BR112016019735B1 - FHBP V2 OR V3 MUTANT, MEMBRANE VESICLES, AND IMMUNOGENIC COMPOSITION - Google Patents

Abstract

FHBP, POLYPEPTIDE, PLASMID OR OTHER NUCLEIC ACID, HOST CELL, MEMBRANE VESICLES, AND, IMMUNOGENIC COMPOSITION The inventors have identified residues in meningococcal fHbp variant 2 and variant 3 that can be modified to increase their stability.

Description

[001] This application claims the benefit of European patent applications 14157399.8 (filed February 28, 2014) and 14177566.8 (filed July 17, 2014), the full contents of both of which are hereby incorporated herein by reference for all purposes.

FIELD OF INVENTION

[002] This invention relates to the field of protein engineering, relating in particular to meningococcal factor H binding protein (fHbp), which is known to be a useful vaccine immunogen.

FUNDAMENTALS OF THE INVENTION

[003] Neisseria meningitidis is an encapsulated Gram-negative bacterium that colonizes the upper respiratory tract of approximately 10% of the human population. Conjugate vaccines are available against serogroups A, C, W135 and Y, but the only vaccine that is available to protect against serogroup B in a two-dose regimen is the BEXSERO™ product that was approved in 2013.

[004] One of the protective immunogens in BEXSERO™ is fHbp, which has also been known as protein ‘741’ (SEQ ID NO: 2536 in ref. i; SEQ ID 1 herein), ‘NMB1870’, ‘GNA1870’ [2 to 4], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [5 to 7]. The 3D structure of this protein is known [8, 9], and the protein has two β-barrels connected by a short linker. Many publications have reported the protective efficacy of this protein in meningococcal vaccines, for example, see refs. 10 to 14. The fHbp lipoprotein is expressed in multiple strains across all serogroups. fHbp sequences have been grouped into three variants [2] (referred to here as v1, v2, and v3), and it has been observed that in general sera that have arisen against a given variant are bactericidal against strains expressing that variant, but are not active against strains expressing one of the other two variants, i.e., there is intravariant cross-protection but not intervariant cross-protection (except for some cross-reactivity of v2 and v3).

[005] To increase interfamily cross-reactivity, the fHbp sequence was engineered to contain specificity for all three variants [15]. Protein engineering has also been used to remove interaction of fHbp with siderophores [16] and with factor H [17–25]. Disruption of the interaction with fH has been reported for all three variants and is postulated to provide a superior vaccine immunogen [22, 26]. For v2 polypeptides, however, references 23 and 24 report an inherent instability that is also seen in mutants with disrupted fH binding. The instability appears to arise from the N-terminal β-barrel domain, and reference 23 cautions that any substitution in this barrel may promote instability.

[006] It is an object of the invention to provide additional v2 and v3 mutants of fHbp, but having enhanced stability.

DESCRIPTION OF THE INVENTION

[007] full-length fHbp of strain 2996 in v2 has the following amino acid sequence (SEQ ID NO: 2): MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDS LIN QRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

[008] The mature lipoprotein is missing the first 19 amino acids of SEQ ID NO: 2 (underlined; gives SEQ ID NO: 4), and the ΔG form of SEQ ID NO: 2 is missing the first 26 amino acids (SEQ ID NO: 5).

[009] full-length fHbp of strain M1239 in v3 has the following amino acid sequence (SEQ ID NO: 3): MNRTAFCCLSLTTALILTACSSGGGGSGGGGVAADIGTGLADALTAP LDHKDKGLKSLTLEDSI PQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEV DGQTITLASGEFQIYKQNHSAVVALQIEKINN PDKTDSLINQRSFLVSG LGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRI EHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFG DRAQEIAGSATVKIGEKVHEIGIAGKQ

[0010] The mature lipoprotein is missing the first 19 amino acids of SEQ ID NO: 3 (underlined; gives SEQ ID NO: 40), and the ΔG form of SEQ ID NO: 3 is missing the first 31 amino acids (SEQ ID NO: 17).

[0011] The inventors have identified residues in SEQ ID NO: 2 and SEQ ID NO: 3 that can be modified to increase polypeptide stability. These residues are generally present throughout the v2 and v3 sequences and thus their modification can provide fHbp v2 and v3 sequences with enhanced stability. Furthermore, the inventors have shown that, as well as increasing stability, mutation of these residues can advantageously decrease binding to human factor H (fH). In addition, however, the mutations described herein can be combined with other mutations, for example, to decrease binding to human factor H (fH), for which several mutations are already known in the art.

[0012] Thus, in general the invention provides a mutant v2 or v3 fHbp that has increased stability relative to a wild-type fHbp (e.g., relative to SEQ ID NOs: 2 or 3) and that, optionally, has lower affinity for human factor H than a wild-type fHbp (e.g., relative to SEQ ID NOs: 2 or 3). The increase in stability and optional reduction in fH affinity preferably result from the same mutation(s), but in some embodiments, they may be attributable to the separate effect of combined mutations. Mutant fHbp proteins with both increased stability and reduced fH affinity are preferred.

[0013] In a first embodiment, the invention provides a polypeptide comprising a mutant fHbp v2 amino acid sequence, wherein: (a) the amino acid sequence has at least k% sequence identity to SEQ ID NO: 5 and/or comprises a fragment of SEQ ID NO: 5; but (b) the amino acid sequence differs from SEQ ID NO: 5 at one or more of the following residues: S32, V33, L39, L41, F69, V100, I113, F122, L123, V124, S125, G126, L127, G128, S151, H239, and/or E240.

[0014] Where feature (a) refers to a fragment, the fragment will include at least one of the residues listed in (b), but this residue will differ when compared to that residue in SEQ ID NO: 5. A fragment of (a) will generally be at least 7 amino acids in length, e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 45, 50, 55, 60 contiguous amino acids or more from SEQ ID NO: 5. The fragment will typically include at least one epitope from SEQ ID NO: 5. Epitope identification and mapping are established for fHbp [11; 27 to 31]. Sharing at least 30 contiguous amino acids with SEQ ID NO: 5 will be typical, and typically a mutant fHbp v2 amino acid sequence will include several (e.g., 2, 3, 4, 5, or more) fragments of SEQ ID NO: 5. In addition, a mutant fHbp v2 amino acid sequence may have at least k% sequence identity to SEQ ID NO: 5, and will include several fragments thereof.

[0015] The value of k may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more. It is preferably 90 (i.e., the v2 amino acid sequence of the mutant fHbp has at least 90% identity to SEQ ID NO: 5) and is more preferably 95.

[0016] The polypeptide may, after administration to a host animal, elicit antibodies that can recognize a wild-type meningococcal polypeptide consisting of SEQ ID NO: 4. Such antibodies are ideally bactericidal (see below). Such antibodies may include some antibodies that do not recognize a v1 or a v3 polypeptide (e.g., a wild-type meningococcal polypeptide consisting of SEQ ID NO: 46 and a wild-type meningococcal polypeptide consisting of SEQ ID NO: 40), although they may also include some antibodies that cross-react with v1 and/or v3 polypeptides.

[0017] The polypeptide has, under the same experimental conditions, a greater stability than the same polypeptide but without the sequence differences of (b), e.g., greater stability than a wild-type meningococcal polypeptide consisting of SEQ ID NO: 4. Enhancement of stability can be assessed using differential scanning calorimetry (DSC), e.g., as discussed in references 32 & 33. DSC has previously been used to assess the stability of v2 fHbp [24]. Suitable conditions for DSC to assess stability can use 20μM polypeptide in a buffered solution (e.g., 25mM Tris) with a pH between 6 and 8 (e.g., 7 to 7.5) with 100 to 200mM NaCl (e.g., 150mM).

[0018] In some embodiments, the polypeptide of the invention is truncated with respect to SEQ ID NO: 5. Compared to the mature wild-type sequence, SEQ ID NO: 5 is already truncated at the N-terminus up to and including the polyglycine sequence (compare SEQ ID NOs: 4 and 5), but SEQ ID NO: 5 may be truncated at the C-terminus and/or further truncated at the N-terminus.

[0019] The increase in stability is ideally at least 5°C, for example at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or higher. These temperatures refer to the increase in the midpoint of the thermal transition (Tm) as assessed by DSC. Wild-type fHbp shows two DSC peaks during unfolding (one for the N-terminal domain and one for the C-terminal domain) and, where a polypeptide of the invention includes both such domains, the increase refers to the stability of the N-terminal domain, which may occur even below 40°C with wild-type v2 sequences [24] (whereas C-terminal domains may have a Tm of 80°C or higher). Thus, the mutant fHbp v2 amino acid sequence of the invention preferably has an N-terminal domain with a Tm of at least 45°C, e.g., >50°C, >55°C, >60°C, >65°C, >70°C, >75°C, or even >80°C.

[0020] In a second embodiment, the invention provides a polypeptide comprising a mutant fHbp v3 amino acid sequence, wherein: (a) the amino acid sequence has at least j% sequence identity to SEQ ID NO: 17 and/or comprises a fragment of SEQ ID NO: 17; but (b) the amino acid sequence differs from SEQ ID NO: 17 at one or more of the following residues: S32, I33, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242, and/or E243.

[0021] Where feature (a) refers to a fragment, the fragment will include at least one of the residues listed in (b), but this residue will differ when compared to that residue in SEQ ID NO: 17. A fragment of (a) will generally be at least 7 amino acids in length, e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 45, 50, 55, 60 contiguous amino acids or more from SEQ ID NO: 17. The fragment will typically include at least one epitope from SEQ ID NO: 17. Epitope identification and mapping are established for fHbp [11; 27 to 31]. Sharing at least 30 contiguous amino acids with SEQ ID NO: 17 will be typical, and typically a mutant fHbp v3 amino acid sequence will include several (e.g., 2, 3, 4, 5, or more) fragments of SEQ ID NO: 17. In addition, a mutant fHbp v3 amino acid sequence may have at least j% sequence identity to SEQ ID NO: 17, and include several fragments thereof.

[0022] The value of j may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more. It is preferably 90 (i.e., the v3 amino acid sequence of the mutant fHbp has at least 90% identity to SEQ ID NO: 17) and is more preferably 95.

[0023] The polypeptide may, after administration to a host animal, elicit antibodies that can recognize a wild-type meningococcal polypeptide consisting of SEQ ID NO: 40. Such antibodies are ideally bactericidal (see below). Such antibodies may include some antibodies that do not recognize a v1 or a v2 polypeptide (e.g., a wild-type meningococcal polypeptide consisting of SEQ ID NO: 46 and a wild-type meningococcal polypeptide consisting of SEQ ID NO: 4), although they may also include some antibodies that cross-react with v1 and/or v2 polypeptides.

[0024] The polypeptide has, under the same experimental conditions, a greater stability than the same polypeptide but without the sequence difference/s of (b), e.g., greater stability than a wild-type meningococcal polypeptide consisting of SEQ ID NO: 40. Enhancement of stability can be assessed using differential scanning calorimetry (DSC), e.g., as discussed in references 32 & 33, DSC was previously used to assess the stability of v3 fHbp [23]. Suitable conditions for DSC to assess stability can use 20μM polypeptide in a buffered solution (e.g., 25mM Tris) with a pH between 6 and 8 (e.g., 7 to 7.5) with 100 to 200mM NaCl (e.g., 150mM).

[0025] In some embodiments, the polypeptide of the invention is truncated with respect to SEQ ID NO: 17. Compared to the mature wild-type sequence, SEQ ID NO: 17 is already truncated at the N-terminus up to and including the polyglycine sequence (compare SEQ ID NOs: 40 and 17), but SEQ ID NO: 17 may be truncated at the C-terminus and/or further truncated at the N-terminus.

[0026] The increase in stability is ideally at least 5°C, for example at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or higher. These temperatures refer to the increase in the midpoint of the thermal transition (Tm) as assessed by DSC. Wild-type fHbp shows two DSC peaks during folding (one for the N-terminal domain and one for the C-terminal domain) and, where a polypeptide of the invention includes both such domains, the increase refers to the stability of the N-terminal domain, which may occur around 60°C or lower with wild-type v3 sequences [24] (whereas C-terminal domains may have a Tm of 80°C or higher). Thus, the mutant fHbp v3 amino acid sequence of the invention preferably has an N-terminal domain with a Tm of at least 65°C, e.g., >70°C, >75°C, or even >80°C.

Mutations related to SEQ ID NO: 5

[0027] Polypeptides of the first embodiment of the invention comprise an amino acid sequence that has at least k% identity to SEQ ID NO: 5 and/or comprise a fragment of SEQ ID NO: 5. Compared to SEQ ID NO: 5, however, this amino sequence has a modification at one or more of amino acid residues S32, V33, L39, L41, F69, V100, I113, F122, L123, V124, S125, G126, L127, G128, S151, H239 and/or E240, for example at 2, 3, 4, 5 or more of these 17 residues. These residues are numbered according to SEQ ID NO: 5; to match the nascent wild-type sequence (SEQ ID NO: 2), the numbering should change +26 (i.e., Ser-32 of SEQ ID NO: 5 is Ser-58 of SEQ ID NO: 2), and to match the mature wild-type sequence (SEQ ID NO: 4) the numbering should change +7 (which also allows easy comparison with ref. 25).

[0028] Preferred residues for mutation are S32, V100, L123, V124, S125, G126, L127, G128, H239 and/or E240. Mutations at these residues give proteins with good stability compared to wild-type v2. Within this subset, preferred residues are S32, L123, V124, S125, G126, L127 and/or G128. The most preferred positions are S32, L123, V124, S125, G126, L127 and/or G128, with residues S32 and/or L123 being particularly preferred, e.g. S32V and/or L123R. Where one or more of V100, S125 and/or G126 is mutated, it is preferred to also mutate a residue outside of this trio.

[0029] The specified residue may be deleted, but preferably is replaced with a different amino acid. For example, Ser-32 may be replaced with any of the other 19 naturally occurring amino acids. When a substitution is made, the amino acid substitution in some embodiments may be a simple amino acid such as glycine or alanine. In other embodiments, the amino acid substitution is a conservative substitution, e.g., is made within the following four groups: (1) acidic, i.e., aspartate, glutamate; (2) basic, i.e., lysine, arginine, histidine; (3) nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is nonconservative. In some embodiments, the substitution does not use alanine.

[0030] Preferred substitutions at the specified residues are as follows: S32V; V33C; L39C; L41C; F69C; V100T; I113S; F122C; L123R; V124I; S125G or S125T; G126D; L127I; G128A; S151C; H239R; E240H.

[0031] Where the mutant fHbp v2 amino acid sequence includes a substitution at E240, this substitution will not be with alanine if only E240 is mutated, although it may be with alanine if an additional amino acid listed in (b) is substituted, for example if residues E240 and H239 are both mutated. Ideally, E240 is not mutated by itself and thus a mutant fHbp v2 amino acid sequence with a substitution at E240 may also include a substitution at a second residue, for example at both E240 and H239 (see mutants #1 and #11).

[0032] Where the mutant fHbp v2 amino acid sequence includes a substitution at F122, this substitution will not be with alanine if only F122 is mutated, although it may be with alanine if an additional amino acid listed in (b) is substituted, for example if residues F122 and S151 are both mutated. Ideally, F122 is not mutated by itself and thus a mutant fHbp v2 amino acid sequence with a substitution at F122 may also include a substitution at a second residue. Where F122 is substituted, it may be preferred that S151 is also substituted, for example both are substituted with cysteine, to allow formation of a disulfide bond (see mutant #10).

[0033] Where the mutant fHbp v2 amino acid sequence includes a substitution at L123, this substitution will not be with alanine if only L123 is mutated, although it may be with alanine if an additional amino acid listed in (b) is substituted, for example, if residues L123 and S32 are both mutated. If L123 is mutated by itself, substitution with arginine is preferred (e.g., see mutant #4). In some embodiments, however, L123 is not mutated by itself, and thus a mutant fHbp v2 amino acid sequence with a substitution at L123 may also include a substitution at a second residue. When L123 is substituted, it may be preferred that: (i) S32 is also substituted, as seen in mutant #3, and optionally S125 is also substituted, as seen in mutants #20 and #22; or (ii) one or more of residues 124 to 128 is/are also substituted, for example, as seen in mutant #12.

[0034] Where the mutant fHbp v2 amino acid sequence includes a substitution at V124, it may be preferred that this substitution not be with alanine if only V124 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted, e.g., if one or more of residues 123 to 128 are also mutated. If V124 is mutated by itself, substitution with isoleucine is preferred. Ideally, however, V124 is not mutated by itself, and thus a mutant fHbp v2 amino acid sequence with a substitution at V124 may also include a substitution at a second residue. When V124 is substituted, it is preferred that one or more of residues 124 to 128 is/are also substituted, e.g., as seen in mutant #12.

[0035] Where the mutant fHbp v2 amino acid sequence includes a substitution at L127, it may be preferred that this substitution not be with alanine if only L127 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted, for example if one or more of residues 123 to 128 are also mutated. If L127 is mutated by itself, substitution with isoleucine is preferred. Ideally, however, L127 is not mutated by itself and thus a mutant fHbp v2 amino acid sequence with a substitution at L127 may also include a substitution at a second residue. When L127 is substituted it is preferred that one or more of residues 124 to 128 is/are also substituted, for example as seen in mutant #12.

[0036] Where the mutant fHbp v2 amino acid sequence includes a substitution at S32, it may be preferred that this substitution be not with alanine if only S32 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted, for example if residues L123 and S32 are both mutated. If S32 is mutated by itself, substitution with valine is preferred. Ideally, however, S32 is not mutated by itself and thus a mutant fHbp v2 amino acid sequence with a substitution at S32 may also include a substitution at a second residue. Where S32 is substituted it may be preferred that (i) L123 is also substituted, for example as seen in mutant #3, and optionally S125 is also substituted, as seen in mutants #20 and #22; or (ii) S125 is also substituted, for example as seen in mutants #19 and #21.

[0037] Where the v2 amino acid sequence of the mutant fHbp includes a substitution at I113, this substitution will not be with alanine if only I113 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted. If I113 is mutated by itself, substitution with serine is preferred, for example as seen in mutant #7.

[0038] Where the v2 amino acid sequence of the mutant fHbp includes a substitution at V33, this is preferably not with isoleucine. Where the v2 amino acid sequence of the mutant fHbp includes a substitution at I113, this is preferably not with threonine or alanine. Where the v2 amino acid sequence of the mutant fHbp includes a substitution at S151, this is preferably not with phenylalanine. Where the v2 amino acid sequence of the mutant fHbp includes a substitution at both H239 and E240, this is preferably not at R239 and Q240.

[0039] Where more than one substitution is made, that substitution can be selected from groups 2A through 2O as follows: 2A: residues 239 and 240, e.g., mutant #1. 2B: residues 32 and 123, e.g., mutant #3. 2C: residues 125 and 126, e.g., mutant #5. 2D: residues 100, 125, and 126, e.g., mutant #6. 2E: residues 33 and 39, e.g., mutant #8. 2F: residues 41 and 69, e.g., mutant #9. 2G: residues 122 and 151, e.g., mutant #10. 2H: residues 100, 125, 126, 239, and 240, e.g., mutant #11. 2I: residues 32 and 125, e.g., mutants #19 and #21. 2J: residues 32, 123, and 125, e.g., mutants #20 and #22. 2K: residues 33 and 39, both replaced by Cys, e.g., mutant #8. 2L: residues 41 and 69, both replaced by Cys, e.g., mutant #9. 2M: residues 122 and 151, both replaced by Cys, e.g., mutant #10. 2N: residues 123, 124, 125, 126, 127, and 128, e.g., mutant #12. 2O: residues 32, 123, 124, 125, 126, 127, and 128.

[0040] Thus, for example, if residue 239 is to be substituted, then a second preferred residue for substitution is 240 (i.e., group 2A); furthermore, residues 100, 125 and 126 may also be modified (i.e., group 2H, which is a combination of groups 2A and 2D). Within groups 2A through 2N & 2O, preferred substitutions at the specified positions are those listed above. For groups 2K, 2L & 2M, the intent is to introduce a disulfide bond. Within groups 2A through 2N, preferred mutants are 2A, 2B, 2C, 2D, 2I, 2J and 2N. Most preferred are 2C, 2I and 2N, with 2N being particularly preferred. Group 2B provides the most preferred mutations, and in particular S32V and L123R (e.g., SEQ ID NOs: 20 and 45). Group 2O is a further set of preferred mutations, which combines 2B, 2C and three additional mutations (e.g., to give SEQ ID NO: 58).

[0041] Amino acid residues noted for mutation in a v2 sequence are numbered relative to SEQ ID NO: 5 which is from strain 2996. The corresponding amino acid residues in a v2 fHbp from any other strain can be readily identified by sequence alignment, e.g., being the amino acid that, when aligned with SEQ ID NO: 5 using a pairwise alignment algorithm (e.g., the Needleman-Wunsch global alignment algorithm, as detailed below), aligns with the amino acid noted therein. Often, the amino acid will be the same as that seen in SEQ ID NO: 5 (e.g., residue 32 will be serine), but alignment will readily identify if this is not the case.

[0042] In addition to the mutation(s) noted above, which are intended to increase stability, a polypeptide of the invention may include one or more additional mutation(s), for example, to disrupt the interaction of the polypeptide with siderophores or, more preferably, to disrupt the ability of the polypeptide to bind fH.

[0043] References 19 and 25 report that the interaction between fH and fHbp of v2 can be disrupted by mutations at residues R80, D211, E218, E248, T220+H222 (double mutation), and G236. Numbered according to SEQ ID NO: 5, these residues are R73, D203, E210, E240, T213+H215, and G228. Of these positions, polypeptides mutated at D203, E210, or T213+H215 are preferred because reference 25 reports no disruption of important epitopes in these mutants. The specific substitutions studied in reference 25 were R73A, D203A, E210A, T213A+H215A, G228I, and E240A; these substitutions are suitable for use in accordance with the invention.

[0044] Reference 24 reports that the interaction between fH and fHbp of v2 can be disrupted by mutations at residues R145, S193, F194, L195, A265, E267, K268, V272, I273, L274, E283, T286, H288, F292, T304, and E313 and E283+T304 (double mutation). Numbered according to SEQ ID NO: 5, these residues are R73, S121, F122, L123, A192, E194, K195, V199, I200, L201, E210, T213, H215, F219, T231, and E240 and E210+T231. Four of these overlap with reference 25 (E210, T213, H215, E240). The specific substitutions studied in reference 24 used alanine (except for A265P and T304E), and these substitutions are suitable for use according to the invention.

[0045] Reference 24 reports that certain substitutions in v2 can increase affinity for fH, and these should be avoided if the intention is to disrupt binding at fH, e.g., E85 in SEQ ID NO: 5 (residue 157 in ref. 24).

[0046] Residues that interact with siderophores can be mutated, using the guidance in references 16 and 34, for example, by aligning SEQ ID NO: 5 here with SEQ ID NO: 4 of reference 16 to identify residues that can interact with siderophores, for example, with catecholates, hydroxamates or carboxylates.

[0047] Additional residues that may be mutated include, but are not limited to, S23, L24, D30, Q31, R34, D95, and/or L102, for example, using the mutations suggested in reference 35.

[0048] The polypeptide of the first embodiment can comprise any of SEQ ID NOs: 18 to 36. Similarly, taking into account the ‘ΔG’ mutation (i.e., truncation of the nascent N-terminus up to and including the native poly-Gly sequence), the polypeptide of the first embodiment can comprise any of SEQ ID NOs: 18 to 36 excluding amino acids 1 to 26 thereof. For example, the polypeptide of the first embodiment can comprise SEQ ID NO: 45, or can comprise SEQ ID NO: 58.

[0049] Considering the possibility of additional point mutations (e.g., to disrupt interactions with siderophores and/or fH), the polypeptide of the first embodiment may comprise any of SEQ ID NOs: 18 to 36 (or any of SEQ ID NOs: 18 to 36 excluding amino acids 1 to 26 thereof, such as SEQ ID NO: 45), but modified by up to 5 single amino acid changes (i.e., 1, 2, 3, 4, or 5 single amino acid substitutions, deletions, and/or insertions), provided that the modified sequence can, after administration to a host animal, elicit antibodies that bind to a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Such amino acid changes must not revert mutations in these sequences with respect to the wild-type sequence, e.g., SEQ ID NO: 45 must not be mutated at residue V32 or R123.

[0050] The invention also provides a polypeptide comprising a v2 amino acid sequence of fHbp, wherein the v2 amino acid sequence is identical to a wild-type v2 amino acid sequence except for a mutation at the amino acid position corresponding to Leu-123 of SEQ ID NO: 5, provided that the mutation is not a substitution for alanine (e.g., wherein the mutation is a substitution for arginine). For example, the polypeptide may comprise SEQ ID NO: 5 but with a mutation (other than L123A) at L123.

[0051] SEQ ID NOs: 59 and 60 are two additional examples of v2 mutants, namely the mature form of mutants #3 &#4 for strain 8047.

Mutations related to SEQ ID NO: 17

[0052] Polypeptides of the second embodiment of the invention comprise an amino acid sequence that has at least j% identity to SEQ ID NO: 17 and/or comprise a fragment of SEQ ID NO: 17. Compared to SEQ ID NO: 17, however, this amino sequence has a modification at one or more of amino acid residues S32, I33, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242 and/or E243, for example at 2, 3, 4, 5 or more of these 17 residues. These residues are numbered according to SEQ ID NO: 17; to match the nascent wild-type sequence (SEQ ID NO: 3), the numbering should swap +31 (i.e., Ser-32 of SEQ ID NO: 17 is Ser-63 of SEQ ID NO: 3), and to match the mature wild-type sequence (SEQ ID NO: 40) the numbering should swap +12.

[0053] Preferred residues for mutation are S32, V103, L126, V127, S128, G129, L130, G131, H242, and/or E243. Within this subset, preferred residues are S32, L126, V127, S128, G129, L130, and/or G131. Most preferred positions are S32, L126, V127, S128, G129, L130, and/or G131, with residues S32 and/or L126 being particularly preferred, e.g., S32V and/or L126R.

[0054] The specified residue may be deleted, but preferably is replaced with a different amino acid. For example, Ser-32 may be replaced with any of the other 19 naturally occurring amino acids. When a substitution is made, the amino acid substitution in some embodiments may be a simple amino acid such as glycine or alanine. In other embodiments, the amino acid substitution is a conservative substitution, e.g., is made within the following four groups: (1) acidic, i.e., aspartate, glutamate; (2) basic, i.e., lysine, arginine, histidine; (3) nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is nonconservative. In some embodiments, the substitution does not use alanine.

[0055] Preferred substitutions at the specified residues are as follows: S32V; I33C; L39C; L41C; F72C; V103T; T116S; F125C; L126R; V127I; S128G or S128T; G129D; L130I; G131A; S154C; H242R; E243H.

[0056] Where the mutant fHbp v3 amino acid sequence includes a substitution at E243, this substitution will not be with alanine if only E243 is mutated, although it may be with alanine if an additional amino acid listed in (b) is substituted, for example if residues E243 and H242 are both mutated. Ideally, E243 is not mutated by itself and thus a mutant fHbp v3 amino acid sequence with a substitution at E243 may also include a substitution at a second residue, for example at both E243 and H242.

[0057] Where the mutant fHbp v3 amino acid sequence includes a substitution at F125, this substitution will not be with alanine if only F125 is mutated, although it may be with alanine if an additional amino acid listed in (b) is substituted, for example if residues F125 and S154 are both mutated. Ideally, F125 is not mutated itself and thus a mutant fHbp v3 amino acid sequence with a substitution at F125 may also include a substitution at a second residue. Where F125 is substituted it may be preferred that S154 is also substituted, for example both are substituted with cysteine, to allow formation of a disulfide bond.

[0058] Where the mutant fHbp v3 amino acid sequence includes a substitution at L126, this substitution will not be with alanine if only L126 is mutated, although it may be with alanine if an additional amino acid listed in (b) is substituted, for example, if residues L126 and S32 are both mutated. If L126 is mutated by itself, substitution with arginine is preferred. In some embodiments, however, L126 is not mutated by itself, and thus a mutant fHbp v3 amino acid sequence with a substitution at L126 may also include a substitution at a second residue. When L126 is substituted it may be preferred that: (i) S32 is also substituted, and optionally S128 is also substituted; or (ii) one or more of residues 127-131 is also substituted.

[0059] Where the mutant fHbp v3 amino acid sequence includes a substitution at V127, it may be preferred that this substitution is not with alanine if only V127 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted, for example if one or more of residues 126-131 are also mutated. If V127 is mutated by itself, substitution with isoleucine is preferred. Ideally, however, V127 is not mutated by itself and thus a mutant fHbp v3 amino acid sequence with a substitution at V127 may also include a substitution at a second residue. When V127 is substituted it is preferred that one or more of residues 127-131 is also substituted.

[0060] Where the mutant fHbp v3 amino acid sequence includes a substitution at L130, it may be preferred that this substitution is not with alanine if only L130 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted, for example if one or more of residues 126-131 are also mutated. If L130 is mutated by itself, substitution with isoleucine is preferred. Ideally, however, L130 is not mutated by itself and thus a mutant fHbp v3 amino acid sequence with a substitution at L130 may also include a substitution at a second residue. When L130 is substituted it is preferred that one or more of residues 127-131 are also substituted.

[0061] Where the mutant fHbp v3 amino acid sequence includes a substitution at S32, it may be preferred that this substitution is not with alanine if only S32 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted, for example if one or more of residues L126 and S32 are both mutated. If S32 is mutated by itself, substitution with valine is preferred. Ideally, however, S32 is not mutated by itself and thus a mutant fHbp v3 amino acid sequence with a substitution at S32 may also include a substitution at a second residue. Where S32 is substituted it may be preferred that (i) L126 is also substituted, and optionally S128 is also substituted, or (ii) S128 is also substituted.

[0062] Where the v3 amino acid sequence of the mutant fHbp includes a substitution at T113, this substitution will not be with alanine if only T113 is mutated, although it may be alanine if an additional amino acid listed in (b) is substituted. If T113 is mutated by itself, substitution with serine is preferred.

[0063] Where the mutant fHbp v3 amino acid sequence includes a substitution at I33, this is preferably not to valine. Where the mutant fHbp v3 amino acid sequence includes a substitution at T116, this is preferably not to isoleucine. Where the mutant fHbp v3 amino acid sequence includes a substitution at G129, this is preferably not to serine. Where the mutant fHbp v3 amino acid sequence includes a substitution at both H242 and E243, this is preferably not to R242 and Q243.

[0064] Where more than one substitution is made, these can be selected from groups 3A to 3O as follows: 3A: residues 242 and 243. 3B: residues 32 and 126. 3C: residues 128 and 129. 3D: residues 103, 128, and 129. 3E: residues 33 and 39. 3F: residues 41 and 72. 3G: residues 125 and 154. 3H: residues 103, 128, 129, 242, and 243. 3I: residues 32 and 128. 3J: residues 32, 126, and 128. 3K: residues 33 and 39, both substituted by Cys. 3L: residues 41 and 72, both substituted by Cys. 3M: residues 125 and 154, both replaced by Cys. 3N: residues 126, 127, 128, 129, 130, and 131. 3O: residues 32, 126, 127, 128, 129, 130, and 131.

[0065] Thus, for example, if residue 242 is to be substituted, then a second preferred residue for substitution is 243 (i.e., group 3A); furthermore, residues 103, 128 and 129 may also be modified (i.e., group 3H, which is a combination of groups 3A and 3D). Within groups 3A through 3N & 3O, preferred substitutions at the specified positions are those listed above. For groups 3K, 3L & 3M, the intent is to introduce a disulfide bond. Within groups 3A through 3N, preferred mutants are 3A, 3B, 3C, 3D, 3I, 3J and 3N. More preferred are 3C, 3I and 3N, with 3N being particularly preferred. Group 3B provides the most preferred mutations, and in particular S32V and L126R (e.g., comprising SEQ ID NO: 44). Group 3O is a further preferred mutation, which combines 3B, 3C and three additional mutations (e.g., to give SEQ ID NO: 61). Mutation L126R alone provides SEQ ID NO: 53.

[0066] Amino acid residues noted for mutation in a v3 sequence are numbered relative to SEQ ID NO: 17 which is from strain M1239. The corresponding amino acid residues in a v3 fHbp from any other strain can be readily identified by sequence alignment, e.g. by being the amino acid that, when aligned with SEQ ID NO: 17 using a pairwise alignment algorithm (e.g. the Needleman-Wunsch global alignment algorithm, as detailed below), aligns with the amino acid noted here. Often the amino acid will be the same as that seen in SEQ ID NO: 17 (e.g. residue 32 will be serine), but alignment will readily identify if this is not the case.

[0067] In addition to the mutation(s) noted above, which are intended to increase stability, a polypeptide of the invention may include one or more additional mutation(s), for example, to disrupt the interaction of the polypeptide with siderophores or, more preferably, to disrupt the ability of the polypeptide to bind fH.

[0068] Reference 24 reports that the interaction between fH and fHbp of v3 can be disrupted by mutations at residues Q107, I147, L156, A157, L195, V196, V272, E283, T286, T304, V311, E313 and E283+T304 (double mutation). Numbered according to SEQ ID NO: 17, these residues are: Q35, I78, L87, A88, L126, V127, V202, E213, T216, T234, V241, E243 and E213+T234. The specific substitutions studied in reference 24 use alanine (except for A157E and T231E), and these substitutions are suitable for use according to the invention. Residues T216 and E243 are also reported in reference 23. Reference 24 reports that the interaction between fH and fHbp of v3 can be disrupted by mutations at residues H288 and G318 (H218 and G248 numbered according to SEQ ID NO: 17), and these substitutions are suitable for use according to the invention, e.g., H218R, G248D.

[0069] Ref. 24 reports that certain substitutions in v3 may increase affinity for fH, and these should be avoided if the intention is to disrupt binding to fH, for example, P44 in SEQ ID NO: 17 (residue 106 in ref. 24).

[0070] Residues that interact with siderophores can be mutated, using the guidance in references 16 and 34, for example, by aligning SEQ ID NO: 17 herein with SEQ ID NO: 4 of reference 16 to identify residues that can interact with siderophores, for example, with catecholates, hydroxamates or carboxylates.

[0071] The polypeptide of the second embodiment can comprise any of SEQ ID NOs: 41 to 44. Considering the possibility of additional point mutations (e.g., to disrupt interactions with siderophores and/or fH) the polypeptide of the first embodiment can comprise any of SEQ ID NOs: 41 to 44 but modified by up to 5 single amino acid changes (i.e., 1, 2, 3, 4, or 5 single amino acid substitutions, deletions, and/or insertions) provided that the modified sequence can, after administration into a host animal, elicit antibodies that bind to a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 40. Such amino acid changes must not revert mutations in these sequences with respect to the wild-type sequence, e.g., SEQ ID NO: 44 must not be mutated at residue V32 or R126.

[0072] The invention also provides a polypeptide comprising a v3 amino acid sequence of fHbp, wherein the v3 amino acid sequence is identical to a wild-type v3 amino acid sequence except for a mutation at the amino acid position corresponding to Leu-126 of SEQ ID NO: 17, provided that the mutation is not a substitution for alanine (e.g., wherein the mutation is a substitution for arginine). For example, the polypeptide may comprise SEQ ID NO: 17, but with a mutation (other than L126A) at L126.

Polypeptides

[0073] Polypeptides of the invention can be prepared by various means, for example, by chemical synthesis (at least in part), by digesting larger polypeptides using proteases, by RNA translation, by purification from cell culture (e.g., from recombinant expression or from N.meningitidis culture), etc. Heterologous expression in an E.coli host is a preferred expression route.

[0074] Polypeptides of the invention ideally have at least 100 amino acids, e.g., 150aa, 175aa, 200aa, 225aa or greater. They include a mutant fHbp v2 and/or v3 amino acid sequence, and the mutant fHbp v2 or v3 amino acid sequence should similarly be at least 100 amino acids in length, e.g., 150aa, 175aa, 200aa, preferred 225aa, or greater.

[0075] fHbp is naturally a lipoprotein in N.meningitidis. It has also been observed to be lipidated when expressed in E.coli with its native leader sequence or with heterologous leader sequences. Polypeptides of the invention may have an N-terminal cysteine residue, which may be lipidated, for example, comprising a palmitoyl group, typically forming tripalmitoyl-S-glyceryl-cysteine. In other embodiments the polypeptides are non-lipidated.

[0076] Polypeptides are preferably prepared in substantially pure or substantially isolated form (i.e., substantially free of other Neisserial or host cell polypeptides). In general, the polypeptides are provided in a non-naturally occurring environment, e.g., they are separated from their naturally occurring environment. In certain embodiments, the polypeptide is present in a composition that is enriched for the polypeptide, compared to a starting material. Thus, purified polypeptide is provided, whereby purified means that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, whereby substantially free means that greater than 50% (e.g., >75%, >80%, >90%, >95%, or >99%) of the total polypeptide in the composition is a polypeptide of the invention.

[0077] Polypeptides can take various forms (e.g., native, fusions, glycosylated, non-glycosylated, lipidated, disulfide bonds, etc.).

[0078] SEQ ID NOs 4, 5, 17 and 40 do not include a methionine at the N-terminus. If a polypeptide of the invention is produced by translation in a biological host, then a stop codon is required, which will provide a methionine at the N-terminus in most hosts. Thus, a polypeptide of the invention, at least at a nascent stage, will include a methionine residue upstream of said sequence SEQ ID NO.

[0079] Cleavage of nascent sequences means that the mutant fHbp v2 or v3 amino acid sequence can itself provide the N-terminus of the polypeptide. In other embodiments, however, a polypeptide of the invention can include an N-terminal sequence upstream of the mutant fHbp v2 or v3 amino acid sequence. In some embodiments, the polypeptide has a single methionine at the N-terminus immediately followed by the mutant fHbp v2 or v3 amino acid sequence; in other embodiments a longer upstream sequence can be used. Such an upstream sequence can be short (e.g., 40 or fewer amino acids, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences that facilitate cloning or purification (e.g., a histidine tag, i.e., Hisn where n = 4, 5, 6, 7, 8, 9, 10, or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art, for example, the native upstream sequences present in SEQ ID NO: 2 or SEQ ID NO: 3.

[0080] A polypeptide of the invention may also include amino acids downstream of the final amino acid of the mutant fHbp v2 or v3 amino acid sequence. Such C-terminal extensions may be short (e.g., 40 or fewer amino acids, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences to direct protein trafficking, short peptide sequences that facilitate cloning or purification (e.g., comprising a histidine tag, i.e., Hisn where n = 4, 5, 6, 7, 8, 9, 10 or more), or sequences that enhance polypeptide stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

[0081] In some embodiments, the invention excludes polypeptides that include a histidine tag (cf. refs. 24 & 25), and in particular a hexa-histidine tag at the C-terminus.

[0082] The term “polypeptide” refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a tagging component. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides may occur as single chains or associated chains.

[0083] Polypeptides of the invention can be affixed or immobilized on a solid support.

[0084] Polypeptides of the invention may comprise a detectable label, for example, a radioactive label, a fluorescent label, or a biotin label. This is particularly useful in immunoassay techniques.

[0085] As described in reference 164, fHbp can be divided into three domains, referred to as A, B, and C. Taking SEQ ID NO: 1, the three domains are (A) 1 to 119, (B) 120 to 183, and (C) 184 to 274: MNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADALTAPLDHKDK GLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKM VAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTI DFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAE KGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ

[0086] The mature form of the ‘A’ domain, from Cys-20 at its N-terminus to Lys-119, is called ‘Mature’.

[0087] Multiple fHbp sequences are known and these can readily be aligned using standard methods. By such alignments one skilled in the art can identify (a) ‘A’ (and ‘Mature’), ‘B’ and ‘C’ domains in any given fHbp sequence by comparison with coordinates in the MC58 sequence, and (b) unique residues in multiple fHbp sequences, e.g., to identify substitutions. For ease of reference, however, the domains are defined as follows: - Domain ‘A’ in a given fHbp sequence is the fragment of that sequence that, when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm, begins with the Met-1-aligned amino acid of SEQ ID NO: 1 and ends with the Lys-119-aligned amino acid of SEQ ID NO: 1. - Domain ‘Mature’ in a given fHbp sequence is the fragment of that sequence that, when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm, begins with the Cys-20-aligned amino acid of SEQ ID NO: 1 and ends with the Lys-119-aligned amino acid of SEQ ID NO: 1. - Domain ‘B’ in a given fHbp sequence is the fragment of that sequence that, when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm, begins with the Gln-120-aligned amino acid of SEQ ID NO: 1. NO: 1 and ends with the amino acid aligned with Gly-183 of SEQ ID NO: 1. - Domain ‘C’ in a given fHbp sequence is the fragment of that sequence that, when aligned with SEQ ID NO: 1 using a pairwise alignment algorithm, begins with the amino acid aligned with Lys-184 of SEQ ID NO: 1 and ends with the amino acid aligned with Gln-274 of SEQ ID NO: 1.

[0088] The preferred pairwise alignment algorithm for defining domains is the Needleman-Wunsch global alignment algorithm [158], using default parameters (e.g., with Gap opening penalty = 10.0, and with Gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [159].

[0089] In some embodiments, a v2 or v3 amino acid sequence of the mutant fHbp of the invention is truncated to remove its A domain. In general, however, it may be preferred that the v2 or v3 amino acid sequence of the mutant fHbp includes both a β-barrel at the N-terminus and a β-barrel at the C-terminus.

[0090] In some embodiments, a polypeptide comprises an amino acid sequence as previously described, except that up to 10 amino acids (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N-terminus and/or up to 10 amino acids (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the C-terminus are deleted.

[0091] Polypeptides of the invention typically consist of an artificial amino acid sequence, namely a sequence that is not present in any naturally occurring meningococcus.

[0092] Affinity for factor H can be quantitatively assessed using surface plasmon resonance, for example, as described in references 18 and 21 to 24 with immobilized human fH. Mutations that provide a reduction in affinity (i.e., an increase in the dissociation constant, KD) of at least 10-fold, and ideally at least 100-fold, are preferred (when measured under the same experimental conditions with respect to the same polypeptide but without the mutation).

Nucleic acids

[0093] The invention provides nucleic acid encoding a polypeptide of the invention as defined above.

[0094] Nucleic acids of the invention can be prepared in many ways, for example, by chemical synthesis (e.g., phosphoramidite DNA synthesis) in whole or in part, by digesting longer nucleic acids using nucleases (e.g., restriction enzymes), by joining shorter nucleic acids or shorter nucleotides (e.g., using ligases or polymerases), from genomic libraries or cDNA, etc.

[0095] Nucleic acids of the invention can take various forms, for example, single-stranded, double-stranded, vectors, oligonucleotide primers, probes, labeled, unlabeled, etc.

[0096] Nucleic acids of the invention are preferably in isolated or substantially isolated form.

[0097] The term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA), etc.

[0098] Nucleic acid according to the invention can be labeled, for example, with a radioactive or fluorescent label.

[0099] The invention also provides vectors (such as plasmids) comprising nucleotide sequences of the invention (e.g., cloning or expression vectors, such as those suitable for nucleic acid immunization) and host cells transformed with such vectors.

Bactericidal responses

[00100] Preferred polypeptides of the invention can elicit antibody responses that are bactericidal against meningococcus. Bactericidal antibody responses are conveniently measured in mice and are a standard indicator of vaccine efficacy (e.g., see endnote 14 of ref. 37 also ref. 38).

[00101] Polypeptides of the first embodiment of the invention can preferably elicit an antibody response that is bactericidal against a strain of N.meningitidis that expresses a fHbp v2 sequence, for example, one or more of strains 961-5945, 2996, 96217, 312294, 11327, a22, gb013 (=M01-240013), e32, m1090, m4287, 860800, 599, 95N477, 90-18311, c11, m986, m2671, 1000, m1096, m3279, bz232, dk353, m3697, ngh38, and/or L93/4286. Bactericidal responses can, for example, be evaluated against var2 strain M2091 (ATCC 13091).

[00102] Preferred polypeptides of the first embodiment of the invention can elicit antibodies in a mouse that are bactericidal against the M2091 strain in a serum bactericidal assay.

[00103] Polypeptides of the second embodiment of the invention may preferably elicit an antibody response that is bactericidal against a strain of N.meningitidis expressing a fHbp v3 sequence, for example one or more of strains M1239, 16889, gb355 (=M01-240355), m3369, m3813, ngp165. Bactericidal responses may for example be assessed against var3 strain M01240355, which is a Neisseria MLST reference strain (id 19265 in ref. 39) that has been fully sequenced (see EMBL ID CP002422 [40j]).

[00104] Preferred polypeptides of the second embodiment of the invention can elicit antibodies in a mouse that are bactericidal against strain M01-240355 in a serum bactericidal assay.

[00105] For example, an immunogenic composition comprising such polypeptides may provide a serum bactericidal titer of >1:4 using the Goldschneider assay with human complement [41-43] and/or provide a serum bactericidal titer of >1:128 using baby rabbit complement.

Immunization

[00106] Polypeptides of the invention can be used as the active ingredient of immunogenic compositions and thus the invention provides an immunogenic composition (e.g. a vaccine) comprising a polypeptide of the invention.

[00107] The invention also provides a method of raising an antibody response in a mammal, comprising administering an immunogenic composition of the invention to the mammal. The antibody response is preferably a protective and/or bactericidal antibody response. The invention also provides polypeptides of the invention for use in such methods.

[00108] The invention also provides a method of protecting a mammal against a Neisseria (e.g., meningococcal) infection, comprising administering to the mammal an immunogenic composition of the invention.

[00109] The invention provides polypeptides of the invention for use as medicaments (e.g., immunogenic compositions or vaccines) or as diagnostic reagents. It also provides the use of nucleic acid or polypeptide of the invention in the manufacture of a medicament for preventing Neisserial (e.g., meningococcal) infection in a mammal.

[00110] The mammal is preferably a human. The human may be an adult or, preferably, a child. Where the vaccine is for prophylactic use, the human is preferably a child (e.g., a child aged 1 to 3 years or a child aged 5 to 7 years); where the vaccine is for therapeutic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults, for example, to assess safety, dosage, immunogenicity, etc.

[00111] The uses and methods are particularly useful for preventing/treating diseases including, but not limited to, meningitis (particularly bacterial, such as meningococcal meningitis) and bacteremia. For example, they are suitable for active immunization of individuals against invasive meningococcal disease caused by N.meningitidis (e.g., in serogroup B).

[00112] Therapeutic treatment efficacy may be tested by monitoring Neisseria infection after administration of the composition of the invention. Prophylactic treatment efficacy may be tested by monitoring immune responses against fHbp after administration of the composition. Immunogenicity of the compositions of the invention may be determined by administering them to test subjects (e.g., children 12 to 16 months of age, or animal models) and then determining standard parameters including serum bactericidal antibodies (SBA) and ELISA titers (GMT). These immune responses will generally be determined around 4 weeks after administration of the composition, and compared with values determined prior to administration of the composition. An increase in SBA of at least 4-fold or 8-fold is preferred. Where more than one dose of the composition is administered, more than one post-administration determination may be made.

[00113] Preferred compositions of the invention may confer an antibody titer in a patient that is superior to the criterion for seroprotection for each antigenic component for an acceptable percentage of human subjects. Antigens with an associated antibody titer above which a host is considered seroconverted against the antigen are well known, and such titers are published by organizations such as the WHO. Preferably, more than 80% of a statistically significant sample of subjects are seroconverted, more preferably more than 90%, even more preferably more than 93% and most preferably 96 to 100%.

[00114] Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or into the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary, or other mucosal administration. Intramuscular administration into the thigh or upper limb is preferred. Injection may be via a needle (e.g., a hypodermic needle), but needleless injection may alternatively be used. A typical intramuscular dose is about 0.5 mL.

[00115] The invention can be used to elicit systemic and/or mucosal immunity.

[00116] Dosing treatment may be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. A primary dose schedule may be followed by a booster dose schedule. Appropriate time between initial doses (e.g., between 4 to 16 weeks), and between initial and booster doses, may be routinely determined.

[00117] The immunogenic composition of the invention will generally include a pharmaceutically acceptable carrier, which may be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in such carriers. A full discussion of suitable carriers is available in ref. 44.

[00118] Neisseria infections affect various areas of the body and thus the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid vehicles prior to injection may also be prepared. The composition may be prepared for topical administration, for example as an ointment, cream or powder. The composition may be prepared for oral administration, for example as a tablet or capsule, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration, for example as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration, for example as drops. Compositions suitable for parenteral injection are most preferred.

[00119] The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered, for example between pH 6 and pH 8, generally around pH 7. Where a composition comprises an aluminium hydroxide salt, it is preferred to use a histidine buffer [45]. Compositions of the invention may be isotonic with respect to humans.

[00120] Immunogenic compositions comprise an immunologically effective amount of immunogen, as well as any of the other specified components, as required. ‘Immunologically effective amount’ means that administration of this amount to a subject, either in a single dose or as part of a series, is effective for treatment or prevention. The term ‘prevention’ means that the progression of the disease is reduced and/or eliminated, or that the onset of the disease is eliminated. For example, a subject’s immune system may be primed (e.g., by vaccination) to trigger an immune response and ward off infection in such a manner that the onset of the disease is eliminated. A vaccinated subject may thus become infected, but is able to ward off infection better than a control subject. This amount will vary depending on the health and physical condition of the individual being treated, age, the taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. The amount is expected to fall within a relatively wide range that can be determined through routine experimentation. Treatment dosing may be a single-dose schedule or a multiple-dose schedule (e.g., including booster doses). The composition may be administered concurrently with other immunoregulatory agents.

[00121] Adjuvants that may be used in compositions of the invention include, but are not limited to, insoluble metal salts, oil-in-water emulsions (e.g., MF59 or AS03, both containing squalene), saponins, non-toxic derivatives of LPS (such as monophosphoryl lipid A or O-deacylated MPL3), immunostimulatory oligonucleotides, detoxified bacterial ADP-ribosylating toxins, microparticles, liposomes, imidazoquinolones, or mixtures thereof. Other substances that act as immunostimulating agents are described in chapter 7 of ref. 46.

[00122] The use of an aluminum hydroxide and/or aluminum phosphate adjuvant is particularly preferred, and polypeptides are generally adsorbed onto these salts. Such salts include oxyhydroxides and hydroxyphosphates (e.g., see chapters 8 & 9 of ref. 46). The salts may take any suitable form (e.g., gel, crystalline, amorphous, etc.).

Additional antigenic components

[00123] Compositions of the invention include mutant fHbp v2 and/or v3 sequence. It is advisable that the composition does not include complex or undefined mixtures of antigens, for example, it is preferred not to include outer membrane vesicles in the composition. Polypeptides of the invention are preferably expressed recombinantly in a heterologous host and then purified.

[00124] As well as including a fHbp polypeptide, a composition of the invention may also include one or more additional neisserial immunogen(s), since a vaccine that targets more than one immunogen per bacterium decreases the possibility of selecting for escape mutants. Thus, a composition may include a second polypeptide that, when administered to a mammal, elicits an antibody response that is bactericidal against meningococcus. The second polypeptide may be a meningococcal fHbp, but will often not be an fHbp, for example, it may be an NHBA sequence, a NadA sequence, etc.

[00125] Any such additional neisserial immunogen may be present as a separate polypeptide in the mutant v2 or v3 fHbp of the invention or may be present as a fusion polypeptide with the modified fHbp. For example, fusion of meningococcal 936 polypeptide and fHbp polypeptides is known [55,56]. Fusion proteins comprising SEQ ID NO: 44 and/or SEQ ID NO: 45 are particularly preferred, optionally wherein SEQ ID NO: 44 and/or 45 is/are modified by up to 5 single amino acid changes (i.e., 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions) as described elsewhere herein.

[00126] A composition of the invention may include an NHBA antigen. The NHBA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB2132 (Gene Bank accession number GI:7227388; SEQ ID NO: 6 herein). The NHBA antigen sequences of many strains have since been published. For example, allelic forms of NHBA can be seen in Figures 5 and 15 of reference 48, and in Example 13 and Figure 21 of reference Error! Indicator not defined. (SEQ IDs 3179 to 3184 therein). Various immunogenic fragments of the NHBA antigen have also been reported. Preferred 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: 6; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 6, 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 of SEQ ID NO: 6. The most useful NHBA antigens of the invention can elicit antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 6. NHBA antigens useful for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[00127] A composition of the invention may include a NadA antigen. The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB1994 (Gene Bank accession number GI:7227256; SEQ ID NO:7 herein). NadA antigen sequences from many strains have since been published, and the activity of the protein as a Neisserial adhesion has been well documented. Several immunogenic fragments of NadA have also been reported. Preferred NadA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 7; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 7, 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).

[00128] Preferred fragments of (b) comprise an epitope of SEQ ID NO: 7. The most useful NadA antigens of the invention may elicit antibodies which, after administration to a subject, may bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 7. NadA antigens useful for use with the invention may elicit bactericidal anti-meningococcal antibodies after administration to a subject. SEQ ID NO: 15 is such a fragment.

[00129] A composition of the invention may include an NspA antigen. The NspA antigen has been included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB0663 (Gene Bank accession number GI:7225888; SEQ ID NO: 8 herein). The antigen was previously known from references 49 & 50. NspA antigen sequences from many strains have since been published. Several immunogenic fragments of NspA have also been reported. Preferred NspA antigens for use with the invention comprise amino acid sequence n: (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: 8; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 8, 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 of SEQ ID NO: 8. The most useful NspA antigens of the invention can elicit antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 8. Suitable NspA antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[00130] Compositions of the invention may include a meningococcal HmbR antigen. The full-length HmbR sequence has been included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB1668 (SEQ ID NO: 9 herein). The invention may use a polypeptide comprising a full-length HmbR sequence, but it will often use a polypeptide comprising a partial HmbR sequence. Thus, in some embodiments, an HmbR sequence used according to the invention may comprise an amino acid sequence having at least i% sequence identity to SEQ ID NO: 9, where the value of i is 50, 60, 70, 80, 90, 95, 99 or more. In other embodiments an HmbR sequence used in accordance with the invention may comprise a fragment of at least j consecutive amino acids of SEQ ID NO: 9, 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 an HmbR sequence used in accordance with the invention may comprise a sequence of amino acids (i) having at least i% sequence identity to SEQ ID NO: 9 and/or (ii) comprising a fragment of at least j consecutive amino acids of SEQ ID NO: 9. Preferred fragments of j amino acids comprise an epitope of SEQ ID NO: 9. Such epitopes will typically comprise amino acids that are located on the surface of HmbR. Useful epitopes include those with amino acids involved in HmbR binding to hemoglobin, since antibodies that bind to these epitopes can block the ability of a bacterium to bind to host hemoglobin. The topology of HmbR, and its critical functional residues, were investigated in reference 51. The most useful HmbR antigens of the invention can elicit antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 9. HmbR antigens useful for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[00131] A composition of the invention may include an NhhA antigen. The NhhA antigen has been included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB0992 (Gene Bank accession number GI:7226232; SEQ ID NO:10 herein). The NhhA antigen sequences of many strains have since been published, e.g. refs 48 & 52, and several immunogenic fragments of NhhA have been reported. It is also known as Hsf. Preferred NhhA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (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 of SEQ ID NO: 10. The most useful NhhA antigens of the invention can elicit antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 10. NhhA antigens useful for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[00132] A composition of the invention may include an App antigen. The App antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB1985 (Gene Bank accession number GI:7227246; SEQ ID NO:11 herein). App antigen sequences from many strains have since been published. Several immunogenic fragments of App have also been reported. Preferred App antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 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 of SEQ ID NO: 11. The most useful App antigens of the invention can elicit antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 11. App antigens useful for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[00133] A composition of the invention may include an Omp85 antigen. The Omp85 antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB0182 (Gene Bank accession number GI:7225401; SEQ ID NO:12 herein). The Omp85 antigen sequences of many strains have since been published. Additional information regarding Omp85 can be found in references 53 and 54. Several immunogenic fragments of Omp85 have also been reported. Preferred Omp85 antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 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 of SEQ ID NO: 12. The most useful Omp85 antigens of the invention can elicit antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 12. Omp85 antigens useful for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

[00134] A composition of the invention may include a 936 antigen. The 936 antigen has been included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as gene NMB2091 (SEQ ID NO: 13 herein). Preferred 936 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 of SEQ ID NO: 13. The most useful 936 antigens of the invention can elicit antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence SEQ ID NO: 13. The 936 antigen is a good fusion partner for fHbp (e.g., see references 55 & 56).

[00135] A composition may comprise: a polypeptide comprising SEQ ID NO: 14; a polypeptide comprising SEQ ID NO: 15; and a polypeptide of the invention comprising a mutant fHbp v2 amino acid sequence and SEQ ID NO: 13 (cf. refs. 55 & 56).

[00136] A composition may comprise: a polypeptide comprising SEQ ID NO: 14; a polypeptide comprising SEQ ID NO: 15; and a polypeptide of the invention comprising a mutant fHbp v3 amino acid sequence and SEQ ID NO: 13 (cf. refs. 55 & 56).

[00137] In some embodiments, a polypeptide of the invention is combined with an additional meningococcal fHbp sequence. In particular, a v2 polypeptide may be combined with a v1 and/or a v3 polypeptide to increase the spectrum of strain coverage [162]. Thus, a composition may comprise: (i) a polypeptide of the invention comprising a mutant fHbp v2 amino acid sequence; and (ii) a v1 fHbp polypeptide and/or a v3 fHbp polypeptide. In other embodiments, a polypeptide of the invention may comprise (i) a mutant fHbp v2 amino acid sequence and (ii) a v1 fHbp amino acid sequence and/or a v3 fHbp amino acid sequence. Thus, the v1 and/or v3 sequences may be combined with the mutant v2 sequence as separate entities in a composition, or in a fusion polypeptide.

[00138] Similarly, a v3 polypeptide can be combined with a v1 and/or a v2 polypeptide to increase the spectrum of strain coverage [162]. Thus, a composition can comprise: (i) a polypeptide of the invention comprising a mutant fHbp v3 amino acid sequence; and (ii) a v1 fHbp polypeptide and/or a v2 fHbp polypeptide. In other embodiments, a polypeptide of the invention can comprise (i) a mutant fHbp v3 amino acid sequence and (ii) a v1 fHbp amino acid sequence and/or a v2 fHbp amino acid sequence. Thus, the v1 and/or v2 sequences can be combined with the mutant v3 sequence as separate entities in a composition, or in a fusion polypeptide.

[00139] Furthermore, mutant v2 and v3 polypeptides can be combined with each other to increase strain coverage. Thus, a composition can comprise: (i) a polypeptide of the invention comprising a mutant fHbp v2 amino acid sequence; (ii) a polypeptide of the invention comprising a mutant fHbp v3 amino acid sequence; and (iii) a fHbp v1 polypeptide. In other embodiments, a polypeptide of the invention can comprise (i) a mutant fHbp v2 amino acid sequence (ii) a mutant fHbp v3 amino acid sequence and (iii) a fHbp v1 amino acid sequence. Thus, mutant v2 and v3 sequences can be combined with a v1 sequence as separate entities in a composition, or in a fusion polypeptide. The v1 sequence can be a wild-type sequence or a mutant sequence.

[00140] A v1 fHbp may comprise (a) an amino acid sequence that has at least k% identity to SEQ ID NO: 16 and/or (b) a fragment of SEQ ID NO: 16. Information about ‘k’ and fragments is given above. The fragment will typically include at least one epitope of SEQ ID NO: 16, and the v1 fHbp polypeptide will include at least one epitope that is not present in the v2 or v3 amino acid sequence of the invention, such that antibodies elicited by the v1 fHbp can recognize v1 strains. Ideally, the v1 fHbp can elicit antibodies that are bactericidal against v1 strains, e.g., against strain MC58 (available from the ATCC as ‘BAA-335’). The v1 fHbp may include an amino acid mutation that disrupts its ability to bind fH.

[00141] A v2 fHbp may comprise (a) an amino acid sequence that has at least k% identity to SEQ ID NO: 5 and/or (b) a fragment of SEQ ID NO: 5. Information about ‘k’ and fragments is given above. The fragment will typically include at least one epitope of SEQ ID NO: 5, and the v2 fHbp polypeptide will include at least one epitope that is not present in the v3 amino acid sequence of the invention, such that antibodies elicited by the v2 fHbp can recognize v2 strains. Ideally, the v2 fHbp can elicit antibodies that are bactericidal against v2 strains, for example against strain M2091 (ATCC 13091). The v2 fHbp can be a polypeptide of the first embodiment.

[00142] A v3 fHbp may comprise (a) an amino acid sequence that has at least k% identity to SEQ ID NO: 17 and/or (b) a fragment of SEQ ID NO: 17. Information about ‘k’ and fragments is given above. The fragment will typically include at least one epitope of SEQ ID NO: 17, and the v3 fHbp polypeptide will include at least one epitope that is not present in the v2 amino acid sequence of the invention, such that antibodies elicited by the v3 fHbp can recognize v3 strains. Ideally, the v3 fHbp can elicit antibodies that are bactericidal against v3 strains, for example against strain M01-240355. The v3 fHbp can be a polypeptide of the second embodiment.

[00143] Thus, for example, the invention provides a polypeptide comprising, within a single polypeptide chain, each of: (i) a fHbp v1 amino acid sequence; (ii) a mutant fHbp v2 amino acid sequence; and (iii) a mutant fHbp v3 amino acid sequence. The polypeptide can, after administration to a host animal, elicit antibodies that bind to each of: a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 46; a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 4; and a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 40. The sequence of (i) may comprise an amino acid sequence having at least k% identity to SEQ ID NO: 16. The sequence of (ii) may comprise an amino acid sequence having at least k% identity to SEQ ID NO: 5 but differing from SEQ ID NO: 5 at one or more of the following residues: S32, V33, L39, L41, F69, V100, I113, F122, L123, V124, S125, G126, L127, G128, S151, H239 and/or E240. The sequence of (iii) may comprise an amino acid sequence having at least k% identity to SEQ ID NO: 17, but differing from SEQ ID NO: 17 at one or more of the following residues: S32, I33, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242 and/or E243. In a preferred embodiment: the sequence of (i) comprises SEQ ID NO: 16, optionally modified by up to 5 single amino acid exchanges (i.e., 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions); the sequence of (ii) comprises SEQ ID NO: 45, optionally modified by up to 5 single amino acid exchanges (i.e., 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions), provided such amino acid exchanges do not revert the mutations in those sequences relative to the wild-type sequence; and the sequence of (iii) comprises SEQ ID NO: 44, optionally modified by up to 5 single amino acid exchanges (i.e., 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions, e.g., to give SEQ ID NO: 53), provided such amino acid exchanges do not revert the mutations in those sequences relative to the wild-type sequence. Amino acid sequences (i), (ii) and (iii) may be arranged in any order from N- to C-terminal to the polypeptide, but are preferably in the order (ii) then (iii) then (i). For example, the invention provides a polypeptide of the formula -A-B-C- wherein: A comprises SEQ ID NO: 45, optionally modified by up to 3 single amino acid substitutions; B comprises SEQ ID NO: 44, optionally modified by up to 3 single amino acid substitutions; and C comprises SEQ ID NO: 16, optionally modified by up to 3 single amino acid substitutions (e.g., substitution(s) to disrupt bonding at fH). A preferred C comprises SEQ ID NO: 49, wherein residue Arg-34 is mutated to Ser as reported for the ‘R41S’ mutation in ref. 21.

[00144] A particularly preferred polypeptide comprises amino acid sequence SEQ ID NO: 47. This sequence includes, from the N-terminus to the C-terminus: the mutant v2 (SEQ ID NO: 45); the mutant v3 (SEQ ID NO: 44); and the mutant v1 (SEQ ID NO: 49). Between these three mutant fHbp sequences there is in each case an adapter sequence, SEQ ID NO: 50. In one embodiment, the polypeptide comprises amino acid sequence SEQ ID NO: 48, which has a methionine at the N-terminus, then SEQ ID NO: 37, and then SEQ ID NO: 47.

[00145] SEQ ID NO: 47 (alone, or within SEQ ID NO: 48) may optionally be modified by up to 5 single amino acid exchanges (i.e., 1, 2, 3, 4, or 5 single amino acid substitutions, deletions, and/or insertions), provided such amino acid exchanges do not revert the mutations in sequences v1, v2, and v3 relative to the wild-type sequence, i.e., amino acid residues V32, R123, V285, R379, and S543 of SEQ ID NO: 47 should not be mutated to S32, L123, S285, L379, and R543. In an exceptional embodiment, however, V285 may revert to S285 and/or V32 may revert to S32.

[00146] The mutant fusion can ideally elicit antibodies that bind to each of: a wild-type meningococcal v1 fHbp polypeptide consisting of the amino acid sequence SEQ ID NO: 46; a wild-type meningococcal v2 fHbp polypeptide consisting of the amino acid sequence SEQ ID NO: 4; and a wild-type meningococcal v3 fHbp polypeptide consisting of the amino acid sequence SEQ ID NO: 40.

[00147] In addition to the Neisserial polypeptide antigens, the composition may include antigens to immunize against other diseases or infections. For example, the composition may include one or more of the following additional antigens: - a saccharide antigen from N.meningitidis serogroup A, C, W135 and/or Y, such as the saccharide described in ref. 57 from serogroup C (see also ref. 58) or in ref. 59. - a saccharide antigen from Streptococcus pneumoniae [e.g., 60, 61, 62]. - a hepatitis A virus antigen, such as inactivated virus [e.g., 63, 64]. a hepatitis B virus antigen, such as surface and/or core antigens [e.g., 64, 65]. - a diphtheria antigen, such as a diphtheria toxoid [e.g., chapter 3 of ref. 67], for example, the CRM197 mutant [e.g., 67]. - a tetanus antigen, such as tetanus toxoid (e.g., chapter 4 of ref. 66). - a Bordetella pertussis antigen, such as pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 (e.g., refs. 68 & 69). - a saccharide antigen from Haemophilus influenzae B [e.g., 58]. - polio antigen(s) [e.g., 70, 71] such as IPV. - measles, mumps, and/or rubella antigens (e.g., chapters 9, 10 & 11 of ref. 66). - influenza antigen(s) (e.g., chapter 19 of ref. 66), such as the hemagglutinin and/or neuraminidase surface proteins. - an antigen from Moraxella catarrhalis [e.g., 72]. - a protein antigen from Streptococcus agalactiae (group B streptococcus) [e.g., 73, 74]. - a saccharide antigen from Streptococcus agalactiae (group B streptococcus). - an antigen from Streptococcus pyogenes (group A streptococcus) [e.g., 74, 75, 76]. - an antigen from Staphylococcus aureus [e.g., 77].

[00148] The composition may comprise one or more such additional antigens.

[00149] Toxic protein antigen can be detoxified where necessary (e.g., detoxification of pertussis toxin by chemical and/or genetic means [69]).

[00150] Where a diphtheria antigen is included in the composition it is preferred to also include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred to also include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred to also include diphtheria and tetanus antigens. Combinations of DTP are thus preferred.

[00151] Saccharide antigens are preferably in the form of conjugates. Carrier proteins for the conjugates are discussed in more detail below.

[00152] Antigens in the composition will typically be present at a concentration of at least 1μg/mL each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.

[00153] Immunogenic compositions of the invention can be used therapeutically (i.e., to treat an existing infection) or prophylactically (i.e., to prevent future infection).

[00154] As an alternative to using protein antigens in the immunogenic compositions of the invention, nucleic acid (which may be RNA, such as an RNA, or self-replicating DNA, such as a plasmid) encoding the antigen may be used.

[00155] In some embodiments, a composition of the invention comprises in addition to the fHbp sequence, conjugated capsular saccharide antigens from 1, 2, 3, or 4 of serogroups A, C, W135, and Y meningococci. In other embodiments, a composition of the invention comprises in addition to the fHbp sequence, at least one conjugated pneumococcal capsular saccharide antigen.

Serogroups Y, W135, C and A meningococci

[00156] Current serogroup C vaccines (Menjugate™ [57, 78], Meningitec™ and NeisVac-C™) include conjugated saccharides. Menjugate™ and Meningitec™ have oligosaccharide antigens conjugated to a CRM197 carrier, while NeisVac-C™ uses the full-length (de-O-acetylated) polysaccharide conjugated to a tetanus toxoid carrier. The Menactra™ vaccine contains conjugated capsular saccharide antigens from each of serogroups Y, W135, C and A.

[00157] Compositions of the present invention may include capsular saccharide antigens from one or more of serogroups Y, W135, C, and A meningococci, wherein the antigens are conjugated to carrier protein(s) and/or are oligosaccharides. For example, the composition may include a capsular saccharide antigen from: serogroup C; serogroups A and C; serogroups A, C, and W135; serogroups A, C, and Y; serogroups C, W135, and Y; or from all four of serogroups A, C, W135, and Y.

[00158] A typical amount of each meningococcal saccharide antigen per dose is between 1 μg and 20 μg, for example, about 1 μg, about 2.5 μg, about 4 μg, about 5 μg, or about 10 μg (expressed as saccharide).

[00159] Where a mixture comprises capsular saccharides from both serogroups A and C, the ratio (w/w) of MenA saccharide:MenC saccharide may be greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1, or greater). Where a mixture comprises capsular saccharides from serogroup Y and either or both of serogroups C and W135, the ratio (w/w) of MenY saccharide:MenW135 saccharide may be greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1, or greater) and/or the ratio (w/w) of MenY saccharide:MenC saccharide may be less than 1 (e.g., 1:2, 1:3, 1:4, 1:5, or less). Preferred ratios (w/w) for saccharides from serogroups A:C:W135:Y are: 1:1:1:1; 1:1:1:2; 2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2; 8:4:1:2; 4:2:2:1; 2:2:1:1; 4:4:2:1; 2:2:1:2; 4:4:1:2; and 2:2:2:1. Preferred ratios (w/w) for saccharides from serogroups C:W135:Y are: 1:1:1; 1:1:2; 1:1:1; 2:1:1; 4:2:1; 2:1:2; 4:1:2; 2:2:1; and 2:1:1. Use of a substantially equal mass of each saccharide is preferred.

[00160] Capsular saccharides may be used in the form of oligosaccharides. These are conveniently formed by fragmentation of purified capsular polysaccharide (e.g. by hydrolysis), which will normally be followed by purification of fragments of the desired size.

[00161] Fragmentation of polysaccharides is preferably carried out to give a final average degree of polymerization (DP) in the oligosaccharide of less than 30 (e.g. between 10 and 20, preferably around 10 for serogroup A; between 15 and 25 for serogroups W135 and Y, preferably around 15 to 20; between 12 and 22 for serogroup C; etc.). DP can conveniently be measured by ion exchange chromatography or by colorimetric assays [79].

[00162] If hydrolysis is performed, the hydrolysate will generally be synthesized in order to remove short length oligosaccharides [58]. This can be achieved in a number of ways, such as ultrafiltration followed by ion exchange chromatography. Oligosaccharides with a degree of polymerization less than or equal to about 6 are preferably removed for serogroup A, and those less than about 4 are preferably removed for serogroups W135 and Y.

[00163] Preferred MenC saccharide antigens are described in reference 78, as used in Menjugate™.

[00164] The saccharide antigen may be chemically modified. This is particularly useful for reducing hydrolysis for serogroup A [80]. De-O-acetylation of meningococcal saccharides may be performed. For oligosaccharides, modification may occur before or after depolymerization.

[00165] Where a composition of the invention includes a saccharide antigen, the MenA antigen is preferably a modified saccharide in which one or more of the hydroxyl groups in the native saccharide has/have been replaced by a blocking group [80]. This modification increases resistance to hydrolysis.

Covalent conjugation

[00166] Capsular saccharides in compositions of the invention will typically be conjugated to carrier protein(s). In general, conjugation enhances the immunogenicity of saccharides since it converts them from T-independent antigens to T-dependent antigens, thus allowing priming for immunological memory. Conjugation is particularly useful for pediatric vaccines and is a well-known technique.

[00167] Typical carrier proteins are bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. Mutant diphtheria toxin CRM197 [81] is useful, and is the carrier in the PREVNAR™ product. Other suitable carrier proteins include the outer membrane protein complex of N.meningitidis [82], synthetic peptides [83, 84], heat shock proteins [85, 86], pertussis proteins [87, 88], cytokines [8], lymphokines [89], hormones [89], growth factors [89], artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens [90] such as N19 [91], protein D of H.influenzae [92, 94], pneumolysin [95] or its nontoxic derivatives [96], PspA of pneumococcal surface protein [97], iron uptake proteins [98], toxin A or B of C.difficile [99], recombinant P.aeruginosa exoprotein A (rEPA) [100], etc.

[00168] Any suitable conjugation reaction may be used, with any suitable adaptor where necessary.

[00169] The saccharide will typically be activated or functionalized prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (e.g., 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [101, 102, etc.]). Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, etc.

[00170] Adapters via an adapter group can be made using any known procedure, for example, the procedures described in references 103 and 104. One type of adapter involves reductive amination of the polysaccharide, coupling the resulting amino group to one end of an adipic acid group adapter, and then coupling a protein to the other end of the adipic acid group adapter [105, 106]. Other linkers include B-propionamido [107], nitrophenylethylamine [108], haloacyl halides [109], glycosidic adapters [110], 6-aminocaproic acid [111], ADH [112], C4 to C12 moieties [113], etc. As an alternative to using an adapter, a direct adapter can be used. Direct protein adaptors may comprise oxidation of the polysaccharide followed by reductive amination with the protein, as described, for example, in references 114 and 115.

[00171] A process involving introduction of the amino groups into the saccharide (e.g., replacing terminal =O groups with -NH2) followed by derivatization with an adipic diester (e.g., N-hydroxysuccinimide adipic acid diester) and reaction with a carrier protein is preferred. Another preferred reaction uses activation of CDAP with a protein D carrier, e.g., for MenA or MenC.

Outer membrane vesicles

[00172] Some compositions of the invention do not include complex or undefined mixtures of antigens, which are typical features of OMVs. However, the invention can be used together with OMVs, since it has been observed that fHbp enhances their efficacy [4], in particular by overexpressing the polypeptides of the invention in the strains used for OMV preparation. See also below.

[00173] This approach can be used in general to improve preparations of N.meningitidis serogroup B microvesicles [116], ‘native OMVs’ [117], blebs or outer membrane vesicles [e.g., refs. 118–123, etc.]. These can be prepared from bacteria that have been genetically engineered [124–127], e.g., to increase immunogenicity (e.g., overexpress immunogens), to reduce toxicity, to inhibit capsular polysaccharide synthesis, to downregulate PorA expression, etc. They can be prepared from hyperbubbling strains [128–131]. Vesicles from a nonpathogenic Neisseria can be included [132]. OMVs can be prepared without the use of detergents [133,134]. They can express non-Neisserial proteins on their surface [1]. They can be depleted of LPS. They can be mixed with recombinant antigens [118,136]. Vesicles from bacteria with different class I outer membrane protein subtypes can be used, for example, six different subtypes [137,138] using two different genetically engineered vesicle populations each exhibiting three subtypes, or nine different subtypes using three different genetically engineered vesicle populations each exhibiting three subtypes, etc. Useful subtypes include: P1.7,16; P1.5-1,2-2; P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1; P1.18-1,3,6.

Host cells

[00174] The invention provides a bacterium that expresses a polypeptide of the invention. The bacterium may be a meningococcus or an E.coli. The bacterium may constitutively express the polypeptide, but in some embodiments, expression may be under the control of an inducible promoter. The bacterium may hyperexpress the polypeptide (cf. ref. 139). Expression of the polypeptide is ideally not phase variable.

[00175] The invention also provides outer membrane vesicles prepared from a bacterium of the invention (particularly from a meningococcus). It also provides a process for producing vesicles from a bacterium of the invention. Vesicles prepared from such strains preferably include the polypeptide of the invention, which should be in an immunoaccessible form in the vesicles, i.e. an antibody that can bind to purified polypeptide of the invention may also be capable of binding to the polypeptide that is present in the vesicles.

[00176] Such outer membrane vesicles include any proteoliposomal vesicle obtained by disruption or bubbling of a meningococcal outer membrane to form vesicles that include outer membrane protein components. Thus, the term includes OMVs (sometimes referred to as ‘blebs’), microvesicles (MVs [116]) and ‘native OMVs’ (‘NOMVs’ [117]).

[00177] MVs and NOMVs are naturally occurring membrane vesicles that form spontaneously during bacterial growth and are released into the culture medium. MVs can be obtained by culturing Neisseria in broth culture medium, separating whole cells from smaller MVs in the broth culture medium (e.g., by filtration or by low-speed centrifugation to precipitate only the cells and not the smaller vesicles), and then collecting the MVs from the cell-depleted medium (e.g., by filtration, by differential precipitation or aggregation of MVs, by high-speed centrifugation to precipitate the MVs). Strains for use in MV production can generally be selected based on the amount of MVs produced in culture, e.g., refs. 130 & 131 describe Neisseria with high MV production.

[00178] OMVs are prepared artificially from bacteria, and can be prepared using detergent treatment (e.g., with deoxycholate), or by non-detergent means (e.g., see reference 134). Techniques for forming OMVs include treating bacteria with a bile acid salt detergent (e.g., salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., with sodium deoxycholate [140 & 141] being preferred for treating Neisseria) at a pH high enough not to precipitate the detergent [142]. Other techniques can be performed substantially in the absence of detergent [134] using techniques such as sonication, homogenization, microfluidization, cavitation, osmotic shock, milling, French press, blending, etc. Methods using no or low detergent can retain useful antigens such as NspA and fHbp [134]. Thus, a method may use an OMV extraction buffer with about 0.5% deoxycholate or less, e.g., about 0.2%, about 0.1%, <0.05%, or zero.

[00179] A useful process for preparing OMVs is described in ref. 143 and involves ultrafiltration on crude OMVs, rather than high-speed centrifugation. The process may involve an ultracentrifugation step after ultrafiltration occurs. OMVs may also be purified using the two-stage size filtration process described in ref. 154.

[00180] Vesicles for use with the invention may be prepared from any meningococcal strain. The vesicles will normally be from a serogroup B strain, but it is possible to prepare them from serogroups other than B (e.g., reference 142 describes a process for serogroup A), such as A, C, W135 or Y. The strain 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.). The meningococcal may be of any suitable lineage, including hyperinvasive and hypervirulent lineages, for example, any of the following seven hypervirulent lineages: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37 complex; group A4; lineage 3.

[00181] Bacteria of the invention may, in addition to encoding a polypeptide of the invention, have one or more additional modifications. For example, they may have a modified fur gene [144]. Expression of nspA expression may be upregulated with concomitant porA and cps knockout. Additional knockout mutants for OMV production are described, for example, in reference 150. Reference 145 describes the construction of vesicles of strains engineered to express six different PorA subtypes. Neisserial mutants with low levels of endotoxin, obtained by knockout of enzymes involved in LPS biosynthesis, may also be used [146, 147]. Neisserial mutants engineered to reduce or switch off expression of at least one gene involved in rendering the lipid A portion of LPS toxic, in particular the lpxl1 gene, may be used with the invention [148]. Similarly, mutant Neisseria engineered to reduce or turn off expression of at least one gene involved in capsular polysaccharide synthesis or export, in particular synX and/or ctrA genes, may be used with the invention. These or other mutants may all be used with the invention.

[00182] Thus, a strain used with the invention may in some embodiments express more than one PorA subtype. 6-valent and 9-valent PorA strains have been previously constructed. The strain may express 2, 3, 4, 5, 6, 7, 8, or 9 of the PorA subtypes: P1.7,16; P1.5-1,2-2; P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1, and/or P1.18-1,3,6. In other embodiments, a strain may have been downregulated for PorA expression, e.g., in which the amount of PorA has been reduced by at least 20% (e.g., >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, etc.), or knocked out, relative to wild-type levels (e.g., relative to strain H44/76).

[00183] In some embodiments, a strain may overexpress certain proteins (relative to the corresponding wild-type strain). For example, strains may overexpress NspA, protein 287 [118], fHbp [139] (including fHbp of the invention), TbpA and/or TbpB [136], Cu,Zn superoxide dismutase, HmbR, etc.

[00184] A gene encoding a polypeptide of the invention may be integrated into the bacterial chromosome or may be present in episomal form, for example within a plasmid.

[00185] Advantageously, for bleb production, a meningococcus may be genetically engineered to ensure that expression of the polypeptide is not subject to phase variation. Methods for reducing or eliminating phase variability of gene expression in meningococcus are described in reference 149. For example, a gene may be placed under the control of a constitutive or inducible promoter, or by removing or replacing the DNA motif that is responsible for its phase variability.

[00186] In some embodiments, a strain can include one or more of the knockout and/or hyperexpression mutations described in references 122, 124, 128, and 150. For example, following the guidance and nomenclature in those four documents, genes useful for downregulation and/or knockout 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; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB; or (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB, SynX and/or SynC.

[00187] Where a mutant strain is used, in some embodiments it may have one or more, or all, of the following features: (i) downregulated or disabled LgtB and/or GalE to truncate the meningococcal LOS; (ii) upregulated TbpA; (iii) upregulated NhhA; (iv) upregulated Omp85; (v) upregulated LbpA; (vi) upregulated NspA; (vii) disabled PorA; (viii) downregulated or disabled FrpB; (ix) downregulated or disabled Opa; (x) downregulated or disabled Opc; (xii) deleted cps gene complex. A truncated LOS may be one that does not include a sialyl-lacto-N-neotetraose epitope, e.g., it may be a galactose deficient LOS. LOS may lack α chain.

[00188] Depending on the meningococcal strain used to prepare the vesicles, they may or may not include the strain's native fHbp antigen [151].

[00189] In a preferred embodiment, a meningococcus does not express a functional MltA protein. As discussed in refs. 152 & 153, knockout of MltA (the membrane-bound lytic transglycosylase, also known as GNA33) in meningococcus provides bacteria that spontaneously release large quantities of membrane vesicles into the culture medium, from which they can be readily purified. For example, the vesicles can be purified using the two-stage size filtration process of ref. 154, comprising: (i) a first filtration step in which the vesicles are separated from the bacteria based on their different sizes, with the vesicles passing into the filtrate; and (ii) a second filtration step in which the vesicles are retained in the retentate. MltA mutation (downregulation or knockout) has been used in ‘GMMA’ vaccines [155], and can conveniently be combined with additional downregulation or knockout of at least one gene involved in rendering the lipid A portion of LPS toxic, particularly lpxl1, and/or at least one gene involved in capsular polysaccharide synthesis or export, particularly the synX and/or ctrA genes. GMMA (Generalized Modules for Membrane Antigens) are genetically detoxified OMVs that are produced from meningococcal strains that have been engineered to release GMMA with reduced reactogenicity and increased immunogenicity. GMMA induce less proinflammatory cytokines than OMVs when tested in the monocyte activation assay (MAT).

[00190] A preferred meningococcal strain for a GMMA vaccine using this approach expresses a mutant v2 fHbp and/or a mutant v3 fHbp of the invention, and expression can be driven by strong promoters. Vesicles released by this strain include the mutant v2 and/or v3 fHbp proteins in immunogenic form, and administration of the vesicles can provide a bactericidal antibody response as discussed in reference 155. The strain can also express a v1 fHbp, or a v1 fHbp can instead be provided as a separate recombinant protein in soluble form (and the v1 fHbp can be a wild-type sequence or a mutant, e.g., mutated to disrupt its ability to bind fH, as discussed above). The invention provides such strains, and also provides the vesicles that these strains release, e.g., as purified from culture medium after growth of the strains. A preferred v2 mutant for expression in these strains has a mutation at S32 and/or L123 as discussed herein, and a preferred v3 mutant for expression in these strains has a mutation at S32 and/or L126 as discussed herein. Thus, vesicles prepared from meningococci expressing these v2 and v3 mutant fHbp sequences are particularly preferred immunogens for use in vaccines of the invention. A wild-type v2 sequence useful for mutagenesis in this manner comprises SEQ ID NO: 51 or SEQ ID NO: 54 (comprising ΔG form SEQ ID NO: 55), and a wild-type v3 sequence useful for mutagenesis in this manner comprises SEQ ID NO: 52.

[00191] Useful promoters for use in such strains include those described in references 156 and 157. For example, the promoter may be: (a) the promoter of a porin gene, preferably porA or porB, particularly from N.meningitidis; or (b) an rRNA gene promoter (such as a 16S rRNA gene), particularly from N.meningitidis. Where a meningococcal porin promoter is used, it is preferably from porA, and even more particularly a -10 region of a meningococcal porA gene promoter and/or a -35 region of a meningococcal porA gene promoter (preferably wherein the -10 region and the -35 region are separated by an intervening sequence of 12 to 20 nucleotides, and wherein the intervening sequence either contains no poly-G sequence or includes a poly-G sequence having no more than eight consecutive G nucleotides). Where an rRNA gene promoter is used, it may more particularly comprise (i) a -10 region of a meningococcal rRNA gene promoter and/or (ii) a -35 region of a meningococcal rRNA gene promoter. It is also possible to use a hybrid of (a) and (b), for example to have a -10 region of a porA promoter and a -35 region of an rRNA promoter (which may be a consensus -35 region). A useful promoter may thus be a promoter which includes both (i) a -10 region of a (particularly meningococcal) rRNA gene and a -35 region of a (particularly meningococcal) porA gene, or (ii) a -10 region of a (particularly meningococcal) porA gene and a -35 region of a (particularly meningococcal) rRNA gene.

[00192] If LOS is present in a vesicle, it is possible to treat the vesicle in this way to bind its LOS and protein components (“intrabubble conjugation” [150]).

General

[00193] The term “comprising” encompasses “including” as well as “consisting,” for example, a composition “comprising” X may consist exclusively of X or may include something additional, for example, X + Y. References to “comprising” (or “comprises,” etc.) may optionally be replaced by references to “consisting of” (or “consists of,” etc.). The phrase “consisting essentially of” limits the scope of a claim to the materials or steps specified “and those which do not materially affect the basic and novel feature(s)” of the claimed invention.

[00194] The expression “about” with respect to a numerical value x is optional and means, for example, x+10%.

[00195] The word “substantially” does not exclude “completely”, for example, a composition that is “substantially free” of Y may be completely free of Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[00196] “Sequence identity” may be determined by the Smith-Waterman homology search algorithm implemented in the MPSRCH program (Oxford Molecular), using an affinity gap search with parameters open gap penalty = 12 and extension gap penalty = 1 but is preferably determined by the Needleman-Wunsch global alignment algorithm [158], using default parameters (e.g., with open gap penalty = 10.0, and with extension gap penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [159]. Where the application concerns sequence identity for a particular SEQ ID, the identity may be calculated over the full length of that SEQ ID.

[00197] The term "fragment" in reference to polypeptide sequences means that the polypeptide is a fraction of a full-length protein. Such fragments may possess qualitative biological activity in common with the full-length protein, for example, a fragment may contain or encode one or more epitopes, such as immunodominant epitopes, which allow a similar immune response to be raised in the fragment as in the full-length sequence. Polypeptide fragments generally have an amino (N)-terminal portion and/or carboxy (C)-terminal portion deleted compared to the native protein, but where the remaining amino acid sequence of the fragment is identical to the amino acid sequence of the native protein. Polypeptide fragments may contain, for example: about 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262 contiguous amino acids, including all integers in between, of a reference polypeptide sequence, for example, between 50 and 260, 50 and 255, 50 and 250, 50 and 200, 50 and 150 contiguous amino acids of a reference polypeptide sequence. The term fragment explicitly excludes full-length fHbp and mature lipoprotein polypeptides thereof.

[00198] After serogroup, meningococcal classification includes serotype, serosubtype, and then immunotype, and the standard nomenclature lists serogroup, serotype, serosubtype, and immunotype, each separated by a colon, e.g., B:4:P1.15:L3,7,9. Within serogroup B, some lineages cause disease frequently (hyperinvasive), some lineages cause more severe forms of disease than others (hypervirulent), and others rarely cause disease at all. Seven hypervirulent lineages are recognized, namely subgroups I, III, and IV-1, ET-5 complex, ET-37 complex, cluster A4, and lineage 3. These were defined by multilocus enzyme electrophoresis (MLEE), but multilocus sequence typing (MLST) has also been used to classify meningococci. The four major hypervirulent clusters are ST32, ST44, ST8, and ST11 complexes.

[00199] In general, the invention does not encompass the various fHbp sequences specifically described in references 2, 3, 5, 6, 7, 160, 161, 162, 163, 164, 165, 166 and 167.

BRIEF DESCRIPTION OF THE DRAWINGS

[00200] Figure 1 shows the different sensitivity of fHbp variants to chymotrypsin cleavage. The arrow shows the position of full-length fHbp proteins.

[00201] Figure 2 shows western blot analysis of v2 mutants. Lanes are: (1 & 2) purified recombinant wild-type v2; (4) wild-type v2 lysate; (5) mutant #1; (6) mutant #2; (7) mutant #4; (8) mutant #5; (9) mutant #7; (10) mutant #8; (11) mutant #12; (12) mutant #14 (13) mutant #15; (14) NΔG-trx control of fHbp var2, i.e., v2 protein where N-terminal sequence GPDSDRLQQRR (SEQ ID NO: 37) is replaced by GSKDISS (SEQ ID NO: 38). Lanes 2 to 14 included chymotrypsin. M are molecular tags.

[00202] Figure 3 shows additional western blot analysis of v2 mutants. Lanes are: (1&2) mutant #3; (3&4) mutant #6; (5&6) mutant #9; (7&8) mutant #10; (9&10) mutant #13; (11&12) Δgono; (13&14) v2 wild type; (15&16) mutant #22. Odd numbered lanes are for proteins that were incubated without chymotrypsin, while even numbered lanes were for proteins incubated with chymotrypsin. The ‘Δgono’ protein is a recombinant v2 where the N-terminal sequence (SEQ ID NO: 37) has been removed.

[00203] Figure 4 shows additional western blot analysis of v2 mutants. Lanes are: (1&2) mutant #11; (3&4) N-trx; (5-7) mutant #19; (8&9) mutant #20; (10&11) mutant #21. Lanes 2, 4, 7, 9 and 11 show proteins that were incubated with chymotrypsin, while the other lanes did not have chymotrypsin. The ‘N-trx’ protein is a recombinant v2 where the N-terminal sequence (SEQ ID NO: 37) is replaced by SEQ ID NO: 39.

[00204] Figure 5 shows DSC results for fHbp from the wild-type v2 and S58V/L149R mutants. The C-terminal domain was unaffected by the mutation, but the Tm of the N-terminal domain was increased by >20°C (marked with the arrow). The y-axis shows Cp (kcal/mol/°C), and the x-axis shows temperature (°C).

[00205] Figure 6 shows the SPR response of fHbp from wild-type (solid) and mutant (dashed) v2.

[00206] Figure 7 shows the SPR response of v3 fHbp, both as wild type (top) and with various mutations.

[00207] Figure 8 shows DSC results for the triple fusion protein of SEQ ID NO: 48. The axes are as in Fig.5.

SPECIFIC EMBODIMENTS OF THE INVENTION

[00208] The invention provides the following specific numbered embodiments: 1. A mutant v2 or v3 fHbp that has increased stability relative to a wild-type fHbp and, preferably, also has lower affinity for human factor H than a wild-type fHbp, for example: (A) a polypeptide comprising a mutant fHbp v2 amino acid sequence, wherein: (a) the amino acid sequence has at least 80% sequence identity to SEQ ID NO: 5 and/or comprises a fragment of SEQ ID NO: 5 that is at least 7 amino acids in length and contains at least one of the residues listed in (b); but (b) the amino acid sequence differs from SEQ ID NO: 5 at one or more of the following residues: 32, 33, 39, 41, 69, 100, 113, 122, 123, 124, 125, 126, 127, 128, 151, 239 and/or 240; provided that: • if the v2 amino acid sequence of the mutant fHbp includes a substitution at residue 32 only, either this substitution is not with alanine or at least one additional residue listed in (b) is substituted; • if the v2 amino acid sequence of the mutant fHbp includes a substitution at residue 113 only, either this substitution is not with alanine or at least one additional residue listed in (b) is substituted. • if the v2 amino acid sequence of the mutant fHbp includes a substitution at residue 122 only, either this substitution is not with alanine or at least one additional residue listed in (b) is substituted; • if the v2 amino acid sequence of the mutant fHbp includes a substitution at residue 123, either this substitution is not with alanine or at least one additional residue listed in (b) is substituted; • if the v2 amino acid sequence of the mutant fHbp includes a substitution at residue 124 only, either this substitution is not with alanine or at least one additional residue listed in (b) is substituted; • if the v2 amino acid sequence of the mutant fHbp includes a substitution at residue 127 only, either this substitution is not with alanine or at least one additional residue listed in (b) is substituted; and • if the v2 amino acid sequence of the mutant fHbp includes a substitution at residue 240 only, either this substitution is not with alanine or at least one additional residue listed in (b) is substituted. or (B) a polypeptide comprising a mutant fHbp v3 amino acid sequence, wherein: (a) the amino acid sequence has at least 80% sequence identity to SEQ ID NO: 17 and/or comprises a fragment of SEQ ID NO: 17 that is at least 7 amino acids in length and contains at least one of the residues listed in (b); but (b) the amino acid sequence differs from SEQ ID NO: 17 at one or more of the following residues: 32, 33, 39, 41, 72, 103, 116, 125, 126, 127, 128, 129, 130, 131, 154, 242, and/or 243; provided that: • if the v3 amino acid sequence of the mutant fHbp includes a substitution at residue 32, either this substitution is not with alanine or at least one additional residue listed in (b) is replaced; • if the v3 amino acid sequence of the mutant fHbp includes a substitution at residue 113, either this substitution is not with alanine or at least one additional residue listed in (b) is replaced. • if the v3 amino acid sequence of the mutant fHbp includes a substitution only at residue 125, either this substitution is not with alanine or at least one additional residue listed in (b) is replaced; • if the v3 amino acid sequence of the mutant fHbp includes a substitution at residue 126, either this substitution is not with alanine or at least one additional residue listed in (b) is replaced; • if the v3 amino acid sequence of the mutant fHbp includes a substitution at residue 127, either this substitution is not with alanine or at least one additional residue listed in (b) is replaced; • if the v3 amino acid sequence of the mutant fHbp includes a substitution at residue 130, or this substitution is not with alanine or at least one additional residue listed in (b) is substituted; and • if the v3 amino acid sequence of the mutant fHbp includes a substitution only at residue 243, or this substitution is not with alanine or at least one additional residue listed in (b) is substituted. 2. The polypeptide of embodiment 1(B), wherein the amino acid sequence differs from SEQ ID NO: 17 by substitution at one or more of the residues listed in (b), for example, wherein the substitution(s) is/are selected from the group consisting of: S32V; I33C; L39C; L41C; F72C; V103T; T116S; F125C; L126R; V127I; S128G or S128T; G129D; L130I; G131A; S154C; H242R; and E243H. 3. The polypeptide of embodiment 1(B) or embodiment 2, comprising more than one substitution at the residues listed in (b), and selected from groups 3A through 3O as noted previously. 4. The polypeptide of embodiment 1(A), wherein the amino acid sequence differs from SEQ ID NO: 5 by substitution at one or more of the residues listed in (b), for example, where the substitution(s) is selected from the group consisting of: S32V; V33C; L39C; L41C; F69C; V100T; I113S; F122C; L123R; V124I; S125G or S125T; G126D; L127I; G128A; S151C; H239R; and E240H. 5. The polypeptide of embodiment 1(A) or embodiment 4, comprising more than one substitution at the residues listed in (b), and selected from groups 2A to 2O as noted above. 6. The polypeptide of any one of embodiments 1 to 5, also including one or more additional mutation(s) that disrupt the ability of the polypeptide to bind human factor H, e.g., in v2 including a substitution at one or more of R73, D203, E210, G228, S121, F122, L123, A192, E194, V199, I200, L201, T213, H215, F219, T231, and E240, or in v3 including a substitution at one or more of Q35, I178, L87, A88, L126, V127, V202, E213, T216, T234, V241, and E243. 7. A polypeptide comprising: • amino acid sequence SEQ ID NO: 44, optionally with 1, 2, 3, 4, or 5 single amino acid substitutions, deletions, and/or insertions, wherein the polypeptide can elicit antibodies that bind to a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 40 (e.g., comprising amino acid sequence SEQ ID NO: 44 with 1, 2, or 3 single amino acid substitutions), but not mutated at residue V32 or R126; • an amino acid sequence of SEQ ID NO: 45, optionally with 1, 2, 3, 4, or 5 single amino acid substitutions, deletions, and/or insertions, wherein the polypeptide can elicit antibodies that bind to a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (e.g., comprising amino acid sequence SEQ ID NO: 45 with 1, 2, or 3 single amino acid substitutions) but not mutated at residue V32 or R123; • an fHbp v3 amino acid sequence, wherein the v3 amino acid sequence is identical to a wild-type v3 amino acid sequence except for a mutation at the amino acid position corresponding to Leu-126 of SEQ ID NO: 17, provided that the mutation is not an alanine substitution (e.g., wherein the mutation is an arginine substitution); • a fHbp v2 amino acid sequence, wherein the v2 amino acid sequence is identical to a wild-type v2 amino acid sequence except for a mutation at the amino acid position corresponding to Leu-123 of SEQ ID NO: 5, provided that the mutation is not a substitution for alanine (e.g., wherein the mutation is a substitution for arginine); or • amino acid sequence SEQ ID NO: 47, optionally with 1, 2, 3, 4, or 5 single amino acid substitutions, deletions, and/or insertions, wherein the polypeptide can elicit antibodies that bind to each of: a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 46; a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 4; and a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 40 (e.g., consisting of the amino acid sequence SEQ ID NO: 48). 8. A plasmid or other nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of embodiments 1 to 7. 9. A host cell transformed with the plasmid of embodiment 8, for example, wherein the cell is a meningococcal bacterium, such as a meningococcal bacterium having downregulation or knockout of mltA and also optionally has downregulation or knockout of: (i) at least one gene involved in rendering the lipid A portion of LPS toxic, particularly lpxl1; and/or (ii) at least one gene involved in capsular polysaccharide synthesis or export, particularly synX and/or ctrA. 10. Membrane vesicles prepared from the host cell of embodiment 9, wherein the vesicles include a polypeptide of any one of embodiments 1 to 7. 11. An immunogenic composition comprising a polypeptide of any one of embodiments 1 to 7, or a vesicle of embodiment 10. 12. The composition of embodiment 11, further comprising a second polypeptide that, when administered to a mammal, elicits an antibody response that is bactericidal against meningococcus. 13. The composition of embodiment 11 or 12, further comprising (i) a conjugated capsular saccharide from N.meningitidis serogroup A, C, W135, and/or Y and/or (ii) a conjugated capsular saccharide from S.pneumoniae. 14. A method of raising an antibody response in a mammal, comprising administering an immunogenic composition of any one of embodiments 11 to 13.

MODES FOR CARRYING OUT THE INVENTION fHbp mutations

[00209] The v2 fHbp is known to be unstable. To analyze the underlying structural reasons for this undesirable property, with the goal of modifying the sequence to improve stability, the inventors analyzed sequence alignments and 3D structures of fHbp polypeptides. One area of particular interest was the structural interface between the N-terminal and C-terminal domains [168].

[00210] The inventors have identified the mutations explained in Table 1. Three of the positions identified for mutation overlap with references 24 and 25, but the invention does not encompass polypeptides reported in the prior art, i.e., where the polypeptides include substitutions only at these positions with alanine. For example, E240 can be substituted with histidine to match v1, and is ideally paired with substitution at residue H239 (mutants #1 and #11). Similarly, if F122 is substituted then it is preferably paired with substitution at S151, both with cysteine to allow formation of a disulfide bond (mutant #10). Also, if L123 is substituted then it may be substituted with arginine (instead of alanine), or it may be paired with substitution at other residues, for example, at S32 (see mutant #3), at S125 (see mutants #20 and #22), or with substitution at residues 124 to 128 (see mutant #12).

Stability studies

[00211] Unstable proteins tend to be less folded and therefore prone to cleavage and degradation by proteases. Figure 1 shows that fHbp of v2 is more sensitive to degradation by chymotrypsin than v1 and v3 and thus this test can be used to assess the stability of mutant proteins.

[00212] For Figure 1, v1, v2, and v3 of wild-type fHbp were prepared at 0.5 mg/μL in 50 mM Tris-HCl, 150 mM NaCl, pH 8. Chymotrypsin was added at a ratio of 1:100 (w/w). Samples were incubated at 24°C, 50 mL volume, without shaking. Samples were extracted and boiled for 0, 1, 3, or 6 hours; then run on a 12% bis-Tris gel (MES buffer). The lane on the left, marked *, indicated a sample incubated for 6 hours without protease.

[00213] E. coli cell lysates expressing the recombinant proteins were incubated with chymotrypsin at a ratio of 1:100 w/w for 3 h at 25 °C. The degradation pattern was analyzed by Western blotting after incubation with a rabbit-extracted polyclonal immune serum against all three fHbp variants. The presence of cleavage products at lower apparent molecular weight (Figures 2 to 4) is interpreted as an indication of instability, while persistence of a band corresponding to an apparent MW of ~30 kDa is interpreted as an indication of increased stability. Mutants #1-6, #12 and #22 all showed increased resistance to chymotrypsin cleavage compared to wild-type v 2.

[00214] DSC was used as a stand-alone approach to assess the effects of mutations on the stability of purified recombinant fHbp v2 proteins. Tm (melting temperature) measured by DSC corresponds to the temperature at which the analyzed protein is 50% in the folded state and 50% in the unfolded state. Mutations that stabilize the conformation of a protein will increase Tm, while mutations that destabilize it will decrease Tm. As seen in Figure 3D of ref. 24, the DSC profile of wild-type fHbp v2 shows two Tm values: Tm1 at ~40°C, which corresponds to the melting temperature of the N-terminal domain, and Tm2 at ~80°C corresponding to the melting temperature of the C-terminal domain. Tm1 and Tm2 values for analyzed mutants are shown in Table 1. Mutants #2, #4, #5, #12, #19 and #21 showed increased Tm of the N-terminal domain with respect to the wild-type protein, and this effect was more marked for mutants #2, #4 and #12.

[00215] Size exclusion chromatography (SEC) was used to assess the percentage of monomeric protein, and results are also shown in Table 1.

Mutants #2, #3, and #4

[00216] Mutant #3 (group 2B) gave the best overall results in v2 stability studies. This protein (SEQ ID NO: 20) includes mutations at Ser-58 (S32 in SEQ ID NO: 5) and Leu-149 (L123 in SEQ ID NO: 5), with substitutions for Val and Arg, respectively. The mutant v2 protein (SEQ ID NO: 20, comprising SEQ ID NO: 45) was analyzed by DSC and, compared to the wild-type sequence, the Tm of its N-terminal domain is >20°C higher (Figure 5).

[00217] In a serum bactericidal assay, this mutant v2 can compete for binding to human antibodies that were raised using a wild-type v2 sequence.

[00218] Although the S58V and L149R mutations were introduced to improve stability, and did not achieve this goal, Figure 6 shows that the mutant v2 polypeptide (dotted line) surprisingly showed greatly reduced fH binding compared to the wild-type v2 sequence (solid line) when measured by surface plasmon resonance against immobilized fH. The S58V mutation itself had little impact on fH binding, and the S58V/L149R double mutant showed greater fH binding than fHbp carrying only the L149R mutant.

[00219] When mutant #3 was further combined with the ‘E313A’ mutation in v2 there was a complete loss of fH binding as assessed by SPR.

[00220] The equivalent mutations were introduced into a v3 sequence (SEQ ID NO: 17) to give v3 mutant SEQ ID NO: 44. The effects of individual S58V and L149R mutations on fH binding were studied in v3 (i.e., the v3 equivalents of v2 mutants #2 and #4). Thus, numbered according to SEQ ID NO: 17, mutation S32V or L126R was introduced into the v3 sequence. These two mutants were compared with two different wild-type v3 sequences, and also with the ‘E313A’ mutant that is known to disrupt fH binding in v3 [23].

[00221] As shown in Figure 7, wild-type v3 binds fH (top two lines). The S58V mutation that was designed to improve stability reduced the SPR peak value by about 2-fold. More surprisingly, the L149R mutation (again, designed to improve stability) reduced fH affinity to a level similar to the known E313A mutant (bottom two lines).

[00222] The S58V and L149R mutations in v3 were also studied by DSC, and were found to increase the Tm at the N-terminus by 5.5°C (S58V) or 6.7°C (L149R) relative to the wild type. The Tm of both mutants was higher than that observed in the v2 double mutant (63.5°C - see Table 1). The v3 L149R mutant also showed an increased Tm value for its C-terminal domain, whereas there was almost no shift here for the v3 S58V mutant. SPR showed that fH binding by mutant #2 was reduced by about half, but for mutant #4 fH affinity was reduced to a level similar to the known E313A mutant (as also observed with v2). When the two mutations were combined (i.e. mutant #3) the increase in Tm compared to the wild type was 11.2°C. When the ‘E313A’ mutation was added to mutant #3 fH binding was almost completely eliminated, although the Tm of the N-terminal domain also decreased by 2.9°C when compared to mutant #3 (still remaining 8.3°C higher than wild type v3). The ‘E313A’ mutation alone was much less stable than wild type, showing a decreased Tm of 6.3°C.

[00223] Thus, mutations #2 and #4 can be used alone, or in combination (i.e., mutant #3), optionally with additional mutations, to stabilize fHbp of v2 or v3, but also to disrupt fH affinity.

[00224] A serum bactericidal assay was used to evaluate the immunogenic efficacy of mutants #3 and #4 in v2 and v3. In addition, the ‘E313A’ mutant was also tested in v2 and v3, either alone or in combination with the #3 mutations. Wild-type v2 and v3 fHbp was also tested for comparison. The proteins were administered at 20μg/dose with an aluminum hydroxide adjuvant and the resulting sera were tested for bactericidal activity against a panel of seven strains (four v2 strains, three v3 strains) including strains expressing the same fHbp as the starting wild-type fHbp sequence (i.e., v2 sequence 2.16 and v3 sequence 3.42).

[00225] Results for v2 proteins were as follows (SEQ ID is for ΔG form; * = homologous fHbp):

[00226] Results for v3 proteins were as follows:

Combination of mutants #2 and #12

[00227] Mutants #2 and #12 each showed improvements in v2 stability, so these two mutants were combined into a single fHbp (SEQ ID NO: 58, ΔG form). Compared to mutant #12, the Tm at the N-terminus of this combined mutant increased by an additional 4.2°C, giving the highest Tm of any of the v2 mutant proteins tested. In addition, it showed greatly reduced fH binding (approximately 8-fold reduced SPR peak value).

Mutant fusion protein

[00228] Mutant v2 and v3 sequences were fused via a GSGGGG (SEQ ID NO: 50) linker, also to a mutant v1 sequence, to give SEQ ID NO: 48. This sequence includes the S58V and L149R mutations for both v2 and v3, and the R41S[21] mutation for v1. SEQ ID NO: 47 includes, from N-terminal to C-terminal: v2 mutant #3 (SEQ ID NO: 45); v3 mutant #3 (SEQ ID NO: 44); and v1 ‘R41S’ mutant (SEQ ID NO: 49), connected by the glycine-rich linker sequence, SEQ ID NO: 50. The fusion protein can conveniently be expressed by adding an N-terminal Met-[SEQ ID NO: 37]- sequence, thus providing a mature protein SEQ ID NO: 48.

[00229] This fusion protein thus takes advantage of the observation that mutant #3 provides for both v2 and v3 a large increase in stability (Tm) and a large decrease in fH affinity. For v1 the R41S mutation has little effect on thermal stability, but strongly reduces fH binding.

[00230] DSC studies of the triple fusion protein (Figure 8) show that the three N-terminal transitions fall together in a broad peak centered at 68°C. The three transitions at the C-terminus also fall together. UPLC showed that the protein was 94.9% pure, and HPLC analysis showed <1.5% oligomers.

Mutant proteins expressed in ‘GMMA’ membrane vesicles

[00231] A meningococcal v1 strain was prepared with mltA, lpxL1 and synX knockouts to provide a genetic background to overexpress v2 and v3 fHbp lipoproteins under the control of the ‘ST2’ promoter [157] in a ‘GMMA’ vaccine. The v2 genes were integrated into the genome at the deleted lpxL1 locus, while the v3 genes were integrated at the synX locus. In addition, the native v1 fHbp gene was deleted so that v2 and v3 could be studied without interference.

[00232] Mutants #3 and #4 were tested for v2, and mutant #4 was tested for v3. In addition, a strain with mutants #4 of both v2 and v3 was prepared. For these bacteria, fHbp expression and fH binding were assessed by FACS.

[00233] For strains expressing only v2 fHbp, FACS showed that the various proteins were expressed at similar levels, at levels 2 log higher than the background Δfhbp strain. In terms of fH binding, however, mutants #3 and #4 showed much less binding with binding in mutant #4 being only slightly above background. These results mirror the SPR data obtained with recombinant v2 proteins.

[00234] For the strain expressing v3 mutant #4, FACS showed full expression of fHbp but its fH binding was abolished (matching the fH binding seen with the H222R’ mutation [19,25]).

[00235] For the strain expressing mutant #4 of v2 and v3, both fHbp proteins could be detected by FACS, but fH binding was only slightly above that seen in the background Δfhbp strain.

[00236] Western blot analysis was used to test the stability of fHbp expression in these bacteria during growth in liquid culture for 6 days. Protein expression of the v2 mutant remained stable over time, even when v3 was coexpressed. Protein expression of the v3 mutant also remained stable, except in the strain expressing both the v2 and v3 mutant, where v3 expression declined over time.

[00237] It should be understood that the invention is described above by way of example only and modifications may be made while remaining within the scope and spirit of the invention. SEQUENCE LISTING >SEQ ID NO: 1 [MC58, v1] MNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADALTAPLDHKDK GLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKM VAKRQFR IGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTI DFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAE KGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ >SEQ ID NO: 2 [2996, v2] [00238] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADA LTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTG KLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKIN NPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAG GKLTYTIDFAAKQGHGKIE HLKTPEQNVELAAAELKADEKSHAVILGD TRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 3 [M1239, v3] MNRTAFCCLSLTTALILTACSSGGGGSGGGGVAADIGTGLADALTAP LDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGK LKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINN PDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPN GRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGD TRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 4 [ 2996 mature] CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLK LAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGE FQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQL PDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVE LAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATV KIGEKVHEIGIAGKQ >SEQ ID NO: 5 [2996 ΔG] VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAE KTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQD HSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAE YHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAEL KADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQ > SEQ ID NO: 6 [NHBA] MFKRSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVSEKET EAKEDAPQAGSQGQGAPSAQGSQDMAAVSEENTGNGGAVTADNP KNEDEVAQNDMPQNAAGTDSSTPNHTPDPNMLAGNMENQATDAGE SSQPANQPDMANAADGMQGDDPSAGGQNAGNTAAQGANQAGNN QAAGSSDPIP ASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCK GDSCSGNNFLDEEVQLKSEFEKLSDADKISNYKKDGKNDKFVGLVAD SVQMKGINQYIIFYKPKPTSFAFRRSARSRRSLPAEMPLIPVNQADT LIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQGEP AKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDFGSKSVDG IIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGE EVAGKYSYRPTDAEKGGFGVFAGKKEQD > SEQ ID NO: 7 [NadA] MSMKHFPSKVLTTAILATFCSGALAATSDDDVKKAATVAIVAAYNNGQ EINGFKAGETI YDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTK TVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNAL NKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDET NTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTA NTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKET RQGLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFRFTE NFAAKAGVAVGTSSGSSAAYHVGVNYEW > SEQ ID NO: 8 [NspA] MKKALATLIALALPAAALAEGASGFYVQADAAHAKASSSLGSAKGFS LRFAVDYTRYKNYKAPSTDFKLYSIGASAIYDFDTQSP VKPYLGARLSLNRASVDLGGSDSFSQTSIGLGVLTGVSYAVTPNVDL DAGYRYNYIGKVNTVKNVRSGELSAGVRVKF > SEQ ID NO: 9 [HmbR] MKPLQMLPIAALVGSIFGNPVLAADEAATETTPVKAEIKAVRVKGQRN APAAVERVNLNRIKQEMIRDNKDLVRYSTDVGLSDSGRHQKGFAVR GVEGNRVGVSIDGVNLPDSEENSLYARYGNFNSSRLSIDPELVRNIEI VKGADSFNTGSGALGGGVNYQTLQGRDLLLDDRQFGVMMKNGYST RNREWTNTLGFGVSNDRVDAALLYSQRRGHETESAGNRGYAVEGE GSGANIRGSARGIPDSSKHKYNHHALG KIAYQINDNHRIGASLNGQQ GHNYTVEESYNLTASSWREADDVNRRRNANLFYEWMPDSNWLSSL KADFDYQKTKVAAVNNKGSFPMDYSTWTRNYNQKDLDEIYNRSMDT RFKRFTLRLDSHPLQLGGGRHRLSFKTFVSRRDFENLNRDDYYFSG RVVRTTSSIQHPVKTTNYGFSLSDQIQWNDVFSSRAGIRYDHTKMTP QELNAECHACDKTPPAANTYKGWSGFVGLAAQLNQAWRVGYDITS GYRVPNASEVYFTYNHGSGNWLPNPNLKAERSTTHTLSLQGRSEKG MLDANLYQSNYRNFLSEEQKLTTSGTPGCTEENAYYGICSDPYKEKL DWQMKNIDKARIRGIELTGRLNVDKVASFVPEGWKLFGSLGYAKSKL SGDNSLLSTQPLKVIAGIDYESPSEKWGVFSRLTYLGAKKVKDAQYT VYENKGWGTPLQKKVKDYPWLNKSAYVFDMYGFYKPAKNLTLRAGV YNLFNRKYTTWDSLRGLYSYSTTNAVDRDGKGLDRYRAPGRNYAVS LEWKF > SEQ ID NO: 10 [NhhA] MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQ ASANNEEQEEDLYLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWA VYFNEKGVLTAREITLKAGDNLKIKQNGTNFTYSLKKDLTDLTSVGTE KLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLNGIGSTLTD TL LNTGATTNVTNDNVTDEKKRAASVKDVLNAGWNIKGVKPGTTASDN VDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKD GKLVTGKDKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANG QTGQADKFETVTSGTNVTFASGKGTTATVSKDDQGNITVMYDVNVG DALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNIN AGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVG SKKDNKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNA RAGIAQAIATAGLVQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDG GNWIIKGTASGNSR GHFGASASVGYQW >SEQ ID NO: 11 [App] MKTTDKRTTETHRKAPKTGRIRFSPAYLAICLSFGILPQAWAGHTYFG INYQYYRDFAENKGKFAVGAKDIEVYNKKGELVGKSMTKAPMIDFSV VSRNGVAALVGDQYIVSVAHNGGYNNVDFGAEGRNPDQHRFTYKIV KRNNYKAGTKGHPYGGDYHMPRLHKFVTDAEPVEMTSYMDGRKYI DQNNYPDRVRIGAGRQYWRSDEDEPNNRESSYHIASAYSWLVGGN TFAQNGSGGGTVNLGSEKIKHSPYGFLPTGGSFGDSGSPMFIYDAQ KQKWLINGVLQTGNPYIGKSNGFQLVRKDWFYDEIFAGDTHSVFYEP RQNGKYSFNDDNNGTGKINA KHEHNSLPNRLKTRTVQLFNVSLSETA REPVYHAAGGVNSYRPRLNNGENISFIDEGKGELILTSNINQGAGGLY FQGDFTVSPENNETWQGAGVHISEDSTVTWKVNGVANDRLSKIGKG TLHVQAKGENQGSISVGDGTVILDQQADDKGKKQAFSEIGLVSGRGT AKVKGDWH LSNHAQAVFGVAPHQSHTICTRSDWTGLTNCVEKTITD DKVIASLTKTDISGNVDLADHAHLNLTGLATLNGNLSANGDTRYTVSH NATQNGNLSLVGNAQATFNQATLNGNTSASGNASFNLSDHAVQNGS LTLSGNAKANVSHSALNGNVSLADKAVFHFESSRFTGQISGGKDTAL HLKDSEWTLPSGTELGNLNLDNATITLNSAYRHDAAGAQTGSATDAP RRRSRRSRRSLLSVTPPTSVESRFNTLTVNGKLNGQGTFRFMSELF GYRSDKLKLAESSEGTYTLAVNNTGNEPASLEQLTVVEGKDNKPLSE NLNFTLQNEHVDAGAWRYQLIRKDGEFRLHNPVKEQELSDKLGKAE AKKQAEKDNAQSLDALIAAG RDAVEKTESVAEPARQAGGENVGIMQ AEEEKKRVQADKDTALAKQREAETRPATTAFPRARRARRDLPQLQP QPQPQPQRDLISRYANSGLSEFSATLNSVFAVQDELDRVFAEDRRNA VWTSGIRDTKHYRSQDFRAIORQQTDLRQIGMQKNLGSGRVGILFSH NRTENTFDDGIGNSARLAHGAVFGQYGIDRFYIGISAGAGFSSGSLSD GIGGKIRRRVLHYGIQARYRAGFGGFGIEPHIGATRYFVQKADYRYEN VNIATPGLAFNRYRAGIKADYSFKPAQHISITPYLSLSYTDAASGKVRT RVNTAVLAQDFGKTRSAEWGVNAEIKGFTLSLHAAAAKGPQLEAQH SAGIKLGYRW >SEQ ID NO: 12 [Omp85] MKLKQIASALMMLGISPLALADFTIQDIRVEGLQRTEPSTVFNYLPVKV GDTYNDTHGSAIIKSLYATGFFDDVRVETADGQLLLTVIERPTIGSLNIT GAKMLQNDAIKKNLESFGLAQSQYFNQATLNQAVAGLKEEYLGRGK LNIQITPKVTKLARNRVDIDITIDEGKSAKITDIEFEGNQVYSDRKLMRQ MSLTEGGIWTWLTRSNQFNEQKFAQDMEKVTDFYQNNGYFDFRILD TDIQTNEDKTKQTIKITVHEGGRFRWGKVSIEGDTNEVPKAELEKLLT MKPGKWYERQQMTAVLGEIQNRMGSAGYAYSEISVQPLPNAETK Tv YDRVNFGLVAEHLTVNTYNKAPKHYADFIKKYGKTDGTDGSFKGWLY KGTVGWGRNKTDSALWPTRGYLTGVNAEIALPGSKLQYYSATHNQT WFFPLSKTFTLMLGGEVGIAGGYGRTKEIPFFENFYGGGLGSVRGYE SGTLGPKVYDEYGEKISYGGNKKANVSAELLFPMPGAKDARTVRLSL GKTYDDNSSSATGGRVQNIYGAGNTHKSTFTNELRYS AGGAVTWLSPLGPMKFSYAYPLKKKPEDEIQRFQFQLGTTF >SEQ ID NO: 13 [NMB2091] MVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNN QTKGYTPQISVVGYDRHLLLLGQVATEGEKQFVGQIARSEQAAEGVY NYITVASLPRTAGDIAGDTWNTSKVRATLLGISPATRARVKIVTYGNVT YVMGILTPEEQAQITQKVSTTVGVQKVITLYQNYVQR >SEQ ID NO: 14 [NHBA merger] QAGSQGQGAPSAQ GGQDMAAVSEENTGNGGAAATDKPKNEDEGAQNDMPQNAADTDS LTPNHTPASNMPAGNMENQAPDAGESEQPANQPDMANTADGMQG DDPSAGGENAGNTAAQGTNQAENNQTAGSQNPASSTNPSATNSGG DFGRTNVGNSVVIDGPSQNITLTHCKGDSCSGNNFLDEEVQLKSEFE KLSDADKISNYKKDGKNDGKNDKFVGLVADSVQMKGINQYIIFYKPKP TSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIF APEGNYRYLTYGAEKLPGGSYALRVQGEPSKGEMLAGTAVYNGEVL HFHTENGRPSPSRGRFAAKVDFGSKSVDGIIDSGDGLHMGTQKFKA AIDGNGF KGTWTENGGGDVSGKFYGPAGEEVAGKYSYRPTDAEKG GFGVFAGKKEQDGSGGGGATYKVDEYHANARFAIDHFNTSTNVGGF YGLTGSVEFDQAKRDGKIDITIPVANLQSGSQHFTDHLKSADIFDAAQ YPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSP MAKTEVCGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDIQIEAAKQ >SEQ ID NO: 15 [NadA fragment] ATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDAT AADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLT TKLADTDAALADTDAALDATTNALNKLGENITTFAEET KTNIVKIDEKLE AVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQN VDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKD NIAKKANSADVYTREESDSKFVRIDGLNATTEKLDTRLASAEKSIADH DTRLNGLDKTVSDLRKETRQGLAEQAALSGLFQPYNVG >SEQ ID NO: 16 [MC58, ΔG] VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQS HSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGR ATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHL KSPELNVDLAAA DIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTV NGIRHIGLAAKQ >SEQ ID NO: 17 [M1239, ΔG] VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEK TFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYK QNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGK AEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAA ELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGE KVHEIGIAGK Q >SEQ ID NO:18 QRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVRHIGIAGKQ >SEQ ID NO: 19 [MUTANT #2] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQS LTLDQVVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVRHIGIAGKQ >SEQ ID NO: 20 [MUTANT #3] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPD KIDSLIN QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDF AAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 21 [MUTANT #4] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDF AAKQGHGKIEHLK TPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 22 [MUTANT #5] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR 23 6] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVTALQIEKINNPDKIDSLINQ RSFLVGDLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAA KQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGT YHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 24 [MUTANT #7] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQS LTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKSDSLIN QRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 25 [MUTANT #8] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSCRKNEKCKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINN PDKIDSLIN QRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 26 [MUTANT #9] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKCAAQGAEKTYGNGDSLNTGKLKNDKVSR CDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTP EQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 27 [MUTANT #10] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSCLVSGLGGEHTAFNQLPDGKAEYHGKAFSCDDAGGKLTYTIDF AAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 28 [MUTANT #11] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVTALQIEKINNPDKIDSLINQ RSFLVGDLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAA KQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGT YHLALFGDRAQEIAGSATVKIGEKVRHIGIAGKQ >SEQ ID NO: 29 [MUTANT #12] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQS LTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFRIGDIAGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 30 [MUTANT #13] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVTALQIEKI NNPDKIDSLINQ RSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAA KQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGT YHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 31 [MUTANT #14] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKCAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTP EQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 32 [MUTANT #15] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSCLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDF AAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 33 [MUTANT #19] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFLVTGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 34 [MUTANT #20] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SL QSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFRVTGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDF AAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 35 [MUTANT #21] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKI NNPDKIDSLIN QRSFLVGGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDF AAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 36 [MUTANT #22] MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDK SLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFRVGGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDF AAKQGHGKIEHLK TPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 37 GPDSDRLQQRR >SEQ ID NO: 38 GSKDISS >SEQ ID NO: 39 GSKDISSGGGG >SEQ ID NO: 40 [M1239, mature] CSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQ NGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDG KADEKSHAVILGDTRYGSEEKGTYHLALFGD RAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 41 [v3(M1239), E243A, ΔG] VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEK TFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYK QNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGK AEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAA ELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGE KVHAIGIAGK Q >SEQ ID NO: 42 [v3(M1239), S32V+E243A, ΔG] VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEK TFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYK QNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGK AEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAA ELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGE KVHAIGIAGKQ >SEQ ID NO: 43 [v3(M1239), S32V+L126R+E2 43A, ΔG] VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEK TFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYK QNHSAVVALQIEKINNPDKTDSLINQRSFRVSGLGGEHTAFNQLPGG KAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAA AELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHAIGIAGKQ >SEQ ID NO: 44 [v3, S32V + L126R, ΔG] VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAE K TFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYK QNHSAVVALQIEKINNPDKTDSLINQRSFRVSGLGGEHTAFNQLPGG KAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAA AELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ >SEQ ID NO: 45 [v2 MUTANT #3 S32V + L123R, ΔG] VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQD HSAVVALQIEKINNPDKIDSLINQRSFRVSGLGGE HTAFNQLPDGKAE YHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAEL KADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQ >SEQ ID NO: 46 [MC58, v1, mature] CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLK LAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGE FQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFD KLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPEL NVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAG SAEVKTV NGIRHIGLAAKQ >SEQ ID NO: 47 [v2-v3-v-1 mutant fusion] VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQD HSAVVALQIEKINNPDKIDSLINQRSFRVSGLGGEHTAFNQLPDGKAE YHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAEL KADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVI PQNGTLTLSAQGAEKTFK AGDKDNSLNTGKLKNDKISRFDFVQKIEV DGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFRVSG LGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRI EHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFG DRAQEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTA PLDHKDKGLQSLTLDQSVSKNEKLKLAAQGAEKTYGNGDSLNTGKL KNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQD SEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDA GGK LTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISG SVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ >SEQ ID NO: 48 [fusion of v2-v3-v-1 mutant, with leader] MGPDSDRLQQRRVAADIGAGLADALTAPLDHKDKSLQSLTLDQVVR KNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLI TLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFRVSGLGGEH TAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQE IAGSATVKIGE KVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHK DKGLKSLTLEDVIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKND KISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKT DSLINQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLH YSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYG SEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQGSGGGGV AADIGAGLADALTAPLDHKDKGLQSLTLDQSVSKNEKLKLAAQGAEK TYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSH SALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRA TYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADI KPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVN GIRHIGLAAKQ >SEQ ID NO: 49 [MC58, ΔG, mutation 'R41S'] VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVSKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQS HSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGR ATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSP ELNVDLAAA DIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTV NGIRHIGLAAKQ >SEQ ID NO: 50 [adapter] GSGGGG >SEQ ID NO: 51 [wild-type v2 sequence, e.g. for GMMA approach] VAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAE > SEQ ID NO: 52 [wild type v3 sequence, e.g. for GMMA approach] VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQS HSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGS ATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAE LKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREK VHEIGIAGKQ >SEQ ID NO: 53 [m1239, L126R mutation] VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEK TFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYK QNHSAVVALQIEKINNPDKTDSLINQRSFRVSGLGGEHTAFNQLPGG KAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAA AELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ >SEQ ID NO: 54 [v2, strain 8047, wild type] MNRTAFCCLSLTTALILTACSSGGGGVAADIGARLADALTAPLDHKDK SLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSR FDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFLVSGLGGEHTAFNQLPDG KAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKG TYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 55 [v2, strain 8047, ΔG] VAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQD HSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAE YHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAEL KADEKSHAVI LGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQ >SEQ ID NO: 56 [v2, mutant 'E313A', ΔG] VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAE > SEQ ID NO: 57 [v2, mutant #3 + 'E313A', ΔG] VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQD HSAVVALQIEKINNPDKIDSLINQRSFRVSGLGGEHTAFNQLPDGKAE YHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAEL KADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HAIGIAGKQ >SEQ ID NO: 58 [MUTANT #2 + #12] QSLTLDQVVRKNEKLKLAAQGAE KTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQD HSAVVALQIEKINNPDKIDSLINQRSFRIGDIAGEHTAFNQLPDGKAEY HGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELK ADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVH EIGIAGKQ >SEQ ID NO: 59 [v2, strain 8047, mutant #4, mature] CSSGGGGVAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLK LAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGE FQ IYKQDHSAVVALQIEKINNPDKIDSLINQRSFRVSGLGGEHTAFNQL PDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVE LAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATV KIGEKVHEIGIAGKQ >SEQ ID NO: 60 [v2, strain 8047, mutant #3, mature] CSSGGGGVAADIGARLADALTAPLDHKDKSLQSLTLDQVVRKNEKLK LAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGE FQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFRVSGLGGEHTAFNQL PDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVE LAAAEL KADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATV KIGEKVHEIGIAGKQ >SEQ ID NO: 61 [v3, mutant #2 + #12, ΔG] VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEK TFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYK QNHSAVVALQIEKINNPDKTDSLINQRSFRIGDIAGEHTAFNQLPGGK AEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAA ELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGE KVHEIGIAGKQ Table 1 * Numbered according to SEQ ID NO: 5; add +26 to match SEQ ID NOs: 18 to 39. ** Resistance to chymotrypsin cleavage. *** % monomeric form

Claims (9)

1. A mutant v2 or v3 fHbp, characterized in that it is: (1) a polypeptide having a mutant fHbp v2 amino acid sequence, wherein: (i) the amino acid sequence consists of SEQ ID NO: 5 and includes a residue corresponding to residue 32 of SEQ ID NO: 5; but (ii) the amino acid sequence differs from SEQ ID NO: 5 at residue 32 by the substitution S32V; or (8) a polypeptide having a mutant fHbp v3 amino acid sequence, wherein: (9) the amino acid sequence consists of SEQ ID NO: 17 and includes a residue corresponding to residue 32 of SEQ ID NO: 17; but (11) the amino acid sequence differs from SEQ ID NO: 17 at residue 32 by the substitution S32V. 2. The polypeptide of claim 1, wherein: (A) the amino acid sequence further differs from SEQ ID NO: 5 by substitution at L123, V124, S125, G126, L127, and/or G128, wherein the substitution(s) is/are selected from the group consisting of: L123R; V124I; S125G or S125T; G126D; L127I; G128A; or (B) the amino acid sequence further differs from SEQ ID NO: 17 by substitution at L126 wherein the substitution is L126R. 3. The polypeptide of claim 1 or 2, comprising a substitution in v2 at one of residues R73, D203, E210, G228, S121, F122, A192, E194, V199, I200, L201, T213, H215, F219, T231, and E240, or in v3 comprising a substitution at one of residues Q35, I78, L87, A88, V127, V202, E213, T216, H218, T234, V241, E243, and G248. 4. Polypeptide according to claim 1, characterized in that it consists of the amino acid sequence of SEQ ID NO: 45. 5. Polypeptide according to claim 1, characterized in that it consists of the amino acid sequence of SEQ ID NO: 44. 6. Membrane vesicles prepared from a host cell, characterized in that the vesicles originating from the host cell include a polypeptide as defined in any one of claims 1 to 5. 7. Immunogenic composition, characterized by the fact that it comprises a polypeptide as defined in any one of claims 1 to 5, or a vesicle as defined in claim 6. 8. Composition according to claim 7, characterized in that it further comprises a second polypeptide, the second polypeptide being a meningococcal fHbp protein, an NHBA polypeptide or a NadA polypeptide. 9. Composition according to claim 7 or claim 8, characterized in that it further comprises (i) a conjugated capsular saccharide from N.meningitidis serogroup A, C, W135 and/or Y and/or (ii) a conjugated capsular saccharide from S.pneumoniae.