio of the TLR agonist to Al.sup.I I I will be less than 5:1 e.g. less than 4:1, less than 3:1, less than 2:1, or less than 1:1. Thus, for example, with an Al.sup.+++ concentration of 0.5 mg/ml the maximum concentration of TLR agonist would be 2.5 mg/ml. But higher or lower levels can be used. A lower mass of TLR agonist than of Al.sup.+++ can be most typical e.g. per dose, 1000 .mu.g of TLR agonist with 0.2 mg Al.sup.+++, etc. For instance, the FENDRIX.TM. product includes 50.mu.g of 3d-MPL and 0.5 mg Al.sup.+++ per dose.

[0137] TLR Agonists

[0138] In some embodiments a composition of the invention includes a TLR agonist i.e. a compound which can agonise a Toll-like receptor. Most preferably, a TLR agonist is an agonist of a human TLR. The TLR agonist can activate any of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR11; preferably it can activate human TLR4 or human TLR7.

[0139] Agonist activity of a compound against any particular Toll-like receptor can be determined by standard assays. Companies such as Imgenex and Invivogen supply cell lines which are stably co-transfected with human TLR genes and NF.kappa.B, plus suitable reporter genes, for measuring TLR activation pathways. They are designed for sensitivity, broad working range dynamics and can be used for high-throughput screening. Constitutive expression of one or two specific TLRs is typical in such cell lines. See also reference 134. Many TLR agonists are known in the art e.g. reference 135 describes certain lipopeptide molecules that are TLR2 agonists, references 136 to 139 each describe classes of small molecule agonists of TLR7, and references 140 & 141 describe TLR7 and TLR8 agonists for treatment of diseases.

[0140] A TLR agonist used with the invention ideally includes at least one adsorptive moiety. The inclusion of such moieties in TLR agonists allows them to adsorb to insoluble aluminium salts (e.g. by ligand exchange or any other suitable mechanism) and improves their immunological behaviour [142]. Phosphorus-containing adsorptive moieties are particularly useful, and so an adsorptive moiety may comprise a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, etc.

[0141] Preferably the TLR agonist includes at least one phosphonate group.

[0142] Thus, in preferred embodiments, a composition of the invention includes a TLR agonist (such as a TLR7 agonist) which includes a phosphonate group. This phosphonate group can allow adsorption of the agonist to an insoluble aluminium salt [142].

[0143] TLR agonists useful with the invention may include a single adsorptive moiety, or may include more than one e.g. between 2 and 15 adsorptive moieties. Typically a compound will include 1, 2 or 3 adsorptive moieties.

[0144] Phosphorus-containing TLR agonists useful with the invention can be represented by formula (A1):

##STR00001## [0145] wherein: [0146] R.sup.X and R.sup.Y are independently selected from H and C.sub.1-C.sub.6 alkyl; [0147] X is selected from a covalent bond, O and NH; [0148] Y is selected from a covalent bond, O, C(O), S and NH; [0149] L is a linker e.g. selected from, C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene, arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; [0150] each p is independently selected from 1, 2, 3, 4, 5 and 6; [0151] q is selected from 1, 2, 3 and 4; [0152] n is selected from 1, 2 and 3; and [0153] A is a TLR agonist moiety.

[0154] In one embodiment, the TLR agonist according to formula (A1) is as follows: R.sup.X and R.sup.Y are H; X is O; L is selected from C.sub.1-C.sub.6 alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 2 halogen atoms; p is selected from 1, 2 and 3; q is selected from 1 and 2; and n is 1. Thus in these embodiments the adsorptive moiety comprises a phosphate group.

[0155] In other embodiments, the TLR agonist according to formula (A1) is as follows: R.sup.X and R.sup.Y are H; X is a covalent bond; L is selected from C.sub.1-C.sub.6 alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 2 halogen atoms; p is selected from 1, 2 or 3; q is selected from 1 or 2; and n is 1. Thus in these embodiments the adsorptive moiety comprises a phosphonate group.

[0156] Useful `A` moieties for formula (A1) include, but are not limited to, radicals of any of the following compounds, defined herein or as disclosed in references 136, 137, 139, 140, 142 & 177:

##STR00002## ##STR00003##

[0157] In some embodiments, the TLR agonist moiety `A` has a molecular weight of less than 1000 Da. In some embodiments, the TLR agonist of formula (A1) has a molecular weight of less than 1000 Da.

[0158] Preferred TLR agonists are water-soluble. Thus they can form a homogenous solution when mixed in an aqueous buffer with water at pH 7 at 25.degree. C. and 1 atmosphere pressure to give a solution which has a concentration of at least 50 .mu.g/ml. The term "water-soluble" thus excludes substances that are only sparingly soluble under these conditions.

[0159] Useful TLR agonists include those having formula (C), (D), (E), (F), (G), (H), (I), (II), (J) or (K) as described in more detail below. Other useful TLR agonists are compounds 1 to 102 as defined in reference 142. Preferred TLR7 agonists have formula (K), such as compound K2 identified below. These can be used as salts e.g. the arginine salt of K2.

[0160] Preferred TLR4 agonists are analogs of monophosphoryl lipid A (MPL), as described in more detail below. For instance, a useful TLR4 agonist is a 3d-MPL.

[0161] A composition of the invention can include more than one TLR agonist. These two agonists are different from each other and they can target the same TLR or different TLRs. Both agonists can be adsorbed to an aluminium salt.

[0162] It is preferred that at least 50% (by mass) of any TLR agonist(s) in the composition is adsorbed to an aluminium salt (if present) e.g. .gtoreq.60%, .gtoreq.70%, .gtoreq.80%, .gtoreq.85%, .gtoreq.90%, .gtoreq.92%, .gtoreq.94%, .gtoreq.95%, .gtoreq.96%, .gtoreq.97%, .gtoreq.98%, .gtoreq.99%, or even 100%.

[0163] Where a composition of the invention includes a TLR agonist adsorbed to a metal salt, and also includes a buffer, it is preferred that the concentration of any phosphate ions in the buffer should be less than 50 mM (e.g. between 1-15 mM) as a high concentration of phosphate ions can cause desorption. Use of a histidine buffer is preferred.

[0164] Formulae (C), (D), (E) and (H)--TLR7 Agonists

[0165] The TLR agonist can be a compound according to any of formulae (C), (D), (E), and (H):

##STR00004##

[0166] wherein: [0167] (a) P.sup.3 is selected from H, C.sub.1-C.sub.6alkyl, CF.sub.3, and --((CH.sub.2).sub.pO)(CH.sub.2).sub.pO.sub.s-- and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); and P.sup.4 is selected from H, C.sub.1-C.sub.6alkyl, --C.sub.1-C.sub.6alkylaryl and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); with the proviso that at least one of P.sup.3 and P.sup.4 is --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y), [0168] (b) P.sup.5 is selected from H, C.sub.1-C.sub.6alkyl, and --Y-L-X--P(OR.sup.X)(OR.sup.Y); P.sup.6 is selected from H, C.sub.1-C.sub.6alkyl each optionally substituted with 1 to 3 substituents selected from C.sub.1-C.sub.4alkyl and OH, and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); and P.sup.7 is selected from H, C.sub.1-C.sub.6alkyl, --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pO.sub.s--, --NHC.sub.1-C.sub.6alkyl and --Y-L-X--P(OR.sup.X)(OR.sup.Y); with the proviso that at least one of P.sup.5, P.sup.6 and P.sup.7 is --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0169] (c) P.sup.8 is selected from H. C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, --NHC.sub.1-C.sub.6alkyl each optionally substituted with OH, and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); and P.sup.9 and P.sup.10 are each independently selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, --NHC.sub.1-C.sub.6alkyl each optionally substituted with OH and C.sub.1-C.sub.6alkyl, and --Y-L-X--P(OR.sup.X)(OR.sup.Y); with the proviso that at least one of P.sup.8, P.sup.9 or P.sup.10 is --Y-L-X--P(OR.sup.X)(OR.sup.Y); [0170] (d) P.sup.16 and each P.sup.18 are each independently selected from H, C.sub.1-C.sub.6alkyl, and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); P.sup.17 is selected from H, C.sub.1-C.sub.6alkyl aryl, heteroaryl, C.sub.1-C.sub.6alkylaryl, C.sub.1-C.sub.6alkyl heteroaryl, C.sub.1-C.sub.6alkylaryl-Y-L-X--P(OR.sup.X)(OR.sup.Y) and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y), each optionally substituted with 1 to 2 substituents selected from C.sub.1-C.sub.6alkyl or heterocyclyl with the proviso that at least one of P.sup.16, P.sup.17 or a P.sup.18 contains a --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y) moiety; [0171] R.sup.X and R.sup.Y are independently selected from H and C.sub.1-C.sub.6alkyl; [0172] R.sup.C, R.sup.D and R.sup.H are each independently selected from H and C.sub.1-C.sub.6alkyl; [0173] X.sup.C is selected from CH and N; [0174] R.sup.E is selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy, C(O)C.sub.1-C.sub.6alkyl, halogen and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p--; [0175] X.sup.E is selected from a covalent bond, CR.sup.E2R.sup.E3 and NR.sup.E4; [0176] R.sup.E2, R.sup.E3 and R.sup.E4 are independently selected from H and C.sub.1-C.sub.6alkyl; [0177] X.sup.H1--X.sup.H2 is selected from --CR.sup.H2R.sup.H3--, --CR.sup.H2R.sup.H3--CR.sup.H2R.sup.H3--, --C(O)CR.sup.H2R.sup.H3--, --C(O)CR.sup.H2R.sup.H3--, --CR.sup.H2R.sup.H3C(O)--, --NR.sup.H4C(O)--, C(O)NR.sup.H4-, CR.sup.H2R.sup.H3S(O).sub.2 and --CR.sup.H2.dbd.CR.sup.H2--; [0178] R.sup.H2, R.sup.H3 and R.sup.H4 are each independently selected from H, C.sub.1-C.sub.6alkyl and P.sup.18; [0179] X.sup.H3 is selected from N and CN; [0180] X is selected from a covalent bond, O and NH; [0181] Y is selected from a covalent bond, O, C(O), S and NH; [0182] L is selected from, a covalent bond C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene, C.sub.1-C.sub.6alkenylene, arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; [0183] m is selected from 0 or 1; [0184] each p is independently selected from 1, 2, 3, 4, 5 and 6; [0185] q is selected from 1, 2, 3 and 4; and [0186] s is selected from 0 and 1.

[0187] Formula (G)--TLR8 Agonist

[0188] The TLR agonist can be a compound according to formula (G):

##STR00005##

[0189] wherein: [0190] P.sup.11 is selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6 alkoxy, NR.sup.VR.sup.W and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0191] P.sup.12 is selected from H. C.sub.1-C.sub.6alkyl, aryl optionally substituted by --C(O)NR.sup.VR.sup.W, and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0192] P.sup.13, P.sup.14 and P.sup.15 are independently selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6 alkoxy and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0193] with the proviso that at least one of P.sup.11, P.sup.12, P.sup.13, P.sup.14 or P.sup.15 is --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0194] R.sup.V and R.sup.W are independently selected from H, C.sub.1-C.sub.6alkyl or together with the nitrogen atom to which they are attached form a 4 to 7 remembered heterocyclic ring; [0195] X.sup.G is selected from C, CH and N; [0196] represents an optional double bond, wherein X.sup.G is C if is a double bond; and [0197] R.sup.G is selected from H and C.sub.1-C.sub.6alkyl; [0198] X is selected from a covalent bond, O and NH; [0199] Y is selected from a covalent bond, O, C(O), S and NH; [0200] L is selected from, a covalent bond C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene, arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; [0201] each p is independently selected from 1, 2, 3, 4, 5 and 6 and [0202] q is selected from 1, 2, 3 and 4.

[0203] Formulae (I) and (II)--TLR7 agonists [137]

[0204] The TLR agonist can be a compound according to formula (I) or formula (II):

##STR00006##

[0205] wherein: [0206] Z is --NH.sub.2 or --OH; [0207] X.sup.1 is alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, carbocyclylene, substituted carbocyclylene, heterocyclylene, or substituted heterocyclylene; [0208] L.sup.1 is a covalent bond, arylene, substituted arylene, heterocyclylene, substituted heterocyclylene, carbocyclylene, substituted carbocyclylene, --S--, --S(O)--, S(O).sub.2, --NR.sup.5--, or --O-- [0209] X.sup.2 is a covalent bond, alkylene, or substituted alkylene; [0210] L.sup.2 is NR.sup.5--, --N(R.sup.5)C(O)--, --O--, --S--, --S(O)--, S(O).sub.2, or a covalent bond; [0211] R.sup.3 is H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, or substituted heterocyclylalkyl; [0212] Y.sup.1 and Y.sup.2 are each independently a covalent bond, --O-- or --NR.sup.5--; or --Y.sup.1--R.sup.1 and --Y.sup.2--R.sup.2 are each independently --O--N.dbd.C(R.sup.6R.sup.7); [0213] R.sup.1 and R.sup.2 are each independently H, alkyl, substituted alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl, substituted heterocyclylalkyl, -alkylene-C(O)--O--R.sup.5, -(substituted alkylene)-C(O)--O--R.sup.5, -alkylene-O--C(O)--R.sup.5, -(substituted alkylene)-O--C(O)--R.sup.5, -alkylene-O--C(O)--O--R.sup.5, or -(substituted alkylene)-O--C(O)--O--R.sup.5 [0214] R.sup.4 is H, halogen, --OH, --O-alkyl, --O-alkylene-O--C(O)--O--R.sup.5, --O--C(O)--O--R.sup.5, --SH, or --NH(R.sup.5); [0215] each R.sup.5, R.sup.6, and R.sup.7 are independently H, alkyl, substituted alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl, or substituted heterocyclylalkyl.

[0216] Formula (J) TLR2 Agonists [143]

[0217] The TLR agonist can be a compound according to formula (J):

##STR00007##

[0218] wherein: [0219] R.sup.1 is H, --C(O)--C.sub.7-C.sub.18alkyl or --C(O)--C.sub.1-C.sub.6alkyl; [0220] R.sup.2 is C.sub.7-C.sub.18alkyl; [0221] R.sup.3 is C.sub.7-C.sub.18alkyl; [0222] L.sub.1 is --CH.sub.2OC(O)--, --CH.sub.2O--, --CH.sub.2NR.sup.7C(O)-- or --CH.sub.2OC(O)NR.sup.7--; [0223] L.sub.2 is --OC(O)--, --O--, --NR.sup.7C(O)-- or --OC(O)NR.sup.7--; [0224] R.sup.4 is -L.sub.3R.sup.5 or -L.sub.4R.sup.5; [0225] R.sup.5 is --N(R.sup.7).sub.2, --OR.sup.7, --P(OR.sup.X)(OR.sup.7).sub.2, --C(O)OR.sup.7, --NR.sup.7C(O)L.sub.3R.sup.8, --NR.sup.7C(O)L.sub.4R.sup.8, --OL.sub.3R.sup.6, --C(O)NR.sup.7L.sub.3R.sup.8, --C(O)NR.sup.7L.sub.4R.sup.8. --S(O).sub.2OR.sup.7, --OS(O).sub.2OR.sup.7, C.sub.1-C.sub.6alkyl, a C.sub.6aryl, a C.sub.10aryl, a C.sub.14aryl, 5 to 14 ring membered heteroaryl containing 1 to 3 heteroatoms selected from O, S and N, C.sub.3-C.sub.8cycloalkyl or a 5 to 6 ring membered heterocycloalkyl containing 1 to 3 heteroatoms selected from O, S and N, wherein the aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R.sup.5 are each unsubstituted or the aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R.sup.5 are cach substituted with 1 to 3 substituents independently selected from --OR.sup.9, --OL.sub.3R.sup.6, --OL.sub.4R.sup.6, --OR.sup.Y, and --C(O)OR; [0226] L.sub.3 is a C.sub.1-C.sub.10alkylene, wherein the C.sub.1-C.sub.10alkylene of L.sub.3 is unsubstituted, or the C.sub.1-C.sub.10alkylene of L.sub.3 is substituted with 1 to 4 R.sup.6 groups, or the C.sub.1-C.sub.10alkylene of L.sub.3 is substituted with 2 C.sub.1-C.sub.6alkyl groups on the same carbon atom which together, along with the carbon atom they are attached to, form a C.sub.3-C.sub.8cycloakyl; [0227] L.sub.4 is --((CR.sup.7R.sup.7).sub.pO).sub.q(CR.sup.10R.sup.10)-- or --(CR.sup.11R.sup.11)((CR.sup.7R.sup.7).sub.pO).sub.q(CR.sup.10R.sup.10).- sub.p--, wherein each R.sup.11 is a C.sub.1-C.sub.6alkyl groups which together, along with the carbon atom they are attached to, form a C.sub.3-C.sub.8cycloakyl; [0228] each R.sup.6 is independently selected from halo, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkyl substituted with 1-2 hydroxyl groups, --OR.sup.7, --N(R.sup.7).sub.2, --C(O)OH, --C(O)N(R.sup.7).sub.2, --P(OR.sup.X)(OR.sup.7).sub.2, a C.sub.6aryl, a C.sub.10aryl and a C.sub.14aryl; [0229] each R.sup.7 is independently selected from H and C.sub.1-C.sub.6alkyl; [0230] R.sup.8 is selected from --SR.sup.7, --C(O)OH, --P(O)(OR.sup.7).sub.2, and a 5 to 6 ring membered heterocycloalkyl containing 1 to 3 heteroatoms selected from O and N; [0231] R.sup.9 is phenyl; [0232] each R.sup.10 is independently selected from H and halo; [0233] each p is independently selected from 1, 2, 3, 4, 5 and 6, and [0234] q is 1, 2, 3 or 4.

[0235] Preferably R.sup.5 is P(O)(OR.sup.7).sub.2, --NR.sup.7C(O)L.sub.3-P(O)(OR.sup.7).sub.2, --NR.sup.7C(O)L.sub.4-P(OR.sup.X)(OR.sup.7).sub.2, --OL.sub.3-P(O)(OR.sup.7).sub.2, --C(O)NR.sup.7L.sub.3-P(O)(OR).sub.2, or --C(O)NR.sup.7L.sub.4-P(O)(OR.sup.7).sub.2.

[0236] In some embodiments of (J), R.sub.1 is H. In other embodiments of (J), R.sub.1 is --C(O)--C.sub.15alkyl;

[0237] In some embodiments of (J): (i) L.sub.1 is --CH.sub.2OC(O)-- and L.sub.2 is --OC(O)--, --O--, --NR.sup.7C(O)-- or --OC(O)NR.sup.7--; or (ii) or L.sub.1 is --CH.sub.2O-- and L.sub.2 is --OC(O)--, --O--, --NR.sup.7C(O)-- or --OC(O)NR.sup.7--; or (iii) L.sub.1 is --CH.sub.2NR.sup.7C(O)-- and L.sub.2 is --OC(O)--, --O--, --NR.sup.7C(O)-- or --OC(O)NR.sup.7--; or (iv) L.sub.1 is --CH.sub.2OC(O)NR.sup.7-- and L.sub.2 is --OC(O)--, --O--, --NR.sup.7C(O)-- or --OC(O)NR.sup.7--.

[0238] In some embodiments of (J): (i) L.sub.1 is --CH.sub.2OC(O)-- and L.sub.2 is --OC(O)--; or (ii) L.sub.1 is --CH.sub.2O-- and L.sub.2 is --O--; or (iii) L.sub.1 is --CH.sub.2O-- and L.sub.2 is --NHC(O)--; or (iv) L.sub.1 is --CH.sub.2OC(O)NH-- and L.sub.2 is --OC(O)NH--.

[0239] In some embodiments of (J), (i) R.sup.2 is --C.sub.11alkyl and R.sup.3 is --C.sub.11alkyl; or (ii) R.sup.2 is --C.sub.16alkyl and R.sup.3 is --C.sub.16alkyl; or (iii) R.sup.2 is --C.sub.16alkyl and R.sup.3 is --C.sub.11alkyl; or (iv) R.sup.2 is --C.sub.12alkyl and R.sup.3 is --C.sub.12alkyl; or (v) R.sup.2 is --C.sub.7alkyl and R.sup.3 is --C.sub.7alkyl; or (vi) R.sup.2 is --C.sub.9alkyl and R.sup.3 is --C.sub.9alkyl; or (vii) R.sup.2 is --C.sub.8alkyl and R.sup.3 is --C.sub.8alkyl; or (viii) R.sup.2 is --C.sub.13alkyl and R.sup.3 is --C.sub.13alkyl; or (ix) R.sup.2 is --C.sub.12alkyl and R.sup.3 is --C.sub.11alkyl; or (x) R.sup.2 is --C.sub.12alkyl and R.sup.3 is --C.sub.12alkyl; or (xi) R.sup.2 is --C.sub.10alkyl and R.sup.3 is --C.sub.10alkyl; or (xii) R.sup.2 is --C.sub.18alkyl and R.sup.3 is --C.sub.18alkyl.

[0240] In some embodiments of (J), R.sup.2 is --C.sub.11alkyl and R.sup.3 is --C.sub.11alkyl.

[0241] In some embodiments of (J), L.sub.3 is a C.sub.1-C.sub.10alkylene, wherein the C.sub.1-C.sub.10alkylene of L.sub.3 is unsubstituted or is substituted with 1 to 4 R.sup.6 groups.

[0242] In some embodiments of (J): L.sub.4 is --((CR.sup.7R.sup.7).sub.pO).sub.q(CR.sup.10R.sup.10).sub.p--; each R.sup.10 is independently selected from H and F; and each p is independently selected from 2, 3, and 4.

[0243] In some embodiments of (J), each R.sup.6 is independently selected from methyl, ethyl, i-propyl, i-butyl, --CH.sub.2OH, --OH, --F, --NH.sub.2, --C(O)OH, --C(O)NH.sub.2, --P(O)(OH).sub.2 and phenyl.

[0244] In some embodiments of (J), each R.sup.7 is independently selected from H, methyl and ethyl.

[0245] TLR4 Agonists

[0246] Compositions of the invention can include a TLR4 agonist, and most preferably an agonist of human TLR4. TLR4 is expressed by cells of the innate immune system, including conventional dendritic cells and macrophages [144]. Triggering via TLR4 induces a signalling cascade that utilizes both the MyD88- and TRIF-dependent pathways, leading to NF-.kappa.B and IRF3/7 activation, respectively. TLR4 activation typically induces robust IL-12p70 production and strongly enhances Th1-type cellular and humoral immune responses.

[0247] Various useful TLR4 agonists are known in the art, many of which are analogs of endotoxin or lipopolysaccharide (LPS). For instance, the TLR4 agonist can be: [0248] (i) 3d-MPL (i.e. 3-O-deacylated monophosphoryl lipid A; also known as 3-de-O-acylated monophosphoryl lipid A or 3-O-desacyl-4'-monophosphoryl lipid A). This derivative of the monophosphoryl lipid A portion of endotoxin has a de-acylated position 3 of the reducing end of glucosamine. It has been prepared from a heptoseless mutant of Salmonella minnesota, and is chemically similar to lipid A but lacks an acid-labile phosphoryl group and a base-labile acyl group. Preparation of 3d-MPL was originally described in ref. 145, and the product has been manufactured and sold by Corixa Corporation. It is present in GSK's `AS04` adjuvant. Further details can be found in references 146 to 149. [0249] (ii) glucopyranosyl lipid A (GLA) [150] or its ammonium salt:

[0249] ##STR00008## [0250] (iii) an aminoalkyl glucosaminide phosphate, such as RC-529 or CRX-524 [151-153]. RC-529 and CRX-524 have the following structure, differing by their R.sub.2 groups:

[0250] ##STR00009## [0251] (iv) compounds containing lipids linked to a phosphate-containing acyclic backbone, such as the TLR4 antagonist E5564 [154,155]:

[0251] ##STR00010## [0252] (v) A compound of formula I, II or III as defined in reference 156, or a salt thereof, such as compounds `ER 803058`, `ER 803732`, `ER 804053`, `ER 804058`, `ER 804059`, `ER 804442`, `ER 804680`, `ER 803022`, `ER 804764` or `ER 804057`. ER 804057 is also known as E6020 and it has the following structure:

[0252] ##STR00011## [0253] whereas ER 803022 has the following structure:

[0253] ##STR00012## [0254] (vi) One of the polypeptide ligands disclosed in reference 157.

[0255] Any of these TLR4 agonists can be used with the invention.

[0256] A composition of the invention can include an aluminium salt to which the TLR4 agonist is adsorbed. TLR4 agonists with adsorptive properties typically include a phosphorus-containing moiety which can undergo ligand exchange with surface groups on an aluminium salt, and particularly with a salt having surface hydroxide groups. Thus a useful TLR4 agonist may include a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, a phosphate, etc. Preferred TLR4 agonists include at least one phosphate group [142] e.g. the agonists (i) to (v) listed above.

[0257] The preferred TLR4 agonist for use with the invention is 3d-MPL. This can be adsorbed to an aluminium phosphate adjuvant, to an aluminium hydroxide adjuvant, or to a mixture of both [158].

[0258] 3d-MPL can take the form of a mixture of related molecules, varying by their acylation (e.g. having 3, 4, 5 or 6 acyl chains, which may be of different lengths). The two glucosamine (also known as 2-deoxy-2-amino-glucose) monosaccharides are N-acylated at their 2-position carbons (i.e. at positions 2 and 2'), and there is also O-acylation at the 3' position. The group attached to carbon 2 has formula --NH--CO--CH.sub.2--CR.sup.1R.sup.1'. The group attached to carbon 2' has formula --NH--CO--CH.sub.2--CR.sup.2R.sup.2'. The group attached to carbon 3' has formula --O--CO--CH.sub.2--CR.sup.3R.sup.3'. A representative structure is:

##STR00013##

[0259] Groups R.sup.1, R.sup.2 and R.sup.3 are each independently --(CH.sub.2).sub.n--CH.sub.3. The value of n is preferably between 8 and 16, more preferably between 9 and 12, and is most preferably 10.

[0260] Groups R.sup.1', R.sup.2' and R.sup.3' can each independently be: (a) --H; (b) --OH; or (c) --O--CO--R.sup.4, where R.sup.4 is either --H or --(CH.sub.2).sub.m--CH.sub.3, wherein the value of m is preferably between 8 and 16, and is more preferably 10, 12 or 14. At the 2 position, in is preferably 14. At the 2' position, m is preferably 10. At the 3' position, m is preferably 12. Groups R.sup.1', R.sup.2' and R.sup.3' are thus preferably --O-acyl groups from dodecanoic acid, tetradecanoic acid or hexadecanoic acid.

[0261] When all of R.sup.1', R.sup.2' and R.sup.3' are --H then the 3d-MPL has only 3 acyl chains (one on each of positions 2, 2' and 3'). When only two of R.sup.1', R.sup.2' and R.sup.3' are --H then the 3d-MPL can have 4 acyl chains. When only one of R.sup.1', R.sup.2' and R.sup.3' is --H then the 3d-MPL can have 5 acyl chains. When none of R.sup.1', R.sup.2' and R.sup.3' is --H then the 3d-MPL can have 6 acyl chains. The 3d-MPL used according to the invention can be a mixture of these forms, with from 3 to 6 acyl chains, but it is preferred to include 3d-MPL with 6 acyl chains in the mixture, and in particular to ensure that the 6 acyl chain form makes up at least 10% by weight of the total 3d-MPL e.g. .gtoreq.20%, .gtoreq.30%, .gtoreq.40%, .gtoreq.50% or more. 3d-MPL with 6 acyl chains has been found to be the most adjuvant-active form.

[0262] Thus the most preferred form of 3d-MPL for use with the invention is:

##STR00014##

[0263] Where 3d-MPL is used in the form of a mixture then references to amounts or concentrations of 3d-MPL in compositions of the invention refer to the combined 3d-MPL species in the mixture.

[0264] Typical compositions include 3d-MPL at a concentration of between 25 .mu.g/ml and 200 .mu.g/ml e.g. in the range 50-150 .mu.g/ml, 75-125 .mu.g/ml, 90-110 .mu.g/ml, or about 100 .mu.g/ml. It is usual to administer between 25-75 .mu.g of 3d-MPL per dose e.g. between 45-55 .mu.g, or about 50 .mu.g 3d-MPL per dose.

[0265] In aqueous conditions, 3d-MPL can form micellar aggregates or particles with different sizes e.g. with a diameter <150 nm or >500 nm. Either or both of these can be used with the invention, and the better particles can be selected by routine assay. Smaller particles (e.g. small enough to give a clear aqueous suspension of 3d-MPL) are preferred for use according to the invention because of their superior activity [159]. Preferred particles have a mean diameter less than 150 nm, more preferably less than 120 nm, and can even have a mean diameter less than 100 nm. In most cases, however, the mean diameter will not be lower than 50 nm. Where 3d-MPL is adsorbed to an aluminum salt then it may not be possible to measure the 3D-MPL particle size directly, but particle size can be measured before adsorption takes place. Particle diameter can be assessed by the routine technique of dynamic light scattering, which reveals a mean particle diameter. Where a particle is said to have a diameter of x nm, there will generally be a distribution of particles about this mean, but at least 50% by number (e.g. .gtoreq.60%, .gtoreq.70%, .gtoreq.80%, .gtoreq.90%, or more) of the particles will have a diameter within the range x.+-.25%.

[0266] Formula (K) [160]

[0267] The TLR agonist can be a compound according to formula (K):

##STR00015##

[0268] wherein: [0269] R.sup.1 is H, C.sub.1-C.sub.6alkyl, --C(R.sup.5).sub.2OH, -L.sup.1R.sup.5, -L.sup.1R.sup.6, -L.sup.2R.sup.5, -L.sup.2R.sup.6, --OL.sup.2R.sup.5, or -OL.sup.2R.sup.6; [0270] L.sup.1 is --C(O)-- or --O--; [0271] L.sup.2 is C.sub.1-C.sub.6alkylene, C.sub.2-C.sub.6alkenylene, arylene, heteroarylene or --((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p, wherein the C.sub.1-C.sub.6alkylene and C.sub.2-C.sub.6alkenylene of L.sup.2 are optionally substituted with 1 to 4 fluoro groups; [0272] each L.sup.3 is independently selected from C.sub.1-C.sub.6alkylene and --((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p--, wherein the C.sub.1-C.sub.6alkylene of L.sup.3 is optionally substituted with 1 to 4 fluoro groups; [0273] L.sup.4 is arylene or heteroarylene; [0274] R.sup.2 is H or C.sub.1-C.sub.6alkyl; [0275] R.sup.3 is selected from C.sub.1-C.sub.4alkyl, -L.sup.3R.sup.5, -L.sup.1R.sup.5, -L.sup.3R.sup.7, -L.sup.3L.sup.4L.sup.3R.sup.7, -L.sup.3L.sup.4R.sup.5, -L.sup.3L.sup.4L.sup.3R.sup.5, --OL.sup.3R.sup.7, --OL.sup.3R.sup.7, --OL.sup.3L.sup.4R.sup.7, --OL.sup.3L.sup.4L.sup.3R.sup.7, --OR, --OL.sup.3L.sup.4R.sup.5, --OL.sup.3L.sup.4L.sup.3R.sup.5 and --C(R.sup.5).sub.2OH; [0276] each R.sup.4 is independently selected from H and fluoro; [0277] R.sup.5 is --P(O)(OR.sup.9).sub.2, [0278] R.sup.6 is --CF.sub.2P(O)(OR.sup.9).sub.2 or --C(O)OR.sup.10; [0279] R.sup.7 is --CF.sub.2P(O)(OR.sup.9).sub.2 or --C(O)OR.sup.10; [0280] R.sup.8 is H or C.sub.1-C.sub.4alkyl; [0281] each R.sup.9 is independently selected from H and C.sub.1-C.sub.6alkyl; [0282] R.sup.10 is H or C.sub.1-C.sub.4alkyl; [0283] each p is independently selected from 1, 2, 3, 4, 5 and 6, and [0284] q is 1, 2, 3 or 4.

[0285] The compound of formula (K) is preferably of formula (K'):

##STR00016##

[0286] wherein: [0287] P.sup.1 is selected from H, C.sub.1-C.sub.6alkyl optionally substituted with COOH and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); P.sup.2 is selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0288] with the proviso that at least one of P.sup.1 and P.sup.2 is --Y-L-X--P(OR.sup.X)(OR.sup.XX)(OR.sup.Y); [0289] R.sup.B is selected from H and C.sub.1-C.sub.6alkyl; [0290] R.sup.X and R.sup.Y are independently selected from H and C.sub.1-C.sub.6alkyl; [0291] X is selected from a covalent bond, O and NH; [0292] Y is selected from a covalent bond, O, C(O), S and NH; [0293] L is selected from, a covalent bond C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene, arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; [0294] each p is independently selected from 1, 2, 3, 4, 5 and 6; and [0295] q is selected from 1, 2, 3 and 4.

[0296] In some embodiments of formula (K'): P.sup.1 is selected from C.sub.1-C.sub.6alkyl optionally substituted with COOH and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); P.sup.2 is selected from C.sub.1-C.sub.6alkoxy and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); R.sup.B is C.sub.1-C.sub.6alkyl; X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; q is selected from 1 and 2.

[0297] A preferred TLR7 agonist of formula K is 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl- )benzo [f]-[1,7]naphthyridin-8-yl)propanoic acid, referred to herein as compound "K2":

##STR00017##

[0298] The K2 compound can also be used as an arginine salt monohydrate.

[0299] Formula (F)--TLR7 Agonists [138]

[0300] The TLR agonist can be a compound according to formula (F):

##STR00018## [0301] wherein: [0302] X.sup.3 is N; [0303] X.sup.4 is N or CR.sup.3 [0304] X.sup.5 is --CR.sub.4.dbd.CR.sub.5--; [0305] R.sup.1 and R.sup.2 are H; [0306] R.sup.3 is H; [0307] R.sup.4 and R are each independently selected from H, halogen, --C(O)OR.sup.7, --C(O)R.sup.7, --C(O)N(R.sup.11R.sup.12), --N(R.sup.11R.sup.12), --N(R.sup.9).sub.2, --NHN(R.sup.9).sub.2, --SR.sup.7, --(CH.sub.2).sub.nOR.sup.7, --(CH.sub.2).sub.nR.sup.7, - LR.sup.8, -LR.sup.10, --OLR.sup.8, --OLR.sup.10, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.4 and R.sup.5 are each optionally substituted with 1 to 3 substituents independently selected from halogen, --CN, --NO.sub.2, --R.sup.7, --OR.sup.8, --C(O)R.sup.8, --OC(O)R.sup.8, --C(O)OR.sup.8, --N(R.sup.9).sub.2, --P(O)(OR.sup.8).sub.2, --OP(O)(OR.sup.8).sub.2, --P(O)(0R.sup.10).sub.2. --OP(OR)(OR.sup.10).sub.2, --C(O)N(R.sup.9).sub.2, --S(O).sub.2R.sup.8, --S(O)R.sup.8, --S(O).sub.2N(R).sub.2, and --NR.sup.9S(O).sub.2R.sup.8; [0308] or, R.sup.1 and R.sup.4, or R.sup.4 and R.sup.5, or R.sup.5 and R.sup.6, when present on adjacent ring atoms, can optionally be linked together to form a 5-6 membered ring, wherein the 5-6 membered ring is optionally substituted with R.sup.7; [0309] each L is independently selected from a bond, --(O(CH.sub.2).sub.m).sub.t--, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenylene and C.sub.2-C.sub.6alkynylene, wherein the C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenylene and C.sub.2-C.sub.6alkynylene of L are each optionally substituted with 1 to 4 substituents independently selected from halogen, --R.sup.8, --OR.sup.8, --N(R.sup.9).sub.2, --P(O)(OR.sup.8).sub.2, --OP(O)(OR.sup.8).sub.2, --P(O)(OR.sup.10).sub.2, and --OP(O)(OR.sup.10).sub.2; [0310] R.sup.7 is selected from H, C.sub.1-C.sub.6alkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, and C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.1-C.sub.6alkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, C.sub.1-C.sub.6heteroalkyl. C.sub.1-C.sub.6haloalkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy, and C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.7 are each optionally substituted with 1 to 3 R.sup.13 groups, and each R.sup.13 is independently selected from halogen, --CN, -LR.sup.9, -LOR.sup.9, --OLR.sup.9, -LR.sup.11, -LOR.sup.10, --OLR.sup.10, -LR.sup.8, -LOR.sup.8, --OLR.sup.8, -LSR.sup.8, -LSR.sup.10, -LC(O)R.sup.8, --OLC(O)R.sup.8, -LC(O)OR.sup.8, -LC(O)R.sup.10, -LOC(O)OR.sup.8, -LC(O)NR.sup.9R.sup.11, -LC(O)NR.sup.9R.sup.8, -LN(R.sup.9).sub.2, -LNR.sup.9R.sup.8, -LNR.sup.9R.sup.10, -LC(O)N(R.sup.9).sub.2, -LS(O).sub.2R.sup.8, -LS(O)R.sup.8, -LC(O)NR.sup.8OH, -LNR.sup.9C(O)R.sup.8, -LNR.sup.9C(O)OR.sup.8, -LS(O).sub.2N(R.sup.9).sub.2, --OLS(O).sub.2N(R.sup.9).sub.2, -LNR.sup.9S(O).sub.2R.sup.8, -LC(O)NR.sup.9LN(R.sup.9).sub.2, -LP(O)(OR.sup.9).sub.2, -LOP(O)(OR.sup.8).sub.2, -LP(O)(OR.sup.10).sub.2 and -OLP(O)(OR.sup.10).sub.2; [0311] each R.sup.8 is independently selected from H, --CH(R.sup.10).sub.2, C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6heteroalkyl, C.sub.3-C.sub.8cycloalkyl, C.sub.2-C.sub.8heterocycloalkyl, C.sub.2-C.sub.6hydroxyalkyl and C.sub.1-C.sub.6haloalkoxy, wherein the C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkene, C.sub.2-C.sub.8alkyne, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy, C.sub.3-C.sub.8cycloalkyl, C.sub.2-C.sub.8heterocycloalkyl, C.sub.1-C.sub.6hydroxyalkyl and C.sub.1-C.sub.6haloalkoxy groups of R.sup.8 are each optionally substituted with 1 to 3 substituents independently selected from --CN, R.sup.11, --OR.sup.11, --SR.sup.11, --C(O)R.sup.11, --OC(O)R.sup.11, --C(O)N(R.sup.9).sub.2, --C(O)OR.sup.11, --NR.sup.9C(O)R.sup.11, --NR.sup.9R.sup.10, --NR.sup.11R.sup.12, --N(R.sup.9).sub.2, --OR.sup.9, --OR.sup.10, --C(O)NR.sup.11R.sup.12, --C(O)NR.sup.11OH, --S(O).sub.2R.sup.11, --S(O)R.sup.11, --S(O).sub.2NR.sup.11R.sup.11, --NR.sup.11S(O).sub.2R.sup.11, --P(O)(OR.sup.11).sub.2, and --OP(O)(OR.sup.11).sub.2; [0312] each R.sup.9 is independently selected from H, --C(O)R.sup.8, --C(O)OR.sup.8, --C(O)R.sup.10, --C(O)OR.sup.10, --S(O).sub.2R.sup.10, --C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl and C.sub.3-C.sub.6 cycloalkyl, or each R.sup.9 is independently a C.sub.1-C.sub.6alkyl that together with N they are attached to form a C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.3-C.sub.8heterocycloalkyl ring optionally contains an additional heteroatom selected from N, O and S, and wherein the C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.3-C.sub.6 cycloalkyl, or C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.9 are each optionally substituted with 1 to 3 substituents independently selected from --CN, R.sup.11, --OR.sup.11, --SR.sup.11, --C(O)R.sup.11, OC(O)R.sup.11, --C(O)0R.sup.11, --NR.sup.11R.sup.12, --C(O)NR.sup.11R.sup.12, --C(O)NR.sup.11OH, --S(O).sub.2R.sup.11, --S(O)R.sup.11, --S(O).sub.2NR.sup.11R.sup.12, --NR.sup.11S(O).sub.2R.sup.11, --P(O)(OR.sup.11) and --OP(O)(OR.sup.11).sub.2; [0313] each R.sup.10 is independently selected from aryl, C.sub.3-C.sub.8cycloalkyl, C.sub.3-C.sub.8heterocycloalkyl and heteroaryl, wherein the aryl, C.sub.3-C.sub.8cycloalkyl, C.sub.3-C.sub.8heterocycloalkyl and heteroaryl groups are optionally substituted with 1 to 3 substituents selected from halogen, --R.sup.8, --OR.sup.8, -LR.sup.9, -LOR.sup.9, --N(R.sup.9).sub.2, --NR.sup.9C(O)R.sup.8, --NR.sup.9CO.sub.2R.sup.8. --CO.sub.2R.sup.8, --C(O)R and --C(O)N(R.sup.9).sub.2, [0314] R.sup.11 and R.sup.12 are independently selected from H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl, wherein the C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6heteroalkyl, C.sub.1-C.sub.6haloalkyl, aryl, heteroaryl, C.sub.3-C.sub.8cycloalkyl, and C.sub.3-C.sub.8heterocycloalkyl groups of R.sup.11 and R.sup.12 are each optionally substituted with 1 to 3 substituents independently selected from halogen, --CN, R.sup.8, --OR.sup.8, C(O)R.sup.8, OC(O)R.sup.8, --C(O)OR.sup.8, --N(R.sup.9).sub.2, --NR.sup.8C(O)R.sup.8, --NR.sup.8C(O)OR.sup.8, --C(O)N(R.sup.9).sub.2, C.sub.3-C.sub.8heterocycloalkyl, --S(O).sub.2R.sup.8, --S(O).sub.2N(R.sup.9).sub.2, --NR.sup.9S(O).sub.2R.sup.8, C.sub.1-C.sub.6haloalkyl and C.sub.1-C.sub.6haloalkoxy; [0315] or R.sup.11 and R.sup.12 are each independently C.sub.1-C.sub.6alkyl and taken together with the N atom to which they are attached form an optionally substituted C.sub.3-C.sub.8heterocycloalkyl ring optionally containing an additional heteroatom selected from N, O and S; [0316] ring A is an aryl or a heteroaryl, wherein the aryl and heteroaryl groups of Ring A are optionally substituted with 1 to 3 R.sup.A groups, wherein each R.sup.A is independently selected from --R.sup.8, --R.sup.7, --OR.sup.7, --OR.sup.8, --R.sup.10, --OR.sup.10, --SR.sup.8, --NO.sub.2, --CN, --N(R.sup.9).sub.2, --NR.sup.9C(O)R.sup.8, --NR.sup.9C(S)R.sup.8, --NR.sup.9C(O)N(R.sup.9).sub.2, --NR.sup.9C(S)N(R.sup.9).sub.2, --NR.sup.9CO.sub.2R.sup.8, --NR.sup.9NR.sup.9C(O)R.sup.8, --NR.sup.9NR.sup.9C(O)N(R.sup.9).sub.2, --NR.sup.9NR.sup.9CO.sub.2R.sup.8, --C(O)C(O)R.sup.8, --C(O)CH.sub.2C(O)R.sup.8, --CO.sub.2R.sup.8, --(CH.sub.2).sub.nCO.sub.2R.sup.8, --C(O)R.sup.8, --C(S)R.sup.8, --C(O)N(R.sup.9).sub.2, --C(S)N(R.sup.9).sub.2, --OC(O)N(R.sup.9).sub.2, --OC(O)R.sup.8, --C(O)N(OR.sup.11)R.sup.8, --C(NOR)R.sup.8, --S(O).sub.2R.sup.8, --S(O).sub.3R.sup.8, --SO.sub.2N(R.sup.9).sub.2, --S(O)R.sup.8, --NR.sup.9SO.sub.2N(R.sup.9).sub.2, --NR.sup.9SO.sub.2R.sup.8, --P(O)(OR.sup.8).sub.2, --OP(O)(OR.sup.8).sub.2, --P(O)(OR.sup.10).sub.2, --OP(O)(OR.sup.10).sub.2, --N(0R.sup.8)R.sup.8, --CH.dbd.CHCO.sub.2R.sup.8, --C(.dbd.NH)--N(R.sup.9).sub.2, and --(CH.sub.2).sub.nNHC(O)R.sup.8 or two adjacent R.sup.A substituents on Ring A form a 5-6 membered ring that contains up to two heteroatoms as ring members; [0317] n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7 or 8; [0318] each m is independently selected from 1, 2, 3, 4, 5 and 6, and [0319] t is 1, 2, 3, 4, 5, 6, 7 or 8.

[0320] Formulae (C), (D), (E), (G) and (H)

[0321] As discussed above, the TLR agonist can be of formula (C), (D), (E), (G) or (H).

[0322] The `parent` compounds of formulae (C), (D), (E) and (H) are useful TLR7 agonists (see references 136-139 and 161-177) but are preferably modified herein by attachment of a phosphorus-containing moiety.

[0323] In some embodiments of formulae (C), (D) and (E) the compounds have structures according to formulae (C'), (D') and (E'), shown below:

##STR00019##

[0324] The embodiments of the invention of formulae (C), (D), (E) and (H) also apply to formulae (C'), (D'), (E') and (H').

[0325] In some embodiments of formulae (C), (D), (E), and (H): X is O; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0326] In other embodiments of formula (C): P.sup.3 is selected from C.sub.1-C.sub.6alkyl, CF.sub.3, and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pO.sub.s-- and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); P.sup.4 is selected from --C.sub.1-C.sub.6alkylaryl and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); X.sup.C is CH; X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3: q is 1 or 2.

[0327] In other embodiments of formulae (C), (D), (E), and (H): X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0328] In other embodiments of formula (C): P.sup.3 is selected from C.sub.1-C.sub.6alkyl, CF.sub.3, and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pO.sub.s-- and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); P.sup.4 is selected from --C.sub.1-C.sub.6alkylaryl and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); X.sup.C is N; X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; q is selected from 1 and 2.

[0329] In other embodiments of formula (D): P.sup.5 is selected from C.sub.1-C.sub.6alkyl, and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y).

[0330] In other embodiments of formula (D): X is O; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0331] In other embodiments of formula (D): X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0332] In other embodiments of formula (E): X is O; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0333] In other embodiments of formula (E): X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0334] In other embodiments of formula (E): X.sup.E is CH.sub.2, P.sup.8 is C.sub.1-C.sub.6alkoxy optionally substituted with --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y).

[0335] In other embodiments of formula (E): P.sup.9 is --NHC.sub.1-C.sub.6alkyl optionally substituted with OH and C.sub.1-C.sub.6alkyl, and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y).

[0336] In some embodiments, a compound of formula (C) is not a compound in which P.sup.4 is --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y).

[0337] In some embodiments, in a compound of formula (C), P.sup.4 is selected from H, C.sub.1-C.sub.6alkyl, --C.sub.1-C.sub.6alkylaryl.

[0338] In some embodiments of formula (H): X.sup.H--X.sup.H2 is CR.sup.H2R.sup.H3, R.sup.H2 and R.sup.H3 are H, X.sup.H3 is N, X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0339] In some embodiments of formula (H): X.sup.H1-X.sup.H2 is CR.sup.H2R.sup.H3, R.sup.H2 and R.sup.H3 are H. X.sup.H3 is N, X is O; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0340] The `parent` compounds of formula (G) are useful TLR8 agonists (see references 140 & 141) but are preferably modified herein by attachment of a phosphorus-containing moiety to permit adsorption. In some embodiments of formula (G), the compounds have structures according to formula (G');

##STR00020##

[0341] In some embodiments of formula (G) or (G'): X.sup.G is C and represents a double bond. In some embodiments of formula (G) or (G'): X is a covalent bond; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0342] In some embodiments of formula (G) or (G'): X is O; L is selected from C.sub.1-C.sub.6alkylene and --((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and --P(O)(OH).sub.2; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

[0343] Immunogenic Compositions

[0344] In addition to the antigen and adjuvant components discussed above, compositions of the invention may comprise further non-antigenic component(s). These can include carriers, excipients, buffers, etc. These non-antigenic components may have various sources. For example, they may be present in one of the antigen or adjuvant materials that is used during manufacture or may be added separately from those components.

[0345] Preferred compositions of the invention include one or more pharmaceutical carrier(s) and/or excipient(s).

[0346] To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml.

[0347] Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 280-320 mOsm/kg. Osmolality has previously been reported not to have an impact on pain caused by vaccination [178], but keeping osmolality in this range is nevertheless preferred.

[0348] Compositions of the invention may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-20 mM range.

[0349] The pH of a composition of the invention will generally be between 6.0 and 7.5. A manufacturing process may therefore include a step of adjusting the pH of a composition prior to packaging. Aqueous compositions administered to a patient can have a pH of between 5.0 and 7.5, and more typically between 5.0 and 6.0 for optimum stability; where a diphtheria toxoid and/or tetanus toxoid is present, the pH is ideally between 6.0 and 7.0.

[0350] Compositions of the invention are preferably sterile.

[0351] Compositions of the invention are preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure; 1 EU is equal to 0.2 ng FDA reference standard Endotoxin EC-2 `RSE`) per dose, and preferably <0.1 EU per dose.

[0352] Compositions of the invention are preferably gluten free.

[0353] If a composition includes adsorbed component then it may be a suspension with a cloudy appearance. This appearance means that microbial contamination is not readily visible, and so the vaccine preferably contains an antimicrobial agent. This is particularly important when the vaccine is packaged in multidose containers. Preferred antimicrobials for inclusion are 2-phenoxyethanol and thimerosal. It is preferred, however, not to use mercurial preservatives (e.g. thimerosal) during the process of the invention. Thus, between 1 and all of the components mixed in a process may be substantially free from mercurial preservative. However, the presence of trace amounts may be unavoidable if a component was treated with such a preservative before being used in the invention. For safety, however, it is preferred that the final composition contains less than about 25 ng/ml mercury. More preferably, the final vaccine product contains no detectable thimerosal. This will generally be achieved by removing the mercurial preservative from an antigen preparation prior to its addition in the process of the invention or by avoiding the use of thimerosal during the preparation of the components used to make the composition. Mercury-free compositions are preferred.

[0354] Compositions of the invention will usually be in aqueous form.

[0355] During manufacture, dilution of components to give desired final concentrations will usually be performed with WFI (water for injection), or with buffer.

[0356] The invention can provide bulk material which is suitable for packaging into individual doses, which can then be distributed for administration to patients. Concentrations discussed above are typically concentrations in final packaged dose, and so concentrations in bulk vaccine may be higher (e.g. to be reduced to final concentrations by dilution).

[0357] Compositions of the invention are administered to patients in unit doses i.e. the amount of a composition given to a single patient in a single administration (e.g. a single injection is a unit dose). Where a composition is administered as a liquid then a unit dose typically has a volume of 0.5 ml. This volume will be understood to include normal variance e.g. 0.5 ml.+-.0.05 ml. For multidose situations, multiple dose amounts will be extracted and packaged together in a single container e.g. 5 ml for a 10-dose multidose container (or 5.5 ml with 10% overfill).

[0358] Residual material from individual antigenic components may also be present in trace amounts in the final vaccine produced by the process of the invention. For example, if formaldehyde is used to prepare the toxoids of diphtheria, tetanus and pertussis then the final vaccine product may retain trace amounts of formaldehyde (e.g. less than 10 .mu.g/ml, preferably <5 .mu.g/ml). Media or stabilizers may have been used during poliovirus preparation (e.g. Medium 199), and these may carry through to the final vaccine. Similarly, free amino acids (e.g. alanine, arginine, aspartate, cysteine and/or cystine, glutamate, glutamine, glycine, histidine, proline and/or hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine and/or valine), vitamins (e.g. choline, ascorbate, etc.), disodium phosphate, monopotassium phosphate, calcium, glucose, adenine sulfate, phenol red, sodium acetate, potassium chloride, etc. may be retained in the final vaccine at .ltoreq.100 .mu.g/ml, preferably <10 .mu.g/ml, each. Other components from antigen preparations, such as neomycin (e.g. neomycin sulfate, particularly from a poliovirus component), polymyxin B (e.g. polymyxin B sulfate, particularly from a poliovirus component), etc. may also be present at sub-nanogram amounts per dose. A further possible component of the final vaccine which originates in the antigen preparations arises from less-than-total purification of antigens. Small amounts of B. pertussis. C. diphtheriae. C. tetani and S. cerevisiae proteins and/or genomic DNA may therefore be present. To minimize the amounts of these residual components, antigen preparations are preferably treated to remove them prior to the antigens being used with the invention.

[0359] Where a poliovirus component is used, it will generally have been grown on Vero cells. The final vaccine preferably contains less than 10 ng/ml, preferably .ltoreq.1 ng/ml e.g. .ltoreq.500 .mu.g/ml or .ltoreq.50 .mu.g/ml of Vero cell DNA e.g. less than 10 ng/ml of Vero cell DNA that is .gtoreq.50 base pairs long.

[0360] Compositions of the invention are presented for use in containers. Suitable containers include vials and disposable syringes (preferably sterile ones). Processes of the invention may comprise a step of packaging the vaccine into containers for use. Suitable containers include vials and disposable syringes (preferably sterile ones).

[0361] The invention also provides a delivery device (e.g. syringe, nebuliser, sprayer, inhaler, dermal patch, etc.) containing a pharmaceutical composition of the invention e.g. containing a unit dose.

[0362] This device can be used to administer the composition to a vertebrate subject.

[0363] The invention also provides a sterile container (e.g. a vial) containing a pharmaceutical composition of the invention e.g. containing a unit dose.

[0364] The invention also provides a unit dose of a pharmaceutical composition of the invention.

[0365] The invention also provides a hermetically scaled container containing a pharmaceutical composition of the invention. Suitable containers include e.g. a vial.

[0366] Where a composition of the invention is presented in a vial, this is preferably made of a glass or plastic material. The vial is preferably sterilized before the composition is added to it. To avoid problems with latex-sensitive patients, vials may be sealed with a latex-free stopper. The vial may include a single dose of vaccine, or it may include more than one dose (a `multidose` vial) e.g. 10 doses. When using a multidose vial, each dose should be withdrawn with a sterile needle and syringe under strict aseptic conditions, taking care to avoid contaminating the vial contents.

[0367] Preferred vials are made of colorless glass.

[0368] A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial (e.g. to reconstitute lyophilised material therein), and the contents of the vial can be removed back into the syringe. After removal of the syringe from the vial, a needle can then be attached and the composition can be administered to a patient. The cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed.

[0369] Where the composition is packaged into a syringe, the syringe will not normally have a needle attached to it, although a separate needle may be supplied with the syringe for assembly and use.

[0370] Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may be provided with peel-off labels on which the lot number and expiration date of the contents may be printed, to facilitate record keeping. The plunger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration. The syringes may have a latex rubber cap and/or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield. Grey butyl rubber is preferred. Preferred syringes are those marketed under the trade name "Tip-Lok".TM..

[0371] Where a glass container (e.g. a syringe or a vial) is used, then it is preferred to use a container made from a borosilicate glass rather than from a soda lime glass.

[0372] After a composition is packaged into a container, the container can then be enclosed within a box for distribution e.g. inside a cardboard box, and the box will be labeled with details of the vaccine e.g. its trade name, a list of the antigens in the vaccine (e.g. `hepatitis B recombinant`, etc.), the presentation container (e.g. `Disposable Prefilled Tip-Lok Syringes` or `10.times.0.5 ml Single-Dose Vials`), its dose (e.g. `each containing one 0.5 ml dose`), warnings (e.g. `For Adult Use Only` or `For Pediatric Use Only`), an expiration date, an indication, a patent number, etc. Each box might contain more than one packaged vaccine e.g. five or ten packaged vaccines (particularly for vials).

[0373] The vaccine may be packaged together (e.g. in the same box) with a leaflet including details of the vaccine e.g. instructions for administration, details of the antigens within the vaccine, etc. The instructions may also contain warnings e.g. to keep a solution of adrenaline readily available in case of anaphylactic reaction following vaccination, etc.

[0374] The packaged vaccine is preferably stored at between 2.degree. C. and 8.degree. C. It should not be frozen.

[0375] Vaccines can be provided in full-liquid form (i.e. where all antigenic components are in aqueous solution or suspension) after manufacture, or they can be prepared in a form where the vaccine can be prepared extemporaneously at the time/point of use by mixing together two components. Such two-component embodiments include liquid/liquid mixing and liquid/solid mixing e.g. by mixing aqueous material with lyophilised material. For instance, in one embodiment a vaccine can be made by mixing: (a) a first component comprising aqueous antigens and/or adjuvant; and (b) a second component comprising lyophilized antigens. In another embodiment a vaccine can be made by mixing: (a) a first component comprising aqueous antigens and/or adjuvant; and (b) a second component comprising aqueous antigens. In another embodiment a vaccine can be made by mixing: (a) a first component comprising aqueous antigens; and (b) a second component comprising aqueous adjuvant. The two components are preferably in separate containers (e.g. vials and/or syringes), and the invention provides a kit comprising components (a) and (b).

[0376] Another useful liquid/lyophilised format comprises (a) an aqueous complex of an aluminium salt and a TLR agonist and (b) a lyophilised component including one or more antigens. A vaccine composition suitable for patient administration is obtained by mixing components (a) and (b). In some embodiments component (a) is antigen-free, such that all antigenic components in the final vaccine are derived from component (b); in other embodiments component (a) includes one or more antigen(s), such that the antigenic components in the final vaccine are derived from both components (a) and (b).

[0377] Thus the invention provides a kit for preparing a combination vaccine, comprising components (a) and (b) as noted above. The kit components are typically vials or syringes, and a single kit may contain both a vial and a syringe. The invention also provides a process for preparing such a kit, comprising the following steps: (i) preparing an aqueous component vaccine as described above; (ii) packaging said aqueous combination vaccine in a first container e.g. a syringe; (iii) preparing an antigen-containing component in lyophilised form; (iv) packaging said lyophilised antigen in a second container e.g. a vial; and (v) packaging the first container and second container together in a kit. The kit can then be distributed to physicians.

[0378] A liquid/lyophilised format is particularly useful for vaccines that include a conjugate component, particularly Hib and/or meningococcal and/or pneumococcal conjugates, as these may be more stable in lyophilized form. Thus conjugates may be lyophilised prior to their use with the invention.

[0379] Where a component is lyophilised it generally includes non-active components which were added prior to freeze-drying e.g. as stabilizers. Preferred stabilizers for inclusion are lactose, sucrose and mannitol, as well as mixtures thereof e.g. lactose/sucrose mixtures, sucrose/mannitol mixtures, etc. A final vaccine obtained by aqueous reconstitution of the lyophilised material may thus contain lactose and/or sucrose. It is preferred to use amorphous excipients and/or amorphous buffers when preparing lyophilised vaccines [179].

[0380] Most compositions of the invention include diphtheria, tetanus and pertussis toxoids. In pediatric-type compositions the composition includes an excess of diphtheria toxoid relative to tetanus toxoid (as measured in Lf units). The excess is ideally at least 1.5:1 e.g. 5 Lf of diphtheria toxoid for every 2 Lf of tetanus toxoid (i.e. a 2.5:1 ratio). These embodiments are most useful in infants and children. In booster-type compositions, which are most useful in adolescents and adults, the composition includes an excess of tetanus toxoid relative to diphtheria toxoid (as measured in Lf units). The excess is ideally at least 1.5:1 e.g. 2 Lf of tetanus toxoid for every 1 Lf of diphtheria toxoid (i.e. a 2:1 ratio). In further embodiments, equal amounts of diphtheria and tetanus toxoids are used (in Lf units). Where one of diphtheria or tetanus is present at an excess, the excess should ideally be at least 1.5-fold e.g. 2-fold or 2.5-fold, but the excess will not usually be more than 5-fold.

[0381] A composition of the invention includes a serogroup B meningococcus immunogen and at least one of a diphtheria toxoid, a tetanus toxoid, and/or a pertussis toxoid. Ideally a composition includes all four of a serogroup B meningococcus immunogen, a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid. In some embodiments a composition of the invention includes no immunogens beyond those in this list; in other embodiments a composition of the invention does include immunogens beyond those in this list. Thus, for example, some compositions include diphtheria, tetanus and pertussis toxoids, inactivated poliovirus for Types 1, 2 & 3, hepatitis B virus surface antigen and a Hib conjugate. The antigenic portion of these compositions may consist of the antigens in this list, or may further include antigens from additional pathogens (e.g. meningococcus). Thus the compositions can be used as vaccines themselves, or as components of further combination vaccines.

[0382] Specific embodiments of the invention include compositions whose immunogens consist of: (a) D-T-aP-MenB; (b) D-T-aP-MenB-IPV; (c) D-T-aP-MenB-HBsAg; (d) D-T-aP-MenB-Hib; (e) D-T-aP-MenB-HBsAg-Hib; (f) D-T-aP-MenB-HBsAg-IPV; (g) D-T-aP-MenB-IPV-Hib; (h) D-T-aP-MenB-IPV-Hib-HBsAg; (i) D-T-MenB; where "D" is diphtheria toxoid, `T` is tetanus toxoid, "aP" is an acellular pertussis antigen or mixture, MenB is a serogroup B meningococcus antigen or mixture, "IPV" is an inactivated poliovirus antigen or mixture, "HBsAg" is a hepatitis B virus surface antigen, and "Hib" is a conjugated H. influenzae type B capsular saccharide.

[0383] Methods of Treatment, and Administration of the Vaccine

[0384] Compositions of the invention are suitable for administration to human patients, and the invention provides a method of raising an immune response in a patient, comprising the step of administering a composition of the invention to the patient.

[0385] The invention also provides a composition of the invention for use in medicine. The composition may be administered as variously described herein e.g. in some embodiments by giving an infant no more than two doses of a combination vaccine.

[0386] The invention also provides the use of a serogroup B meningococcus immunogen, a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid (and, optionally, an adjuvant) in the manufacture of a medicament for raising an immune response in a patient. The medicament is ideally a composition as variously described elsewhere herein, and it can be administered as variously described herein.

[0387] The immune responses raised by these methods, uses and compositions are ideally protective, and immunogenic compositions of the invention are preferably vaccines, for use in the prevention of at least diphtheria, tetanus, and whooping cough. Depending on their antigen components the vaccines may also protect against bacterial meningitis, polio, hepatitis, etc.

[0388] In order to have full efficacy, a typical primary immunization schedule (particularly for a child) may involve administering more than one dose. For example, doses may be at: 0 & 6 months (time 0 being the first dose); at 0, 1, 2 & 6 months; at day 0, day 21 and then a third dose between 6 & 12 months; at 2, 4 & 6 months; at 3, 4 & 5 months; at 6, 10 & 14 weeks; at 2, 3 & 4 months; or at 0, 1, 2, 6 & 12 months.

[0389] Compositions can also be used as booster doses e.g. for children in the second year of life, for an adolescent, or for an adult.

[0390] Compositions of the invention can be administered by intramuscular injection e.g. into the arm or leg.

[0391] Optional Requirements and Disclaimers [180]

[0392] In some embodiments, the invention does not encompass compositions in unit dose form comprising (i) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, and (ii) an aluminium salt adjuvant, wherein the amount of Al.sup.+++ in the unit dose is less than 0.2 mg. In other embodiments, if a composition is in unit dose form and comprises (i) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, and (ii) an aluminium salt adjuvant, but the amount of Al.sup.+++ in the unit dose is less than 0.2 mg, then: (a) the composition includes at least a 1.5-fold excess of diphtheria toxoid to tetanus toxoid, measured in Lf units; or (b) the composition includes at least a 1.5-fold excess of tetanus toxoid to diphtheria toxoid, measured in Lf units; or (c) the composition includes an acellular PT-containing antigen pertussis antigen rather than a whole-cell pertussis antigen.

[0393] In some embodiments, the invention does not encompass compositions comprising (i) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, and (ii) an aluminium salt adjuvant, wherein the concentration of Al.sup.+++ is less than 0.4 mg/ml. In other embodiments, if a composition comprises (i) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, and (ii) an aluminium salt adjuvant, but the concentration of Al.sup.+++ in the unit dose is less than 0.4 mg/ml, then: (a) the composition includes at least a 1.5-fold excess of diphtheria toxoid to tetanus toxoid, measured in Lf units; or (b) the composition includes at least a 1.5-fold excess of tetanus toxoid to diphtheria toxoid, measured in Lf units; or (c) the composition includes an acellular PT-containing antigen pertussis antigen rather than a whole-cell pertussis antigen.

[0394] In some embodiments, the invention does not encompass compositions comprising (i) an aluminium salt adjuvant and (ii) .ltoreq.8 Lf/ml diphtheria toxoid, .ltoreq.3.5 Lf/ml tetanus toxoid, and .ltoreq.5 .mu.g/ml pertussis toxoid. In other embodiments, if a composition comprises (i) an aluminium salt adjuvant and (ii) .ltoreq.8 Lf/ml diphtheria toxoid, .ltoreq.3.5 Lf/ml tetanus toxoid, and .ltoreq.5 .mu.g/ml pertussis toxoid, then: (a) the composition includes at least a 1.5-fold excess of diphtheria toxoid to tetanus toxoid, measured in Lf units; or (b) the composition includes at least a 1.5-fold excess of tetanus toxoid to diphtheria toxoid, measured in Lf units; or (c) the composition includes an acellular PT-containing antigen pertussis antigen rather than a whole-cell pertussis antigen.

[0395] In some embodiments, the invention does not encompass compositions comprising (i) an oil-in-water emulsion adjuvant (ii) a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, and a Hib conjugate, and (iii) a hepatitis B virus surface antigen and/or an inactivated poliovirus antigen. In other embodiments, if a composition comprises (i) an oil-in-water emulsion adjuvant (ii) a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, and a Hib conjugate, then: (a) the composition does not include a hepatitis B virus surface antigen; or (b) the composition does not include an inactivated poliovirus antigen; or (c) the composition includes neither an inactivated poliovirus antigen nor a hepatitis B virus surface antigen; or (d) the composition includes at least a 1.5-fold excess of diphtheria toxoid to tetanus toxoid, measured in Lf units; or (e) the composition includes at least a 1.5-fold excess of tetanus toxoid to diphtheria toxoid, measured in Lf units; or (f) the composition includes an acellular PT-containing antigen pertussis antigen rather than a whole-cell pertussis antigen.

[0396] In some embodiments, the invention does not encompass compositions which comprise a conjugate of a H. influenzae type b capsular saccharide antigen and an outer membrane protein complex from serogroup B meningococcus. In other embodiments, if a composition of the invention includes a conjugate of a H. influenzae type b capsular saccharide antigen and an outer membrane protein complex from serogroup B meningococcus then it must also include a further immunogen from serogroup B meningococcus.

[0397] In some embodiments, the invention does not encompass compositions which include both an aluminium salt adjuvant and a TLR4 agonist.

[0398] General

[0399] 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.

[0400] 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.

[0401] The term "about" in relation to a numerical value x means, for example, x.+-.10%.

[0402] Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

[0403] Where an antigen is described as being "adsorbed" to an adjuvant, it is preferred that at least 50% (by weight) of that antigen is adsorbed e.g. 50%, 60%, 70%, 80%, 90%, 95%, 98% or more. It is preferred that diphtheria toxoid and tetanus toxoid are both totally adsorbed i.e. none is detectable in supernatant. Total adsorption of HBsAg can be used.

[0404] Amounts of conjugates are generally given in terms of mass of saccharide (i.e. the dose of the conjugate (carrier+saccharide) as a whole is higher than the stated dose) in order to avoid variation due to choice of carrier.

[0405] Phosphorous-containing groups employed with the invention may exist in a number of protonated and deprotonated forms depending on the pH of the surrounding environment, for example the pH of the solvent in which they are dissolved. Therefore, although a particular form may be illustrated herein, it is intended, unless otherwise mentioned, for these illustrations to merely be representative and not limiting to a specific protonated or deprotonated form. For example, in the case of a phosphate group, this has been illustrated as --OP(OX)(OH).sub.2 but the definition includes the protonated forms --[OP(O)(OH.sub.2)(OH)].sup.+ and [OP(O)(OH.sub.2).sub.2].sup.2+ that may exist in acidic conditions and the deprotonated forms --[OP(O)(OH)(O)] and [OP(OXO).sub.2].sup.2- that may exist in basic conditions. The invention encompasses all such forms.

[0406] TLR agonists can exist as pharmaceutically acceptable salts. Thus, the compounds may be used in the form of their pharmaceutically acceptable salts i.e. physiologically or toxicologically tolerable salt (which includes, when appropriate, pharmaceutically acceptable base addition salts and pharmaceutically acceptable acid addition salts).

[0407] In the case of TLR agonists shown herein which may exist in tautomeric forms, the compound can be used in all such tautomeric forms.

[0408] Where a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.

[0409] Where animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encephalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE).

[0410] Meningococcal Protein Immunogens

[0411] NHBA (Neisserial Heparin Binding Antigen)

[0412] NHBA [181] was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] as 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 182, and in example 13 and FIG. 21 of reference 183 (SEQ IDs 3179 to 3184 therein). Various immunogenic fragments of NHBA have also been reported.

[0413] 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.

[0414] 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.

[0415] One useful NHBA antigen comprises SEQ ID NO: 4, which is a fusion of NHBA to NMB1030, as present in the BEXSERO.TM. product.

[0416] NadA (Neisserial Adhesin A)

[0417] The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] as gene NMB1994 (GenBank accession number GI:7227256; SEQ ID NO: herein). The sequences of NadA antigen from many strains have been published since then, and the protein's activity as a Neisserial adhesin has been well documented. Various immunogenic fragments of NadA have also been reported.

[0418] 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.

[0419] NadA will usually be present in a composition in oligomeric form e.g. trimers [184].

[0420] 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-mcningococcal antibodies after administration to a subject. SEQ ID NO: 6 is one such fragment, as present in the BEXSERO.TM. product.

[0421] fHbp (Factor H Binding Protein)

[0422] The fHbp antigen has been characterised in detail. It has also been known as protein `741` [SEQ IDs 2535 & 2536 in ref. 183], `NMB1870`, `GNA1870` [185, 186, 207], `P2086`, `LP2086` or `ORF2086` [187-189]. It is naturally a lipoprotein and is expressed across all meningococcal serogroups. The structure of fHbp's C-terminal immunodominant domain (`fHbpC`) has been determined by NMR [190]. This part of the protein forms an eight-stranded .beta.-barrel, whose strands are connected by loops of variable lengths. The barrel is preceded by a short .alpha.-helix and by a flexible N-terminal tail.

[0423] The fHbp antigen falls into three distinct variants [191] 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.

[0424] Where a composition comprises a single fHBP variant, it may include one of the following: [0425] (a) a first polypeptide comprising a first amino acid sequence, where the first amino acid sequence comprises an amino acid sequence (i) having at least a % sequence identity to SEQ ID NO: 1 and/or (ii) consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1; [0426] (b) a second polypeptide, comprising a second amino acid sequence, where the second amino acid sequence comprises an amino acid sequence (i) having at least b % sequence identity to SEQ ID NO: 2 and/or (ii) consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2; [0427] (c) a third polypeptide, comprising a third amino acid sequence, where the third amino acid sequence comprises an amino acid sequence (i) having at least c % sequence identity to SEQ ID NO: 3 and/or (ii) consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3.

[0428] The value of a is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of h is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of c is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The values of a, b and c may be the same or different. In some embodiments, a b and c are identical.

[0429] 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 may be the same or different. In some embodiments, x y and z are identical.

[0430] Fragments preferably comprise an epitope from the respective SEQ ID NO: sequence. Other useful fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the respective SEQ ID NO: while retaining at least one epitope thereof.

[0431] In some embodiments the fragment of at least x contiguous amino acids from SEQ ID NO: 1 is not also present within SEQ ID NO: 2 or within SEQ ID NO: 3. Similarly, the fragment of at least y contiguous amino acids from SEQ ID NO: 2 might not also be present within SEQ ID NO: 1 or within SEQ ID NO: 3. Similarly, the fragment of at least z contiguous amino acids from SEQ ID NO: 3 might not also be present within SEQ ID NO: 1 or within SEQ ID NO: 2. In some embodiments, when said fragment from one of SEQ ID NOs: 1 to 3 is aligned as a contiguous sequence against the other two SEQ ID NOs, the identity between the fragment and each of the other two SEQ ID NOs is less than 75% e.g. less than 70%, less than 65%, less than 60%, etc.

[0432] Where a composition comprises two different meningococcal fHBP antigens, it may include a combination of: (i) a first and second polypeptide as defined above; (ii) a first and third polypeptide as defined above; or (iii) a second and third polypeptide as defined above. A combination of a first and third polypeptide is preferred. Where a composition comprises two different meningococcal fHBP antigens, although these may share some sequences in common, the first, second and third polypeptides have different fHBP amino acid sequences.

[0433] A polypeptide comprising the first amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 20 (MC58). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 21 or to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 22.

[0434] A polypeptide comprising the second amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 21 (2996). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 20 or to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 22.

[0435] A polypeptide comprising the third amino acid sequence will, when administered to a subject, elicit an antibody response comprising antibodies that bind to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 22 (M1239). In some embodiments some or all of these antibodies do not bind to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 20 or to the wild-type meningococcus protein which has nascent amino acid sequence SEQ ID NO: 21.

[0436] A useful first amino acid sequence has at least 85% identity (e.g. >95% or 100%) to SEQ ID NO: 1 (strain MC58). Another useful first amino acid sequence has at least 95% identity (e.g. >98% or 100%) to SEQ ID NO: 23 (strain CDC1573).

[0437] A useful third amino acid sequence has at least 85% identity (e.g. >95% or 100%) to SEQ ID NO: 3 (strain M1239). Another useful third amino acid sequence has at least 95% identity (e.g. >98% or 100%) to SEQ ID NO: 25 (strain M98-250771).

[0438] Combinations comprising a mixture of first and third sequences based around SEQ ID NOs: 23 and 25 (or their close variants) are particularly useful. Thus a composition may comprise a polypeptide comprising amino acid sequence SEQ ID NO: 24 and a polypeptide comprising amino acid sequence SEQ ID NO: 26.

[0439] Where a composition includes two meningococcal fHBP antigens, this may be in a bivalent fl-IBP composition, or there may be more than two different fl-IBP antigens e.g. in a trivalent or tetravalent fHBP composition.

[0440] Another useful fHbp which can be used according to the invention is one of the modified forms disclosed, for example, in reference 192 e.g. comprising SEQ ID NO: 20 or 23 therefrom. These modified forms can elicit antibody responses which are broadly bactericidal against meningococci by recognising multiple fHbp variant. One such modified form is SEQ ID NO: 28 herein (SEQ ID NO: 23 in ref. 192), which can be fused to non-fHbp sequences as disclosed in reference 193 e.g. to give SEQ ID NO: 19 (which contains NMB2091 and two copies of SEQ ID NO: 28), which is used in the examples below.

[0441] SEQ ID NO: 77 from ref. 192 is another useful fHbp sequence which can be used in order to provide broad inter-strain reactivity.

[0442] In some embodiments fHBP polypeptide(s) are lipidated e.g. at a N-terminus cysteine. In other embodiments, however, fHBP polypeptide(s) are not lipidated. For lipidated fHBPs, lipids attached to cysteines will usually include palmitoyl residues e.g. as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys), dipalmitoyl-S-glyceryl cysteine (Pam2Cys), N-acetyl (dipalmitoyl-S-glyceryl cysteine), etc. Examples of mature lipidated f-BP sequences are SEQ ID NO: 24 (including SEQ ID NO: 23) and SEQ ID NO: 26 (including SEQ ID NO: 25). If fHbp protein(s) are located in a vesicle then they will usually be lipidated.

[0443] Administration of a fHBP will preferably elicit antibodies which can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 1, 2 or 3. Advantageous fHBP antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[0444] The total amount of a fHBP polypeptide will usually be between 1 and 500 .mu.g per unit dose e.g. between 60 and 200 .mu.g per unit. An amount of 10, 20, 40, 50, 60, 80, 100 or 200 .mu.g per unit dose for each fHBP polypeptide is typical in a human vaccine dose.

[0445] Where a composition comprises different meningococcal fHBP antigens, these may be present as separate polypeptides as described above (e.g. a first and second polypeptide) or they may be present as part of a single fusion polypeptide i.e. where at least two (e.g. 2, 3, 4, 5, or more) fHBP antigens are expressed as a single polypeptide chain, as disclosed for meningococcal antigens in reference 194. Most usefully, a fusion polypeptide can include each of a first, second and third sequence as discussed above e.g. SEQ ID NO: 27.

[0446] HmbR

[0447] The full-length HmbR sequence was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] as gene NMB1668 (SEQ ID NO: 7 herein). Reference 195 reports a HmbR sequence from a different strain (SEQ ID NO: 8 herein), and reference 196 reports a further sequence (SEQ ID NO: 15 herein). SEQ ID NOs: 7 and 8 differ in length by 1 amino acid and have 94.2% identity. SEQ ID NO: 15 is one amino acid shorter than SEQ ID NO: 7 and they have 99% identity (one insertion, seven differences). The invention can use any such HmbR polypeptide.

[0448] 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.

[0449] 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'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 197. Fragments that retain a transmembrane sequence are useful, because they can be displayed on the bacterial surface e.g. in vesicles. If soluble HmbR is used, however, sequences omitting the transmembrane sequence, but typically retaining epitope(s) from the extracellular portion, can be used.

[0450] 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.

[0451] NspA (Neisserial Surface Protein A)

[0452] The NspA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] as gene NMB0663 (GenBank accession number GI:7225888; SEQ ID NO: 11 herein). The antigen was previously known from references 198 & 199. The sequences of NspA antigen from many strains have been published since then. Various immunogenic fragments of NspA have also been reported.

[0453] 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.

[0454] 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.

[0455] NhhA (Neisseria Hia Homologue)

[0456] The NhhA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] 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 182 & 200, and various immunogenic fragments of NhhA have been reported. It is also known as Hsf.

[0457] Preferred NhhA 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: 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.

[0458] 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.

[0459] Advantageous NhhA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[0460] App (Adhesion and Penetration Protein)

[0461] The App antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] 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.

[0462] 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.

[0463] 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.

[0464] Omp85 (85 kDa Outer Membrane Protein)

[0465] The Omp85 antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] 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 201 and 202. Various immunogenic fragments of Omp85 have also been reported.

[0466] 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.

[0467] 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.

[0468] TbpA

[0469] The TbpA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] 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.

[0470] 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.

[0471] 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.

[0472] TbpB

[0473] The TbpB antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] as gene NMB0460 (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.

[0474] 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.

[0475] 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.

[0476] Cu,Zn-Superoxide Dismutase

[0477] The Cu,Zn-superoxide dismutase antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] 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.

[0478] 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.

[0479] 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.

[0480] ZnuD

[0481] The ZnuD antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [26] as gene NMB0964 (GenBank accession number GI:15676857; SEQ ID NO: 29 herein). The sequences of ZnuD from many strains have been published since then e.g. see references 203 & 204.

[0482] Preferred ZnuD 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: 29; and/or (b) comprising a fragment of at least `n` consecutive amino acids of SEQ ID NO: 29, 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: 29.

[0483] The most useful ZnuD antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 29.

[0484] Advantageous ZnuD antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[0485] Meningococcal Vesicles

[0486] The invention can be used with various types of vesicle which are known for Neisseria meningitidis.

[0487] Reference 22 discloses the construction of vesicles from meningococcal strains modified to express six different PorA subtypes. References 205-207 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 [208]). Reference 209 recently reported a clinical study of five formulations of an OMV vaccine in which PorA & FrpB are knocked-out and Hsf & TbpA are over-expressed. Reference 210 reports a native outer membrane vesicle vaccine prepared from bacteria having inactivated synX, lpxL1, and lgtA genes. All such vesicles can be used herein.

[0488] OMVs can be prepared from meningococci which over-express desired antigen(s) due to genetic modification. In addition to genetic modification(s) which cause over-expression of 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, lgtB, porA, frpB, synX, lgtA, mlt4 and/or lst.

[0489] The bacterium may have low endotoxin levels, achieved by knockout of enzymes involved in LPS biosynthesis [211,212].

[0490] 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.

[0491] 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. 213] e.g. the ET-37 complex is the ST-II complex by MLST, the ET-5 complex is ST-32 (ET-5), lineage 3 is ST-41/44, etc.

[0492] In some embodiments a bacterium may include one or more of the knockout and/or hyper-expression mutations disclosed in references 226 and 214-216. 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 [214]; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, IHtrB/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, PorB, SiaD, SynA, SynB, and/or SynC.

[0493] 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; (xi) deleted cps gene complex; (xi) up-regulated NHBA; (xii) up-regulated NadA; (xiii) up-regulated NHBA and NadA; (xiv) up-regulated fHbp; (xv) down-regulated LpxL1. 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 a chain.

[0494] 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 [216]).

[0495] The vesicles may lack LOS altogether, or they may lack hexa-acylated LOS e.g. LOS in the vesicles may have a reduced number of secondary acyl chains per LOS molecule [217]. For example, the vesicles may from a strain which has a lpxL1 deletion or mutation which results in production of a penta-acylated LOS [206,210]. LOS in a strain may lack a lacto-N-ncotetraose epitope e.g. it may be a lst and/or lgtB knockout strain [209]. LOS may lack at least one wild-type primary O-linked fatty acid [218]. LOS having. The LOS may have no a chain. The LOS may comprise GlcNAc-Hep.sub.2phosphoethanolamine-KDO.sub.2-Lipid A [219].

[0496] As a result of up-regulation mentioned above, vesicles prepared from modified meningococci contain higher levels of the up-regulated antigen(s). The increase in expression in the vesicles (measured relative to a corresponding wild-type strain) is usefully at least 10%, measured in mass of the relevant antigen per unit mass of vesicle, and is more usefully at least 20%, 30%, 40%, 50%, 75%, 100% or more.

[0497] Suitable recombinant modifications which can be used to cause up-regulation 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'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'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 220. These methods include promoter replacement, or the removal or replacement of a DNA motif which is responsible for a gene'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. 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 207, where a bacterium's endogenous chromosomal fHbp gene was deleted and replaced by a plasmid-encoded copy (see also reference 221). Expression from the replacement copy is higher than from the previous copy, thus leading to up-regulation. 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 up-regulated genes can advantageously include a CREN [222].

[0498] A 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.

[0499] In embodiments where NHBA is up-regulated, various approaches can be used. For convenience, the approach already reported in reference 181 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.

[0500] In embodiments where NadA is up-regulated, various approaches can be used. One useful approach involves deletion of the gene encoding NadR (NMB1843), which is a transcriptional repressor protein [223] 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 up-regulation 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.

[0501] Up-regulation of NhhA is already reported in references 209 and 224. Up-regulation of TbpA is already reported in references 209, 224 and 225. Up-regulation of HmbR is already reported in reference 196. Up-regulation of TbpB is already reported in reference 225. Up-regulation of NspA is already reported in reference 226, in combination with porA4 and cps knockout. Up-regulation of Cu,Zn-superoxide dismutase is already reported in reference 225. Up-regulation of fHbp is already reported in references 205-207 & 221, and by a different approach (expressing a constitutively-active mutant FNR) in references 227 & 228.

[0502] In some embodiments each of NHBA, NadA and fHbp are up-regulated. These three antigens are components of the "universal vaccine" disclosed in reference 8 or "4CMenB" [229,230]. 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.

[0503] 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 22 and 23. Conversely, modification to remove PorA is also known e.g. from reference 209.

[0504] The vesicles may be free from one of both of PorA and FrpB. Preferred vesicles are PorA-free.

[0505] The invention may be used with mixtures of vesicles from different strains. For instance, reference 24 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 25 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.

[0506] Another useful combination of vesicles is disclosed in references 231 & 232. A trivalent mixture of this type can include vesicles prepared from each of: (a) a first strain which over-expresses NadA; (b) a second strain which over-expresses a fHbp sequence from variant 1 i.e. a first fHbp polypeptide sequence as defined above; and (c) a third strain which over-expresses a fHbp sequence from variant 2 i.e. a second fHbp polypeptide sequence as defined above. These strains can also have other modifications e.g. knockout of synX and LpxLJ, as disclosed in ref. 231.

BRIEF DESCRIPTION OF DRAWINGS

[0507] FIGS. 1A-B show serum total IgG responses against tetanus toxoid. FIG. 1A shows the serum total IgG responses against tetanus toxoid at day 35. FIG. 1B shows the serum total IgG responses against tetanus toxoid at day 49.

[0508] FIGS. 2A-B show serum total IgG responses against diphtheria toxoid. FIG. 2A shows the serum total IgG responses against diphtheria toxoid at day 35. FIG. 2B shows the serum total IgG responses against diphtheria toxoid at day 49.

[0509] FIGS. 3A-B show serum total IgG responses against pertussis toxoid. FIG. 3A shows the serum total IgG responses against pertussis toxoid at day 35. FIG. 3B shows the serum total IgG responses against pertussis toxoid at day 49.

[0510] FIGS. 4A-B show serum total IgG responses against pertactin. FIG. 4A shows the serum total IgG responses against pertactin at day 35. FIG. 4B shows the serum total IgG responses against pertactin at day 49.

[0511] FIGS. 5A-B show serum total IgG responses against FHA. FIG. 5A shows the serum total IgG responses against FHA at day 35. FIG. 5B shows the serum total IgG responses against FHA at day 49.

[0512] FIG. 6 shows serum total IgG responses against NadA.

[0513] FIG. 7 shows serum total IgG responses against NHBA.

[0514] FIG. 8 shows serum total IgG responses against fHbp.

[0515] The y-axis scale in all cases is 0.01 to 10,000.

MODES FOR CARRYING OUT THE INVENTION

[0516] An immunogen combination was prepared, containing the following components:

TABLE-US-00001 Immunogen Amount (per 0.5 ml) T Tetanus toxoid 5 Lf D Diphtheria toxoid 2 Lf aP Pertussis toxoid, PT-9K/129G 4 .mu.g FHA 4 .mu.g Pertactin 8 .mu.g MenB NHBA (SEQ ID NO: 4) 50 .mu.g NadA (SEQ ID NO: 6) 50 .mu.g fHbp (SEQ ID NO: 19) 50 .mu.g

[0517] For comparison purposes, an equivalent combination was prepared but without the MenB proteins.

[0518] These two immunogen combinations are referred to as "TdaP-MenB" and "TdaP".

[0519] These two combinations were adjuvanted with: [0520] (a) aluminium hydroxide, 1 mg/dose ("AI-H") [0521] (b) aluminium hydroxide, 1 mg/dose, with 100 .mu.g adsorbed `K2` TLR7 agonist [0522] (c) aluminium hydroxide, 1 mg/dose, with 100 .mu.g adsorbed synthetic MPL TLR4 agonist (d) MF59 squalene-containing oil-in-water emulsion.

[0523] All antigens were adsorbed to the Al--H in compositions (a) to (c) for both TdaP and TdaP-MenB, although pertactin was not fully adsorbed in compositions which include the MenB immunogens.

[0524] In addition to these four pairs of adjuvanted compositions, a further pair was unadjuvanted. This gave 10 compositions in total, (C1) to (C10):

TABLE-US-00002 No adjuvant Al--H Al--H/K2 Al--H/MPL MF59 TdaP C1 C2 C3 C4 C5 TdaP-MenB C6 C7 C8 C9 C10

[0525] Furthermore, for comparison the BOOSTRIX.TM. product was also tested ("C"), which contains (per 0.5 ml) 2.5Lf of diphtheria toxoid, 5Lf tetanus toxoid, and 18.5 .mu.g acellular pertussis antigens (a mixture of purified PT, FHA and p69 pertactin), adjuvanted with a mixture of aluminium phosphate and hydroxide salts. Finally, an immunogen-free negative control of buffer alone was also prepared ("C12").

[0526] These 12 compositions were administered to female Balb/C mice (6 weeks old) at 100 .mu.l intramuscular doses (2.times.50 .mu.l) on days 0, 21 and 35. Sera were tested 2 weeks after each dose and assessed for specific IgG responses against each of the 8 immunogens (except that only C6-C10 & C12 were tested for responses against the 3 MenB immunogens). These titers are shown in FIGS. 1-8. FIGS. 1-5 show data for days 35 (1A to 5A) and 49 (1B to 5B), whereas FIGS. 6-8 show data only for day 35.

[0527] The data show that the MenB antigens have no negative impact on IgG responses against the diphtheria, tetanus and acellular pertussis antigens after 2 or 3 doses. Furthermore, the inclusion of a TLR agonist with the Al--H adjuvant improved IgG responses against all antigens. The emulsion adjuvant also gave better results than Al--H alone. In all cases however, the adjuvants did not have a large impact on anti-PT responses.

[0528] The second dose of vaccine (day 21) led to an increase of IgG response against all antigens, but the third dose (day 35) did not provide a further significant increase. Thus the studied adjuvants provide a more rapid response to the re-injected antigens, which can be very useful in booster situations.

[0529] Thus the mixture of D, T, aP and MenB antigens offers a new and effective combination vaccine.

[0530] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

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Sequence CWU 1

1

291248PRTNeisseria meningitidis 1Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser 20 25 30 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 65 70 75 80 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 85 90 95 Ser Ala Leu Thr Ala Phe Gln Thr Glu Gln Ile Gln Asp Ser Glu His 100 105 110 Ser Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala 115 120 125 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr 130 135 140 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr 145 150 155 160 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly Asn Gly Lys Ile Glu His 165 170 175 Leu Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Ala Ala Asp Ile Lys 180 185 190 Pro Asp Gly Lys Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn 195 200 205 Gln Ala Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala 210 215 220 Gln Glu Val Ala Gly Ser Ala Glu Val Lys Thr Val Asn Gly Ile Arg 225 230 235 240 His Ile Gly Leu Ala Ala Lys Gln 245 2247PRTNeisseria meningitidis 2Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser 20 25 30 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 65 70 75 80 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His 85 90 95 Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys 100 105 110 Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly 115 120 125 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 130 135 140 His Gly Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr 145 150 155 160 Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu 165 170 175 Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ala Ala Glu Leu Lys Ala 180 185 190 Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser 195 200 205 Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln 210 215 220 Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys Val His Glu 225 230 235 240 Ile Gly Ile Ala Gly Lys Gln 245 3250PRTNeisseria meningitidis 3Val Ala Ala Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser 20 25 30 Ile Pro Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys 35 40 45 Thr Phe Lys Ala Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu 50 55 60 Lys Asn Asp Lys Ile Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val 65 70 75 80 Asp Gly Gln Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys 85 90 95 Gln Asn His Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn 100 105 110 Pro Asp Lys Thr Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser 115 120 125 Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu Pro Gly Gly Lys 130 135 140 Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg 145 150 155 160 Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile 165 170 175 Glu His Leu Lys Thr Leu Glu Gln Asn Val Glu Leu Ala Ala Ala Glu 180 185 190 Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg 195 200 205 Tyr Gly Ser Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp 210 215 220 Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys 225 230 235 240 Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 4644PRTArtificial SequenceHybrid protein 4Met Ala Ser Pro Asp Val Lys Ser Ala Asp Thr Leu Ser Lys Pro Ala 1 5 10 15 Ala Pro Val Val Ser Glu Lys Glu Thr Glu Ala Lys Glu Asp Ala Pro 20 25 30 Gln Ala Gly Ser Gln Gly Gln Gly Ala Pro Ser Ala Gln Gly Gly Gln 35 40 45 Asp Met Ala Ala Val Ser Glu Glu Asn Thr Gly Asn Gly Gly Ala Ala 50 55 60 Ala Thr Asp Lys Pro Lys Asn Glu Asp Glu Gly Ala Gln Asn Asp Met 65 70 75 80 Pro Gln Asn Ala Ala Asp Thr Asp Ser Leu Thr Pro Asn His Thr Pro 85 90 95 Ala Ser Asn Met Pro Ala Gly Asn Met Glu Asn Gln Ala Pro Asp Ala 100 105 110 Gly Glu Ser Glu Gln Pro Ala Asn Gln Pro Asp Met Ala Asn Thr Ala 115 120 125 Asp Gly Met Gln Gly Asp Asp Pro Ser Ala Gly Gly Glu Asn Ala Gly 130 135 140 Asn Thr Ala Ala Gln Gly Thr Asn Gln Ala Glu Asn Asn Gln Thr Ala 145 150 155 160 Gly Ser Gln Asn Pro Ala Ser Ser Thr Asn Pro Ser Ala Thr Asn Ser 165 170 175 Gly Gly Asp Phe Gly Arg Thr Asn Val Gly Asn Ser Val Val Ile Asp 180 185 190 Gly Pro Ser Gln Asn Ile Thr Leu Thr His Cys Lys Gly Asp Ser Cys 195 200 205 Ser Gly Asn Asn Phe Leu Asp Glu Glu Val Gln Leu Lys Ser Glu Phe 210 215 220 Glu Lys Leu Ser Asp Ala Asp Lys Ile Ser Asn Tyr Lys Lys Asp Gly 225 230 235 240 Lys Asn Asp Gly Lys Asn Asp Lys Phe Val Gly Leu Val Ala Asp Ser 245 250 255 Val Gln Met Lys Gly Ile Asn Gln Tyr Ile Ile Phe Tyr Lys Pro Lys 260 265 270 Pro Thr Ser Phe Ala Arg Phe Arg Arg Ser Ala Arg Ser Arg Arg Ser 275 280 285 Leu Pro Ala Glu Met Pro Leu Ile Pro Val Asn Gln Ala Asp Thr Leu 290 295 300 Ile Val Asp Gly Glu Ala Val Ser Leu Thr Gly His Ser Gly Asn Ile 305 310 315 320 Phe Ala Pro Glu Gly Asn Tyr Arg Tyr Leu Thr Tyr Gly Ala Glu Lys 325 330 335 Leu Pro Gly Gly Ser Tyr Ala Leu Arg Val Gln Gly Glu Pro Ser Lys 340 345 350 Gly Glu Met Leu Ala Gly Thr Ala Val Tyr Asn Gly Glu Val Leu His 355 360 365 Phe His Thr Glu Asn Gly Arg Pro Ser Pro Ser Arg Gly Arg Phe Ala 370 375 380 Ala Lys Val Asp Phe Gly Ser Lys Ser Val Asp Gly Ile Ile Asp Ser 385 390 395 400 Gly Asp Gly Leu His Met Gly Thr Gln Lys Phe Lys Ala Ala Ile Asp 405 410 415 Gly Asn Gly Phe Lys Gly Thr Trp Thr Glu Asn Gly Gly Gly Asp Val 420 425 430 Ser Gly Lys Phe Tyr Gly Pro Ala Gly Glu Glu Val Ala Gly Lys Tyr 435 440 445 Ser Tyr Arg Pro Thr Asp Ala Glu Lys Gly Gly Phe Gly Val Phe Ala 450 455 460 Gly Lys Lys Glu Gln Asp Gly Ser Gly Gly Gly Gly Ala Thr Tyr Lys 465 470 475 480 Val Asp Glu Tyr His Ala Asn Ala Arg Phe Ala Ile Asp His Phe Asn 485 490 495 Thr Ser Thr Asn Val Gly Gly Phe Tyr Gly Leu Thr Gly Ser Val Glu 500 505 510 Phe Asp Gln Ala Lys Arg Asp Gly Lys Ile Asp Ile Thr Ile Pro Val 515 520 525 Ala Asn Leu Gln Ser Gly Ser Gln His Phe Thr Asp His Leu Lys Ser 530 535 540 Ala Asp Ile Phe Asp Ala Ala Gln Tyr Pro Asp Ile Arg Phe Val Ser 545 550 555 560 Thr Lys Phe Asn Phe Asn Gly Lys Lys Leu Val Ser Val Asp Gly Asn 565 570 575 Leu Thr Met His Gly Lys Thr Ala Pro Val Lys Leu Lys Ala Glu Lys 580 585 590 Phe Asn Cys Tyr Gln Ser Pro Met Ala Lys Thr Glu Val Cys Gly Gly 595 600 605 Asp Phe Ser Thr Thr Ile Asp Arg Thr Lys Trp Gly Val Asp Tyr Leu 610 615 620 Val Asn Val Gly Met Thr Lys Ser Val Arg Ile Asp Ile Gln Ile Glu 625 630 635 640 Ala Ala Lys Gln 5434PRTArtificial SequenceHybrid protein 5Met Val Ser Ala Val Ile Gly Ser Ala Ala Val Gly Ala Lys Ser Ala 1 5 10 15 Val Asp Arg Arg Thr Thr Gly Ala Gln Thr Asp Asp Asn Val Met Ala 20 25 30 Leu Arg Ile Glu Thr Thr Ala Arg Ser Tyr Leu Arg Gln Asn Asn Gln 35 40 45 Thr Lys Gly Tyr Thr Pro Gln Ile Ser Val Val Gly Tyr Asp Arg His 50 55 60 Leu Leu Leu Leu Gly Gln Val Ala Thr Glu Gly Glu Lys Gln Phe Val 65 70 75 80 Gly Gln Ile Ala Arg Ser Glu Gln Ala Ala Glu Gly Val Tyr Asn Tyr 85 90 95 Ile Thr Val Ala Ser Leu Pro Arg Thr Ala Gly Asp Ile Ala Gly Asp 100 105 110 Thr Trp Asn Thr Ser Lys Val Arg Ala Thr Leu Leu Gly Ile Ser Pro 115 120 125 Ala Thr Arg Ala Arg Val Lys Ile Val Thr Tyr Gly Asn Val Thr Tyr 130 135 140 Val Met Gly Ile Leu Thr Pro Glu Glu Gln Ala Gln Ile Thr Gln Lys 145 150 155 160 Val Ser Thr Thr Val Gly Val Gln Lys Val Ile Thr Leu Tyr Gln Asn 165 170 175 Tyr Val Gln Arg Gly Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly 180 185 190 Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys 195 200 205 Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys 210 215 220 Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp 225 230 235 240 Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp 245 250 255 Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser 260 265 270 Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe 275 280 285 Gln Thr Glu Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala 290 295 300 Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe 305 310 315 320 Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe 325 330 335 Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala 340 345 350 Ala Lys Gln Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu 355 360 365 Asn Val Asp Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His 370 375 380 Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser 385 390 395 400 Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser 405 410 415 Ala Glu Val Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala 420 425 430 Lys Gln 6327PRTNeisseria meningitidis 6Ala Thr Asn Asp Asp Asp Val Lys Lys Ala Ala Thr Val Ala Ile Ala 1 5 10 15 Ala Ala Tyr Asn Asn Gly Gln Glu Ile Asn Gly Phe Lys Ala Gly Glu 20 25 30 Thr Ile Tyr Asp Ile Asp Glu Asp Gly Thr Ile Thr Lys Lys Asp Ala 35 40 45 Thr Ala Ala Asp Val Glu Ala Asp Asp Phe Lys Gly Leu Gly Leu Lys 50 55 60 Lys Val Val Thr Asn Leu Thr Lys Thr Val Asn Glu Asn Lys Gln Asn 65 70 75 80 Val Asp Ala Lys Val Lys Ala Ala Glu Ser Glu Ile Glu Lys Leu Thr 85 90 95 Thr Lys Leu Ala Asp Thr Asp Ala Ala Leu Ala Asp Thr Asp Ala Ala 100 105 110 Leu Asp Ala Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn Ile Thr 115 120 125 Thr Phe Ala Glu Glu Thr Lys Thr Asn Ile Val Lys Ile Asp Glu Lys 130 135 140 Leu Glu Ala Val Ala Asp Thr Val Asp Lys His Ala Glu Ala Phe Asn 145 150 155 160 Asp Ile Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp Glu Ala 165 170 175 Val Lys Thr Ala Asn Glu Ala Lys Gln Thr Ala Glu Glu Thr Lys Gln 180 185 190 Asn Val Asp Ala Lys Val Lys Ala Ala Glu Thr Ala Ala Gly Lys Ala 195 200 205 Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala Ala Asp Lys Ala Glu Ala 210 215 220 Val Ala Ala Lys Val Thr Asp Ile Lys Ala Asp Ile Ala Thr Asn Lys 225 230 235 240 Asp Asn Ile Ala Lys Lys Ala Asn Ser Ala Asp Val Tyr Thr Arg Glu 245 250 255 Glu Ser Asp Ser Lys Phe Val Arg Ile Asp Gly Leu Asn Ala Thr Thr 260 265 270 Glu Lys Leu Asp Thr Arg Leu Ala Ser Ala Glu Lys Ser Ile Ala Asp 275 280 285 His Asp Thr Arg Leu Asn Gly Leu Asp Lys Thr Val Ser Asp Leu Arg 290 295 300 Lys Glu Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu 305 310 315 320 Phe Gln Pro Tyr Asn Val Gly 325 7792PRTNeisseria meningitidis 7Met Lys Pro Leu Gln Met Leu Pro Ile Ala Ala Leu Val Gly Ser Ile 1 5 10 15 Phe Gly Asn Pro Val Leu Ala Ala Asp Glu Ala Ala Thr Glu Thr Thr 20 25 30 Pro Val Lys Ala Glu Ile Lys Ala Val Arg Val Lys Gly Gln Arg Asn 35 40 45 Ala Pro Ala Ala Val Glu Arg Val Asn Leu Asn Arg Ile Lys Gln Glu 50 55 60 Met Ile Arg Asp Asn Lys Asp Leu Val Arg Tyr Ser Thr Asp Val Gly 65 70 75 80 Leu Ser Asp Ser Gly Arg His Gln Lys Gly Phe Ala Val Arg Gly Val 85 90 95 Glu Gly Asn Arg Val Gly Val Ser Ile Asp Gly Val Asn Leu Pro Asp 100 105 110 Ser Glu Glu Asn Ser Leu Tyr Ala Arg Tyr Gly Asn Phe Asn Ser Ser 115 120

125 Arg Leu Ser Ile Asp Pro Glu Leu Val Arg Asn Ile Glu Ile Val Lys 130 135 140 Gly Ala Asp Ser Phe Asn Thr Gly Ser Gly Ala Leu Gly Gly Gly Val 145 150 155 160 Asn Tyr Gln Thr Leu Gln Gly Arg Asp Leu Leu Leu Asp Asp Arg Gln 165 170 175 Phe Gly Val Met Met Lys Asn Gly Tyr Ser Thr Arg Asn Arg Glu Trp 180 185 190 Thr Asn Thr Leu Gly Phe Gly Val Ser Asn Asp Arg Val Asp Ala Ala 195 200 205 Leu Leu Tyr Ser Gln Arg Arg Gly His Glu Thr Glu Ser Ala Gly Asn 210 215 220 Arg Gly Tyr Ala Val Glu Gly Glu Gly Ser Gly Ala Asn Ile Arg Gly 225 230 235 240 Ser Ala Arg Gly Ile Pro Asp Ser Ser Lys His Lys Tyr Asn His His 245 250 255 Ala Leu Gly Lys Ile Ala Tyr Gln Ile Asn Asp Asn His Arg Ile Gly 260 265 270 Ala Ser Leu Asn Gly Gln Gln Gly His Asn Tyr Thr Val Glu Glu Ser 275 280 285 Tyr Asn Leu Thr Ala Ser Ser Trp Arg Glu Ala Asp Asp Val Asn Arg 290 295 300 Arg Arg Asn Ala Asn Leu Phe Tyr Glu Trp Met Pro Asp Ser Asn Trp 305 310 315 320 Leu Ser Ser Leu Lys Ala Asp Phe Asp Tyr Gln Lys Thr Lys Val Ala 325 330 335 Ala Val Asn Asn Lys Gly Ser Phe Pro Met Asp Tyr Ser Thr Trp Thr 340 345 350 Arg Asn Tyr Asn Gln Lys Asp Leu Asp Glu Ile Tyr Asn Arg Ser Met 355 360 365 Asp Thr Arg Phe Lys Arg Phe Thr Leu Arg Leu Asp Ser His Pro Leu 370 375 380 Gln Leu Gly Gly Gly Arg His Arg Leu Ser Phe Lys Thr Phe Val Ser 385 390 395 400 Arg Arg Asp Phe Glu Asn Leu Asn Arg Asp Asp Tyr Tyr Phe Ser Gly 405 410 415 Arg Val Val Arg Thr Thr Ser Ser Ile Gln His Pro Val Lys Thr Thr 420 425 430 Asn Tyr Gly Phe Ser Leu Ser Asp Gln Ile Gln Trp Asn Asp Val Phe 435 440 445 Ser Ser Arg Ala Gly Ile Arg Tyr Asp His Thr Lys Met Thr Pro Gln 450 455 460 Glu Leu Asn Ala Glu Cys His Ala Cys Asp Lys Thr Pro Pro Ala Ala 465 470 475 480 Asn Thr Tyr Lys Gly Trp Ser Gly Phe Val Gly Leu Ala Ala Gln Leu 485 490 495 Asn Gln Ala Trp Arg Val Gly Tyr Asp Ile Thr Ser Gly Tyr Arg Val 500 505 510 Pro Asn Ala Ser Glu Val Tyr Phe Thr Tyr Asn His Gly Ser Gly Asn 515 520 525 Trp Leu Pro Asn Pro Asn Leu Lys Ala Glu Arg Ser Thr Thr His Thr 530 535 540 Leu Ser Leu Gln Gly Arg Ser Glu Lys Gly Met Leu Asp Ala Asn Leu 545 550 555 560 Tyr Gln Ser Asn Tyr Arg Asn Phe Leu Ser Glu Glu Gln Lys Leu Thr 565 570 575 Thr Ser Gly Thr Pro Gly Cys Thr Glu Glu Asn Ala Tyr Tyr Gly Ile 580 585 590 Cys Ser Asp Pro Tyr Lys Glu Lys Leu Asp Trp Gln Met Lys Asn Ile 595 600 605 Asp Lys Ala Arg Ile Arg Gly Ile Glu Leu Thr Gly Arg Leu Asn Val 610 615 620 Asp Lys Val Ala Ser Phe Val Pro Glu Gly Trp Lys Leu Phe Gly Ser 625 630 635 640 Leu Gly Tyr Ala Lys Ser Lys Leu Ser Gly Asp Asn Ser Leu Leu Ser 645 650 655 Thr Gln Pro Leu Lys Val Ile Ala Gly Ile Asp Tyr Glu Ser Pro Ser 660 665 670 Glu Lys Trp Gly Val Phe Ser Arg Leu Thr Tyr Leu Gly Ala Lys Lys 675 680 685 Val Lys Asp Ala Gln Tyr Thr Val Tyr Glu Asn Lys Gly Trp Gly Thr 690 695 700 Pro Leu Gln Lys Lys Val Lys Asp Tyr Pro Trp Leu Asn Lys Ser Ala 705 710 715 720 Tyr Val Phe Asp Met Tyr Gly Phe Tyr Lys Pro Ala Lys Asn Leu Thr 725 730 735 Leu Arg Ala Gly Val Tyr Asn Leu Phe Asn Arg Lys Tyr Thr Thr Trp 740 745 750 Asp Ser Leu Arg Gly Leu Tyr Ser Tyr Ser Thr Thr Asn Ala Val Asp 755 760 765 Arg Asp Gly Lys Gly Leu Asp Arg Tyr Arg Ala Pro Gly Arg Asn Tyr 770 775 780 Ala Val Ser Leu Glu Trp Lys Phe 785 790 8793PRTNeisseria meningitidis 8Met Lys Pro Leu Gln Met Leu Pro Ile Ala Ala Leu Val Gly Ser Ile 1 5 10 15 Phe Gly Asn Pro Val Phe Ala Ala Asp Glu Ala Ala Thr Glu Thr Thr 20 25 30 Pro Val Lys Ala Glu Val Lys Ala Val Arg Val Lys Gly Gln Arg Asn 35 40 45 Ala Pro Ala Ala Val Glu Arg Val Asn Leu Asn Arg Ile Lys Gln Glu 50 55 60 Met Ile Arg Asp Asn Lys Asp Leu Val Arg Tyr Ser Thr Asp Val Gly 65 70 75 80 Leu Ser Asp Ser Gly Arg His Gln Lys Gly Phe Ala Val Arg Gly Val 85 90 95 Glu Gly Asn Arg Val Gly Val Ser Ile Asp Gly Val Asn Leu Pro Asp 100 105 110 Ser Glu Glu Asn Ser Leu Tyr Ala Arg Tyr Gly Asn Phe Asn Ser Ser 115 120 125 Arg Leu Ser Ile Asp Pro Glu Leu Val Arg Asn Ile Asp Ile Val Lys 130 135 140 Gly Ala Asp Ser Phe Asn Thr Gly Ser Gly Ala Leu Gly Gly Gly Val 145 150 155 160 Asn Tyr Gln Thr Leu Gln Gly Arg Asp Leu Leu Leu Pro Glu Arg Gln 165 170 175 Phe Gly Val Met Met Lys Asn Gly Tyr Ser Thr Arg Asn Arg Glu Trp 180 185 190 Thr Asn Thr Leu Gly Phe Gly Val Ser Asn Asp Arg Val Asp Ala Ala 195 200 205 Leu Leu Tyr Ser Gln Arg Arg Gly His Glu Thr Glu Ser Ala Gly Lys 210 215 220 Arg Gly Tyr Pro Val Glu Gly Ala Gly Ser Gly Ala Asn Ile Arg Gly 225 230 235 240 Ser Ala Arg Gly Ile Pro Asp Pro Ser Gln His Lys Tyr Asn His His 245 250 255 Ala Leu Gly Lys Ile Ala Tyr Gln Ile Asn Asp Asn His Arg Ile Gly 260 265 270 Ala Ser Leu Asn Gly Gln Gln Gly His Asn Tyr Thr Val Glu Glu Ser 275 280 285 Tyr Asn Leu Leu Ala Ser Tyr Trp Arg Glu Ala Asp Asp Val Asn Arg 290 295 300 Arg Arg Asn Thr Asn Leu Phe Tyr Glu Trp Thr Pro Glu Ser Asp Arg 305 310 315 320 Leu Ser Met Val Lys Ala Asp Val Asp Tyr Gln Lys Thr Lys Val Ser 325 330 335 Ala Val Asn Tyr Lys Gly Ser Phe Pro Ile Glu Asp Ser Ser Thr Leu 340 345 350 Thr Arg Asn Tyr Asn Gln Lys Asp Leu Asp Glu Ile Tyr Asn Arg Ser 355 360 365 Met Asp Thr Arg Phe Lys Arg Ile Thr Leu Arg Leu Asp Ser His Pro 370 375 380 Leu Gln Leu Gly Gly Gly Arg His Arg Leu Ser Phe Lys Thr Phe Ala 385 390 395 400 Ser Arg Arg Asp Phe Glu Asn Leu Asn Arg Asp Asp Tyr Tyr Phe Ser 405 410 415 Gly Arg Val Val Arg Thr Thr Ser Ser Ile Gln His Pro Val Lys Thr 420 425 430 Thr Asn Tyr Gly Phe Ser Leu Ser Asp Gln Ile Gln Trp Asn Asp Val 435 440 445 Phe Ser Ser Arg Ala Gly Ile Arg Tyr Asp His Thr Lys Met Thr Pro 450 455 460 Gln Glu Leu Asn Ala Glu Cys His Ala Cys Asp Lys Thr Pro Pro Ala 465 470 475 480 Ala Asn Thr Tyr Lys Gly Trp Ser Gly Phe Val Gly Leu Ala Ala Gln 485 490 495 Leu Asn Gln Ala Trp Arg Val Gly Tyr Asp Ile Thr Ser Gly Tyr Arg 500 505 510 Val Pro Asn Ala Ser Glu Val Tyr Phe Thr Tyr Asn His Gly Ser Gly 515 520 525 Asn Trp Leu Pro Asn Pro Asn Leu Lys Ala Glu Arg Thr Thr Thr His 530 535 540 Thr Leu Ser Leu Gln Gly Arg Ser Glu Lys Gly Thr Leu Asp Ala Asn 545 550 555 560 Leu Tyr Gln Ser Asn Tyr Arg Asn Phe Leu Ser Glu Glu Gln Lys Leu 565 570 575 Thr Thr Ser Gly Asp Val Ser Cys Thr Gln Met Asn Tyr Tyr Tyr Gly 580 585 590 Met Cys Ser Asn Pro Tyr Ser Glu Lys Leu Glu Trp Gln Met Gln Asn 595 600 605 Ile Asp Lys Ala Arg Ile Arg Gly Ile Glu Leu Thr Gly Arg Leu Asn 610 615 620 Val Asp Lys Val Ala Ser Phe Val Pro Glu Gly Trp Lys Leu Phe Gly 625 630 635 640 Ser Leu Gly Tyr Ala Lys Ser Lys Leu Ser Gly Asp Asn Ser Leu Leu 645 650 655 Ser Thr Gln Pro Leu Lys Val Ile Ala Gly Ile Asp Tyr Glu Ser Pro 660 665 670 Ser Glu Lys Trp Gly Val Phe Ser Arg Leu Thr Tyr Leu Gly Ala Lys 675 680 685 Lys Val Lys Asp Ala Gln Tyr Thr Val Tyr Glu Asn Lys Gly Trp Gly 690 695 700 Thr Pro Leu Gln Lys Lys Val Lys Asp Tyr Pro Trp Leu Asn Lys Ser 705 710 715 720 Ala Tyr Val Phe Asp Met Tyr Gly Phe Tyr Lys Pro Val Lys Asn Leu 725 730 735 Thr Leu Arg Ala Gly Val Tyr Asn Val Phe Asn Arg Lys Tyr Thr Thr 740 745 750 Trp Asp Ser Leu Arg Gly Leu Tyr Ser Tyr Ser Thr Thr Asn Ser Val 755 760 765 Asp Arg Asp Gly Lys Gly Leu Asp Arg Tyr Arg Ala Pro Ser Arg Asn 770 775 780 Tyr Ala Val Ser Leu Glu Trp Lys Phe 785 790 9488PRTNeisseria meningitidis 9Met Phe Lys Arg Ser Val Ile Ala Met Ala Cys Ile Phe Ala Leu Ser 1 5 10 15 Ala Cys Gly Gly Gly Gly Gly Gly Ser Pro Asp Val Lys Ser Ala Asp 20 25 30 Thr Leu Ser Lys Pro Ala Ala Pro Val Val Ser Glu Lys Glu Thr Glu 35 40 45 Ala Lys Glu Asp Ala Pro Gln Ala Gly Ser Gln Gly Gln Gly Ala Pro 50 55 60 Ser Ala Gln Gly Ser Gln Asp Met Ala Ala Val Ser Glu Glu Asn Thr 65 70 75 80 Gly Asn Gly Gly Ala Val Thr Ala Asp Asn Pro Lys Asn Glu Asp Glu 85 90 95 Val Ala Gln Asn Asp Met Pro Gln Asn Ala Ala Gly Thr Asp Ser Ser 100 105 110 Thr Pro Asn His Thr Pro Asp Pro Asn Met Leu Ala Gly Asn Met Glu 115 120 125 Asn Gln Ala Thr Asp Ala Gly Glu Ser Ser Gln Pro Ala Asn Gln Pro 130 135 140 Asp Met Ala Asn Ala Ala Asp Gly Met Gln Gly Asp Asp Pro Ser Ala 145 150 155 160 Gly Gly Gln Asn Ala Gly Asn Thr Ala Ala Gln Gly Ala Asn Gln Ala 165 170 175 Gly Asn Asn Gln Ala Ala Gly Ser Ser Asp Pro Ile Pro Ala Ser Asn 180 185 190 Pro Ala Pro Ala Asn Gly Gly Ser Asn Phe Gly Arg Val Asp Leu Ala 195 200 205 Asn Gly Val Leu Ile Asp Gly Pro Ser Gln Asn Ile Thr Leu Thr His 210 215 220 Cys Lys Gly Asp Ser Cys Ser Gly Asn Asn Phe Leu Asp Glu Glu Val 225 230 235 240 Gln Leu Lys Ser Glu Phe Glu Lys Leu Ser Asp Ala Asp Lys Ile Ser 245 250 255 Asn Tyr Lys Lys Asp Gly Lys Asn Asp Lys Phe Val Gly Leu Val Ala 260 265 270 Asp Ser Val Gln Met Lys Gly Ile Asn Gln Tyr Ile Ile Phe Tyr Lys 275 280 285 Pro Lys Pro Thr Ser Phe Ala Arg Phe Arg Arg Ser Ala Arg Ser Arg 290 295 300 Arg Ser Leu Pro Ala Glu Met Pro Leu Ile Pro Val Asn Gln Ala Asp 305 310 315 320 Thr Leu Ile Val Asp Gly Glu Ala Val Ser Leu Thr Gly His Ser Gly 325 330 335 Asn Ile Phe Ala Pro Glu Gly Asn Tyr Arg Tyr Leu Thr Tyr Gly Ala 340 345 350 Glu Lys Leu Pro Gly Gly Ser Tyr Ala Leu Arg Val Gln Gly Glu Pro 355 360 365 Ala Lys Gly Glu Met Leu Ala Gly Ala Ala Val Tyr Asn Gly Glu Val 370 375 380 Leu His Phe His Thr Glu Asn Gly Arg Pro Tyr Pro Thr Arg Gly Arg 385 390 395 400 Phe Ala Ala Lys Val Asp Phe Gly Ser Lys Ser Val Asp Gly Ile Ile 405 410 415 Asp Ser Gly Asp Asp Leu His Met Gly Thr Gln Lys Phe Lys Ala Ala 420 425 430 Ile Asp Gly Asn Gly Phe Lys Gly Thr Trp Thr Glu Asn Gly Ser Gly 435 440 445 Asp Val Ser Gly Lys Phe Tyr Gly Pro Ala Gly Glu Glu Val Ala Gly 450 455 460 Lys Tyr Ser Tyr Arg Pro Thr Asp Ala Glu Lys Gly Gly Phe Gly Val 465 470 475 480 Phe Ala Gly Lys Lys Glu Gln Asp 485 10364PRTNeisseria meningitidis 10Met Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu 1 5 10 15 Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Ser Asp Asp Asp Val 20 25 30 Lys Lys Ala Ala Thr Val Ala Ile Val Ala Ala Tyr Asn Asn Gly Gln 35 40 45 Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Gly Glu 50 55 60 Asp Gly Thr Ile Thr Gln Lys Asp Ala Thr Ala Ala Asp Val Glu Ala 65 70 75 80 Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn Leu Thr 85 90 95 Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala 100 105 110 Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp 115 120 125 Ala Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala 130 135 140 Leu Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys 145 150 155 160 Thr Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr 165 170 175 Val Asp Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp 180 185 190 Glu Thr Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala 195 200 205 Lys Gln Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys 210 215 220 Ala Ala Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala 225 230 235 240 Asn Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp 245 250 255 Ile Lys Ala Asp Ile Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Ser 260 265 270 Ala Arg Ile Asp Ser Leu Asp Lys Asn Val Ala Asn Leu Arg Lys Glu 275 280 285 Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln 290 295 300 Pro Tyr Asn Val Gly Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr 305 310 315 320 Lys Ser Glu Ser Ala Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu 325 330 335 Asn Phe Ala Ala Lys Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser 340

345 350 Ser Ala Ala Tyr His Val Gly Val Asn Tyr Glu Trp 355 360 11174PRTNeisseria meningitidis 11Met Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 20 25 30 His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 50 55 60 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 65 70 75 80 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 85 90 95 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 100 105 110 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile Gly Leu Gly 115 120 125 Val Leu Thr Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 130 135 140 Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys Asn 145 150 155 160 Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 165 170 12591PRTNeisseria meningitidis 12Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp 1 5 10 15 Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala 20 25 30 Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln 35 40 45 Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro 50 55 60 Val Gln Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly 65 70 75 80 Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr 85 90 95 Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala 100 105 110 Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser 115 120 125 Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu 130 135 140 Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys 145 150 155 160 Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr 165 170 175 Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn 180 185 190 Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu 195 200 205 Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn 210 215 220 Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe 225 230 235 240 Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr 245 250 255 Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val 260 265 270 Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu 275 280 285 Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly 290 295 300 Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala 305 310 315 320 Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala 325 330 335 Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser 340 345 350 Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile 355 360 365 Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln 370 375 380 Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser 385 390 395 400 Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met 405 410 415 Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg 420 425 430 Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser 435 440 445 Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp 450 455 460 Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg 465 470 475 480 Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val 485 490 495 Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn 500 505 510 Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala 515 520 525 Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly 530 535 540 Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser 545 550 555 560 Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn 565 570 575 Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp 580 585 590 131457PRTNeisseria meningitidis 13Met Lys Thr Thr Asp Lys Arg Thr Thr Glu Thr His Arg Lys Ala Pro 1 5 10 15 Lys Thr Gly Arg Ile Arg Phe Ser Pro Ala Tyr Leu Ala Ile Cys Leu 20 25 30 Ser Phe Gly Ile Leu Pro Gln Ala Trp Ala Gly His Thr Tyr Phe Gly 35 40 45 Ile Asn Tyr Gln Tyr Tyr Arg Asp Phe Ala Glu Asn Lys Gly Lys Phe 50 55 60 Ala Val Gly Ala Lys Asp Ile Glu Val Tyr Asn Lys Lys Gly Glu Leu 65 70 75 80 Val Gly Lys Ser Met Thr Lys Ala Pro Met Ile Asp Phe Ser Val Val 85 90 95 Ser Arg Asn Gly Val Ala Ala Leu Val Gly Asp Gln Tyr Ile Val Ser 100 105 110 Val Ala His Asn Gly Gly Tyr Asn Asn Val Asp Phe Gly Ala Glu Gly 115 120 125 Arg Asn Pro Asp Gln His Arg Phe Thr Tyr Lys Ile Val Lys Arg Asn 130 135 140 Asn Tyr Lys Ala Gly Thr Lys Gly His Pro Tyr Gly Gly Asp Tyr His 145 150 155 160 Met Pro Arg Leu His Lys Phe Val Thr Asp Ala Glu Pro Val Glu Met 165 170 175 Thr Ser Tyr Met Asp Gly Arg Lys Tyr Ile Asp Gln Asn Asn Tyr Pro 180 185 190 Asp Arg Val Arg Ile Gly Ala Gly Arg Gln Tyr Trp Arg Ser Asp Glu 195 200 205 Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr His Ile Ala Ser Ala Tyr 210 215 220 Ser Trp Leu Val Gly Gly Asn Thr Phe Ala Gln Asn Gly Ser Gly Gly 225 230 235 240 Gly Thr Val Asn Leu Gly Ser Glu Lys Ile Lys His Ser Pro Tyr Gly 245 250 255 Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp Ser Gly Ser Pro Met Phe 260 265 270 Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu Ile Asn Gly Val Leu Gln 275 280 285 Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn Gly Phe Gln Leu Val Arg 290 295 300 Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala Gly Asp Thr His Ser Val 305 310 315 320 Phe Tyr Glu Pro Arg Gln Asn Gly Lys Tyr Ser Phe Asn Asp Asp Asn 325 330 335 Asn Gly Thr Gly Lys Ile Asn Ala Lys His Glu His Asn Ser Leu Pro 340 345 350 Asn Arg Leu Lys Thr Arg Thr Val Gln Leu Phe Asn Val Ser Leu Ser 355 360 365 Glu Thr Ala Arg Glu Pro Val Tyr His Ala Ala Gly Gly Val Asn Ser 370 375 380 Tyr Arg Pro Arg Leu Asn Asn Gly Glu Asn Ile Ser Phe Ile Asp Glu 385 390 395 400 Gly Lys Gly Glu Leu Ile Leu Thr Ser Asn Ile Asn Gln Gly Ala Gly 405 410 415 Gly Leu Tyr Phe Gln Gly Asp Phe Thr Val Ser Pro Glu Asn Asn Glu 420 425 430 Thr Trp Gln Gly Ala Gly Val His Ile Ser Glu Asp Ser Thr Val Thr 435 440 445 Trp Lys Val Asn Gly Val Ala Asn Asp Arg Leu Ser Lys Ile Gly Lys 450 455 460 Gly Thr Leu His Val Gln Ala Lys Gly Glu Asn Gln Gly Ser Ile Ser 465 470 475 480 Val Gly Asp Gly Thr Val Ile Leu Asp Gln Gln Ala Asp Asp Lys Gly 485 490 495 Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu Val Ser Gly Arg Gly Thr 500 505 510 Val Gln Leu Asn Ala Asp Asn Gln Phe Asn Pro Asp Lys Leu Tyr Phe 515 520 525 Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn Gly His Ser Leu Ser Phe 530 535 540 His Arg Ile Gln Asn Thr Asp Glu Gly Ala Met Ile Val Asn His Asn 545 550 555 560 Gln Asp Lys Glu Ser Thr Val Thr Ile Thr Gly Asn Lys Asp Ile Ala 565 570 575 Thr Thr Gly Asn Asn Asn Ser Leu Asp Ser Lys Lys Glu Ile Ala Tyr 580 585 590 Asn Gly Trp Phe Gly Glu Lys Asp Thr Thr Lys Thr Asn Gly Arg Leu 595 600 605 Asn Leu Val Tyr Gln Pro Ala Ala Glu Asp Arg Thr Leu Leu Leu Ser 610 615 620 Gly Gly Thr Asn Leu Asn Gly Asn Ile Thr Gln Thr Asn Gly Lys Leu 625 630 635 640 Phe Phe Ser Gly Arg Pro Thr Pro His Ala Tyr Asn His Leu Asn Asp 645 650 655 His Trp Ser Gln Lys Glu Gly Ile Pro Arg Gly Glu Ile Val Trp Asp 660 665 670 Asn Asp Trp Ile Asn Arg Thr Phe Lys Ala Glu Asn Phe Gln Ile Lys 675 680 685 Gly Gly Gln Ala Val Val Ser Arg Asn Val Ala Lys Val Lys Gly Asp 690 695 700 Trp His Leu Ser Asn His Ala Gln Ala Val Phe Gly Val Ala Pro His 705 710 715 720 Gln Ser His Thr Ile Cys Thr Arg Ser Asp Trp Thr Gly Leu Thr Asn 725 730 735 Cys Val Glu Lys Thr Ile Thr Asp Asp Lys Val Ile Ala Ser Leu Thr 740 745 750 Lys Thr Asp Ile Ser Gly Asn Val Asp Leu Ala Asp His Ala His Leu 755 760 765 Asn Leu Thr Gly Leu Ala Thr Leu Asn Gly Asn Leu Ser Ala Asn Gly 770 775 780 Asp Thr Arg Tyr Thr Val Ser His Asn Ala Thr Gln Asn Gly Asn Leu 785 790 795 800 Ser Leu Val Gly Asn Ala Gln Ala Thr Phe Asn Gln Ala Thr Leu Asn 805 810 815 Gly Asn Thr Ser Ala Ser Gly Asn Ala Ser Phe Asn Leu Ser Asp His 820 825 830 Ala Val Gln Asn Gly Ser Leu Thr Leu Ser Gly Asn Ala Lys Ala Asn 835 840 845 Val Ser His Ser Ala Leu Asn Gly Asn Val Ser Leu Ala Asp Lys Ala 850 855 860 Val Phe His Phe Glu Ser Ser Arg Phe Thr Gly Gln Ile Ser Gly Gly 865 870 875 880 Lys Asp Thr Ala Leu His Leu Lys Asp Ser Glu Trp Thr Leu Pro Ser 885 890 895 Gly Thr Glu Leu Gly Asn Leu Asn Leu Asp Asn Ala Thr Ile Thr Leu 900 905 910 Asn Ser Ala Tyr Arg His Asp Ala Ala Gly Ala Gln Thr Gly Ser Ala 915 920 925 Thr Asp Ala Pro Arg Arg Arg Ser Arg Arg Ser Arg Arg Ser Leu Leu 930 935 940 Ser Val Thr Pro Pro Thr Ser Val Glu Ser Arg Phe Asn Thr Leu Thr 945 950 955 960 Val Asn Gly Lys Leu Asn Gly Gln Gly Thr Phe Arg Phe Met Ser Glu 965 970 975 Leu Phe Gly Tyr Arg Ser Asp Lys Leu Lys Leu Ala Glu Ser Ser Glu 980 985 990 Gly Thr Tyr Thr Leu Ala Val Asn Asn Thr Gly Asn Glu Pro Ala Ser 995 1000 1005 Leu Glu Gln Leu Thr Val Val Glu Gly Lys Asp Asn Lys Pro Leu Ser 1010 1015 1020 Glu Asn Leu Asn Phe Thr Leu Gln Asn Glu His Val Asp Ala Gly Ala 1025 1030 1035 1040Trp Arg Tyr Gln Leu Ile Arg Lys Asp Gly Glu Phe Arg Leu His Asn 1045 1050 1055 Pro Val Lys Glu Gln Glu Leu Ser Asp Lys Leu Gly Lys Ala Glu Ala 1060 1065 1070 Lys Lys Gln Ala Glu Lys Asp Asn Ala Gln Ser Leu Asp Ala Leu Ile 1075 1080 1085 Ala Ala Gly Arg Asp Ala Val Glu Lys Thr Glu Ser Val Ala Glu Pro 1090 1095 1100 Ala Arg Gln Ala Gly Gly Glu Asn Val Gly Ile Met Gln Ala Glu Glu 1105 1110 1115 1120Glu Lys Lys Arg Val Gln Ala Asp Lys Asp Thr Ala Leu Ala Lys Gln 1125 1130 1135 Arg Glu Ala Glu Thr Arg Pro Ala Thr Thr Ala Phe Pro Arg Ala Arg 1140 1145 1150 Arg Ala Arg Arg Asp Leu Pro Gln Leu Gln Pro Gln Pro Gln Pro Gln 1155 1160 1165 Pro Gln Arg Asp Leu Ile Ser Arg Tyr Ala Asn Ser Gly Leu Ser Glu 1170 1175 1180 Phe Ser Ala Thr Leu Asn Ser Val Phe Ala Val Gln Asp Glu Leu Asp 1185 1190 1195 1200Arg Val Phe Ala Glu Asp Arg Arg Asn Ala Val Trp Thr Ser Gly Ile 1205 1210 1215 Arg Asp Thr Lys His Tyr Arg Ser Gln Asp Phe Arg Ala Tyr Arg Gln 1220 1225 1230 Gln Thr Asp Leu Arg Gln Ile Gly Met Gln Lys Asn Leu Gly Ser Gly 1235 1240 1245 Arg Val Gly Ile Leu Phe Ser His Asn Arg Thr Glu Asn Thr Phe Asp 1250 1255 1260 Asp Gly Ile Gly Asn Ser Ala Arg Leu Ala His Gly Ala Val Phe Gly 1265 1270 1275 1280Gln Tyr Gly Ile Asp Arg Phe Tyr Ile Gly Ile Ser Ala Gly Ala Gly 1285 1290 1295 Phe Ser Ser Gly Ser Leu Ser Asp Gly Ile Gly Gly Lys Ile Arg Arg 1300 1305 1310 Arg Val Leu His Tyr Gly Ile Gln Ala Arg Tyr Arg Ala Gly Phe Gly 1315 1320 1325 Gly Phe Gly Ile Glu Pro His Ile Gly Ala Thr Arg Tyr Phe Val Gln 1330 1335 1340 Lys Ala Asp Tyr Arg Tyr Glu Asn Val Asn Ile Ala Thr Pro Gly Leu 1345 1350 1355 1360Ala Phe Asn Arg Tyr Arg Ala Gly Ile Lys Ala Asp Tyr Ser Phe Lys 1365 1370 1375 Pro Ala Gln His Ile Ser Ile Thr Pro Tyr Leu Ser Leu Ser Tyr Thr 1380 1385 1390 Asp Ala Ala Ser Gly Lys Val Arg Thr Arg Val Asn Thr Ala Val Leu 1395 1400 1405 Ala Gln Asp Phe Gly Lys Thr Arg Ser Ala Glu Trp Gly Val Asn Ala 1410 1415 1420 Glu Ile Lys Gly Phe Thr Leu Ser Leu His Ala Ala Ala Ala Lys Gly 1425 1430 1435 1440Pro Gln Leu Glu Ala Gln His Ser Ala Gly Ile Lys Leu Gly Tyr Arg 1445 1450 1455 Trp 14797PRTNeisseria meningitidis 14Met Lys Leu Lys Gln Ile Ala Ser Ala Leu Met Met Leu Gly Ile Ser 1 5 10 15 Pro Leu Ala Leu Ala Asp Phe Thr Ile Gln Asp Ile Arg Val Glu Gly 20 25 30 Leu Gln Arg Thr Glu Pro Ser Thr Val Phe Asn Tyr Leu Pro Val Lys 35 40 45 Val

Gly Asp Thr Tyr Asn Asp Thr His Gly Ser Ala Ile Ile Lys Ser 50 55 60 Leu Tyr Ala Thr Gly Phe Phe Asp Asp Val Arg Val Glu Thr Ala Asp 65 70 75 80 Gly Gln Leu Leu Leu Thr Val Ile Glu Arg Pro Thr Ile Gly Ser Leu 85 90 95 Asn Ile Thr Gly Ala Lys Met Leu Gln Asn Asp Ala Ile Lys Lys Asn 100 105 110 Leu Glu Ser Phe Gly Leu Ala Gln Ser Gln Tyr Phe Asn Gln Ala Thr 115 120 125 Leu Asn Gln Ala Val Ala Gly Leu Lys Glu Glu Tyr Leu Gly Arg Gly 130 135 140 Lys Leu Asn Ile Gln Ile Thr Pro Lys Val Thr Lys Leu Ala Arg Asn 145 150 155 160 Arg Val Asp Ile Asp Ile Thr Ile Asp Glu Gly Lys Ser Ala Lys Ile 165 170 175 Thr Asp Ile Glu Phe Glu Gly Asn Gln Val Tyr Ser Asp Arg Lys Leu 180 185 190 Met Arg Gln Met Ser Leu Thr Glu Gly Gly Ile Trp Thr Trp Leu Thr 195 200 205 Arg Ser Asn Gln Phe Asn Glu Gln Lys Phe Ala Gln Asp Met Glu Lys 210 215 220 Val Thr Asp Phe Tyr Gln Asn Asn Gly Tyr Phe Asp Phe Arg Ile Leu 225 230 235 240 Asp Thr Asp Ile Gln Thr Asn Glu Asp Lys Thr Lys Gln Thr Ile Lys 245 250 255 Ile Thr Val His Glu Gly Gly Arg Phe Arg Trp Gly Lys Val Ser Ile 260 265 270 Glu Gly Asp Thr Asn Glu Val Pro Lys Ala Glu Leu Glu Lys Leu Leu 275 280 285 Thr Met Lys Pro Gly Lys Trp Tyr Glu Arg Gln Gln Met Thr Ala Val 290 295 300 Leu Gly Glu Ile Gln Asn Arg Met Gly Ser Ala Gly Tyr Ala Tyr Ser 305 310 315 320 Glu Ile Ser Val Gln Pro Leu Pro Asn Ala Glu Thr Lys Thr Val Asp 325 330 335 Phe Val Leu His Ile Glu Pro Gly Arg Lys Ile Tyr Val Asn Glu Ile 340 345 350 His Ile Thr Gly Asn Asn Lys Thr Arg Asp Glu Val Val Arg Arg Glu 355 360 365 Leu Arg Gln Met Glu Ser Ala Pro Tyr Asp Thr Ser Lys Leu Gln Arg 370 375 380 Ser Lys Glu Arg Val Glu Leu Leu Gly Tyr Phe Asp Asn Val Gln Phe 385 390 395 400 Asp Ala Val Pro Leu Ala Gly Thr Pro Asp Lys Val Asp Leu Asn Met 405 410 415 Ser Leu Thr Glu Arg Ser Thr Gly Ser Leu Asp Leu Ser Ala Gly Trp 420 425 430 Val Gln Asp Thr Gly Leu Val Met Ser Ala Gly Val Ser Gln Asp Asn 435 440 445 Leu Phe Gly Thr Gly Lys Ser Ala Ala Leu Arg Ala Ser Arg Ser Lys 450 455 460 Thr Thr Leu Asn Gly Ser Leu Ser Phe Thr Asp Pro Tyr Phe Thr Ala 465 470 475 480 Asp Gly Val Ser Leu Gly Tyr Asp Val Tyr Gly Lys Ala Phe Asp Pro 485 490 495 Arg Lys Ala Ser Thr Ser Ile Lys Gln Tyr Lys Thr Thr Thr Ala Gly 500 505 510 Ala Gly Ile Arg Met Ser Val Pro Val Thr Glu Tyr Asp Arg Val Asn 515 520 525 Phe Gly Leu Val Ala Glu His Leu Thr Val Asn Thr Tyr Asn Lys Ala 530 535 540 Pro Lys His Tyr Ala Asp Phe Ile Lys Lys Tyr Gly Lys Thr Asp Gly 545 550 555 560 Thr Asp Gly Ser Phe Lys Gly Trp Leu Tyr Lys Gly Thr Val Gly Trp 565 570 575 Gly Arg Asn Lys Thr Asp Ser Ala Leu Trp Pro Thr Arg Gly Tyr Leu 580 585 590 Thr Gly Val Asn Ala Glu Ile Ala Leu Pro Gly Ser Lys Leu Gln Tyr 595 600 605 Tyr Ser Ala Thr His Asn Gln Thr Trp Phe Phe Pro Leu Ser Lys Thr 610 615 620 Phe Thr Leu Met Leu Gly Gly Glu Val Gly Ile Ala Gly Gly Tyr Gly 625 630 635 640 Arg Thr Lys Glu Ile Pro Phe Phe Glu Asn Phe Tyr Gly Gly Gly Leu 645 650 655 Gly Ser Val Arg Gly Tyr Glu Ser Gly Thr Leu Gly Pro Lys Val Tyr 660 665 670 Asp Glu Tyr Gly Glu Lys Ile Ser Tyr Gly Gly Asn Lys Lys Ala Asn 675 680 685 Val Ser Ala Glu Leu Leu Phe Pro Met Pro Gly Ala Lys Asp Ala Arg 690 695 700 Thr Val Arg Leu Ser Leu Phe Ala Asp Ala Gly Ser Val Trp Asp Gly 705 710 715 720 Lys Thr Tyr Asp Asp Asn Ser Ser Ser Ala Thr Gly Gly Arg Val Gln 725 730 735 Asn Ile Tyr Gly Ala Gly Asn Thr His Lys Ser Thr Phe Thr Asn Glu 740 745 750 Leu Arg Tyr Ser Ala Gly Gly Ala Val Thr Trp Leu Ser Pro Leu Gly 755 760 765 Pro Met Lys Phe Ser Tyr Ala Tyr Pro Leu Lys Lys Lys Pro Glu Asp 770 775 780 Glu Ile Gln Arg Phe Gln Phe Gln Leu Gly Thr Thr Phe 785 790 795 15791PRTNeisseria meningitidis 15Met Lys Pro Leu Gln Met Leu Pro Ile Ala Ala Leu Val Gly Ser Ile 1 5 10 15 Phe Gly Asn Pro Val Leu Ala Ala Asp Glu Ala Ala Thr Glu Thr Thr 20 25 30 Pro Val Lys Ala Glu Ile Lys Ala Val Arg Val Lys Gly Gln Arg Asn 35 40 45 Ala Pro Ala Ala Val Glu Arg Val Asn Leu Asn Arg Ile Lys Gln Glu 50 55 60 Met Ile Arg Asp Asn Lys Asp Leu Val Arg Tyr Ser Thr Asp Val Gly 65 70 75 80 Leu Ser Asp Ser Gly Arg His Gln Lys Gly Phe Ala Val Arg Gly Val 85 90 95 Glu Gly Asn Arg Val Gly Val Ser Ile Asp Gly Val Asn Leu Pro Asp 100 105 110 Ser Glu Glu Asn Ser Leu Tyr Ala Arg Tyr Gly Asn Phe Asn Ser Ser 115 120 125 Arg Leu Ser Ile Asp Pro Glu Leu Val Arg Asn Ile Glu Ile Val Lys 130 135 140 Gly Ala Asp Ser Phe Asn Thr Gly Ser Gly Ala Leu Gly Gly Gly Val 145 150 155 160 Asn Tyr Gln Thr Leu Gln Gly Arg Asp Leu Leu Leu Asp Asp Arg Gln 165 170 175 Phe Gly Val Met Met Lys Asn Gly Tyr Ser Thr Arg Asn Arg Glu Trp 180 185 190 Thr Asn Thr Leu Gly Phe Gly Val Ser Asn Asp Arg Val Asp Ala Ala 195 200 205 Leu Leu Tyr Ser Gln Arg Arg Gly His Glu Thr Glu Ser Ala Gly Asn 210 215 220 Arg Gly Tyr Ala Val Glu Gly Glu Gly Ser Gly Ala Asn Ile Arg Gly 225 230 235 240 Ser Ala Arg Gly Ile Pro Asp Ser Ser Lys His Lys Tyr His Ser Phe 245 250 255 Leu Gly Lys Ile Ala Tyr Gln Ile Asn Asp Asn His Arg Ile Gly Ala 260 265 270 Ser Leu Asn Gly Gln Gln Gly His Asn Tyr Thr Val Glu Glu Ser Tyr 275 280 285 Asn Leu Thr Ala Ser Ser Trp Arg Glu Ala Asp Asp Val Asn Arg Arg 290 295 300 Arg Asn Ala Asn Leu Phe Tyr Glu Trp Met Pro Asp Ser Asn Trp Leu 305 310 315 320 Ser Ser Leu Lys Ala Asp Phe Asp Tyr Gln Lys Thr Lys Val Ala Ala 325 330 335 Val Asn Asn Lys Gly Ser Phe Pro Met Asp Tyr Ser Thr Trp Thr Arg 340 345 350 Asn Tyr Asn Gln Lys Asp Leu Asp Glu Ile Tyr Asn Arg Ser Met Asp 355 360 365 Thr Arg Phe Lys Arg Phe Thr Leu Arg Leu Asp Ser His Pro Leu Gln 370 375 380 Leu Gly Gly Gly Arg His Arg Leu Ser Phe Lys Thr Phe Val Ser Arg 385 390 395 400 Arg Asp Phe Glu Asn Leu Asn Arg Asp Asp Tyr Tyr Phe Ser Gly Arg 405 410 415 Val Val Arg Thr Thr Ser Ser Ile Gln His Pro Val Lys Thr Thr Asn 420 425 430 Tyr Gly Phe Ser Leu Ser Asp Gln Ile Gln Trp Asn Asp Val Phe Ser 435 440 445 Ser Arg Ala Gly Ile Arg Tyr Asp His Thr Lys Met Thr Pro Gln Glu 450 455 460 Leu Asn Ala Glu Cys His Ala Cys Asp Lys Thr Pro Pro Ala Ala Asn 465 470 475 480 Thr Tyr Lys Gly Trp Ser Gly Phe Val Gly Leu Ala Ala Gln Leu Asn 485 490 495 Gln Ala Trp His Val Gly Tyr Asp Ile Thr Ser Gly Tyr Arg Val Pro 500 505 510 Asn Ala Ser Glu Val Tyr Phe Thr Tyr Asn His Gly Ser Gly Asn Trp 515 520 525 Leu Pro Asn Pro Asn Leu Lys Ala Glu Arg Ser Thr Thr His Thr Leu 530 535 540 Ser Leu Gln Gly Arg Ser Glu Lys Gly Met Leu Asp Ala Asn Leu Tyr 545 550 555 560 Gln Ser Asn Tyr Arg Asn Phe Leu Ser Glu Glu Gln Lys Leu Thr Thr 565 570 575 Ser Gly Thr Pro Gly Cys Thr Glu Glu Asn Ala Tyr Tyr Gly Ile Cys 580 585 590 Ser Asp Pro Tyr Lys Glu Lys Leu Asp Trp Gln Met Lys Asn Ile Asp 595 600 605 Lys Ala Arg Ile Arg Gly Ile Glu Leu Thr Gly Arg Leu Asn Val Asp 610 615 620 Lys Val Ala Ser Phe Val Pro Glu Gly Trp Lys Leu Phe Gly Ser Leu 625 630 635 640 Gly Tyr Ala Lys Ser Lys Leu Ser Gly Asp Asn Ser Leu Leu Ser Thr 645 650 655 Gln Pro Leu Lys Val Ile Ala Gly Ile Asp Tyr Glu Ser Pro Ser Glu 660 665 670 Lys Trp Gly Val Phe Ser Arg Leu Thr Tyr Leu Gly Ala Lys Lys Ala 675 680 685 Lys Asp Ala Gln Tyr Thr Val Tyr Glu Asn Lys Gly Trp Gly Thr Pro 690 695 700 Leu Gln Lys Lys Val Lys Asp Tyr Pro Trp Leu Asn Lys Ser Ala Tyr 705 710 715 720 Val Phe Asp Met Tyr Gly Phe Tyr Lys Pro Ala Lys Asn Leu Thr Leu 725 730 735 Arg Ala Gly Val Tyr Asn Val Phe Asn Arg Lys Tyr Thr Thr Trp Asp 740 745 750 Ser Leu Arg Gly Leu Tyr Ser Tyr Ser Thr Thr Asn Ser Val Asp Arg 755 760 765 Asp Gly Lys Gly Leu Asp Arg Tyr Arg Ala Pro Ser Arg Asn Tyr Ala 770 775 780 Val Ser Leu Glu Trp Lys Phe 785 790 16915PRTNeisseria meningitidis 16Met Gln Gln Gln His Leu Phe Arg Phe Asn Ile Leu Cys Leu Ser Leu 1 5 10 15 Met Thr Ala Leu Pro Ala Tyr Ala Glu Asn Val Gln Ala Gly Gln Ala 20 25 30 Gln Glu Lys Gln Leu Asp Thr Ile Gln Val Lys Ala Lys Lys Gln Lys 35 40 45 Thr Arg Arg Asp Asn Glu Val Thr Gly Leu Gly Lys Leu Val Lys Ser 50 55 60 Ser Asp Thr Leu Ser Lys Glu Gln Val Leu Asn Ile Arg Asp Leu Thr 65 70 75 80 Arg Tyr Asp Pro Gly Ile Ala Val Val Glu Gln Gly Arg Gly Ala Ser 85 90 95 Ser Gly Tyr Ser Ile Arg Gly Met Asp Lys Asn Arg Val Ser Leu Thr 100 105 110 Val Asp Gly Val Ser Gln Ile Gln Ser Tyr Thr Ala Gln Ala Ala Leu 115 120 125 Gly Gly Thr Arg Thr Ala Gly Ser Ser Gly Ala Ile Asn Glu Ile Glu 130 135 140 Tyr Glu Asn Val Lys Ala Val Glu Ile Ser Lys Gly Ser Asn Ser Val 145 150 155 160 Glu Gln Gly Ser Gly Ala Leu Ala Gly Ser Val Ala Phe Gln Thr Lys 165 170 175 Thr Ala Asp Asp Val Ile Gly Glu Gly Arg Gln Trp Gly Ile Gln Ser 180 185 190 Lys Thr Ala Tyr Ser Gly Lys Asn Arg Gly Leu Thr Gln Ser Ile Ala 195 200 205 Leu Ala Gly Arg Ile Gly Gly Ala Glu Ala Leu Leu Ile His Thr Gly 210 215 220 Arg Arg Ala Gly Glu Ile Arg Ala His Glu Asp Ala Gly Arg Gly Val 225 230 235 240 Gln Ser Phe Asn Arg Leu Val Pro Val Glu Asp Ser Ser Asn Tyr Ala 245 250 255 Tyr Phe Ile Val Lys Glu Glu Cys Lys Asn Gly Ser Tyr Glu Thr Cys 260 265 270 Lys Ala Asn Pro Lys Lys Asp Val Val Gly Lys Asp Glu Arg Gln Thr 275 280 285 Val Ser Thr Arg Asp Tyr Thr Gly Pro Asn Arg Phe Leu Ala Asp Pro 290 295 300 Leu Ser Tyr Glu Ser Arg Ser Trp Leu Phe Arg Pro Gly Phe Arg Phe 305 310 315 320 Glu Asn Lys Arg His Tyr Ile Gly Gly Ile Leu Glu His Thr Gln Gln 325 330 335 Thr Phe Asp Thr Arg Asp Met Thr Val Pro Ala Phe Leu Thr Lys Ala 340 345 350 Val Phe Asp Ala Asn Lys Lys Gln Ala Gly Ser Leu Pro Gly Asn Gly 355 360 365 Lys Tyr Ala Gly Asn His Lys Tyr Gly Gly Leu Phe Thr Asn Gly Glu 370 375 380 Asn Gly Ala Leu Val Gly Ala Glu Tyr Gly Thr Gly Val Phe Tyr Asp 385 390 395 400 Glu Thr His Thr Lys Ser Arg Tyr Gly Leu Glu Tyr Val Tyr Thr Asn 405 410 415 Ala Asp Lys Asp Thr Trp Ala Asp Tyr Ala Arg Leu Ser Tyr Asp Arg 420 425 430 Gln Gly Ile Gly Leu Asp Asn His Phe Gln Gln Thr His Cys Ser Ala 435 440 445 Asp Gly Ser Asp Lys Tyr Cys Arg Pro Ser Ala Asp Lys Pro Phe Ser 450 455 460 Tyr Tyr Lys Ser Asp Arg Val Ile Tyr Gly Glu Ser His Arg Leu Leu 465 470 475 480 Gln Ala Ala Phe Lys Lys Ser Phe Asp Thr Ala Lys Ile Arg His Asn 485 490 495 Leu Ser Val Asn Leu Gly Phe Asp Arg Phe Gly Ser Asn Leu Arg His 500 505 510 Gln Asp Tyr Tyr Tyr Gln His Ala Asn Arg Ala Tyr Ser Ser Asn Thr 515 520 525 Pro Pro Gln Asn Asn Gly Lys Lys Ile Ser Pro Asn Gly Ser Glu Thr 530 535 540 Ser Pro Tyr Trp Val Thr Ile Gly Arg Gly Asn Val Val Thr Gly Gln 545 550 555 560 Ile Cys Arg Leu Gly Asn Asn Thr Tyr Thr Asp Cys Thr Pro Arg Ser 565 570 575 Ile Asn Gly Lys Ser Tyr Tyr Ala Ala Val Arg Asp Asn Val Arg Leu 580 585 590 Gly Arg Trp Ala Asp Val Gly Ala Gly Leu Arg Tyr Asp Tyr Arg Ser 595 600 605 Thr His Ser Asp Asp Gly Ser Val Ser Thr Gly Thr His Arg Thr Leu 610 615 620 Ser Trp Asn Ala Gly Ile Val Leu Lys Pro Thr Asp Trp Leu Asp Leu 625 630 635 640 Thr Tyr Arg Thr Ser Thr Gly Phe Arg Leu Pro Ser Phe Ala Glu Met 645 650 655 Tyr Gly Trp Arg Ala Gly Val Gln Ser Lys Ala Val Lys Ile Asp Pro 660 665 670 Glu Lys Ser Phe Asn Lys Glu Ala Gly Ile Val Phe Lys Gly Asp Phe 675 680 685 Gly Asn Leu Glu Ala Ser Trp Phe Asn Asn Ala Tyr Arg Asp Leu Ile 690 695 700 Val Arg Gly Tyr Glu Ala Gln Ile Lys Asp Gly Lys Glu Glu Ala Lys 705 710 715 720 Gly Asp Pro Ala Tyr Leu Asn Ala Gln Ser Ala Arg Ile Thr Gly Ile 725 730 735 Asn Ile Leu Gly Lys Ile Asp Trp Asn Gly Val Trp Asp Lys Leu Pro 740 745 750 Glu Gly Trp Tyr Ser Thr Phe Ala Tyr Asn Arg Val Arg

Val Arg Asp 755 760 765 Ile Lys Lys Arg Ala Asp Arg Thr Asp Ile Gln Ser His Leu Phe Asp 770 775 780 Ala Ile Gln Pro Ser Arg Tyr Val Val Gly Leu Gly Tyr Asp Gln Pro 785 790 795 800 Glu Gly Lys Trp Gly Val Asn Gly Met Leu Thr Tyr Ser Lys Ala Lys 805 810 815 Glu Ile Thr Glu Leu Leu Gly Ser Arg Ala Leu Leu Asn Gly Asn Ser 820 825 830 Arg Asn Thr Lys Ala Thr Ala Arg Arg Thr Arg Pro Trp Tyr Ile Val 835 840 845 Asp Val Ser Gly Tyr Tyr Thr Val Lys Lys His Phe Thr Leu Arg Ala 850 855 860 Gly Val Tyr Asn Leu Leu Asn Tyr Arg Tyr Val Thr Trp Glu Asn Val 865 870 875 880 Arg Gln Thr Ala Gly Gly Ala Val Asn Gln His Lys Asn Val Gly Val 885 890 895 Tyr Asn Arg Tyr Ala Ala Pro Gly Arg Asn Tyr Thr Phe Ser Leu Glu 900 905 910 Met Lys Phe 915 17712PRTNeisseria meningitidis 17Met Asn Asn Pro Leu Val Asn Gln Ala Ala Met Val Leu Pro Val Phe 1 5 10 15 Leu Leu Ser Ala Cys Leu Gly Gly Gly Gly Ser Phe Asp Leu Asp Ser 20 25 30 Val Asp Thr Glu Ala Pro Arg Pro Ala Pro Lys Tyr Gln Asp Val Phe 35 40 45 Ser Glu Lys Pro Gln Ala Gln Lys Asp Gln Gly Gly Tyr Gly Phe Ala 50 55 60 Met Arg Leu Lys Arg Arg Asn Trp Tyr Pro Gln Ala Lys Glu Asp Glu 65 70 75 80 Val Lys Leu Asp Glu Ser Asp Trp Glu Ala Thr Gly Leu Pro Asp Glu 85 90 95 Pro Lys Glu Leu Pro Lys Arg Gln Lys Ser Val Ile Glu Lys Val Glu 100 105 110 Thr Asp Ser Asp Asn Asn Ile Tyr Ser Ser Pro Tyr Leu Lys Pro Ser 115 120 125 Asn His Gln Asn Gly Asn Thr Gly Asn Gly Ile Asn Gln Pro Lys Asn 130 135 140 Gln Ala Lys Asp Tyr Glu Asn Phe Lys Tyr Val Tyr Ser Gly Trp Phe 145 150 155 160 Tyr Lys His Ala Lys Arg Glu Phe Asn Leu Lys Val Glu Pro Lys Ser 165 170 175 Ala Lys Asn Gly Asp Asp Gly Tyr Ile Phe Tyr His Gly Lys Glu Pro 180 185 190 Ser Arg Gln Leu Pro Ala Ser Gly Lys Ile Thr Tyr Lys Gly Val Trp 195 200 205 His Phe Ala Thr Asp Thr Lys Lys Gly Gln Lys Phe Arg Glu Ile Ile 210 215 220 Gln Pro Ser Lys Ser Gln Gly Asp Arg Tyr Ser Gly Phe Ser Gly Asp 225 230 235 240 Asp Gly Glu Glu Tyr Ser Asn Lys Asn Lys Ser Thr Leu Thr Asp Gly 245 250 255 Gln Glu Gly Tyr Gly Phe Thr Ser Asn Leu Glu Val Asp Phe His Asn 260 265 270 Lys Lys Leu Thr Gly Lys Leu Ile Arg Asn Asn Ala Asn Thr Asp Asn 275 280 285 Asn Gln Ala Thr Thr Thr Gln Tyr Tyr Ser Leu Glu Ala Gln Val Thr 290 295 300 Gly Asn Arg Phe Asn Gly Lys Ala Thr Ala Thr Asp Lys Pro Gln Gln 305 310 315 320 Asn Ser Glu Thr Lys Glu His Pro Phe Val Ser Asp Ser Ser Ser Leu 325 330 335 Ser Gly Gly Phe Phe Gly Pro Gln Gly Glu Glu Leu Gly Phe Arg Phe 340 345 350 Leu Ser Asp Asp Gln Lys Val Ala Val Val Gly Ser Ala Lys Thr Lys 355 360 365 Asp Lys Pro Ala Asn Gly Asn Thr Ala Ala Ala Ser Gly Gly Thr Asp 370 375 380 Ala Ala Ala Ser Asn Gly Ala Ala Gly Thr Ser Ser Glu Asn Gly Lys 385 390 395 400 Leu Thr Thr Val Leu Asp Ala Val Glu Leu Lys Leu Gly Asp Lys Glu 405 410 415 Val Gln Lys Leu Asp Asn Phe Ser Asn Ala Ala Gln Leu Val Val Asp 420 425 430 Gly Ile Met Ile Pro Leu Leu Pro Glu Ala Ser Glu Ser Gly Asn Asn 435 440 445 Gln Ala Asn Gln Gly Thr Asn Gly Gly Thr Ala Phe Thr Arg Lys Phe 450 455 460 Asp His Thr Pro Glu Ser Asp Lys Lys Asp Ala Gln Ala Gly Thr Gln 465 470 475 480 Thr Asn Gly Ala Gln Thr Ala Ser Asn Thr Ala Gly Asp Thr Asn Gly 485 490 495 Lys Thr Lys Thr Tyr Glu Val Glu Val Cys Cys Ser Asn Leu Asn Tyr 500 505 510 Leu Lys Tyr Gly Met Leu Thr Arg Lys Asn Ser Lys Ser Ala Met Gln 515 520 525 Ala Gly Glu Ser Ser Ser Gln Ala Asp Ala Lys Thr Glu Gln Val Glu 530 535 540 Gln Ser Met Phe Leu Gln Gly Glu Arg Thr Asp Glu Lys Glu Ile Pro 545 550 555 560 Ser Glu Gln Asn Ile Val Tyr Arg Gly Ser Trp Tyr Gly Tyr Ile Ala 565 570 575 Asn Asp Lys Ser Thr Ser Trp Ser Gly Asn Ala Ser Asn Ala Thr Ser 580 585 590 Gly Asn Arg Ala Glu Phe Thr Val Asn Phe Ala Asp Lys Lys Ile Thr 595 600 605 Gly Thr Leu Thr Ala Asp Asn Arg Gln Glu Ala Thr Phe Thr Ile Asp 610 615 620 Gly Asn Ile Lys Asp Asn Gly Phe Glu Gly Thr Ala Lys Thr Ala Glu 625 630 635 640 Ser Gly Phe Asp Leu Asp Gln Ser Asn Thr Thr Arg Thr Pro Lys Ala 645 650 655 Tyr Ile Thr Asp Ala Lys Val Gln Gly Gly Phe Tyr Gly Pro Lys Ala 660 665 670 Glu Glu Leu Gly Gly Trp Phe Ala Tyr Pro Gly Asp Lys Gln Thr Lys 675 680 685 Asn Ala Thr Asn Ala Ser Gly Asn Ser Ser Ala Thr Val Val Phe Gly 690 695 700 Ala Lys Arg Gln Gln Pro Val Arg 705 710 18186PRTNeisseria meningitidis 18Met Asn Met Lys Thr Leu Leu Ala Leu Ala Val Ser Ala Val Cys Ser 1 5 10 15 Val Gly Val Ala Gln Ala His Glu His Asn Thr Ile Pro Lys Gly Ala 20 25 30 Ser Ile Glu Val Lys Val Gln Gln Leu Asp Pro Val Asn Gly Asn Lys 35 40 45 Asp Val Gly Thr Val Thr Ile Thr Glu Ser Asn Tyr Gly Leu Val Phe 50 55 60 Thr Pro Asp Leu Gln Gly Leu Ser Glu Gly Leu His Gly Phe His Ile 65 70 75 80 His Glu Asn Pro Ser Cys Glu Pro Lys Glu Lys Glu Gly Lys Leu Thr 85 90 95 Ala Gly Leu Gly Ala Gly Gly His Trp Asp Pro Lys Gly Ala Lys Gln 100 105 110 His Gly Tyr Pro Trp Gln Asp Asp Ala His Leu Gly Asp Leu Pro Ala 115 120 125 Leu Thr Val Leu His Asp Gly Thr Ala Thr Asn Pro Val Leu Ala Pro 130 135 140 Arg Leu Lys His Leu Asp Asp Val Arg Gly His Ser Ile Met Ile His 145 150 155 160 Thr Gly Gly Asp Asn His Ser Asp His Pro Ala Pro Leu Gly Gly Gly 165 170 175 Gly Pro Arg Met Ala Cys Gly Val Ile Lys 180 185 19686PRTArtificial SequenceHybrid protein 19Met Val Ser Ala Val Ile Gly Ser Ala Ala Val Gly Ala Lys Ser Ala 1 5 10 15 Val Asp Arg Arg Thr Thr Gly Ala Gln Thr Asp Asp Asn Val Met Ala 20 25 30 Leu Arg Ile Glu Thr Thr Ala Arg Ser Tyr Leu Arg Gln Asn Asn Gln 35 40 45 Thr Lys Gly Tyr Thr Pro Gln Ile Ser Val Val Gly Tyr Asp Arg His 50 55 60 Leu Leu Leu Leu Gly Gln Val Ala Thr Glu Gly Glu Lys Gln Phe Val 65 70 75 80 Gly Gln Ile Ala Arg Ser Glu Gln Ala Ala Glu Gly Val Tyr Asn Tyr 85 90 95 Ile Thr Val Ala Ser Leu Pro Arg Thr Ala Gly Asp Ile Ala Gly Asp 100 105 110 Thr Trp Asn Thr Ser Lys Val Arg Ala Thr Leu Leu Gly Ile Ser Pro 115 120 125 Ala Thr Arg Ala Arg Val Lys Ile Val Thr Tyr Gly Asn Val Thr Tyr 130 135 140 Val Met Gly Ile Leu Thr Pro Glu Glu Gln Ala Gln Ile Thr Gln Lys 145 150 155 160 Val Ser Thr Thr Val Gly Val Gln Lys Val Ile Thr Leu Tyr Gln Asn 165 170 175 Tyr Val Gln Arg Gly Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly 180 185 190 Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys 195 200 205 Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys 210 215 220 Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp 225 230 235 240 Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp 245 250 255 Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser 260 265 270 Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe 275 280 285 Gln Thr Glu Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala 290 295 300 Lys Arg Gln Phe Arg Ile Gly Asp Leu Gly Gly Glu His Thr Ala Phe 305 310 315 320 Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr Arg Gly Thr Ala Phe Gly 325 330 335 Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Thr Lys 340 345 350 Lys Gln Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn 355 360 365 Val Glu Leu Ala Ser Ala Glu Ile Lys Ala Asp Gly Lys Ser His Ala 370 375 380 Val Ile Leu Gly Asp Val Arg Tyr Gly Ser Glu Glu Lys Gly Ser Tyr 385 390 395 400 Ser Leu Gly Ile Phe Gly Gly Arg Ala Gln Glu Val Ala Gly Ser Ala 405 410 415 Glu Val Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys 420 425 430 Gln Gly Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala Gly Leu 435 440 445 Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys Gly Leu Gln 450 455 460 Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys Leu Lys Leu 465 470 475 480 Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn 485 490 495 Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg 500 505 510 Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe 515 520 525 Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe Gln Thr Glu 530 535 540 Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala Lys Arg Gln 545 550 555 560 Phe Arg Ile Gly Asp Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu 565 570 575 Pro Asp Gly Lys Ala Glu Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp 580 585 590 Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Thr Lys Lys Gln Gly 595 600 605 Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu Asn Val Glu Leu 610 615 620 Ala Ser Ala Glu Ile Lys Ala Asp Gly Lys Ser His Ala Val Ile Leu 625 630 635 640 Gly Asp Val Arg Tyr Gly Ser Glu Glu Lys Gly Ser Tyr Ser Leu Gly 645 650 655 Ile Phe Gly Gly Arg Ala Gln Glu Val Ala Gly Ser Ala Glu Val Lys 660 665 670 Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala Lys Gln 675 680 685 20274PRTNeisseria meningitidis 20Met Asn Arg Thr Ala Phe Cys Cys Leu Ser Leu Thr Thr Ala Leu Ile 1 5 10 15 Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly 20 25 30 Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys 35 40 45 Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys 50 55 60 Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp 65 70 75 80 Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp 85 90 95 Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser 100 105 110 Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr Ala Phe 115 120 125 Gln Thr Glu Gln Ile Gln Asp Ser Glu His Ser Gly Lys Met Val Ala 130 135 140 Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe 145 150 155 160 Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr Tyr Arg Gly Thr Ala Phe 165 170 175 Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala 180 185 190 Ala Lys Gln Gly Asn Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu 195 200 205 Asn Val Asp Leu Ala Ala Ala Asp Ile Lys Pro Asp Gly Lys Arg His 210 215 220 Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Ala Glu Lys Gly Ser 225 230 235 240 Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala Gln Glu Val Ala Gly Ser 245 250 255 Ala Glu Val Lys Thr Val Asn Gly Ile Arg His Ile Gly Leu Ala Ala 260 265 270 Lys Gln 21273PRTNeisseria meningitidis 21Met Asn Arg Thr Ala Phe Cys Cys Leu Ser Leu Thr Ala Ala Leu Ile 1 5 10 15 Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly 20 25 30 Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys 35 40 45 Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu Lys 50 55 60 Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn Gly Asp 65 70 75 80 Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg Phe Asp 85 90 95 Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu Glu Ser 100 105 110 Gly Glu Phe Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu 115 120 125 Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn 130 135 140 Gln Arg Ser Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe 145 150 155 160 Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser 165 170 175 Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala 180 185 190 Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Thr Pro Glu Gln Asn 195 200 205 Val Glu Leu Ala Ala Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala 210 215 220 Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser Glu Glu Lys Gly Thr Tyr 225 230 235 240 His Leu Ala Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala 245 250 255 Thr Val Lys Ile Gly Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys

260 265 270 Gln 22281PRTNeisseria meningitidis 22Met Asn Arg Thr Ala Phe Cys Cys Leu Ser Leu Thr Thr Ala Leu Ile 1 5 10 15 Leu Thr Ala Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val 20 25 30 Ala Ala Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu 35 40 45 Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile 50 55 60 Pro Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr 65 70 75 80 Phe Lys Ala Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu Lys 85 90 95 Asn Asp Lys Ile Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val Asp 100 105 110 Gly Gln Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln 115 120 125 Asn His Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro 130 135 140 Asp Lys Thr Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly 145 150 155 160 Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu Pro Gly Gly Lys Ala 165 170 175 Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg Leu 180 185 190 His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile Glu 195 200 205 His Leu Lys Thr Leu Glu Gln Asn Val Glu Leu Ala Ala Ala Glu Leu 210 215 220 Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr 225 230 235 240 Gly Ser Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg 245 250 255 Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys Val 260 265 270 His Glu Ile Gly Ile Ala Gly Lys Gln 275 280 23248PRTNeisseria meningitidis 23Val Thr Ala Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser 20 25 30 Ile Ser Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 65 70 75 80 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 85 90 95 Ser Ala Leu Thr Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His 100 105 110 Ser Glu Lys Met Val Ala Lys Arg Arg Phe Arg Ile Gly Asp Ile Ala 115 120 125 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr 130 135 140 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr 145 150 155 160 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His 165 170 175 Leu Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Val Ala Tyr Ile Lys 180 185 190 Pro Asp Glu Lys His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn 195 200 205 Gln Asp Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Glu Lys Ala 210 215 220 Gln Glu Val Ala Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His 225 230 235 240 His Ile Gly Leu Ala Ala Lys Gln 245 24260PRTNeisseria meningitidis 24Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Thr Ala Asp 1 5 10 15 Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys 20 25 30 Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile Ser Gln Asn 35 40 45 Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn 50 55 60 Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg 65 70 75 80 Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu 85 90 95 Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His Ser Ala Leu Thr 100 105 110 Ala Leu Gln Thr Glu Gln Glu Gln Asp Pro Glu His Ser Glu Lys Met 115 120 125 Val Ala Lys Arg Arg Phe Arg Ile Gly Asp Ile Ala Gly Glu His Thr 130 135 140 Ser Phe Asp Lys Leu Pro Lys Asp Val Met Ala Thr Tyr Arg Gly Thr 145 150 155 160 Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp 165 170 175 Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro 180 185 190 Glu Leu Asn Val Asp Leu Ala Val Ala Tyr Ile Lys Pro Asp Glu Lys 195 200 205 His His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys 210 215 220 Gly Ser Tyr Ser Leu Gly Ile Phe Gly Glu Lys Ala Gln Glu Val Ala 225 230 235 240 Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His His Ile Gly Leu 245 250 255 Ala Ala Lys Gln 260 25250PRTNeisseria meningitidis 25Val Ala Ala Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser 20 25 30 Ile Ser Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys 35 40 45 Thr Phe Lys Val Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu 50 55 60 Lys Asn Asp Lys Ile Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val 65 70 75 80 Asp Gly Gln Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys 85 90 95 Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn 100 105 110 Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser 115 120 125 Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu Pro Ser Gly Lys 130 135 140 Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys 145 150 155 160 Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile 165 170 175 Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu 180 185 190 Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg 195 200 205 Tyr Gly Ser Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp 210 215 220 Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys 225 230 235 240 Val His Glu Ile Gly Ile Ala Gly Lys Gln 245 250 26261PRTNeisseria meningitidis 26Cys Ser Ser Gly Ser Gly Ser Gly Gly Gly Gly Val Ala Ala Asp Ile 1 5 10 15 Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp 20 25 30 Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile Ser Gln Asn Gly 35 40 45 Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys Thr Phe Lys Val Gly 50 55 60 Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Ile 65 70 75 80 Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val Asp Gly Gln Thr Ile 85 90 95 Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His Ser Ala 100 105 110 Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys Ile Asp 115 120 125 Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly Gly Glu 130 135 140 His Thr Ala Phe Asn Gln Leu Pro Ser Gly Lys Ala Glu Tyr His Gly 145 150 155 160 Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile 165 170 175 Asp Phe Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Thr 180 185 190 Pro Glu Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala Asp Glu 195 200 205 Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser Glu Glu 210 215 220 Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln Glu Ile 225 230 235 240 Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu Ile Gly 245 250 255 Ile Ala Gly Lys Gln 260 27776PRTArtificial SequenceHybrid protein 27Met Gly Pro Asp Ser Asp Arg Leu Gln Gln Arg Arg Val Ala Ala Asp 1 5 10 15 Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys 20 25 30 Asp Lys Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn 35 40 45 Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn 50 55 60 Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser Arg 65 70 75 80 Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile Thr Leu 85 90 95 Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His Ser Ala Val Val 100 105 110 Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu 115 120 125 Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr 130 135 140 Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr His Gly Lys Ala 145 150 155 160 Phe Ser Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe 165 170 175 Ala Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Thr Pro Glu 180 185 190 Gln Asn Val Glu Leu Ala Ala Ala Glu Leu Lys Ala Asp Glu Lys Ser 195 200 205 His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser Glu Glu Lys Gly 210 215 220 Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly 225 230 235 240 Ser Ala Thr Val Lys Ile Gly Glu Lys Val His Glu Ile Gly Ile Ala 245 250 255 Gly Lys Gln Gly Ser Gly Pro Asp Ser Asp Arg Leu Gln Gln Arg Arg 260 265 270 Val Ala Ala Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro 275 280 285 Leu Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser 290 295 300 Ile Pro Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys 305 310 315 320 Thr Phe Lys Ala Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys Leu 325 330 335 Lys Asn Asp Lys Ile Ser Arg Phe Asp Phe Val Gln Lys Ile Glu Val 340 345 350 Asp Gly Gln Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln Ile Tyr Lys 355 360 365 Gln Asn His Ser Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn 370 375 380 Pro Asp Lys Thr Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser 385 390 395 400 Gly Leu Gly Gly Glu His Thr Ala Phe Asn Gln Leu Pro Gly Gly Lys 405 410 415 Ala Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp Pro Asn Gly Arg 420 425 430 Leu His Tyr Ser Ile Asp Phe Thr Lys Lys Gln Gly Tyr Gly Arg Ile 435 440 445 Glu His Leu Lys Thr Leu Glu Gln Asn Val Glu Leu Ala Ala Ala Glu 450 455 460 Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu Gly Asp Thr Arg 465 470 475 480 Tyr Gly Ser Glu Glu Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp 485 490 495 Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val Lys Ile Gly Glu Lys 500 505 510 Val His Glu Ile Gly Ile Ala Gly Lys Gln Gly Ser Gly Gly Gly Gly 515 520 525 Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 530 535 540 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser 545 550 555 560 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 565 570 575 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 580 585 590 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 595 600 605 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 610 615 620 Ser Ala Leu Thr Ala Phe Gln Thr Glu Gln Ile Gln Asp Ser Glu His 625 630 635 640 Ser Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Ile Ala 645 650 655 Gly Glu His Thr Ser Phe Asp Lys Leu Pro Glu Gly Gly Arg Ala Thr 660 665 670 Tyr Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr 675 680 685 Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly Asn Gly Lys Ile Glu His 690 695 700 Leu Lys Ser Pro Glu Leu Asn Val Asp Leu Ala Ala Ala Asp Ile Lys 705 710 715 720 Pro Asp Gly Lys Arg His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn 725 730 735 Gln Ala Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Lys Ala 740 745 750 Gln Glu Val Ala Gly Ser Ala Glu Val Lys Thr Val Asn Gly Ile Arg 755 760 765 His Ile Gly Leu Ala Ala Lys Gln 770 775 28247PRTArtificial SequenceModified fHbp sequence 28Val Ala Ala Asp Ile Gly Ala Gly Leu Ala Asp Ala Leu Thr Ala Pro 1 5 10 15 Leu Asp His Lys Asp Lys Gly Leu Gln Ser Leu Thr Leu Asp Gln Ser 20 25 30 Val Arg Lys Asn Glu Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys 35 40 45 Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp 50 55 60 Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln 65 70 75 80 Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr Lys Gln Ser His 85 90 95 Ser Ala Leu Thr Ala Phe Gln Thr Glu Gln Ile Gln Asp Ser Glu His 100 105 110 Ser Gly Lys Met Val Ala Lys Arg Gln Phe Arg Ile Gly Asp Leu Gly 115 120 125 Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala Glu Tyr 130 135 140 Arg Gly Thr Ala Phe Gly Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr 145 150 155 160 Thr Ile Asp Phe Thr Lys Lys Gln Gly Asn Gly Lys Ile Glu His Leu 165 170 175 Lys Ser Pro Glu Leu Asn Val Glu Leu Ala Ser Ala Glu Ile Lys Ala

180 185 190 Asp Gly Lys Ser His Ala Val Ile Leu Gly Asp Val Arg Tyr Gly Ser 195 200 205 Glu Glu Lys Gly Ser Tyr Ser Leu Gly Ile Phe Gly Gly Arg Ala Gln 210 215 220 Glu Val Ala Gly Ser Ala Glu Val Lys Thr Val Asn Gly Ile Arg His 225 230 235 240 Ile Gly Leu Ala Ala Lys Gln 245 29758PRTNeisseria meningitidis 29Met Ala Gln Thr Thr Leu Lys Pro Ile Val Leu Ser Ile Leu Leu Ile 1 5 10 15 Asn Thr Pro Leu Leu Ala Gln Ala His Glu Thr Glu Gln Ser Val Asp 20 25 30 Leu Glu Thr Val Ser Val Val Gly Lys Ser Arg Pro Arg Ala Thr Ser 35 40 45 Gly Leu Leu His Thr Ser Thr Ala Ser Asp Lys Ile Ile Ser Gly Asp 50 55 60 Thr Leu Arg Gln Lys Ala Val Asn Leu Gly Asp Ala Leu Asp Gly Val 65 70 75 80 Pro Gly Ile His Ala Ser Gln Tyr Gly Gly Gly Ala Ser Ala Pro Val 85 90 95 Ile Arg Gly Gln Thr Gly Arg Arg Ile Lys Val Leu Asn His His Gly 100 105 110 Glu Thr Gly Asp Met Ala Asp Phe Ser Pro Asp His Ala Ile Met Val 115 120 125 Asp Thr Ala Leu Ser Gln Gln Val Glu Ile Leu Arg Gly Pro Val Thr 130 135 140 Leu Leu Tyr Ser Ser Gly Asn Val Ala Gly Leu Val Asp Val Ala Asp 145 150 155 160 Gly Lys Ile Pro Glu Lys Met Pro Glu Asn Gly Val Ser Gly Glu Leu 165 170 175 Gly Leu Arg Leu Ser Ser Gly Asn Leu Glu Lys Leu Thr Ser Gly Gly 180 185 190 Ile Asn Ile Gly Leu Gly Lys Asn Phe Val Leu His Thr Glu Gly Leu 195 200 205 Tyr Arg Lys Ser Gly Asp Tyr Ala Val Pro Arg Tyr Arg Asn Leu Lys 210 215 220 Arg Leu Pro Asp Ser His Ala Asp Ser Gln Thr Gly Ser Ile Gly Leu 225 230 235 240 Ser Trp Val Gly Glu Lys Gly Phe Ile Gly Val Ala Tyr Ser Asp Arg 245 250 255 Arg Asp Gln Tyr Gly Leu Pro Ala His Ser His Glu Tyr Asp Asp Cys 260 265 270 His Ala Asp Ile Ile Trp Gln Lys Ser Leu Ile Asn Lys Arg Tyr Leu 275 280 285 Gln Leu Tyr Pro His Leu Leu Thr Glu Glu Asp Ile Asp Tyr Asp Asn 290 295 300 Pro Gly Leu Ser Cys Gly Phe His Asp Asp Asp Asn Ala His Ala His 305 310 315 320 Thr His Ser Gly Arg Pro Trp Ile Asp Leu Arg Asn Lys Arg Tyr Glu 325 330 335 Leu Arg Ala Glu Trp Lys Gln Pro Phe Pro Gly Phe Glu Ala Leu Arg 340 345 350 Val His Leu Asn Arg Asn Asp Tyr Arg His Asp Glu Lys Ala Gly Asp 355 360 365 Ala Val Glu Asn Phe Phe Asn Asn Gln Thr Gln Asn Ala Arg Ile Glu 370 375 380 Leu Arg His Gln Pro Ile Gly Arg Leu Lys Gly Ser Trp Gly Val Gln 385 390 395 400 Tyr Leu Gln Gln Lys Ser Ser Ala Leu Ser Ala Ile Ser Glu Ala Val 405 410 415 Lys Gln Pro Met Leu Leu Asp Asn Lys Val Gln His Tyr Ser Phe Phe 420 425 430 Gly Val Glu Gln Ala Asn Trp Asp Asn Phe Thr Leu Glu Gly Gly Val 435 440 445 Arg Val Glu Lys Gln Lys Ala Ser Ile Gln Tyr Asp Lys Ala Leu Ile 450 455 460 Asp Arg Glu Asn Tyr Tyr Asn His Pro Leu Pro Asp Leu Gly Ala His 465 470 475 480 Arg Gln Thr Ala Arg Ser Phe Ala Leu Ser Gly Asn Trp Tyr Phe Thr 485 490 495 Pro Gln His Lys Leu Ser Leu Thr Ala Ser His Gln Glu Arg Leu Pro 500 505 510 Ser Thr Gln Glu Leu Tyr Ala His Gly Lys His Val Ala Thr Asn Thr 515 520 525 Phe Glu Val Gly Asn Lys His Leu Asn Lys Glu Arg Ser Asn Asn Ile 530 535 540 Glu Leu Ala Leu Gly Tyr Glu Gly Asp Arg Trp Gln Tyr Asn Leu Ala 545 550 555 560 Leu Tyr Arg Asn Arg Phe Gly Asn Tyr Ile Tyr Ala Gln Thr Leu Asn 565 570 575 Asp Gly Arg Gly Pro Lys Ser Ile Glu Asp Asp Ser Glu Met Lys Leu 580 585 590 Val Arg Tyr Asn Gln Ser Gly Ala Asp Phe Tyr Gly Ala Glu Gly Glu 595 600 605 Ile Tyr Phe Lys Pro Thr Pro Arg Tyr Arg Ile Gly Val Ser Gly Asp 610 615 620 Tyr Val Arg Gly Arg Leu Lys Asn Leu Pro Ser Leu Pro Gly Arg Glu 625 630 635 640 Asp Ala Tyr Gly Asn Arg Pro Phe Ile Ala Gln Asp Asp Gln Asn Ala 645 650 655 Pro Arg Val Pro Ala Ala Arg Leu Gly Phe His Leu Lys Ala Ser Leu 660 665 670 Thr Asp Arg Ile Asp Ala Asn Leu Asp Tyr Tyr Arg Val Phe Ala Gln 675 680 685 Asn Lys Leu Ala Arg Tyr Glu Thr Arg Thr Pro Gly His His Met Leu 690 695 700 Asn Leu Gly Ala Asn Tyr Arg Arg Asn Thr Arg Tyr Gly Glu Trp Asn 705 710 715 720 Trp Tyr Val Lys Ala Asp Asn Leu Leu Asn Gln Ser Val Tyr Ala His 725 730 735 Ser Ser Phe Leu Ser Asp Thr Pro Gln Met Gly Arg Ser Phe Thr Gly 740 745 750 Gly Val Asn Val Lys Phe 755

Claims

1: An immunogenic composition, comprising: (a) a serogroup B meningococcus immunogen; and (b) a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid, wherein the diphtheria toxoid is present in an excess relative to tetanus toxoid as measured in Lf units.

2: The composition of claim 1, further comprising an adjuvant.