EA043165B1 - MODIFIED MENINGOCOCCAL fHbp POLYPEPTIDES - Google Patents

Description

This application claims the benefit of EP applications 14157399.8 (filed February 28, 2014) and 14177566.8 (filed July 17, 2014), the full contents of which for all purposes are incorporated herein by reference.

Field of invention

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

Prior Art

Neisseria meningitidis is a gram-negative encapsulated bacterium that colonizes the upper respiratory tract in approximately 10% of the population. There are conjugate vaccines against serogroups A, C, W135, and Y, but the only vaccine available to protect against serogroup B in a two-dose regimen is BEXSERO™, which was approved in 2013.

One of the protective immunogens in BEXSERO™ is fHbp, which is also known as protein 741 (SEQ ID NO: 2536 in Source 1; here the sequence is SEQ ID NO: 1), NMB1870, GNA1870 [2-4], P2086, LP2086 or ORF2086 [5-7]. The three-dimensional structure of this protein is known [8, 9], and the protein has two β-barrels linked by a short linker. Numerous publications report the protective efficacy of this protein in meningococcal vaccines, for example see refs. 10-14. Lipoprotein fHbp is expressed in various strains in all serogroups. The fHbp sequences are grouped into three variants [2] (referred to herein as v1, v2 and v3), and in general it was found that the serum obtained against this variant is bactericidal against strains that express this variant, but not active against strains , which express one of the other two variants, i.e. there is cross-protection within a variant, but no cross-protection between variants (except for some cross-reactivity between v2 and v3).

To increase cross-reactivity between families, the fHbp sequence containing specific features for all three variants was constructed [15].

Protein engineering has also been used to remove the interaction of fHbp with siderophores [15] and with factor H [17-25]. Impaired interaction with fH has been reported for all three variants, and an improved vaccine immunogen is expected [22, 26]. However, for the v2 polypeptides in references 23 and 24, an unavoidable instability has been reported, which is also found in fH-defective mutants. The instability appears to arise from the N-terminal β-barrel domain, and reference 23 cautions that any substitutions in this barrel may contribute to instability.

The object of the present invention is to provide additional fHbp v2 and v3 mutants with improved stability.

Description of the invention

Full-length fHbp from strain 2996 in v2 has the following amino acid sequence (SEQ ID NO: 2):

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLOSLTLDQSV

RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQ EIAGSATVKIGEKVHEIGIAGKQ

The mature lipoprotein does not contain the first 19 amino acids of SEQ ID NO: 2 (underlined; SEQ ID NO: 4 is formed), and the ΔG form of SEQ ID NO: 2 does not contain the first 26 amino acids (SEQ ID NO: 5).

Full-length fHbp from strain M1239 in v3 has the following amino acid sequence (SEQ ID NO: 3):

MNRTAFCCLSLTTALILTAC S SGGGGSGGGGVAADIGTGLAD ALTAPLDHKDKGLKSLT

LEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLA SGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYH GKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDT RYGSEKGTYHLALFG DRAQEIAGSATVKIGEKVHEIGIAGKQ

The mature lipoprotein does not contain the first 19 amino acids of SEQ ID NO: 3 (underlined; SEQ ID NO: 40 is formed), and the ΔΘ form of SEQ ID NO: 3 does not contain the first 31 amino acids (SEQ ID NO: 17).

The inventors have identified residues in the sequences of SEQ ID NO: 2 and SEQ ID NO: 3 that can be modified to increase the stability of the polypeptide. These remnants

- 1 043165 are typically presented in v2 and v3 sequences, and thus their modification can lead to v2 and v3 fHbp sequences having increased stability. Moreover, the inventors have demonstrated that, in addition to increasing stability, mutation of these residues can advantageously reduce binding to human factor H (fH). However, the mutations further disclosed herein may be combined with other mutations, for example, to reduce binding to human factor H (fH), for which several mutations are already known in the art.

Thus, in general, the invention provides a v2 or v3 fHbp mutant that has increased stability compared to wild-type fHbp (e.g., compared to SEQ ID NO: 2 or 3) and that may have lower affinity for human factor H compared to wild-type fHbp (for example, compared to the sequence of SEQ ID NO: 2 or 3). The increase in stability and possible decrease in fH affinity preferably result from the same(s) mutation(s), but in some embodiments they may result from separate effects of combined mutations. Mutant fHbp proteins having both increased stability and reduced affinity for fH are preferred.

In a first embodiment of the invention, a polypeptide is provided comprising a mutant fHbp v2 amino acid sequence, wherein: (a) the amino acid sequence has k% identity to the sequence of SEQ ID NO: 5 and/or contains a fragment of the sequence 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.

When feature (a) refers to a fragment, then this fragment includes at least one of the residues listed in (b), but this residue is different compared to the corresponding residue of the sequence SEQ ID NO: 5. Fragment (a), as a rule, has a length of at least 7 amino acids, e.g. consecutive amino acids or more of SEQ ID NO: 5. A fragment typically includes at least one epitope of SEQ ID NO: 5. Epitope identification and mapping has been confirmed for fHbp [11, 27-31]. Having the same at least 30 consecutive amino acids with the sequence of SEQ ID NO: 5 is typical, and usually the mutant fHbp v2 amino acid sequence includes several (for example, 2, 3, 4, 5 or more) fragments of the sequence of SEQ ID NO: 5. In general, the mutant fHbp v2 amino acid sequence may have at least k% identity to the sequence of SEQ ID NO: 5 and include several fragments of the sequence of SEQ ID NO: 5.

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

The polypeptide, upon administration to a host animal, can elicit antibodies that recognize the wild-type meningococcal polypeptide consisting of the sequence of SEQ ID NO: 4. These antibodies are ideally bactericidal (see below). These antibodies may include some antibodies that do not recognize the v1 or v3 polypeptide (for example, the wild-type meningococcal polypeptide of SEQ ID NO: 46 and the wild-type meningococcal polypeptide of SEQ ID NO: 40), although they also may include some antibodies that are cross-reactive with v1 and/or v3 polypeptides.

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

In some embodiments, the polypeptide of the invention is truncated compared to the sequence of SEQ ID NO: 5. Compared to the mature wild-type sequence, SEQ ID NO: 5 is already truncated at the N-terminus and includes a polyglycine sequence (comparison of SEQ ID NO: 4 and 5) , but the sequence of SEQ ID NO: 5 may be truncated at the C-terminus and/or further truncated at the N-terminus.

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 more than 35°C. These temperatures refer to the increase in the midpoint of the temperature transition (Tm) estimated by DSC. fHbp wt de

- 2 043165 shows two DSC peaks during deployment (one for the N-terminal domain and one for the C-terminal domain), and when the polypeptide of the invention includes both such domains, then the increase refers to the stability of the N-terminal domain, for which deployment can occur at even below 40°C for wild-type v2 sequences [24] (whereas C-terminal domains may have a Tm of 80°C or more). Thus, the fHbp v2 mutant amino acid sequence of the invention preferably has an N-terminal domain with a Tm of at least 45°C, such as greater than or equal to 50°C, greater than or equal to 55°C, greater than or equal to 60°C, greater than or equal to 65°C, greater than or equal to 70°C, greater than or equal to 75°C, or even greater than or equal to 80°C.

In a second embodiment of the invention, a polypeptide is provided comprising a mutant fHbp v3 amino acid sequence, wherein: (a) the amino acid sequence has at least j% identity to the sequence of SEQ ID NO: 17, and/or contains a fragment of the sequence 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: S32,133, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242 and/or E243.

When feature (a) refers to a fragment, then the fragment includes at least one of the residues listed in (b), but this residue differs when compared with the corresponding residue in the sequence of SEQ ID NO: 17. Fragment (a), as a rule, has a length of at least 7 amino acids, e.g. consecutive amino acids or more from the sequence of SEQ ID NO: 17. The fragment typically includes at least one epitope of the sequence of SEQ ID NO: 17. Epitope identification and mapping confirmed for fHbp [11; 27-31]. The presence of the same at least 30 consecutive amino acids with the sequence of SEQ ID NO: 17 is typical, and usually the mutant fHbp v3 amino acid sequence includes several (for example, 2, 3, 4, 5 or more than 5) fragments of the sequence of SEQ ID NO: 17. In general, a mutated fHbp v3 amino acid sequence may have at least j% sequence identity to SEQ ID NO: 17 and include several fragments of SEQ ID NO: 17.

The j value 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 fHbp v3 mutant amino acid sequence has at least 90% sequence identity to SEQ ID NO: 17) and more preferably 95.

The claimed polypeptide, upon administration to a host animal, can induce the formation of antibodies that recognize the meningococcal polypeptide consisting of the sequence of SEQ ID NO: 40. These antibodies are ideally bactericidal (see below). These antibodies may include some antibodies that do not recognize the v1 or v2 polypeptide (e.g. wild-type meningococcal polypeptide consisting of SEQ ID NO: 46 and wild-type meningococcal polypeptide consisting of SEQ ID NO: 4), although they also may include some antibodies that are cross-reactive with v1 and/or v2 polypeptides.

The declared polypeptide, under the same experimental conditions, has higher stability than the same polypeptide, but having no sequence difference(s) (b), for example, higher stability than the wild-type meningococcal polypeptide consisting of the sequence of SEQ ID NO: 40. stability can be assessed using differential scanning calorimetry (DSC), for example, as discussed in references 32 and 33. DSC has previously been used to assess the stability of v3 fHbp [23]. Under suitable DSC conditions, 20 μM of the polypeptide in buffered solution (e.g. 25 mM Tris) pH 6 to 8 (e.g. 7-7.5) with 100-200 mM NaCl (e.g. 150 mM) can be used to evaluate stability. .

In some embodiments, the polypeptide of the invention is truncated from SEQ ID NO: 17. Compared to the wild-type mature sequence, SEQ ID NO: 17 is already N-terminally truncated and includes a polyglycine sequence (comparison of SEQ ID NO: 40 and 17 ), but the sequence of SEQ ID NO: 17 may be truncated at the C-terminus and/or further truncated at the N-terminus.

The increase in stability is ideally at least 5°C, such as at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or more. These temperatures refer to the increase in the midpoint of the temperature transition (Tm) estimated by DSC. The wild-type fHbp shows two DSC peaks during deployment (one for the N-terminal domain and one for the C-terminal domain) and when the polypeptide of the invention includes both such domains, then the increase relates to the stability of the N-terminal domain, for which deployment can occur at a temperature of approximately 60°C or less for wild-type v3 sequences [24] (whereas the C-terminal domains may have a Tm of 80°C or more). Thus, the fHbp v3 mutant amino acid sequence of the invention preferably has an N-terminal domain with a Tm of at least 65°C, eg greater than or equal to 70°C, greater than or equal to 75°C, or even greater than or equal to 80°C.

Mutations compared to SEQ ID NO: 5.

- 3 043165

The polypeptides according to the first embodiment of the invention contain an amino acid sequence that has at least k% identity to the sequence of SEQ ID NO: 5, and/or contains a fragment of the sequence of SEQ ID NO: 5. However, compared to the sequence of SEQ ID NO: 5, this amino acid sequence has a modification at one or more of the amino acid residues S32, V33, L39, L41, F69, V100, I113, F122, L123, V124, S125, G126, L127, G128, S151, H239 and/or E240, for example, 2, 3, 4, 5 or more of these 17 residues. These residues are numbered according to the sequence of SEQ ID NO: 5; in order to match the wild-type sequence (SEQ ID NO: 2) in the chain growth step, the numbering should change to +26 (i.e., Ser-32 of SEQ ID NO: 5 is Ser-58 of SEQ ID NO: 2), and in order to match the wild-type mature sequence (SEQ ID NO: 4), the numbering should be renumbered to +7 (which would also allow easy comparison with referenced source 25).

Preferred mutation residues are S32, V100, L123, V124, S125, G126, L127, G128, H239 and/or E240. Mutations at these residues produce 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, where residues S32 and/or L123 are especially preferred, for example S32V and/or L123R. When one or more of V100, S125 and/or G126 has been mutated, then it is preferred that the remainder outside of this trio also be mutated.

A particular residue may be deleted, but preferably replaced with a different amino acid. For example, Ser-32 can be replaced with any of the other 19 naturally occurring amino acids. When making a substitution, the replacement amino acid may, in some embodiments, be a simple amino acid such as glycine or alanine. In other embodiments, the substituting amino acid is a conservative substitution, for example, it is within the following four groups: (1) acidic, ie. aspartate, glutamate; (2) basic, i.e. lysine, arginine, histidine; (3) non-polar, 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 not conservative. In some embodiments, no alanine is used in the substitution.

Preferred substitutions for specific residues are as follows: S32V; V33C; L39C; L41C; F69C; V100T; I113S; F122C; L123R; VI241; S125G or S125T; G126D; L127I; G128A; S151C; H239R; E240H.

When the fHbp v2 mutant amino acid sequence includes a substitution at E240, then that substitution is not an alanine substitution unless E240 is mutated, although the substitution may be alanine if another amino acid listed in (b) is substituted, for example, if both residues E240 and H239 are mutated. Ideally, not only E240 is mutated, and thus the mutant fHbp v2 amino acid sequence at E240 should also include a substitution at a second residue, such as E240 and H239 (see mutants #1 and #11).

When the fHbp v2 mutant amino acid sequence includes a substitution at F122, then that substitution is not an alanine substitution unless F122 is mutated, although the substitution may be alanine if another amino acid listed in (b) is substituted, for example, if both residues F122 and S151 are mutated. Ideally, not only F122 is mutated, and thus the mutated fHbp v2 amino acid sequence at F122 should also include a second residue substitution. When F122 is substituted, then it is preferred that S151 is also substituted, eg both are substituted with cysteine, in order to allow for the formation of a disulfide bridge (see mutant #10).

When the fHbp v2 mutant amino acid sequence includes a substitution at L123, then that substitution is not an alanine substitution unless L123 is mutated, although the substitution may be alanine if another amino acid listed in (b) is substituted, for example, if both residues L123 and S32 are mutated. If only L123 is mutated, then an arginine substitution is preferred (eg, see mutant #4). However, in some embodiments, not only L123 is mutated, 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, then it may be preferred that: (1) S32 is also substituted, as seen in mutant #3, and possibly S125 is also substituted, as seen in mutants #20 and #22; or (2) one or more of the residues 124-128 has also been (were) replaced, for example, as seen in mutant #12.

When the fHbp v2 mutant amino acid sequence includes a substitution at V124, then it is preferred that the substitution is not an alanine substitution unless V124 is mutated, although the substitution may be an alanine if another amino acid listed in (b) is also substituted. for example, if one or more of the residues 123-128 has also been subjected to

- 4 043165 mutations. If only VI24 is mutated, then substitution with isoleucine is preferred. Ideally, however, not only V124 is mutated, and thus a mutated fHbp v2 amino acid sequence with a V124 substitution may also include a second residue substitution. When V124 is substituted, then it may be preferred that one or more of residues 124-128 also be substituted, for example as seen in mutant #12.

When the mutated fHbp v2 amino acid sequence includes a substitution at L127, it is preferred that the substitution is not an alanine substitution if only L127 is mutated, although the substitution may be an alanine substitution if another amino acid listed in (b) is also mutated. , for example, if one or more than one of the residues 123-128 has also undergone a mutation. If only L127 is mutated, then isoleucine is preferred. However, ideally not only L127 is mutated, and thus a mutant fHbp v2 amino acid sequence with a substitution at L127 should also include a substitution at the second residue. When L127 is substituted, then it is preferred that one or more of residues 124-128 also be substituted, for example as seen in mutant #12.

When the mutated fHbp v2 amino acid sequence includes a substitution at S32, then it is preferred that the substitution is not an alanine substitution if only S32 is mutated, although the substitution may be an alanine if another amino acid listed in (b) is also mutated. , for example, if both L123 and S32 residues are mutated. If only S32 is mutated, a valine substitution is preferable. However, ideally not only S32 is mutated, and thus a mutant fHbp v2 amino acid sequence with a substitution at S32 should also include a substitution at the second residue. When S32 is substituted, then preferably (1) L123 is also substituted, eg as seen in mutant #3, and possibly S125 is also substituted as seen in mutants #20 and #22; or (2) S125 has also been substituted, for example as seen in mutants #19 and #21.

When the fHbp v2 mutant amino acid sequence includes a substitution at I113, then that substitution would not be an alanine substitution if only I113 is mutated, although the substitution could be alanine if another amino acid listed in (b) is mutated. If only I113 is mutated, a serine substitution is preferred, eg as seen in mutant #7.

When the fHbp v2 mutant amino acid sequence includes a V33 substitution, it is preferably not an isoleucine substitution. When the fHbp v2 mutant amino acid sequence includes an I113 substitution, then it is preferably not a threonine or alanine substitution. When the fHbp v2 mutant amino acid sequence includes a S151 substitution, then it is preferably not a phenylalanine substitution. When the fHbp v2 mutant amino acid sequence includes a substitution at H239 and E240, then it preferably does not represent a substitution for R239 and Q240.

When more than one substitution is made, then they may be selected from groups 2A-2O according to the following:

2A: residues 239 and 240, eg mutant #1.

2B: residues 32 and 123, eg mutant #3.

2B: residues 125 and 126, eg mutant #5.

2D: residues 100, 125 and 126, eg mutant #6.

2E: residues 33 and 39, eg mutant #8.

2E: residues 41 and 69, eg mutant #9.

2G: residues 122 and 151, eg mutant #10.

23: residues 100, 125, 126, 239 and 240, eg mutant #11.

2I: residues 32 and 125, such as mutants #19 and #21.

2K: residues 32, 123 and 125, for example mutants #20 and #22.

2K: residues 33 and 39, both changed to Cys, eg mutant #8.

2M: residues 41 and 69, both changed to Cys, eg mutant #9.

2H: residues 122 and 151 both changed to Cys, eg mutant #10.

2O: residues 123, 124, 125, 126, 127 and 128, eg mutant #12.

2P: residues 32, 123, 124, 125, 126, 127 and 128.

Thus, for example, if residue 239 is to be replaced, then the preferred second replacement residue is 240 (ie group 2A); in addition, residues 100, 125 and 126 can also be modified (ie group 23, which is a combination of groups 2A and 2D). In groups 2A-2O and 2P, preferred substitutions at the indicated positions are those listed above. For groups 2L, 2M and 2H, a disulfide bridge should be introduced into the invention. Within groups 2A-2O, preferred mutants are 2A, 2B, 2C, 2D, 2I, 2K and 2O. More preferred are 2B, 2I and 2O, with 2O being especially preferred. Group 2B suggests the most preferred mutations, and in particular S32V and L123R (eg, SEQ ID NOS: 20 and 45). Group 2P is another preferred

- 5 043165 set of mutations that combine 2B, 2C and three other mutations (for example, to obtain the sequence of SEQ ID NO: 58).

Amino acid residues indicated for a mutation in the v2 sequence are numbered according to SEQ ID NO: 5 derived from strain 2996. Corresponding amino acid residues in v2 fHbp from any other strain can be easily identified by sequence alignment, e.g. an amino acid that, when aligned with SEQ ID NO: 5 using a pairwise alignment algorithm (eg, the Needleman-Wunsch global alignment algorithm detailed below), is aligned with the amino acid mentioned herein. Often the amino acid is the same as seen in SEQ ID NO: 5 (eg, residue 32 is serine), but the alignment is otherwise easier to achieve.

In addition to the mutation(s) above that serve to increase stability, the polypeptide of the invention may include one or more additional mutations, for example to disrupt the interaction of the polypeptide with siderophores or, more preferably, to disrupt the ability of the polypeptide to bind to fH.

References 19 and 25 report that the interaction between fH and v2 fHbp can be disrupted by mutations at residues R80, D211, E218, E248, T220+H222 (double mutation) and G236. Numbered according to the sequence of 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, since reference 25 does not report damage to important epitopes in these mutants. The specific substitutions investigated in Reference 25 were R73A, D203A, E210A, T213A+H215A, G228I and E240A; these substitutions are suitable for use in accordance with the invention.

Reference 24 reports that the interaction between fH and v2 fHbp 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 the sequence of 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 them overlap with source 25 (E210, T213, H215, E240). The specific substitutions investigated in reference 24 used alanine (with the exception of A265P and T304E) and these substitutions are suitable for use in accordance with the invention.

Reference 24 reports that certain substitutions in v2 may increase affinity for fH, which should be avoided if fH binding is to be disrupted, such as E85 in SEQ ID NO: 5 (residue 157 in reference 24).

Residues that interact with siderophores can be mutated using the guidance given in sources 16 and 34, for example, by aligning SEQ ID NO: 5 with SEQ ID NO: 4 from source 16 to identify residues that can interact with siderophores , for example, with catecholates, hydroxamates or carboxylates.

Other residues that may be mutated include but are not limited to S23, L24, D30, Q31, R34, D95 and/or L102, such as using the mutations proposed in reference 27.

The polypeptide in accordance with the first embodiment may contain any of the sequences of SEQ ID NO: 18-36. Similarly, taking into account the AG mutation (i.e., truncation of the N-terminus at the stage of chain growth and the inclusion of the native sequence of poly-Gly), the polypeptide according to the first embodiment may contain any of the sequences of SEQ ID NO: 18-36, with the exception of amino acids 1-26 in it. For example, a polypeptide according to the first embodiment may contain the sequence of SEQ ID NO: 45 or may contain the sequence of SEQ ID NO: 58.

Considering the possibility of additional point mutations (e.g., to disrupt interactions with siderophores and/or fH), the polypeptide according to the first embodiment may contain any of the sequences of SEQ ID NO: 18-36 (or any of the sequences of SEQ ID NO: 18-36 for excluding their amino acids 1-26, such as the sequence of SEQ ID NO: 45), but be modified by up to 5 single amino acid changes (i.e. 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or inserts), provided that the modified sequence can, upon administration to the host animal, induce the formation of antibodies that bind to the meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 46. Such amino acid changes should not reverse mutations in these sequences relative to the sequence wild-type, for example, the sequence of SEQ ID NO: 45 must not be mutated at residue V32 or R123.

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

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

Mutations compared to SEQ ID NO: 17.

The polypeptides according to the second embodiment of the invention contain an amino acid sequence that has at least j% identity to the sequence of SEQ ID NO: 17, and/or contain a fragment of the sequence of SEQ ID NO: 17. However, compared to the sequence of SEQ ID NO: 17, this amino acid sequence has a modification at one or more of the amino acid residues S32,133, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242 and/or E243, for example, 2, 3, 4, 5 or more of 17 residues. These residues are numbered according to the sequence of SEQ ID NO: 17; in order to match the wild-type sequence (SEQ ID NO: 3) in the chain growth step, the numbering should be changed to +31 (i.e. Ser-32 in SEQ ID NO: 17 is Ser-63 in SEQ ID NO: 3), and in order to match the wild-type mature sequence (SEQ ID NO: 40), the numbering should change to +12.

Preferred mutation residues are S32, V103, L126, V127, S128, G129, L130, G131, H242 and/or E243. Within this subgroup, preferred residues are S32, L126, V127, S128, G129, L130 and/or G131. The most preferred positions are S32, L126, V127, S128, G129, L130 and/or G131, where residues S32 and/or L126 are especially preferred, for example S32V and/or L126R.

A particular residue may be deleted, but preferably replaced with a different amino acid. For example, Ser-32 can be replaced with any of the other 19 naturally occurring amino acids. When making a substitution, the replacement amino acid may, in some embodiments, be a simple amino acid such as glycine or alanine. In other embodiments, the substituting amino acid is a conservative substitution, for example, it is within the following four groups: (1) acidic, ie. aspartate, glutamate; (2) basic, i.e. lysine, arginine, histidine; (3) non-polar, 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 not conservative. In some embodiments, no alanine is used in the substitution.

Preferred substitutions for specific residues are as follows: S32V; I33C; L39C; L41C; F72C; V103T; T116S; F125C; L126R; V127I; S128G OR S128T; G129D; L130I; G131A; S154C; H242R; E243H.

When the fHbp v3 mutant amino acid sequence includes a substitution at E243, then that substitution is not an alanine substitution unless E243 is mutated, although the substitution may be alanine if another amino acid listed in (b) is substituted, for example, if both residues E243 and H242 are mutated. Ideally, not only E243 is mutated, and thus the mutated fHbp v3 amino acid sequence at E243 should also include a substitution at a second residue, eg both E243 and H242.

When the fHbp v3 mutant amino acid sequence includes a substitution at F125, then that substitution is not an alanine substitution unless F125 is mutated, although the substitution may be alanine if another amino acid listed in (b) is substituted, for example, if both residues F125 and S154 are mutated. Ideally, not only F125 is mutated, and thus a mutated fHbp v3 amino acid sequence with a F125 substitution should also include a substitution at the second residue. When F125 is substituted, it is then preferred that S154 is also substituted, eg both are substituted with cysteine, in order to allow for the formation of a disulfide bridge.

When the fHbp v3 mutant amino acid sequence includes a substitution at L126, then that substitution is not an alanine substitution unless L126 is mutated, although the substitution may be alanine if another amino acid listed in (b) is substituted, for example, if both residues L126 and S32 are mutated. If only L126 is mutated, then substitution with arginine is preferred. However, in some embodiments, not only L126 is mutated, 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, then it may be preferable that: (1) S32 is also substituted, and optionally S128 is also substituted; or (2) one or more of residues 127-131 has also been replaced.

When the fHbp v3 mutant amino acid sequence includes a substitution at V127, then it is preferred that the substitution is not an alanine substitution unless V127 is mutated, although the substitution may be alanine if another amino acid listed in (b), for example, if one or more of the residues 126-131 is also mutated. If only V127 is mutated, then isoleucine is preferred. However, ideally not only V127 is mutated, and thus a mutated fHbp v3 amino acid sequence with a V127 substitution should also include a substitution at the second residue. When V127 is substituted, then it is preferred that one or more of residues 127-131 also be substituted.

When the fHbp v3 mutant amino acid sequence includes a substitution at L130, it is preferred that the substitution is not an alanine substitution if only L130 is mutated, although the substitution may be an alanine substitution if another amino acid listed in (b) is also mutated. for example, if one or more of the residues 126-131 has also undergone a mutation. If only L130 is mutated, then isoleucine is preferred. However, ideally not only L130 is mutated, and thus a mutant fHbp v3 amino acid sequence with a change at L130 should also include a change at the second residue. When L130 is substituted, then it is preferred that one or more of residues 127-131 also be substituted.

When the mutated fHbp v3 amino acid sequence includes a substitution at S32, then it is preferred that the substitution is not an alanine substitution if only S32 is mutated, although the substitution may be an alanine substitution if another amino acid listed in (b) is also mutated. for example, if both L126 and S32 residues are mutated. If only S32 is mutated, a valine substitution is preferable. However, ideally not only S32 is mutated, and thus a mutated fHbp v3 amino acid sequence with a substitution at S32 should also include a substitution at the second residue. When S32 is substituted, then it is preferable that (1) L126 is also substituted, and optionally S128 is also substituted; or (2) S128 has also been replaced.

When the fHbp v3 mutant amino acid sequence includes a substitution at T113, then that substitution is not an alanine substitution if only T113 is mutated, although it may be an alanine substitution if another amino acid listed in (b) is mutated. If only T113 is mutated, a serine replacement is preferable.

When the mutant fHbp v3 amino acid sequence includes a substitution at 133, it is preferably not a substitution for valine. When the fHbp v3 mutant amino acid sequence includes a T116 substitution, it is preferably not an isoleucine substitution. When the fHbp v3 mutant amino acid sequence includes a G129 substitution, it is preferably not a serine substitution. When the fHbp v3 mutant amino acid sequence includes a substitution at H242 and E243, then it preferably does not represent a substitution for R242 and Q243.

When more than one substitution is made, then they may be selected from groups 3A-3O according to the following:

3A: residues 242 and 243.

3B: residues 32 and 126.

3B: residues 128 and 129.

3D: residues 103, 128 and 129.

3D: residues 33 and 39.

3E: residues 41 and 72.

3G: residues 125 and 154.

3H: residues 103, 128, 129, 242 and 243.

3I: residues 32 and 128.

3K: residues 32, 126 and 128.

3R: residues 33 and 39, both replaced by Cys.

3M: residues 41 and 72, both replaced by Cys.

3H: residues 125 and 154, both replaced by Cys.

3O: residues 126, 127, 128, 129, 130 and 131.

3P: residues 32, 126, 127, 128, 129, 130 and 131.

Thus, for example, if residue 242 is to be replaced, then the preferred second replacement residue is 243 (ie group 3A); in addition, residues 103, 128 and 129 can also be modified (ie group 33, which is a combination of groups 3A and 3D). In groups 3A-3O and 3P, preferred substitutions at the indicated positions are those listed above. For groups 3L, 3M and 3H, a disulfide bridge should be introduced into the invention. Within the 3A-30 groups, preferred mutants are 3A, 3B, 3C, 3D, 3I, 3K and 3O. More preferred are 3B, 3I and 3O, with 3O being particularly preferred. In group 3B, the most preferred mutations are proposed, and in particular S32V and L126R (eg, containing the sequence of SEQ ID NO: 44). The 3P group is another preferred set of mutations that combine 3B, 3C, and three other mutations (for example, to produce

- 8 043165 sequence SEQ ID NO: 61). The L126R mutation also provides the sequence

SEQ ID NO: 53.

Amino acid residues indicated for mutation in the v3 sequence are numbered according to the sequence of SEQ ID NO: 17 obtained from strain M1239. Corresponding amino acid residues in v3 fHbp from any other strain can be readily identified by sequence alignment, e.g., is an amino acid that, when aligned to SEQ ID NO: 17 using a pairwise alignment algorithm (e.g., the Needleman-Wunsch global alignment algorithm, in detail described below) is aligned with the amino acid mentioned here. Often the amino acid is the same as seen in SEQ ID NO: 17 (eg, residue 32 is serine), but alignment is not easier in this case.

In addition to the mutation(s) above that serve to increase stability, the polypeptide of the invention may include one or more additional mutations, for example to disrupt the interaction of the polypeptide with siderophores or, more preferably, to disrupt the ability of the polypeptide to bind to fH.

Reference 24 reports that the interaction between fH and v3 fHbp can be disrupted by mutations at residues Q107,1147, L156, A157, L195, V196, V272, E283, T286, T304, V311, E313 and E283+T304 (double mutation). Numbered according to the sequence of SEQ ID NO: 17, these residues are Q35,178, L87, A88, L126, V127, V202, E213, T216, T234, V241, E243 and E213+T234. The specific substitutions investigated in reference 24 used alanine (with the exception of A157E and T231E) and these substitutions are suitable for use in accordance with the invention. Residues T216 and E243 were also reported in reference 23. Reference 27 reports that the interaction between fH and v3 fHbp can be disrupted by mutations at residues H288 and G318 (numbering H218 and G248 according to the sequence of SEQ ID NO: 17) , and these substitutions are suitable for use in accordance with the invention, for example, H218R, G248D.

Reference 24 reports that some substitutions in v3 can increase affinity for fH, and the latter should be avoided if fH binding is to be impaired, for example, P44 in SEQ ID NO: 17 (residue 106 in reference 24) .

Residues that interact with siderophores can be mutated using the guidance given in sources 16 and 34, for example, by aligning SEQ ID NO: 17 with SEQ ID NO: 4 from source 16 to identify residues that can interact with siderophores , for example, with catecholates, hydroxamates or carboxylates.

The polypeptide according to the second embodiment may contain any of the sequences of SEQ ID NO: 41-44. Considering the possibility of additional point mutations (e.g., to disrupt interactions with siderophores and/or fH), the polypeptide according to the first embodiment may contain any of the sequences of SEQ ID NOs: 41-44, but be modified by single amino acid changes up to 5 ( i.e. 1, 2, 3, 4, or 5 single amino acid substitutions, deletions, and/or insertions), provided that the modified sequence can, upon administration to the host animal, produce antibodies that bind to the meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 40. Such amino acid changes should not reverse mutations in these sequences relative to the wild-type sequence, for example, the sequence of SEQ ID NO: 44 should not mutate at residue V32 or R126.

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

Polypeptides.

The polypeptides of the invention can be obtained in various ways, for example, by chemical synthesis (at least in part), by cleavage of longer polypeptides using proteases, by translation from RNA, by purification from cell culture (for example, from a culture of recombinant expressing cells or from N. meningitidis cultures), etc. The preferred route of expression is heterologous expression in the E. coli host.

The polypeptides of the invention are theoretically at least 100 amino acids in length, eg 150a, 175a, 200a, 225a, or longer. These include the fHbp v2 and/or v3 mutant amino acid sequence, and the fHbp v2 or v3 mutant amino acid sequence likewise must be at least 100 amino acids in length, eg 150a, 175a, 200a, 225a or longer.

fHbp is a natural N. meningitidis lipoprotein. It has also been found to be lipidized when expressed in E. coli with a native leader sequence or heterologous leader sequences. The polypeptides of the invention may have an N-terminal cysteine residue which may be lipidized, eg, contain a palmitoyl group, typically to form tripalmitoyl-S-glycerylcysteine. In other embodiments, the polypeptides are not lipidized.

The polypeptides are preferably obtained in substantially pure or substantially isolated form (ie, in a form substantially free of other Neisseria polypeptides or host cells). Typically, the polypeptides are provided in a non-natural environment, eg separated from the natural environment. In some embodiments, the polypeptide is presented in a composition that is enriched in the polypeptide compared to the starting material. Thus, a purified polypeptide is provided, wherein purified means that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, wherein substantially free means that more than 50% (e.g., 75% or more, 80% and more than, 90% or more, 95% or more, or 99% or more) of all polypeptides in the composition are a polypeptide of the invention.

The polypeptides can take various forms (eg, native, fused, glycosylated, non-glycosylated, lipidized, disulfide bridged, etc.).

SEQ ID NOs 4, 5, 17 and 40 do not include the N-terminal methionine. If a polypeptide of the invention is produced by translation in a biological host, then a start codon is required that will provide the N-terminal methionine in most hosts. Thus, a polypeptide of the invention will, at least in the chain growth step, include a methionine residue located upstream of the indicated sequence of SEQ ID NO.

Cleavage of sequences during the chain growth step means that the mutant v2 or v3 amino acid sequence can itself represent the N-terminus of the polypeptide. However, in other embodiments, the polypeptide of the invention may include an N-terminal sequence located upstream of the fHbp v2 or v3 mutant amino acid sequence. In some embodiments, the polypeptide has one methionine at the N-terminus immediately followed by a mutated v2 or v3 amino acid sequence; in other embodiments, a longer upstream sequence may be used. Such an upstream sequence 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 leader sequences to direct protein transport or short peptide sequences that facilitate cloning or purification (e.g., a histidine end tag, i.e. His _n where n=4, 5, 6, 7, 8, 9, 10 or more than 10). Other suitable N-terminal amino acid sequences will be understood by those skilled in the art, such as the native upstream sequences shown in SEQ ID NO: 2 or SEQ ID NO: 3.

The polypeptide of the invention may also include amino acids located below the terminal amino acid of the mutated amino acid sequence fHbp v2 or v3. 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 for directing protein transport, short peptide sequences that facilitate cloning or purification (e.g. containing a histidine end tag, i.e. His _n where n=3, 4, 5, 6, 7, 8, 9, 10 or more than 10), or sequences that enhance the stability of the polypeptide. Other suitable C-terminal amino acid sequences will be understood by those skilled in the art.

In some embodiments, the invention excludes polypeptides that include a histidine end tag (see references 24 and 25), and in particular a hexahistidine end tag on the C-End.

The term polypeptide refers to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acid residues. The terms also cover an amino acid polymer that has been naturally or tampered with; for example, by disulfide bridging, glycosylation, lipidation, acetylation, phosphorylation, or any other treatment or modification such as conjugation with a labeling agent. Also included in this definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides may exist as single chains or associated chains.

The polypeptides of the invention may be bound or immobilized on a solid support.

The polypeptides of the invention may contain a detectable label, such as a radioactive label, a fluorescent label, or a biotin label. Such polypeptides are particularly useful in immunoassay methods.

As disclosed in reference 164, fHbp can be divided into three domains, called A, B and C. B

- 10 043165 in relation to the sequence of SEQ ID NO: 1, the three domains are (A) 1-119, (B) 120-183 and (C) 184-274:

MNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSV

RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVY

KOSHSALTAFOTEOIODSEHSGKMVAKRQFRIGDIAGEHTSFKLPEGGRATYRGT

AFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLY

NQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ

The mature form of the A domain from Cys-20 at its N-terminus to Lys-119 is called A _mature .

Many fHbp sequences are known and can be easily aligned using standard techniques. By such alignments, one skilled in the art can identify (a) domains A (and A _mature ), B, and C in any given fHbp sequence by comparison with coordinates in the MC58 sequence, and (b) single residues in multiple fHbp sequences, for example, to identify substitutions. However, for ease of reference, the domains are defined below:

Domain A in this fHbp sequence is a fragment of such a sequence that, when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm, begins with an amino acid aligned with Met-1 of SEQ ID NO: 1 and ends with an amino acid aligned with Lys -119 sequences SEQ ID NO: 1

The _mature A domain in a given fHbp sequence is a fragment of such a sequence that, when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm, begins at an amino acid aligned with Cys-20 of SEQ ID NO: 1 and ends at an amino acid aligned with Lys-119 sequence SEQ ID NO: 1.

Domain B in this fHbp sequence is a fragment of such a sequence that, when aligned with the sequence of SEQ ID NO: 1 using the pairwise alignment algorithm, begins with an amino acid aligned with Gln-120 of SEQ ID NO: 1 and ends with an amino acid aligned with Gly -183 sequences SEQ ID NO: 1.

The C domain in this fHbp sequence is a fragment of such a sequence that, when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm, begins with an amino acid aligned with Lys-184 of SEQ ID NO: 1 and ends with an amino acid aligned with Gln -274 sequences SEQ ID NO: 1.

The preferred pairwise alignment algorithm for determining domains is the Needleman-Wunsch global alignment algorithm [158] using default parameters (e.g. using alignment deletion penalty = 10.0 and deletion extension penalty = 0.5 using a scoring matrix EBLOSUM62). Such an algorithm is conveniently implemented in the needle tool in the EMBOSS package [159].

In some embodiments, the fHbp v2 or v3 mutant amino acid sequence of the invention is truncated to remove its A domain. C-terminal β-barrel.

In some embodiments, the polypeptide contains the amino acid sequence described above, except that 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 (ie 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) at the C-terminus are removed.

The polypeptides of the invention typically consist of an artificial amino acid sequence, namely a sequence that is not present in naturally occurring meningococci.

Affinity for factor H can be quantified using surface plasmon resonance, such as those disclosed in references 18 and 21-24 with immobilized human fH. Mutations are preferred that result in a decrease in affinity (i.e., an increase in the dissociation constant KD) of at least 10-fold, and ideally at least 100-fold (when measured under the same experimental conditions against the same polypeptide, but no mutation).

Nucleic acids.

The invention provides a nucleic acid encoding the above-defined polypeptide according to the invention.

Nucleic acids of the invention can be prepared in various ways, for example, by chemical synthesis (for example, by phosphoramidite DNA synthesis) in whole or in part, by cleavage of longer nucleic acids using nucleases (for example, restriction enzymes), by binding of shorter nucleic acids, or nucleotides (eg.

- 11 043165 using ligases or polymerases), from genomic or cDNA libraries, etc.

The nucleic acids of the invention may take various forms, such as single stranded, double stranded, vectors, primers, probes, labeled, unlabeled, etc.

The nucleic acids of the invention are preferably in isolated or substantially isolated form.

The term nucleic acid includes DNA and RNA, as well as their analogs, such as analogs containing modified skeletons, as well as peptide nucleic acids (PNA), etc.

The nucleic acid of the invention may be labeled, for example with a radioactive or fluorescent label.

The invention also provides vectors (such as plasmids) containing the nucleotide sequences of the invention (eg, cloning or expression vectors, such as those suitable for immunization with nucleic acids) and host cells transformed with such vectors.

Bactericidal responses

Preferred polypeptides of the invention can elicit antibody responses that are bactericidal against meningococci. The production of bactericidal antibodies is commonly measured in mice and is a standard measure of vaccine efficacy (eg see final note 14 in ref. 37; also ref. 38).

The polypeptides according to the first embodiment of the invention may preferably elicit antibody responses that are bactericidal against a strain of N. meningitidis that expresses the v2 fHbp 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, dk35 3, m3697, ngh38, and/ or L93/4286. Bactericidal responses can be determined, for example, against var2 strain M2091 (ATCC (American Type Culture Collection) 13091).

Preferred polypeptides according to the first embodiment of the invention can elicit antibodies in mice that are bactericidal against the M2091 strain in a bactericidal serum assay.

The polypeptides according to the second embodiment of the invention may preferably elicit antibody responses that are bactericidal against a strain of N. meningitidis that expresses the v3 fHbp sequence, for example one or more of the strains M1239, 16889, gb355 (=M01-240355 ), m3369, m3813, ngp165. For example, bactericidal responses can be determined against var3 strain M01-240355, which is a reference strain of Neisseria MLST (identification number 19265 in reference 27) that has been fully sequenced (see EMBL ID CP002422 [27]).

Preferred polypeptides according to the second embodiment of the invention can induce antibodies in mice that are bactericidal against strain M01-240355 in a bactericidal serum assay.

For example, an immunogenic composition containing these polypeptides can provide a bactericidal serum titer of at least 1:4 using the Golypneider assay with human complement [27–28, 29] and/or provide a bactericidal serum titer of at least 1:128 s using rabbit complement.

Immunization.

The polypeptides of the invention can be used as an active ingredient in immunogenic compositions, and thus the invention provides an immunogenic composition (eg, a vaccine) containing a polypeptide of the invention.

The invention also provides a method for inducing an antibody response in a mammal, comprising administering to the mammal an immunogenic composition of the invention. 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.

The invention also provides a method of protecting a mammal from an infection caused by Neisseria (eg, meningococcal infection), comprising administering to the mammal an immunogenic composition of the invention.

The invention provides polypeptides of the invention for use as drugs (eg, as immunogenic compositions or as vaccines) or as diagnostic reagents. The invention also provides the use of a nucleic acid or polypeptide of the invention in the manufacture of a medicament for the prevention of Neisseria infection (eg, meningococcal infection) in a mammal.

The mammal is preferably a human. The person may be an adult or preferably a child. Where the vaccine is for prophylactic use, then the human is preferably a child (eg, a toddler or a newborn); where the vaccine is for therapeutic use, then the human is preferably an adult. A vaccine intended for children may also be administered to adults, for example, to assess safety, dose, immunogenicity, etc.

The uses and methods are particularly useful in the prevention/treatment of diseases including, but not limited to, meningitis (particularly bacterial such as meningococcal meningitis) and bacteremia. For example, they are suitable for the active immunization of individuals against invasive meningococcal disease caused by N. meningitidis (eg, in serogroup B).

The effectiveness of therapeutic treatment can be verified by monitoring Neisseria infection after administration of a composition of the invention.

The effectiveness of prophylactic treatment can be tested by monitoring immune responses against fHbp after administration of the composition. The immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g., children aged 12-16 months or in animal models) and then determining standard parameters including serum bactericidal antibodies (SBA) and ELISA titers (enzyme-linked immunosorbent assay) (GMT ). These immune responses can generally be determined approximately 4 weeks after administration of the composition and compared with values determined prior to administration of the composition. It is preferable to increase the SBA by at least 4 times or 8 times. When more than one dose of the composition is administered, more than one post-administration determination may be made.

Preferred compositions of the invention can result in an antibody titer in a patient that exceeds serological protection criteria for each antigenic component for an acceptable percentage of people. Antigens with an associated antibody titer above which the host is considered seroconverted against the antigen are well known, and such titers are published by organizations such as the WHO (World Health Organization). Preferably, more than 80% of statistically significant samples of subjects are seroconverted, more preferably more than 90%, even more preferably more than 93%, and most preferably 96-100%.

Compositions of the invention will typically be directly administered to a patient. Direct delivery can be by parenteral injection (eg, subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial tissue space) or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary, or other mucosal administration. Preferred is intramuscular injection in the thigh or upper arm. The injection may be by means of a needle (eg, hypodermic needle), but alternatively, a needleless injection may be used. A typical intramuscular dose is approximately 0.5 ml.

The invention can be used to induce systemic and/or mucosal immunity.

Treatment with doses can be done on a single dose schedule or on a split dose schedule. Fractional doses can be used in a primary immunization schedule and/or in a booster immunization schedule. The primary dose schedule may be followed by a booster dose schedule. An appropriate period of time between booster doses (eg, 4 to 16 weeks) and between priming and boosting can be determined by standard methods.

The immunogenic composition of the invention will generally comprise a pharmaceutically acceptable carrier, which may be any substance which does not by itself induce the production of antibodies harmful to the patient receiving the composition and which can be administered without fear of undue toxicity. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerol and ethanol. Adjuvants such as humectants or emulsifiers, pH buffer agents, and the like may also be present in such diluents. A comprehensive discussion of suitable carriers is given in reference 27.

Infections caused by Neisseria affect different areas of the body and, therefore, the compositions of the invention can be prepared in various forms. For example, the compositions may be prepared as injectables, or as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in liquid diluents prior to injection may also be prepared. The composition may be formulated for topical administration, for example as an ointment, cream or powder. The composition may be formulated for oral administration, for example as a tablet or capsule, or as a syrup (possibly flavored). The composition may be formulated for pulmonary administration, for example as an inhaler using a fine powder or spray. The composition may be prepared as a suppository or pessary. The composition may be formulated for nasal, otic or ocular administration, for example in the form of drops. Most preferred are compositions suitable for parenteral injection.

The composition is preferably sterile. Preferably, the composition is pyrogen-free. Preferably, it is buffered, for example at a pH of 6 to 8, typically at about pH 7. When the composition contains an aluminum hydroxide salt, a histidine buffer is preferably used [27]. Compositions of the invention may be isotonic to humans.

- 13 043165

Immunogenic compositions contain an immunologically effective amount of the immunogen, as well as, if necessary, any other specified components. By immunologically effective amount is meant that administration of that amount to an individual, either in a single dose or as part of a series of doses, is effective for treatment or prevention. The term prevention means that the progress of the disease is reduced and/or eliminated, or that the occurrence of the disease is eliminated. For example, the subject's immune system can be primed (eg, by vaccination) to trigger an immune response and suppress infection such that the onset of a disease is eliminated. Thus, the vaccinated subject may be infected, but has a better ability to fight infection compared to the control subject. Such an amount will vary depending on the health and physical condition of the patient being treated, the age, the taxonomic group of the patient being treated (e.g., non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of desired protection, vaccine composition, assessment of the medical situation by the attending physician, and other relevant factors. The amount is expected to be within a relatively wide range, which can be determined in standard tests. Treatment with doses can be done on a single dose schedule or on a split dose schedule (eg, including booster doses). The composition can be administered in combination with other immunoregulatory agents.

Adjuvants that can be used in the compositions of the invention include insoluble metal salts, oil-in-water emulsions (for example, MF59 or AS03, both containing squalene), saponins, non-toxic derivatives of LPS (lipopolysaccharides) (such as monophosphoryl lipid A or 3O- deacylated MPL), but not limited to immunostimulatory oligonucleotides, inactivated bacterial ADP-ribosylating toxins, microparticles, liposomes, imidazoquinolones, or mixtures thereof. Other substances that act as immunostimulatory agents are disclosed in Chapter 7 of Source 46.

The use of an aluminum hydroxide and/or aluminum phosphate adjuvant is particularly preferred, and the polypeptides are generally adsorbed to these salts. These salts include oxyhydroxides and hydroxyphosphates (for example, see chapters 8 and 9 in ref. 46). The salts may take any suitable form (eg, gel, crystalline form, amorphous form, etc.).

Additional antigenic components.

Compositions of the invention include a v2 and/or v3 fHbp mutant sequence. It is useful if the composition does not include complex or indefinite mixtures of antigens, for example, it is preferable not to include outer membrane vesicles in the composition. The polypeptides of the invention are preferably expressed recombinantly in a heterologous host and then purified.

Along with the inclusion of the fHbp polypeptide, the composition of the invention may also include one or more additional Neisseria immunogens, since a vaccine that is directed against more than one bacterial immunogen reduces the chance of selecting escaped mutants. Thus, the 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 is often not an fHbp, for example, it may be an NHBA sequence, a NadA sequence, and the like.

Any such additional Neisseria immunogen may be presented as a polypeptide separate from the v2 or v3 fHbp mutant of the invention, or may be presented as an fHbp-modified fusion polypeptide. For example, a fusion of the meningococcal 936 polypeptide and fHbp polypeptides is known [55, 56]. Especially preferred are fusion proteins containing the sequence of SEQ ID NO: 44 and/or the sequence of SEQ ID NO: 45, optionally when the sequences of SEQ ID NO: 44 and/or 45 are modified(s) with up to 5 single amino acid substitutions (i.e. 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions), as described here.

The composition of the invention may include an NHBA antigen. The NHBA antigen is included in the published genome sequence for meningococcal serogroup B strain MC58 [27] as the NMB2132 gene (GenBank identification number GL7227388; here the sequence is SEQ ID NO: 6). Since then, NHBA antigen sequences have been published from many strains. For example, allelic forms of NHBA can be seen in FIG. 5 and 15 of source 27, and in example 13 and FIG. 21 sources 1 (it has SEQ IDs from 3179 to 3184). Various immunogenic fragments of the NHBA antigen have also been reported. Preferred 287 antigens for use in the invention comprise an amino acid sequence: (a) having 50% or more than 50% identity (e.g., 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more than 99.5%) with the sequence of SEQ ID NO: 6; and/or (b) containing a fragment of at least Y consecutive amino acids of the sequence SEQ ID NO: 6, where n is 7 or greater (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments (b) contain epitopes of the sequence of SEQ ID NO: 6. The most useful NHBA antigens of the invention can induce the formation of antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 6. Favorable NHBA antigens for use in the invention can, upon administration to a subject, induce the formation of bactericidal antibodies against meningococcus.

The composition of the invention may include a NadA antigen. The NadA antigen is included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as the NMB1994 gene (GenBank identification number GI: 7227256; here the sequence is SEQ ID NO: 7). Since then, NadA antigen sequences from many strains have been published, and protein activity as an adhesin of Neisseria has been well documented. Various immunogenic fragments of NadA have also been reported. Preferred NadA antigens for use in the invention comprise an amino acid sequence: (a) having 50% or more than 50% identity (e.g. 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more than 99.5%) with the sequence of SEQ ID NO: 7; and/or (b) containing a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 7, where n is 7 or greater (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 (b) contain an epitope of the sequence SEQ ID NO: 7. The most useful NadA antigens of the invention can elicit antibodies that, upon administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 7. Favorable NadA antigens for use in the invention can, after administration to a subject, cause the formation of bactericidal antibodies against meningococcus. SEQ ID NO: 15 is one such fragment.

The composition of the invention may include an NspA antigen. The NspA antigen is included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as the NMB0663 gene (GenBank identification number GL7225888; here the sequence is SEQ ID NO: 8). This antigen was previously known from References 49 and 50. Since then, NspA antigen sequences have been published from many strains. Various immunogenic fragments of NspA have also been reported. Preferred NspA antigens for use in the invention comprise an amino acid sequence: (a) having 50% or more than 50% identity (e.g., 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more than 99.5%) with the sequence of SEQ ID NO: 8; and/or (b) containing a fragment of at least n consecutive amino acids of the sequence SEQ ID nO: 8, where 'n' is 7 or more than 7 (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments (b) contain an epitope of the sequence of SEQ ID NO: 8. The most useful NspA antigens of the invention can induce the formation of antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 8. Favorable NspA antigens for use in the invention can, after administration to a subject, cause the formation of bactericidal antibodies against meningococcus.

Compositions of the invention may include the meningococcal HmbR antigen. The full-length HmbR sequence is included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as the NMB1668 gene (here, the sequence is SEQ ID NO: 9). The invention can use a polypeptide that contains a full HmbR sequence, but often a polypeptide that contains a partial HmbR sequence is used. Thus, in some embodiments, the HmbR sequence used according to the invention may contain an amino acid sequence having at least i% sequence identity with the sequence of SEQ ID NO: 9, where the value of / is 50, 60, 70, 80, 90, 95, 99 or more. In other embodiments, the HmbR sequence used according to the invention may contain 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, the HmbR sequence used according to the invention may comprise an amino acid sequence (1) having at least i% sequence identity with the sequence of SEQ ID NO: 9, and/or (2) containing a fragment of at least j consecutive amino acids of the sequence SEQ ID NO: 9. Preferred fragments of j amino acids contain an epitope of the sequence SEQ ID NO: 9. Such epitopes typically contain amino acids that are located on the surface of the HmbR. Useful epitopes include epitopes with amino acids involved in HmbR binding to hemoglobin, since antibodies that bind to these epitopes can block the bacterium's ability to bind to host hemoglobin. The topology of HmbR and its critical functional residues was examined in reference 51. The most useful HmbR antigens of the invention can elicit antibodies that, upon administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 9. Favorable HmbR antigens for use in the invention may, upon administration to a subject, induce the formation of bactericidal antibodies against meningococcus.

The composition of the invention may include an NhhA antigen. The NhhA antigen is included in the published genome sequence for the meningococcal serogroup B strain MC58 [47] as a gene

- 15 043165

NMB0992 (GenBank ID GL7226232; here the sequence is SEQ ID NO: 10). Since then, NhhA antigen sequences have been published from many strains, eg sources 48 and 52, and various NhhA immunogenic fragments have been reported. It is also known as Hsf. Preferred NhhA antigens for use in the invention comprise an amino acid sequence: (a) having a sequence identity of 50% or greater (e.g., 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more) with SEQ ID NO: 10; and/or (b) containing a fragment of at least n consecutive amino acids of the sequence SEQ ID NO: 10, where 'n' is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments (b) contain an epitope of the sequence 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 of SEQ ID NO: 10. Favorable NhhA antigens for use in the invention can, after administration to a subject, cause the formation of bactericidal antibodies against meningococcus.

The composition of the invention may include an App antigen. The App antigen is included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as the NMB1985 gene (GenBank identification number GI: 7227246; here the sequence is SEQ ID NO: 11). Since then, App antigen sequences from many strains have been published. Various immunogenic fragments of App have also been reported. Preferred App antigens for use in the invention comprise an amino acid sequence: (a) having a sequence identity of 50% or greater (e.g., 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more) with SEQ ID NO: 11; and/or (b) containing a fragment of at least V consecutive amino acids of the sequence SEQ ID NO: 11, where n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments (b) contain an epitope of the sequence SEQ ID NO: 11. The most useful App antigens of the invention can induce the formation of antibodies that, after administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 11. Favorable App antigens for use in the invention can, after administration to a subject, cause the formation of bactericidal antibodies against meningococcus.

The composition of the invention may include the Omp85 antigen. The Omp85 antigen is included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as the NMB0182 gene (GenBank identification number GL7225401; here the sequence is SEQ ID NO: 12). Since then, Omp85 antigen sequences have been published from many strains. Further information on Omp85 can be found in references 53 and 54. Various immunogenic fragments of Omp85 have also been reported. Preferred Omp85 antigens for use in the invention comprise an amino acid sequence: (a) having a sequence identity of 50% or greater (e.g., 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more) with SEQ ID NO: 12; and/or (b) containing a fragment of at least n consecutive amino acids of the sequence SEQ ID nO: 12, where n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments (b) contain an epitope of the sequence SEQ ID NO: 12. The most useful Omp85 antigens of the invention can elicit antibodies that, upon administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 12. Favorable Omp85 antigens for use in the invention can, after administration to a subject, cause the formation of bactericidal antibodies against meningococcus.

The composition of the invention may include the 936 antigen. The 936 antigen is included in the published genome sequence for meningococcal serogroup B strain MC58 [47] as the NMB2091 gene (here the sequence is SEQ ID NO: 13). Preferred 936 antigens for use in the invention comprise an amino acid sequence: (a) having a sequence identity of 50% or greater (e.g., 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% or more) with SEQ ID NO: 13; and/or (b) containing a fragment of at least n consecutive amino acids of the sequence SEQ ID NO: 13, where n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments (b) contain an epitope of the sequence SEQ ID NO: 13. The most useful 936 antigens of the invention can elicit antibodies that, upon administration to a subject, can bind to a meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 13. The 936 antigen is a good fHbp merger partner (eg see refs 55 and 56).

The composition may contain: a polypeptide containing the sequence of SEQ ID NO: 14; a polypeptide containing the sequence of SEQ ID NO: 15; and a polypeptide of the invention comprising a mutant fHbp v2 amino acid sequence and SEQ ID NO: 13 (see references 55 and 56).

The composition may contain: a polypeptide containing the sequence of SEQ ID NO: 14; a polypeptide containing the sequence of SEQ ID NO: 15; and a polypeptide according to the invention, containing

- 16 043165 mutant amino acid sequence of fHbp v3 and SEQ ID NO: 13 (see references 55 and 56).

In some embodiments, the polypeptide of the invention is combined with another meningococcal fHbp sequence. In particular, the v2 polypeptide can be combined with the v1 and/or v3 polypeptide to increase the strain coverage spectrum [162]. Thus, the composition may comprise: (1) a polypeptide of the invention comprising a mutant fHbp v2 amino acid sequence; and (2) a v1 fHbp polypeptide and/or a v3 fHbp polypeptide. In other embodiments, the polypeptide of the invention may comprise (1) a mutant fHbp v2 amino acid sequence and (2) an fHbp v1 amino acid sequence and/or an fHbp v3 amino acid sequence. Thus, the v1 and/or v3 sequences can be combined with the mutated v2 sequence as separate units in a composition or in a fusion polypeptide.

Similarly, a v3 polypeptide can be combined with a v1 and/or v2 polypeptide to increase strain coverage [162]. Thus, the composition may contain: (1) a polypeptide of the invention comprising a mutant fHbp v3 amino acid sequence; and (2) a v1 fHbp polypeptide and/or a v2 fHbp polypeptide. In other embodiments, the polypeptide of the invention may comprise (1) a mutant fHbp v3 amino acid sequence and (2) an fHbp v1 amino acid sequence and/or an fHbp v2 amino acid sequence. Thus, the v1 and/or v2 sequences can be combined with the mutated v3 sequence as separate entities in a composition or in a fusion polypeptide.

In addition, mutant v2 and v3 polypeptides can be combined with each other to increase strain coverage. Thus, the composition may comprise: (1) a polypeptide of the invention comprising a mutant fHbp v2 amino acid sequence; (2) a polypeptide of the invention comprising a mutant fHbp v3 amino acid sequence; and (3) an fHbp v1 polypeptide. In other embodiments, the polypeptide of the invention may comprise (1) an fHbp v2 mutant amino acid sequence, (2) an fHbp v3 mutant amino acid sequence, and (3) an fHbp v1 amino acid sequence. Thus, the mutant v2 and v3 sequences can be combined with the v1 sequence as separate units in a composition or in a fusion polypeptide. The v1 sequence may be a wild-type sequence or a mutant sequence.

v1 fHbp may contain (a) an amino acid sequence that has at least k% identity with SEQ ID NO: 16, and/or (b) a fragment of SEQ ID NO: 16. Information on k and fragments is given above. The fragment typically includes at least one epitope of SEQ ID NO: 16, and the v1 fHbp polypeptide includes at least one epitope that is not present in the v2 or v3 amino acid sequence of the invention, such that the antibodies that are elicited v1 fHbp can recognize v1. Ideally, v1 fHbp can elicit antibodies that are bactericidal against v1 strains, such as against the MC58 strain (available from the ATCC as 'BAA-335'). v1 fHbp may include an amino acid mutation that impairs its ability to bind to fH.

v2 fHbp may contain (a) an amino acid sequence that has at least k% identity with SEQ ID NO: 5, and/or (b) a fragment of SEQ ID NO: 5. Information on k and fragments is given above. The fragment typically includes at least one epitope of SEQ ID NO: 5, and the v2 fHbp polypeptide includes at least one epitope that is not present in the v3 amino acid sequence of the invention, such that antibodies elicited by v2 fHbp , can recognize strains v2. Ideally, v2 fHbp can induce the formation of antibodies that are bactericidal against v2 strains, for example against strain M2091 (ATCC 13091). v2 fHbp may be a polypeptide according to the first embodiment.

v3 fHbp may contain (a) an amino acid sequence that has at least k% identity with SEQ ID NO: 17, and/or (b) a fragment of SEQ ID NO: 17. Information on k and fragments is given above. The fragment typically includes at least one epitope of SEQ ID NO: 17, and the v3 fHbp polypeptide includes at least one epitope that is not present in the v2 amino acid sequence of the invention, such that antibodies elicited by v3 fHbp , can recognize strains v3. Ideally, v3 fHbp can induce antibodies that are bactericidal against v3 strains, eg M01-240355 strain. v3 fHbp may be a polypeptide according to the second embodiment.

Thus, for example, the invention provides a polypeptide containing in a single polypeptide chain each of: (1) the amino acid sequence fHbp v1; (2) a mutant fHbp v2 amino acid sequence; and (3) a mutant fHbp v3 amino acid sequence. This polypeptide can, upon administration to a host animal, induce the formation of antibodies that bind to each of: a meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 46; meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 4; and meningococcal polypeptide fHbp, consisting of the amino acid

- 17 043165 sequence of SEQ ID NO: 40. Sequence (1) may contain an amino acid sequence that has at least k% identity to the sequence of SEQ ID NO: 16. Sequence (2) may contain an amino acid sequence that has at least k% sequence identity to SEQ ID NO: 5, but different 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. Sequence (3) may contain an amino acid sequence that has at least k% identity to SEQ ID NO: 17 but differs from SEQ ID NO: 17 at one or more of the following residues: S32,133, L39, L41 , F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242 and/or E243. In a preferred embodiment: sequence (1) contains the sequence SEQ ID NO: 16, optionally modified with up to 5 single amino acid changes (ie 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions); sequence (2) contains the sequence of SEQ ID NO: 45, optionally modified with 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 such amino acid changes do not reverse mutations in these sequences relative to the wild-type sequence; and sequence (3) contains the sequence of SEQ ID NO: 44, optionally modified to 5 single amino acid changes (i.e. 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions, for example to obtain the sequence of SEQ ID NO: 53), provided that such amino acid changes do not reverse mutations in these sequences relative to the wild-type sequence. The amino acid sequences (1), (2) and (3) may be in any order from N- to C-terminus in the polypeptide, but preferably in the order (2), then (3), then (1). For example, the invention provides a polypeptide of the formula -A-B-C-, where: A contains the sequence of SEQ ID NO: 45, possibly modified by up to 3 single amino acid substitutions; B contains the sequence of SEQ ID NO: 44, optionally modified with up to 3 single amino acid substitutions; and C contains the sequence of SEQ ID NO: 16, optionally modified with up to 3 single amino acid substitutions (eg substitution(s) to disrupt fH binding). Preferred C contains the sequence of SEQ ID NO: 49, where the Arg-34 residue is mutated to Ser as reported for the 'R41S' mutation in reference 21.

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

The sequence of SEQ ID NO: 47 (by itself or in the sequence of SEQ ID NO: 48) can optionally be 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 such amino acid changes do not reverse the mutations in the v1, v2, and v3 sequences relative to the wild-type sequence, ie. amino acid residues V32, R123, V285, R379, and S543 of SEQ ID NO: 47 must not mutate to S32, L123, S285, L379, and R543. However, in one exclusive embodiment, V285 may change back to S285 and/or V32 may change back to S32.

Fusion of the mutant sequences can ideally generate antibodies that bind to each of the following: fHbp wild type v1 meningococcal polypeptide consisting of the amino acid sequence of SEQ ID NO: 46; a meningococcal wild type v2 fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO: 4; and a meningococcal v3 fHbp wild-type polypeptide consisting of the amino acid sequence of SEQ ID NO: 40.

In addition to Neisseria polypeptide antigens, the composition may include antigens for immunization against other diseases or infections. For example, the composition may include one or more of the following additional antigens:

saccharide antigen N. meningitidis serogroups A, C, W135 and/or Y, such as the saccharide disclosed in reference 57 of serogroup C (see also reference 58) or reference 59;

saccharide antigen of Streptococcus pneumoniae [eg, 60, 61, 62];

hepatitis A virus antigen, such as an inactivated virus [eg, 63, 64];

hepatitis B virus antigen, such as surface and/or core antigens [eg 64, 65];

a diphtheria antigen such as diphtheria toxoid [eg chapter 3 of source 66], eg CRM ₁₉₇ mutant [eg 67];

tetanus antigen, such tetanus toxoid [eg chapter 4 of source 66];

Bordetellapertussis antigen, such as pertussis cholotoxin (PT) and filamentous haemagglutinin

- 18 043165 (FHA) from B. pertussis, possibly also in combination with pertactin and/or agglutinogens 2 and 3 (eg sources 68 and 69);

Haemophilus influenzae B saccharide antigen [eg 58];

poliovirus(e) antigen(s) (eg 70, 71], such as IPV;

measles, mumps and/or rubella antigens (eg chapters 9, 10 and 11 of source 66);

influenza antigen(s) (eg chapter 19 of source 66), such as the surface proteins hemagglutinin and/or neuraminidase;

Moraxella catarrhalis antigen [eg 72];

protein antigen Streptococcus agalactiae (group B streptococcus) [eg, 73, 74];

saccharide antigen of Streptococcus agalactiae (group B streptococcus);

Streptococcus pyogenes antigen (group A streptococcus) [eg, 74, 75, 76];

Staphylococcus aureus antigen [eg, 77].

The composition may contain one or more of these additional antigens.

If necessary, toxic protein antigens can be neutralized (for example, neutralization of pertussis toxin using chemical and/or genetic methods [69]).

Where diphtheria antigen is included in the composition, then it is preferred to also include tetanus antigen and pertussis antigens. Similarly, when a tetanus antigen is included, then diphtheria and pertussis antigens are also preferably included. Similarly, when a pertussis antigen is included, then diphtheria and tetanus antigens are also preferably included. Thus, combinations of DTP are preferred.

The saccharide antigens are preferably in the form of conjugates. Carrier proteins for conjugates are discussed in more detail below.

The antigens in the composition will typically be present at a concentration of at least 1 μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.

The immunogenic compositions of the invention may be used therapeutically (ie, to treat an existing infection) or prophylactically (ie, to prevent a future infection).

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

In some embodiments, the composition of the invention comprises, in addition to the fHBP sequence, conjugated capsular saccharide antigens from 1, 2, 3, or 4 meningococcal serogroups A, C, W135, and Y. antigen

Meningococcal serogroups Y, W135, C and A.

Current serogroup C vaccines (Menjugate™ [57,78], Meningitec™ and NeisVac-C™) include conjugated saccharides. Menjugate™ and Meningitec™ have oligosaccharide antigens conjugated to a CRM ₁₉₇ carrier, while NeisVac-C™ uses the full polysaccharide (de-Oacetylated) conjugated to a tetanus toxoid carrier. The Menactra™ vaccine contains conjugated capsular saccharide antigens from each of the Y, W135, C and A serogroups.

Compositions of the present invention may include capsular saccharide antigens from one or more of the Y, W135, C and A meningococcal serogroups, wherein the antigens are conjugated to a 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 serogroups A, C, W135 and Y.

A typical amount of each meningococcal saccharide antigen per dose is from 1 μg to 20 μg, eg, about 1 μg, about 2.5 μg, about 4 μg, approximately 5 μg, or approximately 10 μg (expressed as saccharide).

When the mixture contains capsular saccharides from both serogroups A and C, then the ratio (w/w) MenY saccharide:MenC saccharide may be greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10 :1 or higher). When the mixture contains capsular saccharides from serogroup Y and one or both serogroups C and W135, then the ratio (w/w) Men Y saccharide: MenW135 saccharide may be greater than 1 (e.g., 2:1, 3:1, 4:1 , 5:1, 10:1 or higher) and/or such a ratio (wt./wt.) MenY saccharide: MenC saccharide may be less than 1 (for example, 1:2, 1:3, 1:4, 1:5 or below). Preferred ratios (wt./wt.) for saccharides of 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) of 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. Preferably, substantially equal weights of each saccharide are used.

Capsular saccharides can be used in the form of oligosaccharides. They are usually formed by fragmentation of the purified capsular polysaccharide (for example, by hydrolysis), after which

- 19 043165 usually followed by purification of fragments of the desired size.

The fragmentation of the polysaccharides is preferably carried out to obtain a final average degree of polymerization (DP) in the oligosaccharide of less than 30 (e.g., 10 to 20, preferably about 10 for serogroup A; 15 to 25 for serogroups W135 and Y, preferably about 15-20; from 12 to 22 for serogroup C, etc.).

DP can usually be measured using ion exchange chromatography or colorimetric assays [79].

If hydrolysis is carried out, the hydrolyzate will typically be sized to remove short oligosaccharides [58]. This can be done in various 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 in the case of serogroup A, and those with a degree of polymerization of less than about 4 are preferably removed in the case of serogroups W135 and Y.

Preferred MenC saccharide antigens are disclosed in reference 78, such as those used in Menjugate™.

The saccharide antigen may be chemically modified. The latter is especially useful for reductive hydrolysis in the case of serogroup A [80]. De-Oacetylation of meningococcal saccharides can be carried out. In the case of oligosaccharides, the modification may occur before or after depolymerization.

When the composition according to the invention includes a MenA saccharide antigen, then the antigen is preferably a modified saccharide in which one or more hydroxyl groups of the native saccharide are replaced by a blocking group [80]. This modification increases the resistance to hydrolysis.

covalent conjugation.

The capsular saccharides in the compositions of the invention can generally be conjugated to a carrier protein(s). Generally, conjugation enhances the immunogenicity of saccharides by converting them from T-independent antigens to T-dependent antigens, thus allowing immunological memory priming. Conjugation is particularly useful for pediatric vaccines and is a well known technique.

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

Any suitable conjugation reaction may be used, optionally with any suitable linker.

The saccharide can usually be activated or functionalized prior to conjugation. For activation, for example, cyanylating reagents such as CDAP (eg, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [101, 102, etc.]) can be used. Other suitable methods use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide), TSTU (O-(Nsuccinimidyl )-1,1,3,3-tetramethyluronium tetrafluoroborate), etc.

Linking through the linker group can be accomplished using any known method, such as those described in References 103 and 104. One type of linkage is to reductively aminate the polysaccharide, link the resulting amino group to one end of the adipic acid linker group, and then link the protein to the other end of the linker group based on adipic acid [105, 106]. Other linkers include B-propionamido [107], nitrophenylethylamine [108], haloacyl halides [109], glycosidic bonds [110], 6-aminocaproic acid [111], ADH [112], C ₄ -C ₁₂ moieties [113], etc. . As an alternative to using a linker, direct linking can be used. Direct protein binding may involve oxidation of the polysaccharide followed by reductive amination using the protein, as described, for example, in references 114 and 115.

Preferred is a method involving the introduction of amino groups into the saccharide (for example, by replacing the terminal =O-groups with a -NH ₂ group) followed by derivatization with adipic acid diesters (for example, N-hydroxysuccinimidodiester of adipic acid) and by interaction with a carrier protein. Another preferred reaction uses activation

- 20 043165

CDAP carrier protein D, such as in the case of MenA or MenC.

Vesicles of the outer membrane.

Some compositions of the invention do not include the complex or undefined mixtures of antigens that are typical characteristics of OMVs (Outer Membrane Vesicles). However, the invention can be used in combination with OMVs, as it has been found that fHbp enhances their effectiveness [4], in particular as a result of overexpression of the polypeptides of the invention in the strains used to produce OMVs. See also below.

This approach can generally be applied to improve preparations of N. meningitidis serogroup B microvesicles [116], native OMVs [117], outer membrane vesicles or vesicles [eg refs. 118-123, etc.]. They can be obtained from bacteria that have been genetically manipulated [124-127], for example, to increase immunogenicity (for example, overexpression of immunogens), to reduce toxicity, to inhibit the synthesis of capsular polysaccharides, to down-regulate PorA expression, etc. Vesicles can be obtained from hyperblebbing strains [128-131]. Vesicles from non-pathogenic Neisseria may be included [132]. OMV can be obtained without the use of detergents [133, 134]. They can express proteins that differ from those of Neisseria on their surface [135]. They can be depleted in LPS (lipopolysaccharides). They can be mixed with recombinant antigens [118, 136]. It is possible to use vesicles from bacteria with different class I outer membrane protein subtypes, for example six different subtypes [137,138] using two different populations of genetically engineered vesicles each presenting three subtypes, or nine different subtypes using three different populations of genetically engineered vesicles. vesicles, each representing three subtypes, and so on. Useful subtypes include P1.7.16, P1.5-1.2-2, P1.19.15-1, P1.5-2.10, P1.12-1.13, P1.72.4, P1 .22.14, P1.7-1.1, P1.18-1.3.6.

Host cells.

The invention provides a bacterium that expresses a polypeptide of the invention. The bacterium may be meningococcus or 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 can overexpress the polypeptide (see reference 139). The expression of the polypeptide ideally may not vary depending on the phase.

The invention also provides outer membrane vesicles derived from the bacterium of the invention (particularly meningococcus). The invention also provides a method for producing vesicles from the bacterium of the invention. The vesicles obtained from these strains preferably include the polypeptide of the invention, which must be present in the vesicles in an immunoavailable form, ie. an antibody that can bind to a purified polypeptide of the invention must also be able to bind to a polypeptide that is present in the vesicles.

These outer membrane vesicles include any proteoliposome vesicle resulting from the disruption or blistering of the outer membrane of meningococcus to form vesicles therefrom that include the protein components of the outer membrane. Thus, the term includes OMVs (sometimes referred to as vesicles), microvesicles (MVs [116]), and native OMVs (NOMVs [117]).

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

OMVs are produced artificially from bacteria and can be obtained using detergent treatment (eg deoxycholate) or non-detergent means (eg see ref. 134). Methods for the formation of OMV consist in treating bacteria with a detergent based on bile salts (for example, salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., while sodium deoxycholate [140 and 141] is preferred for treating Neisseria) at a pH high enough not to precipitate the detergent [142]. Other methods can be carried out essentially in the absence of detergent [134] using methods such as sonication, homogenization, microfluidization, cavitation, osmotic shock, milling, French press, mixing, etc. Detergent-free or low-detergent methods can retain useful antigens such as NspA and fHbp [134]. Thus, an OMV extraction buffer containing about 0.5% or less deoxycholate, such as about 0.2%, about 0.1%, less than 0.05%, or no deoxycholate can be used in the process.

- 21 043165

A useful method for obtaining OMVs is described in reference 143 and involves ultrafiltration of crude OMVs instead of high speed centrifugation. The method may include the step of ultracentrifugation after ultrafiltration. OMVs can also be purified using the two-stage gel filtration method described in Reference 154.

Vesicles for use in the invention can be obtained from any meningococcal strain. Typically, vesicles are obtained from a strain of serogroup B, but they can be obtained from other serogroups other than serogroup B (for example, reference 142 describes the method for serogroup A), such as A, C, W135 or Y. The strain can have any serotype (for example , 1, 2a, 2b, 4, 14, 15, 16, etc.), any serosubtype and any immunotype (eg, L1; L2; L3; L3,3,7; L10, etc.). Meningococci may be from any suitable strain, including hyperinvasive and hypervirulent strains, for example from any of the following seven hypervirulent strains: subgroup I; subgroup III; subgroup IV-1; complex ET-5; complex ET-37; cluster A4; line 3.

The bacteria of the invention, in addition to encoding the polypeptide of the invention, may have one or more additional modifications. For example, they may have a modified fur gene [144]. nspA expression can be upregulated with concomitant knockout of por A and cps. Additional N. meningitidis knockout mutants for producing OMV are disclosed, for example, in reference 150. Reference 145 discloses the construction of vesicles from strains modified to express six different PorA subtypes. It is also possible to use a Neisseria mutant with low endotoxin levels achieved as a result of knockout of enzymes involved in LPS biosynthesis [146, 147]. The invention can use a Neisseria mutant designed to reduce or disable the expression of at least one gene involved in conferring toxicity to the lipid A fragment in LPS, in particular the lpxl1 gene [148]. Similarly, a Neisseria mutant engineered to reduce or disable the expression of at least one gene involved in capsular polysaccharide synthesis or export, in particular the synX and/or ctrA genes, can be used in the invention. All of these or other mutants can be used in the invention.

Thus, the strain used in the invention may, in some embodiments, express more than one PorA subtype. Previously, 6-valent and 9-valent PorA strains were constructed. The strain can express 2, 3, 4, 5, 6, 7, 8 or 9 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, the strain may be down-regulated for PorA expression, e.g., in which PorA is reduced by at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least less than 70%, not less than 80%, not less than 90%, not less than 95%, etc.), or it is even knocked out compared to wild-type levels (for example, compared to strain H44/76).

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

The gene encoding the polypeptide of the invention may be integrated into the bacterial chromosome or may be present in episomal form, such as in a plasmid.

Favorably for vesicle production, meningococcus can be genetically engineered to ensure that polypeptide expression does not undergo phase variations. Methods for reducing or eliminating phase variability in gene expression in meningococci are disclosed in reference 149. For example, a gene can be placed under the control of a constitutive or inducible promoter, or can be removed or replaced with a DNA motif that is responsible for its phase variability.

In some embodiments, the strain may include one or more knockout and/or overexpression mutations disclosed in references 122, 124, 128, and 150. regulation 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; (6) 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.

If a mutant strain is used, in some embodiments it may have one or more or all of the following characteristics: (1) downregulation or knockout of LgtB and/or GalE to truncate LOS (lipooligosaccharide) meningococcus; (2) upregulation of TbpA; (3) upregulation of NhhA; (4) upregulation of Omp85; (5) upregulation of LbpA; (6) upregulation of NspA; (7) PorA knockout; (8) downregulation or knockout of FrpB; (9) down regulation or knockout of Ora; (10) down regulation or knockout ORS; (11) deletion of the cps gene complex. The truncated LOS may be a LOS that does not include the sialyllacto-M-neotetraose epitope, for example, it may be a galactose-deficient LOS. LOS may not have an α chain.

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

In one preferred embodiment, the meningococcus does not express a functional MltA protein. As discussed in references 152 and 153, knockout of MltA (membrane-bound lytic transglycosylase, also known as GNA33) in meningococcus results in bacteria that spontaneously release large numbers of membrane vesicles into the culture medium, from which they can be easily purified. For example, vesicles can be purified using a two-stage filtration by size method according to source 154, including: (1) a first filtration stage in which vesicles are separated from bacteria based on their size difference, with the vesicles passing into the filtrate; and (2) a second filtration step in which the vesicles remain in the concentrate. The MltA (downregulation or knockout) mutation is used in GMMA vaccines [155] and can conveniently be combined with additional downregulation or knockout of in particular at least one gene involved in conferring toxicity on a lipid A fragment in LPS, in particular lpxl1 , and/or at least one gene involved in the synthesis or export of the capsular polysaccharide, in particular the synX and/or ctrA genes. GMMAs (generalized modules for membrane antigens) genetically deactivate OMVs, which are derived from meningococcal strains engineered to release GMMA with reduced reactivity and increased immunogenicity. GMMAs induce fewer pro-inflammatory cytokines than OMVs when tested in the monocyte activation test (MAT).

A preferred meningococcal strain for a GMMA vaccine expresses a v2 fHbp mutant and/or a v3 fHbp mutant of the invention using this approach, and expression can be driven by strong promoters. The vesicles released by this strain include mutant v2 and/or v3 fHbp proteins in an immunogenic form, and administration of the vesicles can provide a bactericidal antibacterial response, as discussed in reference 155. The strain can also express v1 fHbp, or instead v1 fHbp can be proposed in as a single recombinant protein in soluble form (and v1 fHbp may be wild-type or a mutated sequence, eg mutated to disrupt its ability to bind to fH, as discussed above). The invention provides such strains and also provides vesicles that release these strains, for example, purified from culture media after growth of the strains. The preferred v2 mutant for expression in these strains has a mutation at S32 and/or L123 as discussed herein, and the preferred v3 mutant for expression in these strains has a mutation at S32 and/or L126 as discussed here. Thus, vesicles derived from meningococci expressing these v2 and v3 fHbp mutant sequences are particularly preferred immunogens for use in the vaccines of the invention. Thus, a mutagenesis-useful wild-type v2 sequence contains SEQ ID NO: 51 or SEQ ID NO: 54 (containing the AG form of SEQ ID NO: 55), and thus a mutagenesis-useful sequence of wild-type v3 contains SEQ ID NO: 52.

Promoters useful for use in such strains include those discussed in references 156 and 157. For example, a promoter can be: (a) a porin gene promoter, preferably porA or porB, in particular from N. meningitidis; or (b) an rRNA gene promoter (such as the 16S rRNA gene), in particular from N. meningitidis. When a meningococcal porin promoter is used, then it is preferably derived from porA, and more particularly the -10 region of the meningococcal porA gene promoter and/or the -35 region of the meningococcal porA gene promoter (preferably where the -10 region and the -35 region are separated by an intermediate sequence of 12- 20 nucleotides, and where the intermediate sequence either does not contain a poly-G sequence, or includes a poly-G sequence having no more than eight consecutive G nucleotides). When an rRNA gene promoter is used, then it may comprise specifically (1) the -10 region of the meningococcal rRNA gene promoter and/or (2) the -35 region of the meningococcal rRNA gene promoter. It is also possible to use a hybrid of (a) and (b), for example having the -10 region of the porA promoter and the -35 region of the rRNA promoter (which may be the -35 consensus region). Thus, a useful promoter can be a promoter that includes either (1) the -10 region (particularly meningococcal) rRNA gene and the -35 region (particularly meningococcal) PorA gene, or (2) the -10 region (particularly meningococcal) ) of the PorA gene and region -35 (in particular meningococcal) of the rRNA gene.

If LOS are present in the vesicle, then the vesicle can be processed in such a way as to bind its LOS and protein components (conjugation within the vesicle [150]).

General information.

The term containing encompasses including as well as consisting, for example, a composition containing X may consist solely of X or may include something in addition, for example, X+Y. References to containing (or contains, etc.) may optionally be replaced by consisting of (or consists of, etc.). The term essentially consisting of limits the scope of the claims to specific materials or steps and those that do not materially affect the essential(s) and novel feature(s) of the claimed invention.

The term approximately in relation to the numerical value of x is optional and means, for example, x±10%.

The word essentially does not exclude completely, for example, a composition that essentially does not contain Y may not contain Y at all. If necessary, the word essentially can be excluded from the definition of the invention.

Sequence identity can be determined using the Smith-Waterman homology search algorithm implemented in MPSRCH (Oxford Molecular) using an affine gap search with alignment deletion penalty = 12 and deletion extension penalty = 1, but is preferably determined at using the Needleman-Wunsch global alignment algorithm [158] using default parameters (e.g. alignment deletion penalty = 10.0 and deletion continuation penalty = 0.5 using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [159]. When the application relates to sequence identity to a particular SEQ ID, then the identity must be calculated over the entire length of that SEQ ID.

The term fragment in relation to polypeptide sequences means that the polypeptide is a fraction of a full-length protein. Such fragments may exhibit qualitative biological activity, the same as a full length protein, for example, a fragment may contain or encode one or more epitopes, such as immunodominant epitopes, which allow for a similar immune response to the fragment as well as to the full length sequence. Polypeptide fragments typically have a deletion at the N-terminal fragment and/or C-terminal fragment 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: example 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, 258, 258, 258 259, 260, 261, 262 consecutive amino acids, including all integer values in between, of the reference polypeptide sequence, e.g. 50 to 260, 50 to 255, 50 to 250, 50 to 200, 50 to 150 consecutive amino acids of the reference polypeptide sequences. The term fragment unambiguously excludes full-length fHbp polypeptides and their mature lipoproteins.

Once the serogroup is determined, the classification of meningococci includes serotype, serosubtype, and then immunotype, and standard nomenclature indicates serogroup, serotype, serosubtype, and immunotype, each separated by a colon, eg B:4:P1.15:L3,7,9. In serogroup B, some lines often cause disease (hyperinvasive), some lines cause more severe disease than others (hypervirulence), and others rarely cause disease. Seven hypervirulent lineages have been identified, namely subgroups I, III and IV-1, ET-5 complex, ET-37 complex, A4 cluster and line 3. Such lines were identified using multilocus enzymatic electrophoresis (MLEE), but meningococcal classification was also used multilocus sequence typing (MLST). The four major hypervirulent clusters are the ST32, ST44, ST8, and ST11 complexes.

In general, the invention does not cover the various fHbp sequences, in particular those disclosed in references 2, 3, 5, 6, 7, 160, 161, 162, 163, 164, 165, 166 and 167.

Brief description of the figures

Fig. 1 shows the differing sensitivity of fHbp variants to chymotrypsin digestion. The arrow shows the position of the full-length fHbp proteins.

Fig. 2 shows western blot analysis of v2 mutants. Lanes are: (1 and 2) recombinant purified v2 wild type; (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) fHbp var2 NAG-trx control, i.e. protein v2, where the N-terminal sequence GPDSDRLQQRR (SEQ ID NO: 37) is replaced by GSKDISS (SEQ ID NO: 38). Lanes 2-14 included chymotrypsin. M are molecular markers.

Fig. 3 shows additional Western blot analysis of v2 mutants. Lanes are: (1 and 2) mutant #3; (3 and 4) mutant #6; (5 and 6) mutant #9; (7 and 8) mutant #10; (9 and 10) mutant #13; (11 and 12) Agono; (13 and 14) v2 wild type; (15 and 16) mutant #22. Odd lanes refer to proteins that have been incubated without chymotrypsin, while even lanes refer to proteins that have been incubated with chymotrypsin. The Agono protein is recombinant v2 where the N-terminal sequence (SEQ ID NO: 37) has been removed.

Fig. 4 shows additional Western blot analysis for v2 mutants. Lanes are: (1 and 2) mutant #11; (3 and 4) N-trx; (5-7) mutant #19; (8 and 9) mutant #20; (10 and 11) mutant #21. Lane 2, 4, 7, 9 and 11 for proteins that were incubated with chymotrypsin, while the other lanes were without chymotrypsin. The N-trx protein is a recombinant v2, where the N-terminal

- 24 043165 the sequence (SEQ ID NO: 37) is replaced by SEQ ID NO: 39.

Fig. 5 shows DSC results for wild-type v2 fHbp and S58V/L149R mutant. The C-terminal domain was not affected by the mutation, but the Tm of the N-terminal domain increased by >20°C (indicated by an arrow). The y-axis shows Cp (kcal/mol/°C) and the x-axis shows temperature (°C).

Fig. 6 shows the response in SPR (surface plasmon resonance) for v2 fHbp wild type (solid line) and mutant (dashed line).

Fig. 7 shows the response in SPR for v3 fHbp as wild type (top) or with various mutations.

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

Specific embodiments of the invention

The invention provides the following specific numbered embodiments.

1. A v3 or v2 mutant of fHbp that is more stable than wild-type fHbp and preferably also has less affinity for human factor H than wild-type fHbp;

For example:

(A) a polypeptide containing 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 contains a fragment of SEQ ID NO: 5 that is at least 7 amino acids and which contains at least one of the residues specified 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 fHbp v2 mutant amino acid sequence includes a substitution at residue 32 only, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) is replaced;

if the mutant fHbp v2 amino acid sequence includes a substitution at residue 113 only, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) has been replaced.

if the fHbp v2 mutant amino acid sequence includes a substitution at residue 122 only, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) has been replaced;

if the fHbp v2 mutant amino acid sequence includes a substitution at residue 123, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) is replaced;

if the fHbp v2 mutant amino acid sequence includes a substitution at residue 124 only, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) has been replaced;

if the mutant fHbp v2 amino acid sequence includes a substitution at residue 127 only, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) has been replaced; and if the fHbp v2 mutant amino acid sequence includes a substitution at residue 240 only, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) is replaced;

or (B) a polypeptide containing a mutant fHbp v3 amino acid sequence, wherein: (a) the amino acid sequence shares at least 80% sequence identity with SEQ ID NO: 17 and/or contains a fragment of SEQ ID NO: 17 that is at least 7 amino acids and which 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 fHbp v3 mutant amino acid sequence includes a substitution at residue 32, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) is replaced;

if the fHbp v3 mutant amino acid sequence includes a substitution at residue 113, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) is replaced;

if the fHbp v3 mutant amino acid sequence includes a substitution at residue 125 only, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) has been replaced;

if the fHbp v3 mutant amino acid sequence includes a substitution at residue 126, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) is replaced;

if the fHbp v3 mutant amino acid sequence includes a substitution at residue 127, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) has been replaced;

if the fHbp v3 mutant amino acid sequence includes a substitution at residue 130, then either the substitution is not an alanine substitution, or at least one additional residue listed in (b) is replaced; and if the fHbp v3 mutant amino acid sequence includes a substitution at residue 243 only, then either the substitution is not an alanine substitution or at least one additional residue listed in (b) is replaced.

2. The polypeptide in accordance with embodiment 1(B), where the amino acid sequence differs from SEQ ID NO: 17 substitution at one or more than one of the residues listed in (b); for example, where the replacement(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. A polypeptide according to embodiment 1(B) or embodiment 2 containing more than one substitution at the residues listed in (b) and selected from groups 3A-3P as above.

4. The polypeptide in accordance with embodiment 1(A), where the amino acid sequence differs from SEQ ID NO: 5 substitution at one or more than one of the residues listed in (b); for example, when the replacement(s) is(are) 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. A polypeptide according to embodiment 1(A) or embodiment 4 containing more than one substitution at the residues listed in (b) and selected from groups 2A-2P as above.

6. A polypeptide according to any of embodiments 1-5, also comprising one or more additional(s) mutation(s) that(s) violate(s) the ability of the polypeptide to bind to human factor H; e.g., v2, including a replacement of one or more of R73, D203, E210, G228, S121, F122, L123, A192, E194, V199, I200, L201, T213, H215, F219, T231, and E240, or v3 , which includes replacing one or more of Q35, I178, L87, A88, L126, V127, V202, E213, T216, T234, V241, and E243.

7. A polypeptide containing:

the amino acid sequence of SEQ ID NO: 44, possibly with 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions, where the polypeptide can cause the formation of antibodies that bind to the meningococcal fHbp polypeptide, consisting of the amino acid sequence of SEQ ID NO : 40 (eg, containing the amino acid sequence of SEQ ID NO: 44 with 1, 2, or 3 single amino acid substitutions), but no mutation at residue V32 or R126;

the amino acid sequence of SEQ ID NO: 45, possibly with 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions, where the polypeptide can cause the formation of antibodies that bind to the meningococcal fHbp polypeptide, consisting of the amino acid sequence of SEQ ID NO : 2 (eg, containing the amino acid sequence of SEQ ID NO: 45 with 1, 2, or 3 single amino acid substitutions), but no mutation at residue V32 or R123;

amino acid sequence of fHbp v3 that is identical to the amino acid sequence of wild-type v3, except for a mutation at the amino acid position corresponding to Leu-126 in SEQ ID NO: 17, provided that the mutation is not a substitution for alanine (e.g., when the mutation is a substitution for arginine)

the amino acid sequence of fHbp v2 that is identical to the wild-type v2 amino acid sequence, except for the mutation at the amino acid position corresponding to Leu-123 in SEQ ID NO: 5, provided that the mutation is not a substitution for alanine (e.g., when the mutation is a substitution for arginine) or the amino acid sequence of SEQ ID NO: 47, optionally with 1, 2, 3, 4 or 5 single amino acid substitutions, deletions and/or insertions, where the polypeptide can cause the formation of antibodies that bind to each of: meningococcal fHbp polypeptide, consisting of the amino acid the sequences of SEQ ID NO: 46; 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 (eg, consisting of the amino acid sequence of SEQ ID NO: 48).

8. A plasmid or other nucleic acid containing a nucleotide sequence encoding a polypeptide according to any one of embodiments 1-7.

9. Host cell transformed with a plasmid according to embodiment 8; for example, where the cell is a meningococcal bacterium, such as the bacterium meningococcus, downregulating or knocking out the mltA gene and also possibly downregulating or knocking out: (1) at least one gene involved in conferring toxicity on a lipid A fragment in LPS, in particular lpxl1 gene; and/or (2) at least one gene involved in synthesis or export

- 26 043165 capsular polysaccharide or, in particular, synX and/or ctrA.

10. Membrane vesicles derived from a host cell according to embodiment 9, which comprise a polypeptide according to any one of embodiments 1-7.

11. An immunogenic composition comprising a polypeptide according to any of embodiments 1 to 7 or a vesicle according to embodiment 10.

12. A composition according to embodiment 11 further comprising a second polypeptide which, when administered to a mammal, elicits an antibody response that is bactericidal against meningococcus.

13. A composition according to embodiment 11 or 12, further comprising (1) N. meningitidis serogroup A, C, W135 and/or Y conjugated capsular saccharide and/or (2) S. pneumoniae conjugated capsular saccharide.

14. A method of inducing an antibody response in a mammal, comprising administering an immunogenic composition according to any one of embodiments 11-13.

Methods for implementing the invention

fHbp mutations.

It is believed that v2 fHbp is unstable. To analyze the structural causes underlying this undesirable property with the intent to design a sequence to improve stability, the inventors analyzed the sequence alignment and 3D structure of the fHbp polypeptides. One area of particular interest was the structural boundary between the N-terminal and C-terminal domains [168].

The inventors identified the mutations explained in the table. 1. Three of the positions identified for the mutation overlap with references 24 and 25, but the invention does not cover the polypeptides reported in the prior art, ie. when the polypeptides include substitutions exclusively at these positions with alanine. For example, E240 can be replaced with histidine to match v1, and is preferably in combination with a substitution at the H239 residue (mutants #1 and #11). Similarly, if F122 is substituted, it is preferably in combination with the S151 substitution, both with cysteine, to allow for the formation of a disulfide bridge (mutant #10). In addition, if L123 is substituted, it may be substituted for arginine (rather than alanine), or it may be combined with a substitution at other residues, such as at S32 (see mutant #3), at S125 (see mutants #20 and #22), or with a substitution at residues 124-128 (see mutant #12).

Stability studies.

Unstable proteins tend to fold less and are therefore prone to cleavage and degradation by proteases. Fig. 1 demonstrates that v2 fHbp 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.

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

Cell lysates of E. coli expressing recombinant proteins were incubated with chymotrypsin at a ratio of 1:100 w/w for 3 hours at 25°C. The degradation pattern was analyzed by Western blotting followed by incubation with immune polyclonal blood serum generated in a rabbit against all three fHbp variants. The presence of cleavage products with lower apparent molecular weight (FIGS. 2-4) is interpreted as an indicator of instability, while the presence of a band corresponding to an apparent MW of approximately 30 kDa is interpreted as an indicator of increased stability. All mutants #1-6, #12 and #22 showed increased resistance to chymotrypsin digestion compared to wild type v2.

DSC was used as an independent approach to evaluate the effects of mutations on the stability of purified recombinant fHbp v2 proteins. T _m (melting point), measured using DSC, corresponds to the temperature at which the analyzed protein is 50% folded and 50% unfolded. Changes that stabilize protein conformation increase Tm, while destabilizing changes decrease Tm. As seen in FIG. 3D in Reference 24, the DSC profile for wild type v2 fHbp shows two Tm values: T _m1 at approximately 40°C, which corresponds to the melting point of the N-terminal domain, and T _m2 at approximately 80°C, corresponding to the melting point of the C-terminal domain. . The values of T _m1 and T _m2 for the analyzed mutants are presented in table. 1. Mutants #2, #4, #5, #12, #19 and #21 showed a reduced Tm for the N-terminal domain compared to the wild-type protein, and this effect was more significant for mutants #2, #4 and # 12.

Size exclusion chromatography (SEC) was used to evaluate the percentage of monomeric protein.

- 27 043165 ka, and the results are also presented in table. 1.

Mutants #2, #3 and #4.

Mutant #3 (Group 2B) showed the best overall results in v2 stability studies. This protein (SEQ ID NO: 20) includes mutations in Ser-58 (S32 in SEQ ID NO: 5) and Leu149 (L123 in SEQ ID NO: 5), with substitutions for Val and Arg, respectively. The mutant v2 protein (SEQ ID NO: 20 containing SEQ ID NO: 45) was analyzed by DSC and, compared to the wild-type sequence, the Tm for its N-terminal domain was more than 20° C. higher (FIG. 5).

In a bactericidal serum assay, this v2 mutant can compete for binding with human antibodies that result from the use of the wild type v2 sequence.

Although the S58V and L149R mutations were introduced to improve stability and did achieve this goal, FIG. 6 shows that the mutant v2 polypeptide (dashed line) unexpectedly showed much reduced binding to fH compared to the wild-type v2 sequence (solid line) when measured by surface plasmon resonance against immobilized fH. The S58V mutation itself has little effect on fH binding, and the S58V/L149R double mutant showed higher binding to fH than to fHbp carrying only the L149R mutant.

When mutant #3 was further combined with the E313A mutation in v2, a complete loss of fH binding was then detected as measured by SPR.

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

As shown in FIG. 7, wild-type v3 binds to fH (top two bands). The S58V mutation, which was designed to improve stability, reduced the magnitude of the SPR peak by approximately 2-fold. Most surprisingly, the L149R mutation (again engineered to improve stability) reduced fH affinity to a level close to that of the known E313A mutant (bottom two bands).

Mutations of S58V and L149R in v3 were also examined by DSC and found to increase the N-terminal Tm by 5.5°C (S58V) or 6.7°C (L149R) compared to wild type. The Tm for both mutants was higher than that found in the v2 double mutant (63.5° C. - see Table 1). The L149R v3 mutant also showed a higher T _m value for its C-terminal domain, while there was almost no shift for the S58V v3 mutant. SPR showed that mutant #2 fH binding was approximately halved, but for mutant #4 the fH affinity was reduced to a level close to that of the known E313A mutant (consistent with that also found for v2). When the two mutations were combined (ie, mutant #3), then the Tm increase compared to wild type was 11.2°C. When the E313 A mutation was added to mutant #3, then binding to fH was completely abolished, although the Tm for the N-terminal domain also decreased by 2.9°C when compared to mutant #3 (remaining 8.3°C higher than for v3 wild type). The E313A mutation itself was much less stable than the wild type, showing a decrease in Tm by 6.3°C.

Thus, mutations #2 and #4 can be used alone or in combination (ie, mutant #3), possibly with additional mutations, to stabilize v2 or v3 fHbp as well as to break fH affinity.

Serum bactericidal assay was used to assess the immunogenic efficacy of mutants #3 and #4 in v2 and v3. In addition, the E313A mutant was also tested in v2 and v3, alone or in combination with mutations #3. For comparison, v2 and v3 wild-type fHbp were also tested. Proteins were administered at 20 μg/dose with aluminum hydroxide as an adjuvant, and the resulting serum was tested for bactericidal activity against a panel of seven strains (four v2 strains, three v3 strains) including strains that expressed the same fHbp, as the original wild-type fHbp sequences (i.e. v2 sequence 2.16 and v3 sequence 3.42).

The results for v2 proteins were also as follows (SEQ ID for AG form; * = homologous fHbp):

- 28 043165

Protein SEQ ID SBA vs Strains v2 SBA vs Strains v3 v2.16* v2.19 v2.21 v2.24 v3.42* v3.28 v3.30 wild type 5 32768 32 32 1024 >32768 <16 32 #4 21 32768 <16 <16 128 1024 <16 16 #3 45 4096 32 <16 <16 >32768 128 <16 #3+E313A 57 4096 16 <16 <16 >32768 <16 <16 E313A 56 128 16 <16 <16 4096 <16 <16

two mutants were combined in one fHbp (SEQ ID NO: 58, ΔG form). Compared to mutant #12, the Tm for the N-terminus of this combination mutant increased by an additional 4.2°C, resulting in the highest Tm for any of the v2 mutant proteins tested. In addition, the mutant showed a significantly reduced binding to fH (the magnitude of the SPR peak decreased by approximately 8-fold).

Mutant fusion protein.

The v2 and v3 mutant sequences were fused via a GSGGGG linker (SEQ ID NO: 50), also to the v1 mutant sequence to give SEQ ID NO: 48. This sequence includes the S58V and L149R mutations for both v2 and v3, and the R41S mutation [21] for v1. SEQ ID NO: 47 includes N-terminal to C-terminal: mutant v2 #3 (SEQ ID NO: 45); mutant v3 #3 (SEQ ID NO: 44); and a v1R41S mutant (SEQ ID NO: 49) linked by a glycine-rich linker sequence of SEQ ID NO: 50. The fusion protein can conveniently be expressed by adding the sequence Met[SEQ ID NO: 37]- at the N-terminus, thus providing mature protein SEQ ID NO: 48.

Thus, this fusion protein has the advantage that mutant #3 was found to confer on both v2 and v3 a significant increase in stability (Tm) and a significant decrease in fH affinity. For v1, the R41S mutation has little effect on temperature stability, but significantly reduces binding to fH.

DSC studies of the triple fusion protein (FIG. 8) demonstrate that the three N-terminal junctions are together within a broad peak centered at 68°C. Three End crossings also ended up together. UPLC (Ultra High Performance Liquid Chromatography) showed that the protein was 94.9% pure, and HPLC (High Performance Liquid Chromatography) analysis showed less than 1.5% oligomers.

Mutant proteins expressed in GMMA membrane vesicles.

The v1 meningococcal strain was obtained with knockouts for the mltA, lpxL1, and synX genes and obtained a genetic background for overexpression of v2 and v3 fHbp lipoproteins under the control of the ST2 promoter [157] in the GMMA vaccine. The v2 genes were inserted into the genome at the deleted lpxL1 locus, while the v3 genes were inserted at the synX locus. In addition, the native v1 fHbp gene was deleted so that v2 and v3 could be examined without interference.

- 29 043165

Mutants #3 and #4 were tested for v2 and mutant #4 were tested for v3. In addition, received a strain with both mutants v2 and v3 #4. For these bacteria, fHbp expression and fH binding were assessed using FACS (fluorescence activated cell sorter).

For strains expressing only v2 fHbp, FACS demonstrated that different proteins were expressed at similar levels, with levels 2 log higher than the base AfHbp strain. However, in terms of fH binding, mutants #3 and #4 showed much less binding, with mutant #4 binding only slightly above background. These results reflect SPR data obtained with recombinant v2 proteins.

For the strain expressing the v3 #4 mutant, FACS showed full expression of fHbp, but its binding to fH was abolished (corresponding to the fH binding found for the H222R mutation [19,25]).

For the strain expressing mutant #4 from v2 and v3, both fHbp proteins could be detected by FACS, but binding to fH was only slightly higher than that found for the base AfHbp strain.

Western blot analysis was used to test the stability of fHbp expression in these bacteria grown in liquid culture for 6 days. Expression of the v2 mutant proteins remained stable over time even when v3 was co-expressed. Expression of the v3 mutant proteins also remained stable except for the strain expressing both v2 and v3 mutants where v3 expression decreased over time.

It is understood that the invention has been described above solely by way of example, and modifications may be made while remaining within the scope and spirit of the invention.

Sequence Listing >SEQ ID NO: 1 [MC58, vl]

MNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSV

RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVY KQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFG SDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQA EKGSYSLG IFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ >SEQ ID NO: 2 [2996, v2]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGI<LTYTIDFA <QGHGI<IEHLI<TPEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI< GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 3 [M1239, v3]

MNRTAFCCLSLTTALILTACSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLT

LEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLA SGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYH GKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDT RYGSEKGTYHLALFG DRAQEIAGSATVKIGEKVHEIGIAGKQ

- 30 043165 >SEQ ID NO: 4 [mature 2996]

KIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGH GKIEHLKTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGS ATVKIGEKVHEIGIAGKQ >SEQ ID NO: 5 [2996 A G]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQ RSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELAAAEL KADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEK VHEIGIAGKQ >SEQ ID NO: 6 [NHBA]

MFKRSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQ GQGAPSAQGSQDMAAVSEENTGNGGAVTADNPKNEDEVAQNDMPQNAAGTDSSTPN HTPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGGQNAGNTAA QGANQAGNNQAAGSSDPIPASNPAPANGGSNFGR VDLANGVLIDGPSQNITLTHCKGDS C SGNNFLDEEVQLKSEFEKLSD ADKISNYKKDGKNDKF VGLVAD S VQMKGINQ YIIF YK PKPTSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLT YGAEKLPGGSYALRVQGEPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRAFAAKV D FGSKSVDGHDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAG KYSYRPTDAEKGGFGVFAGKKEQD >SEQIDNO: 7 [NadA]

MSMKHFPSKVLTTAILATFCSGALAATSDDDVKKAATVAIVAAYNNGQEINGFKAGETI YDIGEDGTITQKDATAADVEADDFKGLGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEI EKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKIDEKLEAVADT VDI<HAEAFNDIADSLDETNTI<ADEAVI< TANEAI<QTAEETI<QNVDAI<VI<AAETAAGI<A EAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQ GLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFRFTENFAAKAGVAVGTS SGS SAAYHVGVNYEW >SEQ ID NO: 8 [NspA]

- 31 043165

MKKALATLIALALPAAALAEGASGFYVQADAAHAKASSSLGSAKGFSPRISAGYRINDL RFAVDYTRYKNYKAPSTDFKLYSIGASAIYDFDTQSPVKPYLGARLSLNRASVDLGGSD SFSQTSIGLGVLTGVSYAVTPNVDLDAGYRYNYIGKVNTVKNVRSGELSAGVRVKF >SEQ ID NO: 9 [HmBR]

MKPLQMLPIAALVGSIFGNPVLAADEAATETTPVKAEIKAVRVKGQRNAPAAVERVNL NRIKQEMIRDNKDLVRYSTDVGLSDSGRHQKGFAVRGVEGNRVGVSIDGVNLPDSEEN SLYARYGNFNSSRLSIDPELVRNIEIVKGADSFNTGSGALGGGVNYQTLQGRDLLLDDR QFGVMMKNGYSTRNREWTNTLGFGVSND RVDAALLYSQRRGHETESAGNRGYAVEG EGSGANIRGS ARGIPD S SKHKYNHHALGKIAYQINDNHRIGASLNGQQGHNYTVEES YN LTASSWREADDVNRRRNANLFYEWMPDSNWLSSLKADFDYQKTKVAAVNNKGSFPM DYSTWTRNYNQKDLDEIYNRSMDTRFKRFTLRLDSHPLQLGGGRHRLSFKTFVSR RDFE NLNRDD YYF SGRVVRTT S SIQHP VKTTN YGF SLSDQIQWND VF S SRAGIRYDHTKMTPQ ELNAECHACDKTPPAANTYKGWSGFVGLAAQLNQAWRVGYDITSGYRVPNASEVYFT YNHGSGNWLPNPNLKAERSTTTHTLSLQGRSEKGMLDANLYQSNYRNFLSEEQKLTTSG TPGCTEENAY YGICSDPYKEKLDWQMKNIDKARIRGIELTGRLNVDKVASFVPEGWKLF GSLGYAKSKLSGDNSLLSTQPLKVIAGIDYESPSEKWGVFSRLTYLGAKKVKDAQYTVY ENKGWGTPLQKKVKDYPWLNKSAYVFDMYGFYKPAKNLTLRAGVYNLFNRKYTTWD SLRGLYSYSTTNAVDRDGKGLDRY RAPGRNYAVSLEWKF >SEQ ID NO: 10 [NhhA]

MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEE DLYLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNL KIKQNGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDT TVHLNGIGSTLTDTLLNGATTNVTN DNVTDDEKKRAASVKDVLNAGWNIKGVKPGTT ASDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTG KDKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNV TFASGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLD SKAVAGSSGKVI SGNVSPSKGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDG DALNVGSKKDNKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIA QAIATAGLVQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHF GASASVGY QW >SEQ ID NO: 11 [App]

- 32 043165

MKTTDKRTTETHRKAPKTGRIRFSPAYLAICLSFGILPQAWAGHTYFGINYQYYRDFAE NKGKFAVGAKDIEVYNKKGELVGKSMTKAPMIDFSVVSRNGVAALVGDQYIVSVAHN GGYNNVDFGAEGRNPDQHRFTYI<IVI<RNNYI<AGTI<GHPYGGDYHMPRLHI<FVTDAEP VEMTSYMDGR KYIDQNNYPDRVRIGAGRQYWRSDEDEPNNRESSYHIASAYSWLVGG NTFAQNGSGGGTVNLGSEKIKHSPYGFLPTGGSFGDSGSPMFIYDAQKQKWLINGVLQT GNPYIGKSNGFQLVRKDWFYDEIFAGDTHSVFYEPRQNGKYSFNDDNNGTGKINAKHE HNSLPNRLKTRTVQLFNVSLSETAREPVYHAAG GVNSYRPRLNNGENISFIDEGKGELIL TSNINQGAGGLYFQGDFTVSPENNETWQGAGVHISEDSTVTWKVNGVANDRLSKIGKG NKDIATTGNN SLDSKKEIAYNGWFGEKDTTKTNGRLNLVYQPAAEDRTLLLSGGTNLNGNITQTNGKLF FSGRPTPHAYNHLNDHWSQKEGIPRGEIVWDNDWINRTFKAENFQIKGGQAVVSRNVA KVKGDWHLSNHAQAVFGVAPHQSHTICTRSDWTGLTNCVEKTITDDKVIASLTKTDISG NVDLADHAHLN LTGLATLNGNLSANGDTRYTVSHNATQNGNLSLVGNAQATFNQATL NGNTSASGNASFNLSDHAVQNGSLTLSGNAKANVSHSALNGNVSLADKAVFHFESSRF TGQISGGKDTALHLKDSEWTLPSGTELGNLNLDNATITLNSAYRHDAAGAQTGSATDAP RRRSRRSRRSLLSVTPPTSVESRFNTLTVNGKLNGQGTFRFMS ELFGYRSDKLKLAESSE GTYTLAVNNTGNEPASLEQLTVVEGKDNKPLSENLNFTLQNEHVDAGAWRYQLIRKDG EFRLHNPVKEQELSDKLGKAEAKKQAEKDNAQSLDALIAAGRDAVEKTESVAEPARQA GGENVGIMQAEEEKKRVQADKDTALAKQREAETRPATTAFPRARRARRDLPQLQPQPQ PQPQRDLISRYANSGLSEFSATLNSVFAVQDELDRVFAEDRRNAVWTSGIRDTKHYRSQ DFRAYRQQTDLRQIGMQKNLGSGRVGILFSHNRTENTFDDGIGNSARLAHGAVFGQYGI DRFYIGISAGAGFSSGSLSDGIGGKIRRRVLHYGIQARYRAGFGGFGIEPHIGATRYFVQK ADYRYENVNIATPGLAFNRYRAGI KADYSFKPAQHISITPYLSLSYTDAASGKVRTRVNT AVLAQDFGKTRSAEWGVNAEIKGFTLSLHAAAAKGPQLEAQHSAGIKLGYRW >SEQ ID NO: 12 [Omp85]

MKLKQIASALMMLGISPLALADFTIQDIRVEGLQRTEPSTVFNYLPVKVGDTYNDTHGS AIIKSLYATGFFDDVRVETADGQLLLTVIERPTIGSLNITGAKMLQNDAIKKNLESFGLAQ SQYFNQATLNQAVAGLI<EEYLGRGI<LNIQITPI<VTI<LARNRVDIDITIDEGI<SAI<ITDIEF EGNQV YSDRKLMRQMSLTEGGIWTWLTRSNQFNEQKFAQDMEKVTDFYQNNGYFDFR ILDTDIQTNEDKTKQTIKITVHEGGRFRWGKVSIEGDTNEVPKAELEKLLTMKPGKWYE RQQMTAVLGEIQNRMGSAGYAYSEISVQPLPNAETKTVDFVLHIEPGRKIYVNEIHITGN NKTRDEVVRRELRQMESAPYDTSK LQRSKERVELLGYFDNVQFDAVPLAGTPDKVDLN MSLTERSTGSLDLSAGWVQDTGLVMSAGVSQDNLFGTGKSAALRASRSKTTLNGSLSF

- 33 043165

TDPYFTADGVSLGYDVYGKAFDPRKASTSIKQYKTTTAGAGIRMSVPVTEYDRVNFGL VAEHLTVNTYNKAPKHYADFIKKYGKTDGTDGSFKGWLYKGTVGWGRNKTDSALWP TRGYLTGVNAEIALPGSKLQYYSATHNQTWFFPLSKTFTLMLGGEVGIAGGYGRTKEIP FFENFYGGGLGSVRGY ESGTLGPKVYDEYGEKISYGGNKKANVSAELLFPMPGAKDAR TVRLSLF AD AGS VWDGKT YDDNS S S ATGGRVQNIYGAGNTHKSTFTNELRYS AGGAVT WLSPLGPMKFSYAYPLKKKPEDEIQRFQFQLGTTF >SEQ ID NO: 13 [NMB2091]

MVSAVIGSAAVGAKSAVDRRTTGAQTDDNVMALRIETTARSYLRQNNQTKGYTPQISV VGYDRHLLLLGQVATEGEKQFVGQIARSEQAAEGVYNYITVASLPRTAGDIAGDTWNT SKVRATLLGISPATRARVKIVTYGNVTYVMGILTPEEQAQITQKVSTTVGVQKVITLYQN YVQR >SEQ ID NO: 14 [NHBA merger]

MASPDVKSADTLSKPAAPVVSEKETEAKEDAPQAGSQGQGAPSAQGGQDMAAVSEEN TGNGGAAATDKPKNEDEGAQNDMPQNAADTDSLTPNHTPASNMPAGNMENQAPDAG ESEQPANQPDMANTADGMQGDDPSAGGENAGNTAAQGTNQAENNQTAGSQNPASST NPSATNSGGDFGRTNVGNSVVIDGPSQNI TLTHCKGDSCSGNNNFLDEEVQLKSEFEKLSD ADKISNYKDGKNDGKNDKFVGLVADSVQMKGINQYIIFYKPKPTSFARFRRSARSRRS LPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLPGGSYALRVQ GEPSKGEMLAGTAVYNGEVLHFHTENGRPSPSRGRFAAKVDFGSKSVDGIIDSGD HGK TAPVKLKAEKFNCYQSPMAKTEVCGGDFSTTIDRTKWGVDYLVNVGMTKSVRIDI QIEAAKQ >SEQ ID NO: 15 [NadA fragment]

ATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDATAADVEADDF KGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDA TTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADE AVKTANEAKQTAEETKQNVDAKVKAAETA AGKAEAAAGTANTAADKAEAVAAKVT DIKADIATNKDNIAKKANSADVYTREESDSKFVRIDGLNATTEKLDTRLASAEKSIADHD TRLNGLDKTVSDLRKETRQGLAEQAALSGLFQPYNVG

- 34 043165 >SEQ ID NO: 16 [MC58, ΔG]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMV AKRQFRIGDIAGEHTSFKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEH LKSPELNVDL AAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKT VNGIRHIGLAAKQ >SEQ ID NO: 17 [M1239, Δ G]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL KTLEQNVELAAAELKADE KSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ >SEQ ID NO: 18 [MUTANT #1

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVRHIGIAGKQ >SEQ ID NO: 19 [MUTANT #2]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQVV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVRHIGIAGKQ >SEQ ID NO: 20 [MUTANT #3]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQVV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQffiKINNPDKIDSLINQRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTID FAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

- 35 043165 >SEQ ID NO: 21 [MUTANT #4]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 22 [MUTANT #5]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVGDLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTID FAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 23 [MUTANT #6]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVTALQIEKINNPDKIDSLINQRSFLVGDLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTID FAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 24 [MUTANT #7]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKSDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 25 [MUTANT #8]

MNRTAFCCLSLT AALILT AC S SGGGGVAADIGAGLAD ALTAPLDHKDKSLQ SLTLDQSC RKNEKCKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTID FAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

- 36 043165 >SEQ ID NO: 26 [MUTANT #9]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKCAAQGAEKTYGNGDSLNTGKLKNDKVSRCDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 2 7 [MUTANT #10]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSCLVSGLGGEHTAFNQLPDGKAEYHGKAFSC DDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 28 [MUTANT #11]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVTALQIEKINNPDKIDSLINQRSFLVGDLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTID FAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVRHIGIAGKQ >SEQ ID NO: 29 [MUTANT #12]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFRIGDIAGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAKQ GHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 30 [MUTANT #13]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVTALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

- 37 043165 >SEQ ID NO: 31 [MUTANT #14]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKCAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 32 [MUTANT #15]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSCLVSGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 33 [MUTANT #19]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQVV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVTGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 34 [MUTANT #20]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQVV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFRVTGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTIDFA AKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 35 [MUTANT #21]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQVV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVGGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTID FAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

- 38 043165 >SEQ ID NO: 36 [MUTANT #22]

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQVV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFRVGGLGGEHTAFNQLPDGKAEYHGKAFSS DDAGGKLTYTID FAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEE KGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 37

GPDSDRLQQRR >SEQIDNO: 38

GSKDISS >SEQIDNO:39

GSKDISSGGGG >SEQ ID NO: 40 [Mature M1239]

TKKQGYGRIEHLK TLEQNVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDR AQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 41 [v3(M1239), E243A, A G]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL KTLEQNVELAAAELKADE KSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHAIGIAGKQ >SEQ ID NO: 42 [v3(M1239), S32V+E243A, AG]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL

- 39 043165

KTLEQNVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIG EKVHAIGIAGKQ >SEQ ID NO: 43 [v3(M1239), S32V+L126R+E243A, ΔG]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL KTLEQNVELAAAELKADE KSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHAIGIAGKQ >SEQ ID NO: 44 [v3, S32 V + LI26R, Δ GJ

VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL KTLEQNVELAAAELKADE KSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ >SEQ ID NO: 45 [v2 MUTANT #3 S32 V + L123R, Δ GJ

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHL KTPEQ NVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ >SEQ ID NO: 46 [mature MC58, vl]

CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTY GNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDS EHSGKMVAKRQFRIGDIAGEHTSFKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQ GNGKIEHLKSPELN VDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVA GSAEVKTVNGIRHIGLAAKQ >SEQID NO: 47 [mutant merged v2-v3-v-l]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHL

- 40 043165

KTPEQNVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDK TDSLINQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEKGTYH LALFGDRAQEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHKD KGLQSLTLDQSVSKNEKLKLAAQGAEKTYGNG DSLNTGKLKNDKVSRFDFIRQIEVDG QLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPE GGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKR HAVISGSVLYNQAEKGSYSLGIFGKAQEVAGSAEVKTVNGIRHIGLA AKQ >SEQID NO: 48 [mutant fusion, leader sequence v2-v3-v-l]

MGPDSDRLQQRRVAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQG AEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIE KINNPDKIDSLINQRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFA AKQGHGKIE HLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRA QEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTL EDVIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLAS GEFQIYKQNHSAVVALQIEKINNPDK TDSLINQRSFRVSGLGGEHTAFNQLPGGKAEYH GKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDT RYGSEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLA DALTAPLDHKDKGLQSLTLDQSVSKNEKLKLAAQGAEKTYGNGD SLNTGKLKNDKVS RFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIA GEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTroFAAKQGNGKIEHLKSPELNVDLA AADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGL AA KQ >SEQ ID NO: 49 [MC58, ΔG, ‘R41S’ mutation]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVSKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMV AKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEH LKSPELNVDL AAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKT VNGIRHIGLAAKQ >SEQ ID NO: 50 [linker]

- 41 043165

GSGGGG >SEQ ID NO: 51 [wild-type v2 sequence, e.g. for GMMA approach]

VAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKroSLINQ RSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELAAA ELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIGEK VHEIGIAGKQ >SEQ ID NO: 52 [wild-type v3 sequence, e.g. for GMMA approach]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKM VAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNV ELATAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVK IREK VHEIGIAGKQ >SEQ ID NO: 53 [ml239, mutation L126R]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL KTLEQNVELAAAELKADE KSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ >SEQ ID NO: 54 [v2, wt strain 8047]

MNRTAFCCLSLTTALILTACSSGGGGVAADIGARLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY KQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSD DAGGKLTYTIDFAAK QGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEK GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ >SEQ ID NO: 55 [v2, strain 8047, A G]

VAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLINQ RSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT

- 42 043165

PEQNVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIGEK VHEIGIAGKQ >SEQ ID NO: 56 [v2, mutant ‘E313A A G]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQ RSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKT PEQNVELAAAEL KADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIGEK VHAIGIAGKQ >SEQ ID NO: 5 7 [v2, mutant #3 + Έ313Α A G]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHL KTPEQ NVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIG EKVHAIGIAGKQ >SEQ ID NO: 58 [MUTANT #2 + #12]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFRIGDIAGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLK TPEQNVELAAAELKA DEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGSATVKIGE TO VHEIGIAGKQ >SEQ ID NO: 59 [v2, strain 8047, mutant #4, mature]

KIDSLINQRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGH GKIEHLKTPE QNVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAGS ATVKIGEKVHEIGIAGKQ >SEQ ID NO: 60 [v2, strain 8047, mutant #3, mature]

CSSGGGGVAADIGARLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTY GNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNP DKIDSLINQRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQG HGKIEHLKTPE QNVELAAAELKADEKSHAVILGDTRYGSEKGTYHLALFGDRAQEIAG SATVKIGEKVHEIGIAGKQ >SEQ ID NO: 61 [v3, mutant #2 + #12, AG]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFRIGDIAGEHTAFNQLPGGKAEYHGKAF S SDDPNGRLHYSIDFTKKQG YGRIEHL KTLEQNVELAAAELKADE KSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ

- 43 043165

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Table 1

# Balance(s)* Mutation(s) Directions SEQ ID NO Him yy Tsh1 °C Tsh2 °C Mon yyy 1 H239+E240 R, H Surface between N- and C-terminal domains. The challenge is to mimic vl. An additional transition to DSC was found at 100.3° C. and some aggregation was found. 18 Yes N/A (no data) 83.5 34.2 9 2 S32 V N-terminal domain. The hydrophilic side chain of S32 is directed into the hydrophobic cavity. The task is to increase the hydrophobicity and stabilize the cavity. 19 Yes 57.0 84.2 80.3 8 3 S32+L123 V, R Mutants #2 + #4 20 Yes 63.5 83.8 4 76.1 4 L123 R N-terminal domain. In vl, the reverse change reduced the stability of [I]. 21 Yes 54D 84.1 89.9 7 5 S125+G126 G, D N-terminal domain. The challenge is to mimic vl 22 Yes 52.3 83.3 90.4 8

- 51 043165

6 V100+S125 +G126 T, G, D N-terminal domain. The challenge is to mimic vl 23 Yes 52 83.7 86.4 1 7 1113 S Surface loop of the N-terminal domain. Removal from the surface of a possible protease cleavage site. 24 No n/a n/a n/a 8 V33+L39 C, C Core of the N-terminal domain. Introduction of S-S bridging. Some aggregation detected. 25 No 55.9 85 28.7 1 9 L41+F69 with, with — 33.5 Core of the N-terminal domain. Introduction of S-S bridging. 26 No 46.4 84.4 + 53.2 2 10 F122+S151 with, with Core of the N-terminal domain. Introduction of S-S bridging. 27 No - - - AND V100+S125 T, G, D, — +G126+H2 R, N Mutants #1 + #6 28 No 47.9 82.2 85.1 39+E240 12 L123-G128 RIGDIA N-terminal domain. The task is to mimic vl in the entire area 123-128 29 Yes 62.8 84.4 71.2 8 13 V100 T Partial Mutant #6 thirty No 43 84.3 91.0 6 14 L41 WITH Partial mutant #9. Some aggregation detected. 31 No - 85 24.8 2 15 F122 WITH Partial mutant #10. Some aggregation detected. 32 No - 84.4 16.6 3 19 S32+S125 V, T N-terminal domain. Additional increase in hydrophobicity compared to #2 33 No 50.6 83.5 81.3 20 S32+S125+ L123 V, T, R Combination #4 + #19 34 No - - - 21 S32+S125 V, G N-terminal domain. Additional increase in hydrophobicity compared to #2 35 No 52.8 84 75.3 22 S32+S125+ L123 V, G, R Combination #4 + #21 36 Yes - - - 23 S32+L123- G128 RIGDIA S Combination #2 + #12 62 Yes 66.3 84.7 -

* Numbered according to SEQ ID NO: 5; +26 added to match SEQ ID NO: 18-39.

** Resistance to cleavage by chymotrypsin.

***% monomeric form.

Claims (15)

1) v1 fHbp polypeptide; 1) a polypeptide having the amino acid sequence of SEQ ID NO: 45, possibly modified by up to 5 single amino acid substitutions, deletions and/or insertions; and/or 1) the amino acid sequence of the mutant v2 fHbp polypeptide further differs from SEQ ID NO: 5 by a substitution at one or more of L123, V124, S125, G126, L127 and/or G128, where the substitution(s) is(are) selected from the group, consisting of: L123R; V124I; S125G or S125T; G126D; L127I; G128A; and/or 1) mutant v2 fHbp polypeptide contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5, and/or contains a fragment of SEQ ID NO: 5, which is at least 7 amino acids long and which contains a residue corresponding to residue 32 in SEQ ID NO: 5; where the amino acid sequence of the mutant v2 fHbp differs from SEQ ID NO: 5 at residue 32 by substitution S32V; And 1. A fusion polypeptide containing a mutant variant 2 of the factor H binding polypeptide (v2 fHbp) and a mutant variant 3 of the factor H binding polypeptide (3 fHbp), where: 2) mutant v2 fHbp polypeptide; And 2) a polypeptide having the amino acid sequence of SEQ ID NO: 44, possibly modified by up to 5 single amino acid substitutions, deletions and/or insertions. 2) the amino acid sequence of the mutant v3 fHbp polypeptide further differs from SEQ ID NO: 17 by a substitution at L126, where the substitution is L126R. 2. The polypeptide according to claim 1, where: 2) the mutant v3 fHbp polypeptide contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 17 and/or contains a fragment of SEQ ID NO: 17 that is at least 7 amino acids long and contains a residue corresponding to residue 32 in SEQ ID NO: 17; where the amino acid sequence of the mutant v3 fHbp differs from SEQ ID NO: 17 at residue 32 by substitution S32V. - 52 043165 3) a mutant v3 fHbp polypeptide, wherein the polypeptides are in the order v2 fHbp polypeptide, v3 fHbp polypeptide, and v1 fHbp polypeptide from N-terminus to C-terminus. 3. A polypeptide according to any one of claims 1, 2, containing: 4. A polypeptide according to any one of claims 1 to 3, wherein the v2 fHbp mutant polypeptide and/or the v3 fHbp mutant polypeptide is further combined with a v1 fHbp polypeptide. 5. The polypeptide of claim 4, wherein the v1 fHbp polypeptide contains an amino acid sequence having at least 80% identity with SEQ ID NO: 16 and/or contains a fragment of SEQ ID NO: 16. 6. A polypeptide according to any one of claims 3-5, which contains: 7. A polypeptide according to any one of claims 4 to 6, wherein the v2 fHbp mutant, v3 fHbp mutant, and v1 fHbp mutant polypeptides are joined by a linker having the amino acid sequence of SEQ ID NO: 50 between each of the sequences. 8. A plasmid or other nucleic acid containing a nucleotide sequence encoding a polypeptide according to any one of claims 1-7. 9. Membrane vesicles obtained from a host cell transformed with a plasmid according to claim 8, where the vesicles comprise a polypeptide according to any one of claims 1-7. 10. An immunogenic composition containing a polypeptide according to any one of claims 1 to 7 or membrane vesicles according to claim 9. 11. The composition of claim 10, further comprising (1) a conjugated capsular saccharide from N. meningitidis serogroup A, C, W135 and/or Y, and/or (2) a conjugated capsular saccharide from S. pneumoniae. 12. A polypeptide according to any one of claims 1 to 7 as a drug. 13. Composition according to any one of claims 10, 11 as a medicine. 14. A polypeptide according to any one of claims 1 to 7 for preventing Neisseria infection. 15. Composition according to any one of claims 10, 11 for preventing infection caused by Neisseria.