EA046480B1 - MODIFIED MENINGOCOCCAL fHbp POLYPEPTIDES - Google Patents

Description

Technical field

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

Prior Art

Invasive meningococcal disease (IMD) is caused by the bacterial pathogen Neisseria meningitidis. Of the five serogroups predominantly associated with IMI worldwide (MenA, B, C, W, and Y), MenB is the predominant serogroup, causing IMI in a number of regions, including Canada, the United States of America, Australia, New Zealand, and Europe. MenB causes a severe and often fatal disease, mainly affecting children and young adults. It is often misdiagnosed, can cause death within 24 hours of onset, and can lead to permanent disability despite treatment.

There are currently two licensed vaccines against meningococcal serogroup B: BEXSERO from GSK and TRUMENBA from Pfizer.

BEXSERO (also known by the generic name 4CMenB) contains an outer membrane vesicle (OMV) formulation from the epidemic group B meningococcal strain NZ98/254 along with five meningococcal antigens: Neisseria heparin binding protein A (NHBA), factor H binding protein (fHbp) variant 1.1 , Neisseria adhesion protein A (NadA) and accessory proteins GNA1030 and GNA2091. Four of these antigens are in the form of fusion proteins (NHBA-GNA1030 fusion protein and GNA2091-fHbp fusion protein). 4CMenB is described in the literature (for example, see Bai et al. (2011), Expert. Opin. Biol. Ther. 11:969-85, Su & Snape (2011), Expert. Rev. Vaccines, 10:575-88) . The terms BEXSERO and 4CMenB are used interchangeably in this document.

TRUMENBA contains two lipidated fHbp MenB antigens (v1.55 and v3.45) adsorbed on aluminum phosphate.

fHbp (also known in the art as Neisseria genome-derived antigen (GNA) 1870, LP2086, and protein 741) binds to human factor H (hfH), which is a large (180 kDa) multidomain soluble glycoprotein consisting of 20 complement-regulating proteins elements (SSR) connected by short linker sequences. hfH circulates in human plasma and regulates the alternative complement pathway. Functional binding of fHbp to hfH is predominantly dependent on CCP 6-7 elements (or domains) of hfH and increases bacterial resistance to complement-mediated cytolysis. Thus, fHbp expression mediates survival in human blood and serum ex vivo.

Various fHbp classification schemes have been proposed; there is a specialized database with a unified fHbp nomenclature to designate new subvariants: (http)://neisseria.org/nm/typing/fhbp (and also (https)://pubmlst.org/neisseria/fHbp /).

In the fHbp classification, there are three (main) variants 1, 2 and 3, which are further subdivided into subvariants fHbp-1.x, fHbp-2.x and Shbp-3.x, where x denotes a specific peptide subvariant. Unlike v2 and v3, fHbp v1 is highly heterogeneous and includes several subvariants. Another nomenclature groups subvariants into subfamily A (corresponding to variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity.

BEXSERO is predicted to provide broad overlap among MenB strains circulating worldwide (Medini D. et al., Vaccine, 2015; 33:2629-2636; Vogel U. et al., Lancet Infect Dis. 2013; 13:416-425; Knzova et al., Epidemiol. Mikrobiol. Imunol. 2014;63:103-106; Tzanakaki G. et al., BMC Microbiol. 2014;14:111; Wasko I. et al., Vaccine, 2016;34:510- 515; 6. Simoes M. J. et al., PLoS ONE, 12(5):e0176177; and Parikh S. R. et al., Lancet Infect. Dis. 2017; 17:754-62). Additionally, following the introduction of BEXSERO into the UK national childhood immunization program in September 2015, 10-month data showed 83% vaccine efficacy against all MenB strains after two doses (Parikh S.R. et al., Lancet, 2016;388:2775 -82).

However, bactericidal activity is variant specific; antibodies against one variant will not necessarily provide cross-protection against the other variants, although cross-activity has been described for fHbp v2 and v3 (Masignani V. et al., J. Exp. Med. 2003; 197:789799). Antibodies against the fHbpv1.1 subvariant included in the 44CMenB vaccine are highly cross-reactive with the most common fHbp v1 subvariants, but less cross-reactive with v1 subvariants that have the lowest affinity to v1.1. In addition, antibodies against the fHbpv1.1 subvariant included in the 44CMenB vaccine have poor cross-reactivity with fHbp v2 and v3 (Brunelli B et al., Vaccine, 2011; 29:1072-1081). This means that 44CMenB does not overlap with some meningococcal strains carrying fHbp v2, v3, or strains carrying some v1 subvariants.

Thus, despite the effectiveness of licensed serogroup B meningococcal vaccines, such as BEXSERO, there remains a need to develop meningococcal vaccines that maintain efficacy and are as effective as existing approved vaccines, such as 44CMenB, but have better coverage of meningococcal strains harboring fHbp variants, which

- 1 046480 are poorly covered by existing vaccines.

Summary of the invention

In a first aspect of the invention, there is provided a mutant meningococcal polypeptide fHbp v1.13 comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the amino acid sequence of said mutant meningococcal polypeptide fHbp v1.13 includes the substitution mutation E211, S216, E232 in the sequence SEQ ID NO: 2 or combinations thereof.

A second aspect of the invention provides a mutant fHbp v1.15 meningococcal polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6, wherein the amino acid sequence of said fHbp v1.15 mutant meningococcal polypeptide includes a substitution mutation E214, S219, E235 in the sequence of SEQ ID NO: 6 or combinations thereof.

In a third aspect of the invention, a fusion polypeptide is provided comprising all three meningococcal fHbp polypeptides v1, v2 and v3, wherein the fHbp sequence variants are arranged in the order v2-v3-v1 from N- to C-terminus and wherein the v1 polypeptide is a mutant fHbp v1 polypeptide. 13 according to the first aspect of the invention or the mutant fHbp v1.15 polypeptide according to the second aspect of the invention.

A fourth aspect of the invention provides an isolated nucleic acid molecule, possibly a plasmid, comprising a nucleotide sequence encoding a mutant fHbp v1.13 polypeptide according to the first aspect of the invention, a mutant fHbp v1.15 polypeptide according to the second aspect of the invention, or a fusion polypeptide according to the third aspect of the invention.

A fifth aspect of the invention provides a recombinant host cell transformed with a nucleic acid molecule according to the fourth aspect of the invention.

The sixth aspect of the invention provides outer membrane vesicles obtained or prepared from a recombinant host cell according to the fifth aspect of the invention.

A seventh aspect of the invention provides an immunogenic composition comprising a mutant fHbp v1.13 polypeptide according to the first aspect of the invention, a mutant fHbp v1.15 polypeptide according to the second aspect of the invention, a fusion polypeptide according to the third aspect of the invention, or an outer membrane vesicle according to the sixth aspect of the invention. Said immunogenic composition is useful for immunizing a mammal, preferably a human, against infection caused by Neisseria meningitidis.

Description of graphic materials

Fig. 1A is a graph showing the occurrence of meningococcal B strains expressing fHbp v1.x subvariants and showing which of these v1.x subvariants are overlapped (black) or not overlapped (white) by the 44CMenB vaccine.

Fig. 1B is a graph showing the occurrence of meningococcal B strains carrying fHbp v2 and showing which of these strains overlap (black) or do not overlap (white) with the 44CMenB vaccine.

Fig. 1C is a graph showing the occurrence of meningococcal B strains carrying fHbp v3 and showing which of these strains are overlapped (black) or not overlapped (white) by the 44CMenB vaccine.

Fig. 2 includes four graphs (thermograms) comparing differential scanning calorimetry (DSC) data for fHbp v1.1, fHbp v1.13 (wild type) and fHbp v.1.13 E211A (Fig. 2A); fHbp v.1.13 S216R (Fig. 2B); fHbp v.1.13 E211A/E232A (Fig. 2C) and fHbp v.1.13 E211A/S216R (Fig. 2D).

Fig. 3 includes four graphs (sensorgrams) comparing the binding of factor H domains 6-7 to fHbp v1.1, fHbp v1.13 (wild type) and fHbp v.1.13 E211A (Fig. 3A); fHbp v.1.13 S216R (Fig. 3B); fHbp v.1.13 E211A/E232A (Fig. 3C) and fHbp v.1.13 E211A/S216R (Fig. 3D).

Fig. 4A-D includes four graphs (sensorgrams) comparing the binding of full-length factor H protein to fHbp v1.1, fHbp v1.13 (wild type) and fHbp v.1.13 E211A (Fig. 4A); fHbp v.1.13 S216R (Fig. 4B); fHbp v.1.13 E211A/E232A (Fig. 4C) and fHbp v.1.13 E211A/S216R (Fig. 4D).

Fig. 5A-D includes four graphs (sensorgrams) comparing the binding of factor H domains 6-7 to fHbp v1.1, fHbp v1.15 (wild type) and fHbp v.1.15 E214A (Fig. 5A); fHbp v.1.15 S219R (Fig. 5B); fHbp v.1.15 E214A/E235A (Fig. 5C) and fHbp v.1.15 E214A/S219R (Fig. 5D).

Fig. 6A-D includes four graphs (sensorgrams) comparing the binding of full-length factor H protein to fHbp v1.1, fHbp v1.15 (wild type) and fHbp v.1.15 E214A (Fig. 6A); fHbp v.1.15 S219R (Fig. 6B); fHbp v.1.15 E214A/E235A (Fig. 6C) and fHbp v.1.15 E214A/S219R (Fig. 6D).

Fig. 7A, B includes two graphs (sensorgrams) showing the binding of factor H domains 6-7 to two different fHbp fusion proteins of the invention in comparison with known fHbp fusion proteins. In fig. 7A compares the binding of wild-type fHbp 231, fHbp 231S, and fHbp 231.13 E211A/S216R fusion. In fig. 7B compares the binding of wild-type fHbp 231, fHbp 231S, and the fHbp 231.13 E211A/E232A fusion.

In fig. Figure 8 compares the binding of factor H domains 6-7 to wild-type fHbp 231, fHbp 231S, and sly.

- 2 046480 tym fHbp 231.15 E214A/E235A according to the invention.

Fig. 9A,B includes two graphs showing the binding of full-length factor H protein to two different fHbp fusion proteins of the invention compared to known fHbp fusion proteins.

In fig. 9A compares the binding of wild-type fHbp 231, fHbp 231S, and fHbp 231.13 E211A/S216R fusion.

In fig. 9B compares the binding of wild-type fHbp 231, fHbp 231S, and the fHbp 231.13 E211A/E232A fusion.

In fig. 10 compares the binding of full-length factor H protein to wild-type fHbp 231, fHbp 231S, and the fHbp 231.15 E214A/E235A fusion of the invention.

In fig. 11A shows serum bactericidal titers measured in the presence of rabbit complement (rSBA) for each of the six fHbp-related antigens and the BEXSERO-like composition against a number of meningococcal strains expressing fHbp v1.x. Each dot represents the serum bactericidal titer against an individual strain, measured in pooled serum.

In fig. 11B shows serum bactericidal titers measured in the presence of human complement (hSBA) for each of the six fHbp-related antigens and a BEXSERO-like composition against the same set of strains expressing fHbp v1.x.

In fig. 12A shows serum bactericidal titers measured in the presence of rabbit complement (rSBA) for each of the six fHbp-related antigens and the BEXSERO-like composition against a number of meningococcal strains expressing fHbp v2 or v3. Each dot represents the serum bactericidal titer against an individual strain, measured in pooled serum.

In fig. 12B shows serum bactericidal titers measured in the presence of human complement (hSBA) for each of the six fHbp-related antigens and a BEXSERO-like composition against the same set of strains expressing fHbp v2 or v3.

In fig. 13 shows the bactericidal activity of pooled serum measured in the presence of human complement (hSBA) for each of the compositions against strains of types var2/3 (Fig. 13A) and v1.x (Fig. 13B). Serum obtained from vaccinated mice was tested in pooled form in the presence of human plasma as a source of complement (hSBA) on 50 MenB strains, grouped as var1 strains (30 strains) and var2/3 strains (20 strains). In this figure, fHbp 2-3-1.13 refers to the fusion protein containing wild type v1.13, fHbp 2-3-1.13 NB ES refers to the 231.13_E211A/S216R fusion, fHbp 2-3-1.13 NB EE refers to the 231.13_E211A fusion /E232A, fHbp 2-3-1.15 refers to the fusion protein containing wild type v1.15, and fHbp 2-3-1.15 NB EE refers to the 231.15_E214A/E235A fusion protein.

In fig. 14 shows the percentage overlap between strains expressing fHbp var2/3 (A) and fHbp var1 (B) with the vaccine compositions tested in a mouse study. In this figure, fHbp 2-3-1.13 refers to the fusion protein containing wild type v1.13, fHbp 2-3-1.13 NB ES refers to the 231.13_E211A/S216R fusion, fHbp 2-3-1.13 NB EE refers to the 231.13 E211A fusion /E232A, fHbp 2-3-1.15 refers to the fusion protein containing wild type v1.15, and fHbp 2-3-1.15 NB EE refers to the 231.15_E214A/E235A fusion protein.

In fig. 15 shows mouse serum bactericidal titers measured in the presence of human complement (hSBA) against 11 strains, including the BEXSERO reference strains and fHbp variant 1.1 and 1.4 strains. In this figure, fHbp 2-3-1.13 refers to the fusion protein containing wild type v1.13, fHbp 2-3-1.13 NB ES refers to the 231.13_E211A/S216R fusion, fHbp 2-3-1.13 NB EE refers to the 231.13_E211A fusion /E232A, fHbp 2-3-1.15 refers to the fusion protein containing wild type v1.15, and fHbp 2-3-1.15 NB EE refers to the 231.15_E214A/E235A fusion protein.

In fig. 16, compositions of the invention containing the BEXSERO + fHbp231.13_E211A/S216R fusion protein (indicated in the graphs as BEXSERO PLUS PLUS) are compared with a standard BEXSERO composition against a panel of four BEXSERO indicator strains: M14459 for fHbp var1.l (Fig. 16A) ; NZ98/254 for PorA P1.4 (Fig. 16B); M4407 for NHBA (Fig. 16C) and 96217 for NadA (Fig. 16D).

In fig. 17 shows the bactericidal activity of pooled serum measured in the presence of human complement (hSBA) for each of the compositions against strains of types var2/3 (Fig. 17A) and v1.x (Fig. 17B). Serum obtained from vaccinated rabbits was tested in pooled form against MenB strains, grouped as var1 and var2/3 strains. In this graph, fHbp 2-3-1.13 refers to the fusion protein containing wild type v1.13, fHbp 2-3-1.13 NB ES refers to the fusion protein 231.13_E211A/S216R, fHbp 2-3-1.15 refers to the fusion protein containing v1 .15 is wild type, and fHbp 2-3-1.15 NB EE refers to the 231.15_E214A/E235A fusion.

In fig. 18 shows the percentage of overlap between strains expressing fHbp var2/3 (A) and fHbp var1 (B) provided by the vaccine compositions in a rabbit study. In this figure, fHbp 2-3-1S refers to the prior art fusion protein 231.1R41S, fHbp 2-3-1.13 refers to the fusion protein containing wild type v1.13, fHbp 2-3-1.13 NB ES refers to the fusion protein

- 3 046480

231.13_E211A/S216R, fHbp 2-3-1.13 NB EE refers to the 231.13_E211A/E232A fusion, fHbp 2-3-1.15 refers to a fusion protein containing wild-type v1.15, and fHbp 2-3-1.15 NB EE refers to merged 231.15_E214A/E235A.

In fig. 19 shows rabbit serum bactericidal titers measured in the presence of human complement (hSBA) against 11 strains, including the BEXSERO reference strains and fHbp variant 1.1 and 1.4 strains. In this figure, fHbp 2-3-1.13 refers to the fusion protein containing wild type v1.13, fHbp 2-3-1.13 NB ES refers to the fusion protein 231.13_E211A/S216R, fHbp 2-3-1.15 refers to the fusion protein containing v1 .15 is wild type, and fHbp 2-3-1.15 NB EE refers to the 231.15_E214A/E235A fusion.

In fig. Figure 20 shows the dynamics of bacterial infection (bioluminescent MC58 cc32/var. 1) in immunized and non-immunized mice. Group A mice were immunized with wild-type 4CMenB+fHbp 23(S)1.13, while group B mice were immunized with 4CMenB+fHbp 23(S)1.13_E211A/S216R. The nonimmunized mouse received phosphate-buffered saline (PBS) alone as a control.

In fig. 21A and B present a quantitative and comparative assessment of the dynamics of bacterial infection (bioluminescent MC58 cc32/var. 1) in immunized and non-immunized mice. Comparisons were made using raw total signals (in photons per second per mouse at each time point) (Fig. 21A) or the ratio of signals at 30 minutes and 6 hours after bacterial challenge (Fig. 21B).

Detailed Description of the Invention

Lipoprotein factor H binding protein (fHbp) is expressed on the surface of all MenB strains. fHbp binds to human complement factor H protein (hfH), forming a complex that protects bacteria from complement-mediated cytolysis and provides a mechanism to ensure the survival of N. meningitidis in the human bloodstream. Antibodies against fHbp perform a dual function: they are bactericidal in themselves, and by preventing binding to hfH, they make bacterial strains more susceptible to cytolysis. Reducing or eliminating the ability of fHbp to bind to hfH increases the immunogenicity of the fHbp antigen by preventing the formation of protective complexes between fHbp and hfH that can mask fHbp epitopes and prevent antibody binding.

There are three different genetic and immunogenic variants of fHbp (v1, v2 and v3) and many subvariants. Most MenB strains not overlapping 44CMenB express fHbp v2 or v3 or v1 subvariants with low relatedness to var1.1.

Modern epidemiology using the MATS approach (as described, for example, by Medini et al. Vaccine 2015; 33(23); 2629-36) shows that strains with v1.1 and v1.4 are the most common, followed by v1. 15, v1.14 and v1.13, as shown in FIG. 1A. Antibodies against the fHbp v1.1 subvariant included in the 44CMenB vaccine have strong cross-reactivity with the most common fHbp v1 subvariants (v1.1 and v1.4), but less cross-reactivity with the v1 subvariants that have the lowest affinity to v1 .1 (for example, v1.15 and v1.13). This is shown in Fig. 1, since meningococcal B strains expressing fHbp v1.15 and v1.13 are the most common strains that are not overlapped by the 44CMenB vaccine.

In addition, antibodies against the fHbp v1.1 subvariant included in the 44CMenB vaccine have poor cross-reactivity with fHbp v2 and v3 (Brunelli B et al., Vaccine, 2011; 29:1072-1081). In fig. 1B and C show gaps in the 44CMenB overlap of some of the most common strains expressing fHbp v2 or v3.

This means that 44CMenB does not overlap with some meningococcal strains carrying fHbp v2, v3, or strains carrying some sub-variants v1.

The present invention provides mutated fHbp variant 1.13 or variant 1.15 polypeptides (v1.13 or v1.15) that are immunogenic and that can be combined with existing meningococcal vaccines to provide better overlap between N. meningitidis strains.

In particular, the v1 polypeptides of the invention are subvariants of fHbp variant 1 that are genetically distinct from the fHbp v1.1 antigen included in 44CMenB.

In addition, the v1 polypeptides of the invention are mutated to reduce binding to hfH compared to the corresponding wild-type v1 polypeptide. In contrast, the fHbp v1.1 antigen included in BEXSERO and the fHbp antigens v1.55 and v3.45 included in TRUMENBA bind to hfH.

The v1 polypeptides of the invention may be present alone or as a component of a fusion protein, along with mutant forms of fHbp variants 2 and 3 that have been modified to improve stability as well as reduce binding to fHbp. By providing a single fusion protein containing said v2 and v3 antigens along with the v1 antigen of the invention, we have achieved better strain overlap compared to existing licensed meningococcal B vaccines. To be clear, neither v2 nor v3 antigen is present in, for example, 44CMenB . The presence of v2 and v3 antigens in the fusion proteins of this invention improves

- 4 046480 strain overlap compared to, for example, 44CMenB.

The v1 polypeptides and fusion proteins of the invention can be used alone or in combination with meningococcal antigen NHBA, meningococcal antigen NadA, meningococcal antigen fHbp and meningococcal outer membrane vesicle (for example, in combination with the BEXSERO composition) to obtain a combination immunogenic composition having increased immunogenicity ( due to the addition/inclusion of non-binding forms of fHbp variants), and better N. meningitidis strain overlap (due to the addition of new fHbp variants/subvariants) compared to BEXSERO alone.

Mutant polypeptides of meningococcal fHbp v1.13.

The present inventors have identified residues in the fHbp v1.13 sequence that can be modified to reduce binding to hfH. Such mutants are referred to herein as non-binding (NB) mutants. We also discovered combinations of mutations in the v1.13 sequence that are particularly effective in reducing hfH binding. fHbp v1.13 is also known in the art as fHbp B09 variant.

The wild-type mature lipoprotein fHbp v1.13 from strain M982 (GenBank accession number AAR84475.1) has the following amino acid sequence, with the N-terminal polyglycine signal sequence underlined:

CSSGGGGVAADIGAGLADALTAPLDHKDKGLOSLTLDOSVRKNEKLKLAAOGAEKT

YGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQV QDSEDSGI<MVAI<RQFRIGDIAGEHTSFDI<LPI<GGSATYRGTAFGSDDAGGI<LTYTID FAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGG QAQEVAGS AEVET ANGIHHIGLAAKQ (SEQ Ш NO: 1)

Mature lipoprotein v1.13 differs from the full-length wild-type sequence in that the full-length polypeptide has an additional 19 residues of N-terminal leader sequence, which is cleaved from the mature polypeptide. Thus, full-length wild-type fHbp v1.13 has the following amino acid sequence (with the N-terminal leader sequence shown in bold):

MNRTAFCCFSLTAALILTACSSGGGVAADIGAGLADALTAPLDHKDKGLQSLTLD

QSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGE

FQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATY

RGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISG SVLYNQDEKGSYSLGIFGGQAQEV AGS AEVETANGIHHIGLAAKQ (SEQ ID NO: 31)

The ΔG form of the mature lipoprotein v1.13 lacks the N-terminal polyglycine sequence of the mature polypeptide, i.e. it lacks the first 7 amino acids of SEQ ID NO: 1, and it lacks the first 26 amino acids of SEQ ID NO: 31:

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSL

NTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSG

I<MVAI<ROFRIGDIAGEHTSFDI<LPI<GGSATYRGTAFGSDDAGGI<LTYTIDFAAI<QGH

GKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAG

SAEVETANGIHHIGLAAKQ (SEQ ID NO: 2)

Thus, the first aspect of the invention provides a mutant fHbp v1.13 meningococcal polypeptide comprising an amino acid sequence having at least k% sequence identity to SEQ ID NO: 2, provided that the amino acid sequence of said fHbp v1.13 mutant meningococcal polypeptide includes the mutation with the substitution of one or more of the E211, S216 or E232 residues of the sequence SEQ ID NO: 2.

The value of k may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. Preferably it is 80 (i.e. amino acid the sequence of the mutant fHbp v1.13 is at least 80% identical to SEQ ID NO: 2), and more preferably is 85, more preferably is 90, and more preferably is 95. Most preferably, the amino acid sequence of the mutant fHbp v1.13 is at least identical 97%, at least 98% or at least 99% SEQ ID NO: 2.

Preferably, the amino acid sequence differs from SEQ ID NO: 2 by at least one or more than one substitution E211A, S216R or E232A. More preferably, the amino acid sequence contains substitutions of multiple residues selected from the following: (i) E211A and E232A or (ii) E211A and S216R. Preferably, the amino acid sequence contains substitutions of residues E211A and S216R relative to SEQ ID NO: 2.

Without being limited by theory, substitution of glutamic acid (E) for alanine (A) at position 211 of SEQ ID NO: 2 removes a negatively charged residue involved in hfH recruitment, thereby eliminating fH binding. Substitution of arginine (R) for serine (S) at position 216 of SEQ ID NO: 2 replaces the wild type amino acid corresponding

- 5 046480 relevant residue from N. gonorrhoeae that does not bind to hfH.

In preferred embodiments, the mutant polypeptide of the invention has the amino acid sequence of SEQ ID NO: 3 (v1.13 ΔG E211A/E232A) or SEQ ID NO: 4 (v1.13 ΔG (E211A/S216R). More preferably, the v1.13 mutant polypeptide of the invention has the amino acid sequence SEQ ID NO: 4.

Upon administration to an animal host, preferably a mammal and more preferably a human, the v1.13 mutant polypeptide of the invention can elicit the production of antibodies that are capable of recognizing wild-type meningococcal fHbp polypeptides of the sequence SEQ ID NO: 1. Ideally, such antibodies are bactericidal (see . below).

Mutant polypeptides of meningococcal fhbp v1.15.

The present inventors also identified residues in the fHbp v1.15 sequence that could be modified to prevent binding to hfH. Such mutants are referred to herein as non-binding (NB) mutants. We also discovered combinations of mutations in the v1.15 sequence that are particularly effective in preventing hfH binding. fHbp v1.15 is also known in the art as the fHbp B44 variant.

The wild-type mature lipoprotein fHbp v1.15 from strain NM452 (GenBank accession number ABL14232.1) has the following amino acid sequence, with the N-terminal polyglycine signal sequence underlined:

CSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLTLEDSISONGTLTLSAQG

AERTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTA LQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGG KLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSY SLGIFGGQAQEVAG SAEVETANGIRHIGLAAKQ (SEQ Ш NO: 5)

Mature lipoprotein v1.15 differs from the full-length wild-type sequence in that the full-length polypeptide has an additional 19 residues of N-terminal leader sequence, which is cleaved from the mature polypeptide. Thus, full-length wild-type fHbp v1.15 has the following amino acid sequence (with the N-terminal leader sequence shown in bold):

MNRTTFCCLSLTAALILTACSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLK

SLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEVDGQL ITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPK DVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEK HHAVISGSVLYNQAEKGSY SLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ (SEQ Ш NO: 32)

The ΔG form of mature lipoprotein v1.15 lacks the N-terminal polyglycine sequence, i.e. it lacks the first 12 amino acids of SEQ ID NO: 5 and it lacks the first 31 amino acids of SEQ ID NO: 32:

VAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDN

SLNTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHS GKMVAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQ GHGKIEHLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEV AGSAEVETANGIRHI GLAAKQ (SEQ ID NO: 6)

Thus, the second aspect of the invention provides a mutant fHbp v1.15 meningococcal polypeptide comprising an amino acid sequence having at least k% sequence identity to SEQ ID NO: 6, provided that the amino acid sequence of said fHbp v1.15 mutant meningococcal polypeptide includes the mutation with substitution of one or more of the E214, S219 or E235 residues of the sequence SEQ ID NO: 6.

The value of k may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. Preferably it is 80 (i.e. amino acid the sequence of the mutant fHbp v1.15 is at least 80% identical to SEQ ID NO: 6), more preferably 85, more preferably 90, and more preferably 95. Most preferably, the amino acid sequence of the mutant fHbp v1.15 is at least 97% identical , at least 98% or at least 99% SEQ ID NO: 6.

Preferably, the amino acid sequence differs from SEQ ID NO: 6 in at least one or more than one of E214A, S219R or E235A substitutions. More preferably, the amino acid sequence contains substitutions of residues selected from the following: (i) S219R, (ii) E214A and S219R, and (iii) E214A and E235A.

In preferred embodiments, the v1.15 mutant polypeptide of the invention has the amino acid sequence of SEQ ID NO: 7 (v.1.15_S219R), SEQ ID NO: 8 (v1.15_E214A/S219R) or SEQ ID NO: 9 (v1.15_E214A/E235A) .

After administration to a host animal, preferably a mammal and more preferably

- 6 046480 to humans, the v1.15 mutant polypeptide of the invention can produce antibodies that are capable of recognizing wild-type meningococcal fHbp polypeptides of SEQ ID NO: 5. Ideally, such antibodies are bactericidal (see below).

Fusion polypeptide.

The invention also provides a fusion polypeptide containing all three meningococcal fHbp polypeptides v1, v2 and v3, where the fHbp sequence variants are arranged in the order v2-v3-v1 from N- to C-terminus. In a preferred embodiment, the fHbp fusion polypeptide has an amino acid sequence of the formula NH ₂ -A-[-XL] ₃ -B-COOH, where each X is a different variant of the fHbp sequence, L is the amino acid sequence of a possible linker, A is a possible N-terminal amino acid sequence and B represents a possible C-terminal amino acid sequence.

The fHbp v1 polypeptide component of a fusion polypeptide of the invention is either a mutant fHbp v1.13 polypeptide or a mutant fHbp v1.13 polypeptide described above.

The fHbp v2 and v3 polypeptide components of the invention are preferably mutant v2 and v3 polypeptides having improved stability and reduced ability to bind to hfH compared to wild-type v2 and v3 polypeptides. As explained above, reducing the binding of fHbp to hfH is advantageous because it prevents the formation of protective complexes between fHbp and hfH that can mask fHbp epitopes, and thus increases the immunogenicity of the polypeptide antigen.

The inventors have previously identified residues in the v2 and v3 sequences that can be modified to increase the stability of the polypeptide as well as reduce binding to hfH. These mutated v2 and v3 sequences are described in detail in WO 2015/128480.

Full-length wild-type fHbp v2 from strain 2996 has the following amino acid sequence (leader sequence shown in bold and polyglycine sequence underlined):

MNRTAFCCLSLTAALILTACSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLD

QSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGE

FQIYI <QDHSAVAVAVALQIEI <Innpdi <idslinqrsflvsghehtafnqlpdgi <AFSSDAGI <ltyTidfaai <qhhli <iehli <iehlilaaaeli GSEEKGTYHLALLFGDRAQEAGSATVKIGEKVHEIGIGKQ (SEQ SO: 10)

The mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 10:

CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKT

YGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKIN

NPDK ID SLINQRSFLVSGLGGEHT AFNQLPDGKAEYHGKAF S SDDAGGKLT YTIDF AA KQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRA

QEIAGSATVKIGEKVHEIGIAGKQ (SEQ ID NO: 11)

The ΔG form SEQ ID NO: 10 lacks the first 26 amino acids:

VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSL

NTGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSL

INQRSFLVSGLGGEHT AFNQLPDGKAEYHGKAF S SDD AGGKLTYTIDF AAKQGHGKI EHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSA TVKIGEKVHEIGIAGKQ (SEQ Ш NO: 12)

In a preferred embodiment, the fusion polypeptide of the invention comprises a mutant fHbp v2 polypeptide comprising an amino acid sequence having at least k% sequence identity to SEQ ID NO: 12, provided that the fHbp v2 amino acid sequence includes a mutation substituting one or more of the residues S32 and L123 of SEQ ID NO: 12. Preferably, the substitutions are S32V and L123R.

The k value may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. Preferably it is 80 (i.e. amino acid the sequence of the mutant fHbp v2 is at least 80% identical to SEQ ID NO: 12), more preferably 85, more preferably 90, and more preferably 95.

In some embodiments, the fHbp v2 polypeptide included in the fusion protein of the invention is truncated relative to SEQ ID NO: 12. Compared to the mature wild type sequence, SEQ ID NO: 12 is already truncated at the N terminus and does not include a polyglycine sequence. (compare SEQ ID NO: 11 and 12), however, SEQ ID NO: 12 may be truncated at the C-terminus and/or further truncated at the N-terminus.

In a preferred embodiment, the fHbp v2 polypeptide included in the fusion protein contains or consists of the amino acid sequence SEQ ID NO: 16.

The fHbp v2 polypeptide included in the fusion protein of the invention has higher stability under the same experimental conditions than the same polypeptide without sequence differences.

- 7 046480 residues S32 and L123, for example, higher stability than the wild-type meningococcal polypeptide consisting of SEQ ID NO: 10. The S32V mutation stabilizes the structure due to the occurrence of favorable hydrophobic interactions. The L123R mutation abolishes fH binding by introducing inconsistencies with fH and interfering charges with binding.

Improvement in stability can be assessed using differential scanning calorimetry (DSC), for example, as discussed by Johnson (2013), Arch. Biochem. Biophys. 531:100-9 and Bruylants et al., Current Medicinal Chemistry, 2005; 12:2011-20. DSC has previously been used to assess the stability of fHbp v2 (Johnson et al. PLoS Pathogen 2012;8:e1002981). Suitable conditions for assessing stability using DSC may be to use 20 µM polypeptide in a buffer solution (eg 25 mM Tris) with a pH between 6 and 8 (eg 7-7.5) with 100-200 mM NaCl (eg 150 mm).

An increase in stability is indicated by an increase in the phase transition temperature ( _Tm ) for at least one peak in the DSC curve by at least 5°C, for example, by at least 10, 15, 20, 25, 30, 35°C or more compared to the wild type. When wild-type fHbp is unfolded by DSC, two peaks are detected (one for the N-terminal domain and the other for the C-terminal domain) and, when the v2 polypeptide included in the fusion protein of the invention includes both such domains, the increase in stability is attributed to increasing T _m for the N-terminal domain by at least 5°C. T _m for the N-terminal domain in wild-type v2 sequences can be 40°C or even lower (Johnson et al., (2012), PLoS Pathogen, 8: e1002981), while C-terminal domains can have a T _{m of} 80° C or higher. Thus, the amino acid sequence of fHbp v2 included in the fusion protein of the invention preferably has an N-terminal domain with a T _m of at least 45°C, for example >50, >55, >60, >65, >70, >75 or even >80°C.

Full-length wild-type fHbp v3 from strain M1239 has the following amino acid sequence (leader sequence shown in bold and polyglycine sequence underlined):

MNRTAFCCLSLTTALILTACSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLK

SLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQ TITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPG GKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKS HAVILGDTRYGSEEKGTY HLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ (SEQ Ш NO: 13)

The mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 13: CSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQG

AEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVV ALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRL HYSIDFTI<I<QGYGRIEHL1<TLEQNVELAAAEL1<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRA QEIAGSATVKIGEKVHEIGIAGKQ (SEQ ID NO: 14)

The AG form of SEQ ID NO: 13 lacks the first 31 amino acids (i.e., lacks the signal sequence and polyglycine sequence):

VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDN SLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKT DSLINQRSFLVSGLGGEHTAFNQLPGGI<AEYHGI<AFSSDDPNGRLHYSIDFTI<I<QGYG RIEHLK TLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGS ATVKIGEKVHEIGIAGKQ (SEQ Ш NO: 15)

In a preferred embodiment, the fusion polypeptide of the invention comprises a mutant fHbp v3 polypeptide comprising an amino acid sequence having at least k% sequence identity to SEQ ID NO: 15, provided that the fHbp v3 amino acid sequence includes a mutation substituting one or more of the residues S32 and L126 of SEQ ID NO: 15. Preferably, the substitutions are S32V and L126R.

The value of k may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. Preferably it is 80 (i.e. amino acid the sequence of the mutant fHbp v2 is at least 80% identical to SEQ ID NO: 15) and more preferably is 85, more preferably is 90, and more preferably is 95.

In some embodiments, the fHbp v3 polypeptide included in the fusion protein of the invention is truncated relative to SEQ ID NO: 15. Compared to the mature wild-type sequence, SEQ ID NO: 15 is already truncated at the N-terminus and does not include a polyglycine sequence. (compare SEQ ID NO: 14 and 15), however, SEQ ID NO: 15 may be truncated at the C-terminus and/or further truncated at the N-terminus.

In a preferred embodiment, the fHbp v3 polypeptide included in the fusion protein of the invention contains or consists of the amino acid sequence SEQ ID NO: 17.

The fHbp v3 polypeptide included in the fusion protein of the invention has, under the same experimental conditions, greater stability than the same polypeptide without sequence differences.

- 8 046480 residues S32 and L126, for example, higher stability than the wild-type meningococcal polypeptide consisting of SEQ ID NO: 13. The S32V mutation stabilizes the structure due to the occurrence of favorable hydrophobic interactions. The L126R mutation abolishes fH binding by introducing mismatches with fH and interfering charges.

Improvement in stability can be assessed using differential scanning calorimetry (DSC), for example, as discussed by Johnson (2013), Arch. Biochem. Biophys. 531:100-9 and Bruylants et al. (2005), Current Medicinal Chemistry, 12:2011-20. DSC has previously been used to assess the stability of fHbp v3 (van der Veen et al. (2014), Infect. Immun. PMID, 24379280). Suitable conditions for assessing stability using DSC may be to use 20 µM polypeptide in a buffer solution (eg 25 mM Tris) with a pH between 6 and 8 (eg 7-7.5) with 100-200 mM NaCl (eg 150 mm).

An increase in stability is indicated by an increase in the phase transition temperature ( _Tm ) for at least one peak in the DSC curve by at least 5°C, for example, by at least 10, 15, 20, 25, 30, 35°C or more compared to the wild type. When wild-type fHbp is unfolded by DSC, two peaks are detected (one for the N-terminal domain and the other for the C-terminal domain) and, when the v3 polypeptide included in the fusion protein of the invention includes both such domains, the increase in stability is attributed to increasing T _m for the N-terminal domain by at least 5°C. The _Tm for the N-terminal domain in wild-type v3 sequences can be 60°C or lower (Johnson et al. (2012), PLoS Pathogen, 8:e1002981), while the C-terminal domains can have a _{Tm of} 80°C or less higher. Thus, the amino acid sequence of the mutant fHbp v3 according to the invention preferably has an N-terminal domain with a T _m of at least 65°C, for example >70, >75 or even >80°C.

As described above, in a preferred embodiment, the fHbp fusion polypeptide has an amino acid sequence of the formula NH ₂ -A-[-XL] ₃ -B-COOH, where each X represents a different variant of the fHbp sequence and L represents the amino acid sequence of a possible linker. In a preferred embodiment, the amino acid sequence of linker L is a glycine or glycine-serine polymer linker. Examples of linkers include, but are not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG and GSGGGG. Other suitable glycine or glycineserine polymer linkers will be apparent to one skilled in the art. In a preferred fusion polypeptide of the invention, the v2 and v3 sequences and the v3 and v1 sequences are linked by a glycine-serine polymer linker GSGGGG.

In a preferred embodiment, the fusion polypeptide of the invention contains or consists of one of the following amino acid sequences (glycine-serine linker sequences are underlined and mutated residues are indicated in bold):

fHbp 23S1.13 E211A/E232A (SEQ ID NO: 18)

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKroSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKOGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLINQRSFR VSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSL NTGKLKNDKVSRFDFIRQIEVD GKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFKL PKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEK RHAVISGSVLYNQDAKGSYSLGIFGGQAQEVAGSAAVETANGIHHIGLAAKQ f Hbp 23S1.13 E211A/S216R (SEQ ID NO: 19)

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKOGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLIN QRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLQSLTLDQSVRKNEKLKLAAQGAEKTYG NGDSLNTGKLKNDKVSRFDFIRQIEVD

- 9 046480

GKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKL PKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEK RHAVISGSVLYNQDAKGSYRLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ fHbp_23S_1.15 S23 1R (SEQ ID NO: 20)

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKroSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKOGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLINQRSFR VSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKL KNDKISRFDFIRQIEV DGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGK LPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAEKGSYRLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ fflbp _23S_1.15 E214A/S219R (SEQ ID NO: 21)

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHL KTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLINQ RSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAQEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNT GKLKNDKISRFDFIRQIEV DGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGK LPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAAKGSYRLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ fH bp_23S_1.15 E214A/E235A (SEQ ID NO: 22)

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLINQRS FRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGK LKNDKISRFDFIRQIEV DGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGK LPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAAKGSYSLGIFGGQAQEVAGSAAVETANGIRHIGLAAKQ

In a preferred embodiment, the fusion polypeptide of the invention contains the amino acid sequence of SEQ ID NO: 19. In an alternative embodiment, the fusion polypeptide of the invention contains the amino acid sequence of SEQ ID NO: 18.

Upon administration to an animal host, preferably a mammal and more preferably a human, a fusion polypeptide of the invention can elicit the production of antibodies that are capable of recognizing wild-type meningococcal fHbp polypeptides, in particular polypeptides with the sequences SEQ ID NO: 31, 32, 10 and/or 13. Ideally, such antibodies are bactericidal (see below).

As described above, in a preferred embodiment, the fHbp fusion polypeptide of the invention has the amino acid sequence of the formula NH ₂ -A-[-XL] ₃ -B-COOH, where each X represents a different variant of the fHbp sequence; A represents a possible N-terminal amino acid sequence. In a preferred embodiment, the fusion polypeptides described herein further comprise the following N-terminal amino acid sequence

- 10 046480 activity that is preferred to ensure good expression of the fusion protein: MGPDSDRLQQRR (SEQ ID NO: 34).

Any of the fusion proteins described herein (eg, SEQ ID NO: 18-22, 29 and 30) can be modified to include the amino acid sequence of SEQ ID NO: 34 at the N-terminus of the fusion polypeptide, i.e. the amino acid sequence SEQ ID NO: 34 is added to the N-terminus of the fHbp v2 component of the fusion polypeptide.

Germicidal responses.

Preferred v1.13, v1.15 and/or fusion peptides of the invention can induce the production of antibodies that are bactericidal against meningococci. The bactericidal antibody response is conveniently measured in mice and serves as an indicator of vaccine efficacy (eg, see note 14 in Pizza et al. (2000) Science 287:1816-1820; also WO 2007/028408).

The polypeptides of the first embodiment of the invention can preferably elicit an antibody response that is bactericidal against the N. meningitidis strain expressing the v1.13 fHbp sequence.

Preferred polypeptides of a first embodiment of the invention can cause mice to produce antibodies that are bactericidal in a serum bactericidal test against a strain of N. meningitidis expressing the v1.13 fHbp sequence.

The polypeptides of the second embodiment of the invention can preferably elicit an antibody response that is bactericidal against a strain of N. meningitidis expressing the v1.15 fHbp sequence.

Preferred polypeptides of a second embodiment of the invention can cause mice to produce antibodies that are bactericidal in a serum bactericidal test against a strain of N. meningitidis expressing the v1.15 fHbp sequence.

For example, an immunogenic composition containing these polypeptides can provide a bactericidal serum titer of >1:4 when tested with human complement, as proposed by Goldschneider [Goldschneider et al. (1969), J. Exp. Med. 129:1307-26, Santos et al. (2001), Clinical and Diagnostic Laboratory Immunology, 8:616-23, and Frasch et al. (2009), Vaccine, 27S:B112-6] and/or provide a bactericidal serum titer of >1:128 when using rabbit complement.

Polypeptides.

The polypeptides of the invention can be produced in a variety of ways, for example by chemical synthesis (at least in part), by cutting longer polypeptides with proteases, by RNA translation, by purification from cell culture (for example, by recombinant expression or from culture N. meningitidis), etc. Heterologous expression in the E. coli host is the preferred mode of expression.

The polypeptides of the invention ideally have a length of at least 100 amino acids, for example, 150 amino acids, 175 amino acids, 200 amino acids, 225 amino acids or more. These include the sequence of mutant fHbp v1, v2 and/or v3, and the sequence of mutant fHbp v1, v2 and/or v3 should likewise be at least 100 amino acids in length, for example 150 amino acids, 175 amino acids, 200 amino acids, 225 amino acids or more.

In nature, fHbp is a lipoprotein of N. meningitidis. It has also been found to lipidate when expressed in E. coli with its native leader sequence or with heterologous leader sequences. The polypeptides of the invention may have an N-terminal cysteine residue which may be lipidated, for example containing a palmitoyl group, typically forming tripalmitoyl-glyceryl cysteine. In other embodiments, the polypeptides are not lipidated.

The polypeptides are preferably obtained in substantially pure or substantially isolated form (ie, substantially free from other Neisseria or host cell polypeptides). Typically, polypeptides are found in a non-natural environment, for example, they are separated from their natural environment. In some embodiments, the polypeptides are in a composition enriched in polypeptide relative to the starting material. Thus, a purified polypeptide is provided, where purified means that the polypeptide is in a composition that is substantially free from other expressed polypeptides, where substantially free means that more than 50% (for example, >75, >80, >90, > 95 or >99%) of all polypeptides in the composition are polypeptides of the invention.

Polypeptides can be in various forms (eg, native, fused, glycosylated, non-glycosylated, lipidated, disulfide bridged, etc.).

If the polypeptide of the invention is produced by translation in a biological host, a start codon is required which will provide an N-terminal methionine in most hosts. Thus, the polypeptide of the invention will, at least at the formation stage, include a methionine residue located in the 5' direction of the specified sequence SEQ ID NO.

Cleavage of a nascent sequence means that the amino acid sequence

- 11 046480 the identity of mutant fHbp v1, v2 or v3 may itself provide the N-terminus of the polypeptide. However, in other embodiments, the polypeptide of the invention may include an N-terminal sequence located in the 5' direction of the amino acid sequence of fHbp v1, v2 or v3. In some embodiments, the polypeptide has a single methionine at the N-terminus, immediately followed by the amino acid sequence of mutant fHbp v1, v2 or v3; in other embodiments, a longer sequence located in the 5' direction may be used. Such 5' sequence may be short (i.e., 40 amino acids or less, 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 isolation (eg, histidine tags, ie Hisn, where n=4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art.

The polypeptide of the invention may also include amino acids located in the 3' direction of the amino acid sequence of mutant fHbp v1, v2 or v3. Such C-terminal extensions may be short (i.e., 40 amino acids or less, 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 isolation (eg, containing histidine tags, i.e. Hisn, where n=4, 5, 6, 7, 8, 9, 10 or more), or sequences that improve the stability of the polypeptide. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

In some embodiments, the invention does not cover polypeptides that include a histidine tag (cf. Johnson et al. (2012), PLoS Pathogen 8:e1002981, and Pajon et al. (2012), Infect. Immun. 80:2667-77) and, in particular, a hexahistidine tag at the C-terminus.

The term polypeptide refers to amino acid polymers of any length. The polymer may be linear or branched and may contain modified amino acids and be interrupted by non-amino acids. The terms also cover amino acid polymer that has been naturally or artificially modified; for example, by formation of a disulfide bond, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as, for example, conjugation to a tag moiety. The definition also covers, for example, polypeptides containing one or more amino acid analogues (including, for example, amino acids not found in nature, etc.), as well as other modifications known in the art. Polypeptides can be single chain or consist of several associated chains.

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

The polypeptides of the invention may contain a detectable label, for example a radioactive label, a fluorescent label or a biotin label. This is particularly useful in immunoassay methods.

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

Affinity for factor H can be quantified using surface plasmon resonance (eg, as described by Schneider et al. (2009), Nature, 458:890-5) with immobilized human fH. Mutations that result in a decrease in affinity (e.g., an increase in the dissociation constant K _D ) by at least 10-fold and ideally by at least 100-fold (compared to the same peptide, but without mutation, when performing the experiment in the same conditions) are preferred. the most conditions).

Nucleic acids.

A fourth aspect of the invention provides a plasmid or other nucleic acid comprising a nucleotide sequence encoding a mutant fHbp v1.13 polypeptide, a mutant fHbp v1.15 polypeptide, or a fusion polypeptide as defined above.

The nucleic acids of the invention can be prepared in a variety of ways, for example by chemical synthesis (eg phosphoramidite DNA synthesis) in whole or in part, by cutting longer nucleic acids with nucleases (eg restriction enzymes), by combining shorter nucleic acids or nucleotides (for example, using ligases or polymerases), from genomic or cDNA libraries, etc.

The nucleic acids of the invention can be in various forms, for example single-stranded, double-stranded, vectors, primers, probes, labeled, unlabeled, etc.

Preferably, the nucleic acids of the invention are in isolated or substantially isolated form.

The term nucleic acid covers DNA and RNA, as well as their analogues, such as those containing modified backbones, as well as peptide nucleic acids (PNAs), etc.

- 12 046480

The nucleic acids 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.

Host cells.

A fifth aspect of the invention provides a host cell transformed with a plasmid containing the nucleotide sequences of the invention described above.

The host cell of the invention is preferably a bacterium that expresses the polypeptide of the invention. The bacterium may, for example, be meningococcus or E. coli. The bacterium may constitutively express the polypeptide, but in some embodiments the expression may be under the control of an inducible promoter. The bacterium can overexpress the polypeptide (cf. WO 2006/081259). Ideally, polypeptide expression is not subject to phase variation.

The cell may be a meningococcus bacterium, such as a meningococcus bacterium, having reduced expression or knockout of mltA, and also possibly having reduced expression or knockout of: (i) at least one gene involved in conferring toxicity on the lipid A portion of LPS, in particularly Ipxl1; and/or (ii) at least one gene involved in the synthesis or export of capsular polysaccharide, in particular synX and/or ctrA.

Vesicles.

A sixth aspect of the invention provides an outer membrane vesicle derived from a host cell according to the invention, in particular from a meningococcal host cell.

Vesicles obtained from a host cell according to the invention preferably include a polypeptide according to the invention, which should be in an immunoavailable form in the vesicles, i.e. an antibody that can bind to a purified polypeptide of the invention must also be able to bind to the polypeptide contained in the vesicles.

Such membrane vesicles include any proteoliposomal vesicles produced by disruption or desquamation of the outer membrane of the meningococcus to form vesicles that include the protein components of the outer membrane. Thus, the term covers outer membrane vesicles (OMV; sometimes referred to as vesicles), microvesicles (MV [WO 02/09643]) and native OMVs (NOMV [Katial et al. (2002), Infect. Immun. 70:702-707] ).

MV and NOMV are naturally occurring membrane vesicles that are formed spontaneously during bacterial growth and released into the culture medium. MVs can be obtained by culturing Neisseria in liquid culture medium, separating whole cells from smaller MVs in liquid culture medium (e.g., by filtration or low-speed centrifugation to pellet only cells and not smaller vesicles), and then collecting MVs from the depleted media cells (eg, by filtration, differential precipitation or aggregation of MVs, high-speed centrifugation to pellet MVs). Strains for use in MV production can generally be selected based on the amount of MV produced in culture, for example US 6,180,111 and WO 01/34642 describe Neisseria with high MV production.

OMVs are produced artificially from bacteria and can be produced by treatment with detergents (eg deoxycholate) or detergent-free processes (eg see WO 2004/019977). Methods for producing OMVs involve treating bacteria with a bile salt detergent (e.g. salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., with sodium deoxycholate [see EP 0011243 and Fredriksen et al (1991), NIPH Ann 14(2):67-80], which is preferred for treating Neisseria) at pH values high enough not to cause detergent precipitation [WO 01/91788]. Other techniques can be carried out essentially in the absence of detergent [WO 2004/019977] using techniques such as ultrasonication, homogenization, microfluidization, cavitation, osmotic shock, grinding, French press, blending, etc. Methods without or using small amounts of detergent can preserve beneficial antigens such as NspA and fHbp. Thus, the method may use an OMV extraction buffer with about 0.5% deoxycholate or less, such as about 0.2%, about 0.1%, <0.05%, or 0.

An efficient process for obtaining OMVs is described in WO 2005/004908 and involves ultrafiltration of primary OMVs, instead of high-speed centrifugation. The process may include an ultracentrifugation step after ultrafiltration. OMV can also be purified using the two-step gel filtration process described in WO 2011/036562.

Vesicles for use according to the invention can be obtained from any strain of meningococcus. The vesicles will usually be derived from a serogroup B strain, but it is possible to obtain them from serogroups other than B, such as A, C, W135 or Y (eg WO 01/91788 describes a process for serogroup

- 13 046480 group A). The strain can be of any serotype (for example, 1, 2a, 2b, 4, 14, 15, 16, etc.), any serosubtype and any immunotype (for example, L1; L2; L3; L3,3,7; L10 and etc.). Meningococci can be any suitable lineage, including hyperinvasive and hypervirulent lineages, for example any of the following seven hypervirulent lineages: subgroup I; subgroup III; subgroups IV-1; complex ET-5; complex ET-37; cluster A4; lines 3.

In addition to encoding the polypeptide of the invention, the bacteria of the invention may have one or more of the following modifications. For example, they may have a modified fur gene [WO 98/56901]. nspA expression may be increased when porA and cps are knocked out simultaneously. Additional N. meningitidis knockout mutants for producing OMV are described, for example, in WO 2004/014417. Claassen et al. (1996), 14(10):1001-8 describes the production of vesicles from strains modified to express six different PorA subtypes. It is also possible to use mutant Neisseria with low endotoxin levels, which is achieved by knocking out the enzymes involved in LPS biosynthesis. The invention can also use mutant Neisseria modified to reduce or disable the expression of at least one gene involved in conferring toxicity on the lipid A portion of LPS, in particular the lpxl1 gene [Fisseha et al. (2005), Infect. Immun. 73:4070-80]. Likewise, mutant Neisseria modified to reduce or disable the expression of at least one gene involved in the synthesis or export of capsular polysaccharide, in particular the synX and/or ctrA genes, can also be used in the invention. All of these or other mutants may be used in the invention.

Thus, a strain used in the invention may, in some embodiments, express more than one PorA subtype. Previously, 6-valent and 9-valent strains with PorA were constructed. The strain can express PorA 2, 3, 4, 5, 6, 7, 8 or 9 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 have reduced PorA expression, for example, when the amount of PorA is reduced by at least 20% (e.g., >30, >4%, >50, >60, >70, >80, >90, >95% etc.) compared to levels in wild-type strains (for example, compared to strain H44/76) or even knocked out.

In some embodiments, the strain may overexpress certain proteins (compared to the corresponding wild-type strain). For example, strains may overexpress NspA, protein 287 [WO 01/52885], fHbp [WO 2006/081259] (including the fHbp of the invention), TbpA and/or TbpB [WO 00/25811], Cu,Zn superoxide dismutase, HmbR and etc.

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

Preferably, to obtain the vesicles, the meningococcus can be genetically modified so that there is no phase variation in the expression of the polypeptide. Methods for reducing or eliminating phase variation in gene expression in meningococcus are described in WO 2004/015099. For example, a gene may be placed under the control of a constitutive or inducible promoter, or the DNA motif that is responsible for its phase variation may be deleted or replaced.

In some embodiments, a strain may include one or more knockout and/or overexpression mutations set forth in WO 02/09746, WO 01/09350, WO 02/062378 and WO 2004/014417. For example, according to the instructions and nomenclature presented in these four documents, useful genes for downregulation and/or knockout include:

(a) Cps, CtrA, CtrB, CtrC, CtrD, FrpB,

GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB ; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB; or (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB, SynX and/or SynC.

When a mutant strain is used, in some embodiments it may have one, more than one, or all of the following characteristics: (i) reduced expression or knockout of LgtB and/or GalE to shorten the LOS of meningococcus; (ii) increased expression of TbpA; (iii) increased expression of NhhA; (iv) increased expression of Op85; (v) increased expression of LbpA; (vi) increased expression of NspA; (vii) PorA knockout; (viii) decreased expression or knockout of FrpB; (ix) decreased expression or knockout of Ora; (x) decreased expression or knockout of Opc; (xii) deletion of the cps gene complex. The truncated LOS may be one that does not include the sialyl-lacto-I-neotetraose tag, for example it may be a galactose-deficient LOS. LOS may not have an α chain.

Depending on the strain of meningococcus used to obtain the vesicles, they may or may not include the native fHbp antigen of the strain (WO 2004/046177).

In one preferred embodiment, the meningococcus does not express functional MltA protein. As discussed in WO 2006/046143 and Adu-Bobie et al. (2004), Infect. Immun. 72:1914-19, knockout

- 14 046480

MltA (membrane-bound lytic transglycosylase, also known as GNA33) in meningococcus produces bacteria that spontaneously release large quantities of membrane vesicles into the culture medium, from which they can be easily isolated. For example, vesicles can be isolated using a two-stage gel filtration process according to WO 2011/036562, comprising: (i) a first filtration stage in which vesicles are separated from bacteria based on differences in their size, with the vesicles passing into the filtrate and (ii) the second stage of filtration, in which the vesicles remain in the retentate. The MltA mutation (reduction or knockout) has been used in GMMA vaccines [Koeberling et al. (2014), Vaccine, 32:2688-95], and can be easily combined with additional downregulation or knockout of, in particular, at least one gene involved in conferring toxicity to the lipid A portion of LPS, in particular Ipxl1, and/or at least one gene involved in the synthesis or export of capsular polysaccharide, in particular the synX and/or ctrA genes. GMMA (generalized modules for membrane antigens) are genetically neutralized OMVs that are derived from meningococcal strains that have been genetically modified to release GMMA with reduced reactogenicity and increased immunogenicity. When tested in a monocyte activation test (MAT), GMMA induces fewer proinflammatory cytokines than OMVs.

A preferred meningococcal strain for creating a GMMA vaccine using this approach expresses the mutant fHbp v2 and/or mutant fHbp v3 of the invention, and expression may be under the control of strong promoters. The vesicles released by this strain include mutant fHbp v2 and/or v3 proteins in an immunogenic form, and bactericidal antibodies can be produced in response to the vesicles, as discussed by Koeberling et al. (2014), Vaccine, 32:2688-95. The strain may also express fHbp v1, or instead fHbp v1 may be presented as a separate recombinant protein in soluble form (and fHbp v1 may be a wild-type sequence or a mutant sequence, e.g. mutated to impair its ability to bind fH, as discussed above) . The invention proposes such strains, and also proposes vesicles that these strains release, for example, isolated from the culture medium after growing the strains. The preferred v2 mutant for expression in these strains has the S32 and/or L123 mutation, as discussed herein, and the preferred v3 mutant for expression in these strains has the S32 and/or L126 mutation, as discussed herein. Thus, vesicles derived from meningococci expressing such mutant fHbp v2 and v3 sequences are the most preferred immunogens for use in the vaccines of the invention.

Effective promoters for use in such strains include those described in WO 2013/033398 and WO 2013/113917. For example, the promoter may be (a) a promoter from a porin gene, preferably porA or porB, in particular from N. meningitidis; or (b) an rRNA gene promoter (such as a 16S rRNA gene), particularly from N. meningitidis. When a meningococcal porin promoter is used, preferably it is derived from porA, and, more specifically, the -10 region from the meningococcal porA gene promoter, and/or the -35 region from the meningococcal porA gene promoter (preferably, where the -10 region and -35 region are separated by an intermediate sequence of 12-20 nucleotides and where the intervening 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, in particular it may comprise (i) a -10 meningococcal rRNA gene promoter region and/or (ii) a -35 meningococcal rRNA gene promoter region. A hybrid of (a) and (b) can also be used, for example having a -10 region from the porA promoter and a -35 region from the rRNA promoter (which may be a consensus -35 region). Thus, an effective promoter may be a promoter that includes either (i) the -10 region from an rRNA gene (particularly meningococcal) and the -35 region from a porA gene (particularly meningococcal) or (ii) the -10 region from a gene porA (specifically meningococcal) and -35 region from the rRNA gene (specifically meningococcal).

If LOS is present in the vesicle, it is possible to treat the vesicle in such a way as to bind its LOS and protein components (intravesicular conjugation [WO 2004/014417]).

Immunogenic compositions.

The polypeptides of the invention can be used as active ingredients of immunogenic compositions, therefore, the seventh aspect of the invention provides an immunogenic composition comprising a mutant fHbp v1.13 polypeptide according to the first aspect of the invention, a mutant fHbp v1.15 polypeptide according to the second aspect of the invention, a fusion polypeptide according to the third aspect of the invention or an outer membrane vesicle according to the sixth aspect of the invention. These immunogenic compositions may find use in immunizing a mammal, preferably a human, against infection caused by Neisseria meningitidis.

In a preferred embodiment of the invention, in addition to the polypeptide antigens of the first, second or third aspects of the invention, the immunogenic composition of the invention further comprises one or more 44CMenB antigenic components.

As described above, the 4CMen product (BEXSERO) contains the epidemic strain OMV preparation

- 15 046480 meningococcus group B NZ98/254, B:4:P1.7b,4. The same OMVs are found in the MeNZB vaccine and are referred to herein as OMVnz. In addition, 44CMenB contains five meningococcal antigens: NHBA (287; subvariant 1.2), fHbp (741; subvariant 1.1), NadA (961; subvariant 3.1), GNA1030 (953), and GNA2091 (936). Four of these antigens are in the form of fusion proteins (NHBA-GNA1030 (287-953) fusion protein and GNA2091-fHbp (936-741) fusion protein).

A 0.5 ml dose of the finished product 44CMenB is formulated to contain 50 µg each of NHBA-GNA1030, NadA and GNA2091-fHbp adsorbed onto 1.5 mg aluminum hydroxide adjuvant and 25 µg OMV from N. meningitidis strain NZ98 /254. In addition, each 0.5 ml dose of the drug contains 3.125 mg of sodium chloride, 0.776 mg of histidine and 10 mg of sucrose.

In another preferred embodiment, the immunogenic composition of the invention further comprises the finished vaccine product 44CMenB, marketed under the trade name BEXSERO.

In another preferred embodiment, the immunogenic composition of the invention comprises an fHbp fusion polypeptide of the sequence SEQ ID NO: 19 (fHbp 23S_1.13_E211A/S216R) and the final composition 44CMenB.

Meningococci serogroups A, C, W135 and Y.

The compositions of this invention may also include meningococcal capsular saccharide antigens from one or more of serogroups Y, W135, C and A, where the antigens are conjugated to carrier protein(s) and/or are oligosaccharides. Capsular saccharides can be used in the form of oligosaccharides. They are conveniently prepared by fragmentation of the purified capsular polysaccharide (eg by hydrolysis), which is usually followed by isolation of fragments of the required size.

Modern serogroup C vaccines [Costantino et al.] (1992), Vaccine, 10:691-698, Jones (2001), Curr Opin Investig Drugs, 2:47-49], MENINGITEC and NEISVAC-C) include conjugated saccharides. MENJUGATE and MENINGITEC have oligosaccharide antigens conjugated to a CRM197 carrier, while NEISVAC-C uses a full-length polysaccharide (de-O-acetylated) conjugated to a tetanus toxoid carrier.

The vaccine products marketed under the trade names MENVEO, MENACTRA and NIMENRIX all contain conjugated capsular polysaccharide antigens from each of serogroups Y, W135, C and A.

In MENVEO (also known by its generic name as meningococcal oligosaccharide (groups A, C, Y and W-135) diphtheria conjugated CRM197 vaccine), the A, C, W135 and Y antigens are each conjugated to a CRM197 carrier.

In MENACTRA (also known by its generic name as diphtheria toxoid-conjugated meningococcal polysaccharide vaccine (groups A, C, Y, and W-135), the A, C, W135, and Y antigens are each conjugated to a diphtheria toxoid-conjugated carrier.

In NIMENRIX (also known by its generic name as meningococcal polysaccharide conjugate vaccine (groups A, C, Y and W-135), the A, C, W135 and Y antigens are each conjugated to a tetanus toxoid carrier.

In a preferred embodiment of the invention, in addition to the polypeptide antigens of the first, second or third aspects of the invention, the immunogenic composition of the invention further comprises one or more conjugated capsular saccharide antigen of N. meningitidis serogroups A, C, W135 and/or Y.

In a preferred embodiment of the invention, in addition to the polypeptide antigens of the first, second or third aspects of the invention, the immunogenic composition of the invention further comprises a 44CMenB finished product along with one or more conjugated capsular saccharide antigens of N. meningitidis serogroups A, C, W135 and/or Y .

In a preferred embodiment, the immunogenic composition of the invention, in addition to the polypeptide antigens of the first, second or third aspects of the invention, contains the finished product 44CMenB along with conjugated capsular saccharide antigens of N. meningitidis of each of serogroups A, C, W135 and/or Y, forming a pentavalent immunogenic composition , containing antigens against each of the meningococcal serotypes A, B, C, W135 and Y.

In preferred embodiments, the composition comprises conjugates of antigens A, C, W135 and Y that are present in MENVEO, conjugates of antigens A, C, W135 and Y that are present in MENACTRA, or conjugates of antigens A, C, W135 and Y that are present in NIMENRIX .

In another preferred embodiment, the immunogenic composition of the invention comprises an fHbp fusion polypeptide of the sequence SEQ ID NO: 19 (fHbp 23S_1.13_E211A/S216R), the 44CMenB product, and conjugates of the A, C, W135 and Y antigens that are present in MENVEO.

Alternatively, an immunogenic composition of the invention comprising the polypeptide antigens of the first, second or third aspects of the invention may be co-administered with one or more of BEXSERO and MENVEO, MENACTRA or NIMENRIX. Preferably

- 16 046480 the immunogenic composition according to the invention is administered together with BEXSERO and MENVEO.

The term co-administered means that the different immunogenic compositions/vaccines can be administered either separately or in combination.

When vaccines are given separately, they are usually given at different sites, such as one vaccine in the left forearm and a second vaccine in the right forearm. Thus, the two vaccines can be administered contralaterally (eg, both forearms, or both legs, or an arm and leg on opposite sides) or ipsilaterally (eg, an arm and leg on the same side). Although the vaccines can be administered separately, they are administered at substantially the same time (eg, during the same medical consultation or visit to a healthcare professional or vaccination center), for example within 1 hour.

However, administration is performed as a combination rather than co-immunization with separate administration. Thus, during joint immunization, a combination vaccine can be used, i.e. a single composition in which various immunogens are mixed. The advantage of combination vaccines is that fewer injections are administered to the subject, which may increase compliance, which is an advantage in clinical practice.

Use of immunogenic compositions according to the invention

The immunogenic compositions of the invention are suitable for use in medicine, and in particular can be used to immunize a mammal against infection and/or disease caused by Neisseria meningitidis such that recipients of the immunogenic composition mount an immune response that provides protection against infection and/or disease caused by the bacteria Neisseria meningitidis.

In a preferred embodiment, the immunogenic compositions of the invention may find use in immunizing a mammal against an infection or disease caused by meningococcus B. However, in embodiments of the invention where meningococcal serogroup B antigens are combined with meningococcal antigens of other serogroups (e.g., A.C, W and/or or Y), the immunogenic compositions may find use in immunizing a mammal against infection or disease caused by meningococcus A, B, C, W and/or Y.

Thus, the immunogenic compositions of the invention may find use in prophylactic methods for immunizing subjects against infection and/or disease caused by Neisseria meningitidis. Immunogenic compositions can also be used in methods of treatment (ie, for the treatment of infection caused by Neisseria meningitidis).

The invention also provides a method of generating an in vivo immune response in a mammal against an infection caused by Neisseria meningitidis, comprising administering to the mammal an immunogenic composition of the invention. The invention also provides the polypeptides of the invention for use in these methods.

The immune response is preferably protective and preferably includes antibodies and/or cell-mediated immunity. Preferably, the immune response is a bactericidal antibody response. The method may cause a booster response. By producing an immune response in vivo, the mammal may be protected against Neisseria disease (particularly meningococcal disease).

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

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

The immunological compositions of the invention are preferably formulated as vaccine products that are suitable for therapeutic (ie, treating an infection) or prophylactic (ie, preventing an infection) use. As a rule, vaccines are preventative.

Preferably the mammal is human. The person may be an adult, an adolescent, or a child (for example, a toddler or infant). A vaccine intended for children may also be administered to adults, for example to evaluate safety, dosage, immunogenicity, etc.

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

Protection against N. meningitidis can be assessed epidemiologically, for example in a clinical trial, but it is convenient to use indirect methods to confirm that the immunogenic composition elicits a serum bactericidal antibody (SBA) response in recipients. At

- 17 046480 SBA study, serum from recipients of the composition is incubated with target bacteria (in this invention, N. meningitidis) in the presence of complement (preferably human complement, although rabbit complement is often used instead) and bacterial cytolysis is assessed at various dilutions of the serum to determine SBA activity . The results of the SBA assay can be reinforced by performing a competitive SBA test to further indirectly confirm the immunogenic activity of the antigen(s) of interest. In the SBA competition test, sera from recipients of an immunogenic composition containing antigen(s) are preincubated with said antigen(s) and then incubated with target bacteria in the presence of human complement. Bacterial cytolysis is then assessed, which will be reduced or absent if, during the preincubation phase, bactericidal antibodies in the recipient serum bind to the antigens of interest and thus become unavailable for binding to the bacterial surface antigen.

The composition does not necessarily need to protect against every strain of N. meningitidis, nor does every recipient of the composition need to be protected. Such universal protection is not a generally accepted standard in the art. Rather, protection against a panel of reference laboratory strains, often selected separately for each country and possibly varying over time, is usually assessed and tested in the recipient population.

Preferred compositions of the invention can provide a patient with an antibody titer that is superior in seroprotection to each antigenic component in a reasonable percentage of individuals. The antigens and associated antibody titers above which the host is considered to have seroconverted against the pathogen are well known, and such titers have been published by organizations such as the WHO. Preferably, more than 80% of a statistically significant sample of subjects are seroconverted, more preferably more than 90%, even more preferably more than 93%, and most preferably 96-100%.

Immunogenic compositions contain an immunologically effective amount of an immunogen, and, if necessary, any other of the other specified components.

By immunologically effective amount is meant that administration of the amount to an individual, either as a single dose or as part of a series of administrations, is effective for treatment or prevention.

The term warning means that the progression of the disease is slowed and/or stopped, or that the disease does not start. For example, a subject's immune system may be primed (eg, through vaccination) to initiate an immune response and resist infection such that disease does not occur. Thus, a vaccinated subject may be infected but is better able to resist infection than a control subject. This amount will vary depending on the health and physical condition of the individual being treated, the age, the taxonomic group of the individual being treated (eg, non-human primate, etc.), the ability of the individual's immune system to synthesize antibodies, the the desired degree of protection, on the composition of the vaccine, on the assessment of the medical situation by the attending physician and other significant factors. The amount is expected to be within a relatively wide range, which can be determined by routine testing. The composition can be administered in combination with other immunoregulatory agents.

Vaccine effectiveness.

Immunogenic compositions for use according to this invention preferably provide vaccine efficacy against at least one strain of N. meningitidis of at least 10%, for example, >20, >30, >40, >50, >60, >70, >80, > 85, >90% or more.

Vaccine efficacy is determined by the reduction in the relative risk of developing meningococcal disease in subjects who receive a composition of the invention compared with subjects who do not receive such a composition (eg, are not immunized or receive a placebo or negative control). Thus, the number of cases of meningococcal disease in a population immunized with the invention is compared with the number of cases in a control population that was not immunized with the invention to obtain a relative risk and vaccine efficacy equal to 100% minus this number.

Vaccine effectiveness is determined on a population basis rather than on an individual basis. Therefore, it is a useful epidemiological indicator but does not predict individual protection. For example, an individual subject may be exposed to a very large dose of an infectious agent or may have other risk factors that make him or her more susceptible to infection, but this will not affect the reliability or practical value of the effectiveness measure. The size of the population that is immunized according to the invention and in which the effectiveness of the vaccine is determined is ideally at least 100 and may be larger, for example at least 500 subjects. The size of the control group should also be at least 100, for example at least 500.

- 18 046480

Introduction.

The compositions of the invention are typically administered directly to the patient. Direct administration may be by parenteral injection (eg, subcutaneous, intraperitoneal, intravenous, intramuscular, interstitial tissue) or by rectal, oral, vaginal, superficial, transdermal, intranasal, ocular, auricular, pulmonary, or other mucosal application. Preferably intramuscular injection into the thigh or forearm. The injection can be performed using a needle (eg, a hypodermic needle), but alternatively, a needle-free injection can be performed. The typical intramuscular dose is approximately 0.5 ml.

Neusserial infections affect various parts of the body, so the compositions of the invention can be prepared in various forms. For example, the compositions can be formulated as injectables, either as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection can also be prepared. Compositions can be formulated for surface application, for example in the form of an ointment, cream or powder. The compositions may be formulated for oral administration, for example in the form of tablets or capsules, or in the form of a syrup (optionally flavored). Compositions can be formulated for administration into the lungs, for example by inhalation, using a fine powder or spray. The compositions can be prepared in the form of a suppository or pessary. Compositions can be formulated for nasal, ear or ocular administration, for example in the form of drops. Most preferred are compositions suitable for parenteral injection.

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

As used herein, a dose of a composition is a volume of the composition suitable for administration to a subject through a single immunization. Human vaccines are usually administered in a dose volume of approximately 0.5 ml, although fractional doses may be administered (eg, to children). The dose volume may further vary depending on the concentration of antigens in the composition.

In addition, the composition can be presented in the form of a multi-dose kit, i.e. one container contains an amount of the composition sufficient for several immunizations. Multi-dose embodiments may include a preservative or the multi-dose container may have an aseptic device for dispensing individual doses of the composition.

Administration may involve a single-dose regimen, but will typically involve a multiple-dose regimen. A schedule of at least three dosing administrations is preferred. Suitable intervals between priming doses can be determined routinely, eg 4 to 16 weeks, eg 1 month or two months. For example, BEXSERO may be administered at 2, 4, and 6 months of age, or 2, 3, and 4 months, with a possible fourth dose at 12 months of age.

The subject to be immunized is a human being, who may be of any age, for example, 0-12 months, 1-5 years, 5-18 years, 18-55 years, or over 55 years. Preferably, the subject being immunized is an adolescent (eg, 12-18 years of age) or an adult (18 years of age or older).

Optionally, the subject is an adolescent or adult who was immunized against N. meningitidis as a child (eg, before the age of 12 years) and receives a booster dose of an immunogenic composition of the invention.

When the invention relates to co-immunization, the different immunogenic compositions/vaccines can be administered either individually or in combination.

When vaccines are given separately, they are usually given at different sites, such as one vaccine in the left forearm and a second vaccine in the right forearm.

Thus, the two vaccines can be administered contralaterally (eg, both forearms, or both legs, or an arm and leg on opposite sides) or ipsilaterally (eg, an arm and leg on the same side). Although the vaccines can be administered separately, they are administered at substantially the same time (for example, during the same medical consultation or visit to a health care provider or vaccination center), for example, within 1 hour.

However, administration is carried out as a combination rather than co-immunization with separate administration. Thus, during joint immunization, a combination vaccine can be used, i.e. a single composition in which various immunogens are mixed. The advantage of combination vaccines is that fewer injections are administered to the subject, which may increase compliance, which is an advantage in clinical practice.

Components that are not antigenic.

The immunogenic composition of the invention will generally include a pharmaceutically acceptable carrier, which may be any substance that does not itself produce antibodies harmful to the patient receiving the composition and that does not exhibit nonspecific toxicity when administered. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerin and ethanol. In such carrier environments, they can also

- 19 046480 there are no auxiliary substances such as wetting or emulsifying agents, buffering agents to maintain pH, etc. Suitable carriers are discussed in detail in Gennaro (2000), Remington: The Science and Practice of Pharmacy. 20th ed., ISBN: 0683306472.

Preferably the composition is sterile. Preferably it is pyrogen-free. Preferably it is buffered, for example having a pH of 6 to 8, typically around pH 7. When the composition contains an aluminum hydroxide salt, it is preferable to use a histidine buffer [WO 03/009869]. The compositions of the invention may be isotonic in humans.

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 LPS derivatives (such as monophosphoryl lipid A or 3-O-deacetylated monophosphoryl lipid), immunostimulating oligonucleotides, detoxified bacterial ADP-ribosylating toxins, liposomes, imidazoquinolones or mixtures thereof, but are not limited to the above. Other substances that act as immunostimulating agents are described in Chapter 7 of Vaccine Design. (1995), ed. Powell & Newman. ISBN: 030644867X. Plenum.

The use of aluminum hydroxide and/or aluminum phosphate adjuvants is particularly preferred and, as a rule, the polypeptides are adsorbed onto these salts. These salts include oxyhydroxides and hydroxyphosphates (for example, see chapters 8 and 9 of Vaccine Design. (1995), ed. Powell & Newman. ISBN: 030644867X. Plenum). The salts can take any suitable form (eg, gel, crystalline, amorphous, etc.).

Additional antigenic components.

The immunogenic compositions of the invention may include antigens for immunization against other diseases or infections. For example, the composition may include one or more of the following antigens:

saccharide antigen from Streptococcus pneumoniae [eg Watson (2000), Pediatr. Infect. Dis. J. 19:331-332, Rubin (2000), Pediatr. Clin. North Am. 47:269-285 and Jedrzejas (2001), Microbiol. Mol. Biol. Rev. 65:187-207];

hepatitis A virus antigen, such as inactivated virus [eg, Bell (2000), Pediatr. Infect. Dis J 19:1187–1188, Iwarson (1995) APMIS 103:321–326];

hepatitis B virus antigen, such as surface and/or core antigens [eg, Gerlich et al. (1990), Vaccine, 8 Suppl: S3-68 &79-80];

diphtheria antigen, such as diphtheria toxoid [eg, Chapter 3 of Vaccines (1988), ed. Plotkin & Mortimer. ISBN 0-7216-1946-0] e.g. the CRM197 mutant [e.g. Del Guidice et al. (1998), Molecular Aspects of Medicine, 19:1-70];

a tetanus antigen, such as tetanus toxoid (eg, Chapter 4 of Vaccines (1988), edited by Plotkin & Mortimer. ISBN 0-7216-1946-0);

Bordetella pertussis antigen, such as pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) of B. pertussis, possibly also in combination with pertactin and/or agglutinogens 2 and 3 (for example, Gustafsson et al. (1996), N. Engl. J Med 334:349-355, and Rappuoli et al (1991), TIBTECH 9:232-238);

saccharide antigen from Haemophilus influenzae B [eg, Costantino et al. (1999) Vaccine 17:12511263];

polio pathogen antigens [eg, Sutter et al. (2000) Pediatr Clin North Am 47:287-308 and Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126], such as inactivated polio virus;

measles, mumps and/or rubella antigens (for example, chapters 9, 10 and 11 of Vaccines (1988) edited by Plotkin & Mortimer. ISBN 0-7216-1946-0);

influenza antigens (eg, Chapter 19 of Vaccines (1988), edited by Plotkin & Mortimer. ISBN 0-7216-1946-0), such as hemagglutinin and/or neuraminidase surface proteins;

Moraxella catarnhalis antigen [eg, McMichael (2000), Vaccine 19 Suppl 1: S101-107];

protein antigen from Streptococcus agalactiae (group B streptococcus) [eg, Schuchat (1999), Lancet, 353 (9146):51-6, WO02/34771];

saccharide antigen from Streptococcus agalactiae (group B streptococcus);

Streptococcus pyogenes (group A streptococcus) antigen [eg WO 02/34771, Dale (1999), Infect. Dis. Clin. North Am. 13:227-43, Ferretti et al. (2001), PNAS USA, 98: 4658-4663];

Staphylococcus aureus antigen [eg, Kuroda et al. (2001), Lancet, 357(9264):1225-1240; see also p. 1218-1219].

If necessary, toxic protein antigens can be detoxified (eg, detoxification of pertussis toxin by chemical and/or genetic means [Rappuoli et al. (1991), TIBTECH, 9:232-238]).

When the composition includes diphtheria antigen, it is preferable to also include tetanus antigen and pertussis antigen. Likewise, when tetanus antigen is included, it is preferable to also include diphtheria and pertussis antigens. Likewise, when pertussis anti is turned on

- 20 046480 gene, preferably also including diphtheria and tetanus antigens. Thus, combinations of diphtheria, tetanus and pertussis antigens are preferred.

Saccharide antigens are preferably in the form of conjugates. In general, conjugation enhances the immunogenicity of saccharides because it converts them from T-independent antigens to T-dependent antigens, thereby allowing priming for immunological memory. Conjugation is particularly effective for vaccines used in pediatrics and is a well-known technique.

Typical carrier proteins are bacterial toxins such as diphtheria or tetanus toxins or toxoids or mutant forms thereof. The diphtheria toxin mutant CRM197 [Research Disclosure, 453077 (Jan 2002)] is effective and is the carrier in the Streptococcus pneumoniae vaccine marketed under the trade name PREVNAR. Other suitable carrier proteins include N. meningitidis outer membrane protein complex [EP-A-0372501], synthetic peptides [EP-A-0378881, EP-A-0427347], heat shock proteins [WO 93/17712, WO 94/03208 ], pertussis virus proteins [WO 98/58668, EP-A-0471177], cytokines [WO 91/01146], lymphokines [WO 91/01146], hormones [WO 91/01146], growth factors [WO 91/01146] , artificial proteins containing multiple human CD4+ T-cell epitopes of various antigens of pathogenic origin [Falugi et al. (2001), Eur. J. Immunol. 31:3816-3824], such as N19 [Baraldo et al. (2004), Infect. Immun. 72(8):4884-7], protein D from H. influenzae [EP-A-0594610, Ruan et al. (1990), J. Immunol. 145:33793384], pneumolysin [Kuo et al. (1995), Infect. Immun. 63:2706-13] or its non-toxic derivatives [Michon et al. (1998), Vaccine. 16:1732-41], pneumococcal surface protein PspA [WO 02/091998], iron uptake proteins [WO 01/72337], toxin A or B from C. difficile [WO 00/61761], recombinant exoprotein A from P. aeruginosa (rEPA) [WO 00/33882] etc.

Any suitable conjugation reaction can be used, if necessary, with any suitable linker.

Activation or functionalization of the saccharide is usually carried out before conjugation. Activation may include, for example, cyanylating reagents such as CDAP (eg, 1-cyano-4dimethylaminopyridine tetrafluoroborate [Lees et al. (1996), Vaccine, 14:190-198, WO 95/08348]). Other suitable methods use carbodiimides, hydrazides, active esters, norborane, nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, etc.

Attachment via a linker group can be carried out using any known method, for example, the methods described in US 4882317 and US 4695624. Other types of additions include reductive amination of the polysaccharide, attaching the resulting amino group to one end of the adipic acid that is the linker group, and then attaching the protein to the other end of the adipic acid, which is the linker group [Porro et al. (1985), Mol. Immunol. 22:907-919, EP 0208375]. Other linkers include B-propionamido [WO 00/10599], nitrophenyl-ethylamine [Gever et al. Med. Microbiol. Immunol, 165:171-288 (1979)], halides [US 4057685], glycosidic bonds [US 4673574; US 4761283; US 4808700], 6-aminocaproic acid [US 4459286], ADH [US 4965338], C ₄ -C ₁₂ groups [US 4663160], etc. An alternative to using a linker is direct linking. Direct protein binding may involve oxidation of the polysaccharide followed by reductive amination, as described, for example, in US 4,761,283 and US 4,356,170.

A preferred process involves introducing amino groups into the saccharide (eg, by replacing the =O end groups with -NH2), followed by derivatization with an adipic diester (eg, N-hydroxysuccinimide adipic diester) and reaction with a protein carrier. Another preferred reaction utilizes CDAP activation using a protein D vehicle, for example for MenA or MenC.

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

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

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

General information.

The term containing covers the terms including and consisting of, for example a composition containing X may consist solely of X or may include something additional, for example X+Y. The mention containing (or contains, etc.) can be replaced by the mention consisting of (or consists of, etc.). The term essentially consisting of limits the scope of the claim to specific materials or steps and those that do not significantly affect the essential and novel characteristics of the invention.

In relation to the digital value x, it is possible to use the term approximately, which means, for example, x+10%.

- 21 046480

The term essentially does not exclude meaning entirely, for example, a composition that is essentially free of Y may be completely free of Y. The term essentially can be ignored in the definition of the invention where necessary.

When the invention concerns an epitope, the epitope may be a B cell epitope and/or a T cell epitope, but is typically a B cell epitope. Such epitopes can be detected experimentally (eg, using PEPSCAN (see, for example, Geysen et al. (1984), PNAS USA, 81:3998-4002 and Carter (1994), Methods Mol. Biol. 36:207-23 ) or similar methods) or can be predicted (eg, using the Jameson-Wolf antigenic index (Jameson, B.A. et al. 1988, CABIOS, 4(1):181-186), matrix-based approaches (Raddrizzani & Hammer (2000 ), Brief Bioinform. 1(2):179-89), MAPITOPE (Bublil et al. (2007), Proteins, 68(1):294-304), TEPITOPE (De Lalla et al. (1999), J. Immunol. 163:1725-29 and Kwok et al. (2001), Trends Immunol. 22:583-88), neural networks (Brusic et al. (1998), Bioinformatics, 14(2):121-30), OptiMer & EpiMer (Meister et al. (1995), Vaccine, 13(6):581-91 and Roberts et al. (1996), AIDS Res. Hum. Retroviruses, 12(7):593-610), ADEPT (Maksyutov & Zagrebelnaya (1993), Comput. Appl. Biosci. 9(3):291-7), T-sites (Feller & de la Cruz (1991), Nature, 349(6311):720-1), hydrophilicity (Norr (1993), Peptide Research, 6:183-190) or antigenic index (Welling et al. (1985), FEBS Lett. 188:215-218)). Epitopes are the part of an antigen that is recognized and binds to the antigen-binding sites of antibodies or T-cell receptors, and may also be designated antigenic determinants.

As used herein, references to percent sequence identity between a searched amino acid sequence and a found similar amino acid sequence are understood to indicate an identity value that is calculated using a suitable pairwise sequence alignment algorithm or software known in the art.

The desired amino acid sequence can be described by the amino acid sequence described in one or more claims. The searched sequence may be 100% identical to the found similar sequence, or it may include some integer number of amino acid substitutions (e.g., point mutations, substitutions, deletions, insertions, etc.) compared to the found similar amino acid sequence, so that the % identity is less than 100%. For example, a searched sequence may be at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to a found similar sequence.

The preferred alignment tools used for alignment and calculation of percent (%) sequence identity are local alignment tools such as BLAST (Basic Local Alignment Search Tool) algorithms. Programs for performing BLAST analysis are made publicly available by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Alignment can be performed using the Smith-Waterman homology search algorithm, using an affine gap search with a gap opening penalty of 12, a gap continuation penalty of 2, and the BLOSUM matrix, 62. The Smith-Waterman homology search algorithm is described by Smith & Waterman (1981), Adv. Appl. Math. 2:482-489. Other preferred alignment tools are Water (EMBOSS) and Marcher (EMBOSS). Alternatively, the preferred alignment tools used for alignment and calculation of percent (%) sequence identity are best-fit alignment tools such as GENEPAST, also known as the KERR algorithm.

To calculate percent identity, the searched and found similar sequence can be compared and aligned with the maximum match in a designated region (e.g., a region of at least 40, 45, 50, 55, 60, 65 or more amino acids in length and which may extend to the entire length of the found similar amino acid sequence). Said designated region must include a region of the search sequence containing any specific point mutation of the amino acid sequence. Alternatively, percent sequence identity can be calculated over the entire length of the search sequence. Any N-terminal or C-terminal amino acid regions that may be present in the target sequence, such as signal peptides or leader peptides or C-terminal or N-terminal tags, should be excluded from the alignment.

The term fragment, when applied to a polypeptide sequence, means that the polypeptide is a section of a full-length protein. As used herein, the mutant polypeptide fragment also contains mutation(s). The fragments may have qualitative biological activity along with the full-length protein, for example, the immunogenic fragment contains or encodes one or more epitopes, such as immunodominant epitopes, which allows the production of the same or comparable immune response to the fragment as to the full-length sequence. Polypeptide fragments typically have a deletion of the amino-terminal and/or carboxy-terminal portions of the native protein, but the remainder of the amino acid sequence of the fragment is identical to the amino acid sequence of the native protein. Polypeptide fragments

- 22 046480 may contain, for example: approximately 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 45, 50, 55, 60 , 70, 80, 90, 100, 150, 200, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258 , 259, 260, 261, 262 consecutive amino acids of the reference polypeptide sequence, including all intervening integers, e.g. 50 to 260, 50 to 255, 50 to 250, 50 to 200, 50 to 150 after each other amino acid of the reference polypeptide sequence. The term fragment completely excludes full-length fHbp polypeptides and its mature lipoproteins.

In the classification of meningococci, serogroup is followed by serotype, serosubtype and then immunotype, and standard nomenclature lists serogroup, serotype, serosubtype and immunotype separated by a colon, for example B:4:P1.15:L3,7,9. Within serogroup B, some lineages cause disease frequently (hyperinvasive), some lineages cause more severe disease than others (hypervirulent), and others rarely cause disease. There are seven generally recognized hypervirulent lineages, namely subgroups I, III and IV-1, ET-5 complex, ET-37 complex, cluster A4 and lineage 3. These have been characterized using multilocus enzyme electrophoresis (MLEE), but multilocus enzymatic electrophoresis (MLEE) has also been used to classify meningococci. sequence typing (MLST). The four major hypervirulence clusters are the ST32, ST44, ST8 and ST11 complexes.

When referenced herein to increased stability, or higher stability, or increased stability, it is meant that the mutant polypeptides set forth herein have a higher thermal stability (in kcal/mol) compared to the unmutated polypeptide (wild type) under the same experimental conditions . Increased stability can be assessed using differential scanning calorimetry (DSC), for example, as discussed in Bruylants et al. (Differential Scanning Calorimetry in Life Sciences: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design, 2005, Curr. Med. Chem. 12:2011-2020) and Calorimetry Sciences Corporation's Characterizing Protein stability by DSC (Life Sciences Application Note, Doc No. 20211021306, February 2006) or using differential scanning fluorimetry (DSF). An increase in stability can be characterized as an increase in the phase transition temperature (Tm) of at least 5°C when assessed by the DSC or DSF method. See, for example, Thomas et al., Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen, 2013 Hum. Vaccin. Immunother. 9(4):744-752.

It should be understood that the description of the invention is given above by way of example only and any modifications are possible provided they remain within the scope and spirit of the invention.

Sequences

SEQ ID NO: 1 [vl.13 mature polypeptide from strain M982]

CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTY GNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQD SEDSGKMVAKRQFRIGDIAGEHTSFKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAK QGHGKIEHLKSPEL NVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEV AGSAEVETANGIHHIGLAAKQ

SEQ ID NO: 2 [vL13 AG]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKM VAI<RQFRIGDIAGEHTSFDI<LPI<GGSATYRGTAFGSDDAGGI<LTYTIDFAAI<QGHGI<IE

- 23 046480

HLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVE TANGIHHIGLAAKQ

SEQ ID NO: 3 [vl.13 AG (E211A/E232A)]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKM VAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVEL ATAYIKPDEKRHAVISGSVLYNQDAKGSYSLGIFGGQAQEVAGSAAVE TANGIHHIGLAAKQ

SEQ ID NO: 4 [vl.13 AG (E211A/S216R)]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKM VAI<RQFRIGDIAGEHTSFDI<LPI<GGSATYRGTAFGSDDAGGI<LTYTIDFAAI<QGHGI<IE HLKSPEL NVELATAYIKPDEKRHAVISGSVLYNQDAKGSYRLGIFGGQAQEVAGSAEVE TANGIHHIGLAAKQ

SEQ ID NO: 5 [vl.15 mature polypeptide from strain NM452]

CSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAE RTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQT EQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYT IDFAAKQGHGKIEHLK SPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSYSLGIFGG QAQEVAGSAEVETANGIRHIGLAAKQ

SEQ ID NO: 6 [vl.15 AG]

VAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSL NTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKM VAI<RQFRIGDIVGEHTSFGI<LPI<DVMATYRGTAFGSDDAGGI<LTYTIDFAAI<QGHGI<IE HLKSPELNVDLAAADI KPDEKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVE TANGIRHIGLAAKQ

SEQ ID NO: 7 [vl.15 AG (S219R)]

- 24 046480

VAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSL NTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKM VAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAEKGSYRLGIFGGQAQEVAGSAEVE TANGIRHIGLAAKQ

SEQ ID NO: 8 [vl.15 AG (E214A/S219R)]

VAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSL NTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKM VAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAAKGSYRLGIFGGQAQEVAGSAEV ETANGIRHIGLAAKQ

SEQ ID NO: 9 [vl.15 AG (E214A/E235A)]

VAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSL NTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKM VAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAAKGSYSLGIFGGQAQEVAGSAAVE TANGIRHIGLAAKQ

SEQ ID NO: 10 [v2 wild type from strain 2996]

MNRTAFCCLSLTAALILTACSSGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIY I<QDHSAVVALQIEI<INNPDI<IDSLINQRSFLVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSD DAGGI< LTYTIDFAAI<QGHGI<IEHLI<TPEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI< GTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ

SEQ ID NO: 11 [v2 mature polypeptide]

CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYG NGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPD I<IDSLINQRSFLVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGH

- 25 046480

GKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGS ATVKIGEKVHEIGIAGKQ

SEQ ID NO: 12 [v2 AG]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLINQ RSFLVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL I<T PEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEK VHEIGIAGKQ

SEQ ID NO: 13 [v3 wild type from strain M1239]

MNRTAFCCLSLTTALILTACSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLT LEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLA SGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYH GKAF S SDDPNGRLHY S I DFTI<I<QGYGRIEHLI<TLEQN VELA A AELK A DEK SHA VILGDT RYGSEEKGTYHLALFGDRAQEIAGS ATVKIGEKVHEIGIAGKQ

SEQ ID NO: 14 [v3 mature]

CSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAE KTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQI EI<INNPDI<TDSLINQRSFLVSGLGGEHTAFNQLPGGI<AEYHGI<AFSSDDPNGRLHYSIDF TKKQGY GRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDR AQEIAGSATVKIGEKVHEIGIAGKQ

SEQ ID NO: 15 [v3 AG]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL KTLEQNVELAAA ELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ

SEQ ID NO: 16 [v2 AG S32V/L123R]

- 26 046480

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKroSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ

SEQ ID NO: 17 [v3 AG S32V/L126R]

VAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLSAQGAEKTFKAGDKDNSL NTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLI NQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHL KTLEQNVELAAA ELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQ

SEQ ID NO: 18 [(23S_1.13 E211A/E232A)]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSL INQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTG KLKNDKVSRFDFIRQIEVD GKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKL PKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEK RHAVISGSVLYNQDAKGSYSLGIFGGQAQEVAGSAAVETANGIHHIGLAAKQ

SEQ ID NO: 19 [23S_1.13 E211A/S216R]

- 27 046480

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKroSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKOGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLINQRSFR VSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKOGSGGGGVAADIGAGLADALTAPLDHK DKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GKLKNDKVSRFDFIRQIEVD GKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFKL PKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEK RHAVISGSVLYNQDAKGSYRLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ

SEQ ID NO: 20 [23S_1.15 S219R]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN QRSFRVSGLGGEHTAFNQLPDGI<AEYHGI<AFSSDDAGGI<LTYTIDFAAI<QGHGI<IEHL KTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKOGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLIN QRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKOGSGGGGVAADIGAGLADALTAPLDHK DKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTG KLKNDKISRFDFIRQIEV DGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGK LPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAEKGSYRLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ

SEQ ID NO: 21 [23S_1.15 E214A/S219R]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN

- 28 046480

QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHL KTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTL SAQGAEKTFKAGDKDNSLNTGKL KNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLINQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGI AGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEV DGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGK LPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDL AAADIKPD EKHHAVISGSVLYNQAAKGSYRLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ

SEQ ID NO: 22 [23S_1.15 E214A/E235A]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSRFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLIN QRSFRVSGLGGEHTAFNQLPDGKAEYHGKAF S SDD AGGKLTYTIDF AAKQGHGKIEHL KTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKOGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPONGTLTL SAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHS AVVALQIEKINNPDKTDSLIN QRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNG RLHYSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYH LALFGDRAOEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHK DKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNT GKLKNDKISRFDFIRQIEV DGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGK LPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAAKGSYSLGIFGGQAQEVAGSAAVETANGIRHIGLAAKQ

SEQ ID NO: 23 [vl.l AG + His tag]

VAADIGAGL AD ALT APLDHKDKGLQ SLTLDQ S VRKNEKLKL AAQGAEKT YGNGD SLN TGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMV AKRQFRIGDIAGEHTTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEH L KSPELGLAAKQLNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVA

- 29 046480

GSAEVKTVNGIRHLEHHHHHH

SEQ ID NO: 24 [vl.13 AG + His tag]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKM VAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVEL ATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVE TANGIHHIGLAAKQLEHHHHHH

SEQ ID NO: 25 [vl.13 AG (E211A)]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKM VAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVEL ATAYIKPDEKRHAVISGSVLYNQDAKGSYSLGIFGGQAQEVAGSAEVE TANGIHHIGLAAKQLEHHHHHH

SEQ ID NO: 26 [vl.13 AG (S216R)]

VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLN TGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKM VAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVEL ATAYIKPDEKRHAVISGSVLYNQDEKGSYRLGIFGGQAQEVAGSAEVE TANGIHHIGLAAKQLEHHHHHH

SEQ ID NO: 27 [vl.15 AG + His tag]

VAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSL NTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKM VAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVDLAAADIKPD EKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVE TANGIRHIGLAAKQLEHHHHHH

SEQ ID NO: 28 [vl.15 AG (E214A) + His tag]

VAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSL

- 30 046480

NTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKM VAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIE HLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAAKGSYSLGIFGGQAQEVAGSAEVE TANGIRHIGLAA KQLEHHHHHH

SEQ ID NO: 29 [fHbp fusion polypeptide 231 wild type]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNT GI<LI<NDI<VSFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLINQRS FLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPE QNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKV HEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQ GAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVV ALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLH YSIDFTI<I<QGYGRIEHLI<TLEQNVELAAAELI<ADEI<SHAVILGDTRYGSEEI<GTYHLAL FGDRAQEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHKDKGL QSLTLDQSVRKNEKLKLAAQGAEKTYGNGSLNTGKLKNDKVSRFDFIRQIEVDGQLITL ESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRA TYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAAIKPDGKRHAVISG SVLYNQAEKGSYSLGIFGKAQEVAGSAEVKTVNGIRHIGLAAKQ

SEQ ID NO: 30 [fHbp 231S fusion polypeptide]

VAADIGAGLADALTAPLDHKDKSLQSLTLDQVVRKNEKLKLAAQGAEKTYGNGDSLN TGI<LI<NDI<VSFDFIRQIEVDGQLITLESGEFQIYI<QDHSAVVALQIEI<INNPDI<IDSLINQ RSFRVSGLGGEHTAFNQLPDGKAEYHGKAF S SDD AGGKLT YTIDF AAKQGHGKIEHL K TPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGE KVHEIGIAGKQGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDVIPQNGTLTLS AQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSA VVALQIEKINNPDK TDSLINQRSFRVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGR LHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHL ALFGDRAQEIAGSATVKIGEKVHEIGIAGKQGSGGGGVAADIGAGLADALTAPLDHKDK GLQSLTLDQSVSKNEKLKLAAQGAEKTYGNG SLNTGKLKNDKVSRFDFIRQIEVDGQLI TLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGR

- 31 046480

ATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAAIKPDGKRHAVIS

GSVLYNQAEKGSYSLGIFGKAQEVAGSAEVKTVNGIRHIGLAAKQ

SEQ ID NO: 31 [vl.13 full-length wild-type sequence]

MNRTAFCCFSLTAALILTACSSGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSV RKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVY KQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFKLPKGGSATYRGTAFG SDDAGGKLTYTIDFAAK QGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQD EKGSYSLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ

SEQ ID NO: 32 [vl.15 full-length wild-type sequence]

MNRTTFCCLSLTAALILTACSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLT

LEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEVDGQLITLES GEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPKDVMAT YRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEKHHAVISG SVLYNQAEKGSYSL GIFGGQAQEVAGSAEVETANGIRHIGLAAKQ

SEQ ID NO: 33 [mature fHbp vl.l]

CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTY GNGDSLNTGKLKNDKVSRFDFIQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSE HSGI<MVAI<RQFRIGDIAGEHTSFDI<LPEGGRATYRGTAFGSDDAGGI<LTYTIDFAAI<Q GNGKIEHLKSP ELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVA GSAEVKTVNGIRHIGLAAKQ

SEQ ID NO: 34 [possible N-terminal amino acid sequence]

MGPDSDRLQQRR

Embodiments of the Invention

The invention will now be described in more detail using examples, which are not exhaustive.

Examples

Example 1. Stability study of stabilized fHbp 231 fusion containing variant 1.13 mutants.

As discussed above, differential scanning calorimetry (DSC) provides information on the thermal stability and domain folding of proteins and is described in the literature, e.g. Johnson (2013), Arch. Biochem. Biophys. 531:100-9 and Bruylants et al., Current Medicinal Chemistry, 2005; 12:2011-20. DSC has previously been used to assess the stability of fHbp v2 (Johnson et al., PLoS Pathogen, 2012;8:e1002981). Suitable conditions for assessing stability using DSC may be to use 20 µM polypeptide in a buffer solution (eg 25 mM Tris) with a pH between 6 and 8 (eg 7-7.5) with 100-200 mM NaCl (eg 150 mm).

The present inventors used this technique to study the effect of fHbp variant 1 subvariant mutations on the stability of the 231S fusion protein.

The stabilized fHbp fusion protein 231 (designated 231S) used in this example includes variant 2 and variant 3 sequences containing stabilizing mutations. Specifically, the v2 component of the 231S fusion protein has the sequence SEQ ID NO: 16 containing both the S32V and L123R mutations. The v3 component of the 231S fusion protein has the sequence SEQ ID NO: 17 containing both the S32V and L126R mutations. The inventors investigated the effect of variations in the v1 component of the 231S fusion on stability.

Each of the fHbp polypeptide variants contains an N-terminal and C-terminal domain, which can be clearly distinguished in DSC thermograms in the form of two independent transitions (peaks). While the T _m values corresponding to the transitions of the C-terminal domains of most fHbp variants are around 90°C, for the N-terminal domains they vary widely between different fHbp variants, with the lowest values observed in wild-type variant 2.1 (42°C ), and the highest are in option 1.1 (70°C).

In fig. Figure 2 shows four different thermograms comparing DSC data, where option 1, which is a component of the fHbp fusion 231S, is:

fHbp v1.1 or fHbp v1.13 or (A) fHbp v.1.13 E211A (Fig. 2A);

fHbpv1.1 or fHbp v1.13 or (B) fHbp v1.13 S216R (Fig. 2B);

- 32 046480 fHbp v1.1 or fHbp v1.13 or fHbp v.1.13 E211A/E232A (Fig. 2C) and fHbp v1.1 or fHbp v1.13 or fHbp v.1.13 E211A/S216R (Fig. 2D).

Based on each of these four thermograms, it can be concluded that (i) the fHbp units in the fusion constructs are properly folded and ii) that the v1 mutations are effective in stabilizing the fusion constructs, resulting in increased transition temperatures for the C-terminal domains compared to the fusion constructs. containing wild-type v1 sequences.

Example 2. Binding of mutants of one fHbp variant to hfH.

The following polypeptides were created, shown in table. 1 (with the addition of C-terminal His ₆ tags where they were not part of the reference sequence) to examine the effect of substitution mutations on the ability of fHbp variant 1 subvariants 1.13 and 1.15 to reduce binding to human factor H (hfH).

Table 1

Protein SEQ ID # Purification Purity % (exclusive

UHPLC)

fHbp vl.l 23 MATS3 98 fHbp vl.13 24 MenB 664 95 fHbp V 1.13 E211A 25 MenB 672 98 fHbp V 1.13 S216R 26 MenB 673 98 fHbp VI.13 E211A/E232A 3 MenB 687 92 fHbp VI.13 E211A/S216R 4 MenB 686 91 fHbp vl.15 27 MenB 669 82 fHbp vl.15 E214A 28 MenB 679 85 fHbpvl.15 E214A/E235A 9 MenB 680 86 fHbp vl.15 E214A/S219R 8 MenB 682 82 fHbpvl.15 S219R 7 MenB 683 83

We examined the binding of mutants of one hfH variant by surface plasmon resonance (SPR), which is a technique that allows detailed and quantitative characterization of protein-protein interactions and determination of their equilibrium and kinetic parameters (as described, for example, by Karlsson et al. (1994) , Methods, 6:99-110).

The SPR-based binding method involves immobilizing a ligand on the surface of a sensor chip. The ligand of interest is immobilized on the surface of the sensor chip using strictly defined chemical reactions, which makes it possible to pass solutions with different concentrations of the analyte over its surface and characterize their interactions with the immobilized ligand. The SPR signal is caused by changes in the refractive index on the surface of the gold sensor chip.

By tracking changes in the SPR signal over time, a sensogram is obtained, a graph of binding (in RU units) versus time, which allows the different stages of the binding event to be visualized and assessed.

When an analyte is injected, the response increases due to the formation of analyte-ligand complexes on the surface, and the association phase predominates in the sensorgram. After injection, a stationary state is reached in which the binding and dissociating molecules are in equilibrium. After the end of analyte injection, the response decreases due to the dissociation of complexes, which characterizes the dissociation phase. Fitting the sensorgram data using an appropriate binding kinetics model allows one to calculate kinetic parameters such as the degree of association ( _ka ) and dissociation ( _kd ) constants, as well as the binding affinity for the interaction being studied.

The inventors conducted experiments under the following conditions:

Chip: SM-5 with immobilization by amino groups of domains 6-7 of factor H, 10 μg/ml in acetate buffer with pH 4.0 to a level of ~400 RU, and immobilization by amino groups of human factor H (Merck Millipore), 20 μg/ml in acetate buffer with pH 4.0 to a level of ~ 2500 RU.

Flow buffer: 1xHBS-P.

Antigens studied: are given in table. 1.

Antigens were applied at a constant concentration of 250 nM.

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Interaction time: 120 s, flow rate: 30 µl/min.

Dissociation time: 120 s.

Regeneration buffer: 100 mM glycine-HCl, 3 M NaCl, pH 2.0.

The data shown in Fig. 3A-D compare the binding of factor H (hfH) domains 6-7 to fHbp v1.1 (fHbp antigen present in BEXSERO®), fHbp v1.13 (wild type) and fHbp v.1.13 E211A (Fig. 3A) ; fHbp v.1.13 S216R (Fig. 3B); fHbp v.1.13 E211A/E232A (Fig. 3C) and fHbp v.1.13 E211A/S216R (Fig. 3D).

An hfH fragment containing only domains 6-7 has been shown to be sufficient to mimic the fHbp-hfH interaction (Schneider et al. (2009), Nature, 458:890-893), thus providing a simplified model system for assessing the affinity of mutants and fHbp to hfH constructs.

While fHbp v1.13 single mutants show reduced binding to fH compared to wild-type v.1.1 and v1.13 (see Fig. 3A and B), v1.13 E211A/E232A and v1.13 E211A/S216R double mutants show a significant decrease in binding activity (see Fig. 3C and D).

The data shown in Fig. 4A-D compare the binding of full-length factor H protein to fHbp v1.1, fHbp v1.13 (wild type), and fHbp v.1.13 E211A (Fig. 4A); fHbp v1.13 S216R (Fig. 4B); fHbp v.1.13 E211A/E232A (Fig. 4C) and fHbp v.1.13 E211A/S216R (Fig. 4D).

While fHbp v1.13 single mutants show reduced binding to fH compared to wild-type v.1.1 and v1.13 (see Fig. 4A and B), v1.13 E211A/E232A and v1.13 E211A/S216R double mutants show a significant decrease in binding activity (see Fig. 4C and D).

The data shown in Fig. 5A-D, allow us to compare the binding of domains 6-7 of factor H with fHbp v1.1, fHbp v1.15 (wild type) and fHbp v.1.15 E214A (Fig. 5A), fHbp v.1.15 S219R (Fig. 5B), fHbp v.1.15 E214A/E235A (Fig. 5C) and fHbp v.1.15 E214A/S219R (Fig. 5D).

While fHbp v1.15 single mutants show reduced binding to fH compared to wild-type v.1.1 and v1.15 (see Fig. 5A and B), v1.15 E214A/E235A and v1.15 E214A/S219R double mutants demonstrate no significant binding to the fH6-7 subdomains of factor H (see Fig. 5C and D).

The data shown in Fig. 6A-D allow us to compare the binding of full-length factor H protein to fHbp v1.1, fHbp v1.15 (wild type) and fHbp v.1.15 E214A (Fig. 6A), fHbp v.1.15 S219R (Fig. 6c), fHbp v .1.15 E214A/E235A (Fig. 6C) and fHbp v.1.15 E214A/S219R (Fig. 6D).

While the fHbp v1.15 E214A single mutant exhibits reduced fH binding compared to wild-type v.1.1 and v1.15 (see Fig. 6A), the v1.15 S219R single mutant and the v1.15 E214A/E235A double mutant are essentially do not bind to the full-length fH protein (see Fig. 6B and C).

The v1.15 E214A/S219R double mutant shows some residual binding activity to the full-length fH protein (see Fig. 6D).

Thus, in summary, the most promising candidates for reducing or eliminating binding to hfH are the fHbp mutants v1.13 E211A/E232A, v1.13 E211A/S216R, v1.15 S219R, and v1.15 E214A/E235A.

Example 3. Binding of fHbp fusion mutants 23(S) 1.13 and 23(S) 1.15 to hfH.

The following fusion polypeptides (Table 2) were created (with the addition of C-terminal His ₆ tags where they were not part of the reference sequence) to study the effect of mutations in the var1 components of the fHbp 231 fusion proteins on the ability of the fusion protein to bind to human factor H (hfH).

table 2

Protein SEQ ID# Cleaning Purity % (SEC UHPLC) fHbp 231wt 29 MENB593 85 fHbp 231S thirty MENB665 92 fHbp 23S_1.13_E211A/S216R 19 MENB689 93 fHbp-23S_1.15 22 MENB702 91 E214A/E235A fHbp- 18 MENB703 90

23S_1.13_E211A/E232A

We examined the binding of fHbp fusion mutants 23(S)1.13 and 23(S)1.15 to hfH by SPR as described in Example 2.

The fHbp fusion protein 231S (SEQ ID NO: 30) used in this example includes variant 2 and variant 3 sequences containing the S32V/L123R and S32V/L126R stabilizing mutations, respectively, described in detail above. In particular, the v2 component of the 213S fusion protein has after

- 34 046480 sequence SEQ ID NO: 16, and the v3 component of the 213S fusion protein has the sequence SEQ ID NO: 17. The v1.1 component of the 231S fHbp fusion protein includes a point mutation (R^S) that prevents fH binding, which is shown in bold in SEQ ID NO: 30 above and corresponds to the R41S mutation described in WO 2011/126863.

The wild-type fHbp 231 fusion (SEQ ID NO: 29) used in this example corresponds to the fusion protein of SEQ ID NO: 30, but without introducing stabilizing mutations in v2 and v3 and without the anti-binding mutation in v1.1.

The fHbp fusion 23S_1.13E211A/S216R (SEQ ID NO: 19) corresponds to the fusion protein of SEQ ID NO: 30, however, the v1 component of the fusion protein is a nonbinding v1 mutant. 13 E211A/S216R according to this invention.

The fHbp 23S_1.15 E214A/E235A fusion protein (SEQ ID NO: 22) corresponds to the fusion protein of SEQ ID NO: 30, however, the v1 component of the fusion protein is the v1.15 E214A/E235A nonbinding mutant of the present invention.

The fHbp-23S_1.13E211A/E232A fusion protein (SEQ ID NO: 18) corresponds to the fusion protein of SEQ ID NO: 30, however, the v1 component of the fusion protein is a non-binding v1 mutant. 13 E211A/S216R according to this invention.

The wild type 231 (SEQ ID NO: 29) and fHbp 231S (SEQ ID NO: 30) fusion proteins serve as controls in this experiment.

Sensograms shown in Fig. 7A, B), allow us to compare the binding of domains 6-7 of factor H with wild-type fHbp 231, fHbp 231S and fHbp 23S_1.13E211A/S216R (Fig. 7A), as well as fHbp 23S_1.13E211A/E232A (Fig. 7B).

Sensograms shown in Fig. 8 allow us to compare the binding of factor H domains 6-7 to wild-type fHbp 231, fHbp 231S and fHbp 23S_1.15 E214A/E23 5A.

An hfH fragment containing only domains 6-7 has been shown to be sufficient to mimic the fHbp - hfH interaction (Schneider et al. (2009), Nature, 458:890-893), thus providing a simplified model system for assessing the affinity of mutants and fHbp to hfH constructs.

From fig. 7 and 8, it is clear that all three V1.13/1.15 mutants exhibit a strong reduction in binding activity to factor H domains 6-7. Compared to the 2fHbp 31S fusion, the binding activity exhibited by the v1.13 and v1.15 mutant fusion proteins is clearly reduced.

Sensograms shown in Fig. 9A,B compare the binding of full-length factor H protein to wild-type fHbp 231, fHbp 231S and fHbp 23S_1.13E211A/S216R (Fig. 9A), as well as fHbp 23S_1.13E211A/E232A (Fig. 9B).

Sensograms shown in Fig. 10 allow us to compare the binding of the full-length factor H protein to wild-type fHbp 231, fHbp 23S and fHbp 23S_1.15 E214A/E235A.

These data indicate that all three V1.13/1.15 mutants tested exhibit a marked reduction in binding activity to full-length factor H compared to the fHbp 231S fusion.

Example 4. Study of the immunogenicity caused by the fHbp fusion proteins of the invention against meningococcal strains expressing many different fHbp v1 (v1.x) subvariants.

The main task:

To investigate the immunogenicity caused by 231.13 fusion proteins containing mutations that reduce or eliminate hfH binding against meningococcal strains expressing fHbp as multiple different fHbp v1 subvariants, in comparison with existing fHbp antigens/fusion proteins and the licensed 44CMenB vaccine. Immunogenicity was determined by the bactericidal activity of serum in the presence of rabbit complement (rSBA) and bactericidal activity of serum in the presence of human complement (hSBA) against the following subvariants of the fHbp v1 (v1.x) strain: v1.1, v1.10, v1.13, v1 .14 and v1.15.

The objective of these experiments is to evaluate whether the fHbp fusion proteins of the present invention demonstrate comparable (non-inferior) immunogenicity against a panel of fHbp v1.x expressing strains when compared to existing antigen/fusion protein compositions and the licensed 44CMenB vaccine.

Immunization schedule:

Seven groups of 10 mice (female CD1 6-8 weeks old) received three separate 200 μl doses of one of seven different antigen compositions, as detailed in Table 1. 3.

Mice were immunized intraperitoneally (i.p.) on days 1, 22, and 36.

Mice were bled at days 0, 35, and 50.

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Table 3

Group Number of animals Impact Antigen Antigen dose Adjuvant dose Volume and method of injection 1 10 231.13E21 1A/S216R (SEQ Ш NO: 19) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 2 10 231.13E21 1A/E211A/E 232A (SEQ IDNO: 18) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 3 10 231.13 (SEQ Ш NO:29) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 4 10 231.IS (SEQ ID NO:30) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 5 10 fHbp vl.l (SEQ ID NO: 33) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 6 10 936-741* 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 7 10 BEXSERO-like* * 20 µg antigen + 10 µg OMV A1(OH) ₃ 3 mg/ml 200 µl WB

*936-741 is a GNA2091-fHbp fusion included in the 44CMenB vaccine.

^BEXSERO-like means a finished BEXSERO product, but not necessarily from a batch approved for release.

End point:

Production of total IgG against fHbp two weeks after the third immunization.

Examination of pooled serum in rSBA and hSBA tests using a panel of Neisseria meningitidis strains expressing fHbp v1.1, v1.10, v1.13, v1.14 and v1.15.

Results:

In fig. 11A shows serum bactericidal titers measured in the presence of rabbit complement (rSBA) against each of 7 antigen compositions in a study of different meningococcal strains expressing fHbp v1.x. When displaying SBA results, each dot represents the serum bactericidal titer against an individual strain, measured in pooled serum. In fig. 11B shows serum bactericidal titers measured in the presence of human complement (hSBA) against each of 7 antigen compositions in a study against the same set of strains expressing fHbp v1.x.

The results show that the fHbp fusion proteins of the invention (groups 1 and 2) demonstrate comparable (non-inferior) immunogenicity to the v1.x panel when compared to the licensed BEXSERO product and the BEXSERO fHbp antigen (936-741).

Interestingly, these results also indicate that the fHbp fusion proteins of the invention (Groups 1 and 2) are more immunogenic than existing fusion proteins known in the art (specifically the 231.1S fusion (herein SEQ ID NO: 30) ), against the v1 .x panel of strains, both as determined using rSBA and hSBA.

Example 5: Study of the immunogenicity caused by the fHbp fusion proteins of the invention against meningococcal strains expressing fHbp v2/v3.

The main task:

To investigate the immunogenicity caused by 231.13 fusion proteins containing mutations that reduce or eliminate hfH binding against meningococcal strains expressing

- 36 046480 fHbp v2 or v3, compared to existing fHbp antigens/fusion proteins and the licensed 44CMenB vaccine. The objective of these experiments was to evaluate whether the inclusion of three different mutated fHbp variants would allow greater strain overlap compared to existing antigen compositions.

Immunogenicity was determined by serum bactericidal activity in the presence of rabbit complement (rSBA) and serum bactericidal activity in the presence of human complement (hSBA) against the following fHbp v2 and v3 strains: v2.16, v3.31 and v3.42.

Immunization schedule:

Seven groups of 10 mice (female CD1 6-8 weeks old) received three separate 200 μl doses of one of seven different antigen compositions, as detailed in Table 1. 4 below.

Mice were immunized intraperitoneally (i.p.) on days 1, 22, and 36.

Mice were bled at days 0, 35, and 50.

Table 4

Group Number of animals Impact Antigen Antigen dose Adjuvant dose Volume and method of injection 1 10 231.13E21 1A/S216R (SEQ Ш NO: 19) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 2 10 231.13E21 1A/E211A/E 232A (SEQ IDNO: 18) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 3 10 231.13 (SEQ Ш NO:29) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 4 10 231.IS (SEQ IDNO:30) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 5 10 fHbp vl.l (SEQ ID NO: 33) 20 µg antigen A1(OH) ₃ 3 mg/ml 200 µl WB 6 10 936-741* 20 µg antigen A1(OH) ₃ 200 µl WB 3 mg/ml 7 10 BEXSERO-like** 20 µg antigen + 10 µg OMV A1(OH) ₃ 3 mg/ml 200 µl WB

*93 6-741 is a GNA2091-fHbp fusion included in the 44CMenB vaccine.

^BEXSERO-like means a finished BEXSERO product, but not necessarily from a batch approved for release.

End point:

Production of total IgG against fHbp two weeks after the third immunization.

Examination of pooled serum in rSBA and hSBA tests against a panel of Neisseria meningitidis strains expressing fHbp in option 2 or option 3.

Results:

In fig. 12A shows serum bactericidal titers measured in the presence of rabbit complement (rSBA) against each of 7 antigen compositions in a study of different meningococcal strains expressing fHbp v2 or v3. When displaying SBA results, each dot represents the serum bactericidal titer against an individual strain measured in pooled serum. In fig. 12B shows serum bactericidal titers measured in the presence of human complement (hSBA) against each of 7 antigen compositions in a study against the same set of strains expressing fHbp v2 or v3.

The results show that fusion proteins containing all three fHbp variants lead to much higher titers in the rSBA and hSBA assays compared to fHbp v1.1 alone, with fusion protein 936-741 included in BEXSERO. or in fact with the BEXSERO product itself. This indicates a significant improvement in the immune response against strains expressing fHbp v2 or v3 compared to the existing vaccine or vaccine components.

Example 6: Evaluation of the additional benefits of incorporating the non-binding fHbp231.x mutants of the invention into the existing 44CMenB vaccine.

The main task:

a) Assess strain overlap (defined as titers >64 in the hSBA test) of the 44CMenB+fHbp231.13_E211A/S216R fusion protein composition of the invention compared to the existing 44CMenB vaccine in two different animal models (mice and rabbits) two weeks after the third dose , when measuring titers against 20 strains of fHbp variants 2 and 3 of selected serogroup B of Neisseria meningitidis in the hSBA test.

b) Assess strain overlap (defined as titers >64 in the hSBA test) of the 44CMenB+fHbp231.13_E211A/S216R fusion protein composition of the invention compared to the existing 44CMenB vaccine in two different animal models (mice and rabbits) two weeks after the third dose , when measuring titers against 30 strains of fHbp variants 1.x of selected serogroup B of Neisseria meningitidis in the hSBA test.

c) Assess the strain overlap of the 44CMenB+fHbp231.13_E211A/S216R fusion protein composition of the invention (to prove that it is non-inferior) when compared with the existing 44CMenB vaccine in two different animal models (mice and rabbits) two weeks after the third dose, when measuring titers against 11 serogroup B variants of Neisseria meningitidis, including reference strains 44CMenB and strains carrying fHbp variants 1.1 and 1.4 overlapped by 44CMenB, in the hSBA test.

Endpoint: Titers of pooled serum collected two weeks after the third injection in the hSBA test (one pool per group).

Mouse study design.

Female CD1 mice, 4–6 weeks of age, received three intraperitoneal (IP) injections of 200 μl of aluminum hydroxide-adsorbed 44CMenB (Bexsero) or aluminum hydroxide-adsorbed 44CMenB (BEXSERO) plus various fHbp 231 fusion proteins (as shown in Table 5) on days 1, 22 and 36. Blood samples were taken before the 1st injection (day 0) and the last blood draw was performed two weeks after the third injection (day 49).

Table 5

Group Number of animals Impact Immunization schedule (days) Sample collection (organs, blood volume) and days Vaccine Antigen dose Adjuvant dose Volume and method of injection 1 10 4SMepV 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49 2 10 4CMenB+fHbp 231.13_wt 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49 3 10 4CMenB + fHbp 231.13-E211A /S16R 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49 4 10 4CMenB + fHbp 231.13-E211A / E232A 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49 5 10 4CMenB + fHbp 23IS* 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49 6 10 4CMenB + fHbp 231.15_wt 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49 7 10 4CMenB + fHbp 231.15_E214A / E235A 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49

- 38 046480

8 10 4СМepВ + fHbp 23IS* 20 mcg protein +10 mcg OMV A1(OH)3 3 mg/ml 200 µl WB 1,22,36 Blood on days 0.49

*The fHbp 231S fusion protein includes stabilizing mutations in components v2 and v3, in addition to the point mutation (R^S) in component v1.1 that prevents fH binding, shown in bold in SEQ ID NO: 30, which corresponds to the R41S mutation, described in WO 2011/126863.

Design of the study on rabbits.

Female New Zealand rabbits aged 9 weeks received three intramuscular (IM) injections of 500 μl of 44CMenB adsorbed on aluminum hydroxide (BEXSERO) or 44CMenB adsorbed on aluminum hydroxide (BEXSERO) plus various fHbp 231 fusion proteins (as shown in Table 6) at 1, 21 and 35 days. The last blood draw was performed two weeks after the third injection.

Table 6

Group Number about animals Vaccine Impact Immunization schedule (days) Sample collection (organs, blood volume) and days Antigen dose Adjuvant dose Volume and method of injection 1 3 4SMepV 50 + 100 mcg protein + 25 mcg OMV A1(OH)3 3 mg/ml 500 µl VM 1,22,36 Blood on days 0.49 2 3 4CMenB+fHbp 231.13_wt 50+100 mcg protein + 25 mcg OMV A1(OH)3 3 mg/ml 500 µl VM 1,22,36 Blood on days 0.49 3 3 4CMenB + fHbp 231.13-E211A /S16R 50+100 mcg protein + 25 mcg OMV A1(OH)3 3 mg/ml 500 µl VM 1,22,36 Blood on days 0.49 4 3 4CMenB + fHbp 231.15_wt 50+100 mcg protein + 25 mcg OMV A1(OH)3 3 mg/ml 500 µl VM 1,22,36 Blood on days 0.49 5 3 4CMenB + fHbp 23IS* 250+ 100 mcg protein + 25 mcg OMV A1(OH)3 3 mg/ml 500 µl VM 1,22,36 Blood on days 0.49 6 3 4CMenB + fHbp 231.15_ E214A/ E235A 50+100 mcg protein + 25 mcg OMV A1(OH)3 3 mg/ml 500 µl VM 1,22,36 Blood on days 0.49

*The fHbp 231S fusion protein includes stabilizing mutations in components v2 and v3, in addition to the point mutation (R^S) in component v1.1 that prevents fH binding, shown in bold in SEQ ID NO: 30, which corresponds to the R41S mutation, described in WO 2011/126863.

Determining sample size.

Ten mice and 3 rabbits per group are the minimum number of animals that will produce enough pooled serum to perform the hSBA test against a large panel of strains (approximately 73), allowing for 20% of repeat tests or additional titrations.

A total of 61 strains carrying fHbp variants 1, 2 and 3 of the selected serogroup B of Neisseria meningitides were tested using hSBA.

Readable immunological parameters.

Functional antibodies were determined by serum bactericidal activity using human complement complement (hSBA) against 61 N. meningitidis strains carrying fHbp variant 1.x, variant 2, or variant 3 and a panel of Bexsero reference antigenic strains.

SBA is the only generally accepted correlate of protection against N. meningitidis in humans. Results.

- 39 046480

Humoral responses: functional antibodies measured using rSBA.

To measure functional antibodies produced in response to various BEXSERO formulations capable of initiating complement-mediated cytolysis of N. meningitidis strains, pooled serum collected two weeks after the third vaccination was tested for serum bactericidal activity using human rabbit serum as a source of complement ( hSBA) against a panel of approximately 60 N. meningitidis strains.

The total number of Neisseria strains selected was divided into three different panels of strains: fHbp variants 2 and 3, fHbp variants 1.x, as well as BEXSERO reference strains and fHbp variants 1.1 and 1.4 strains.

To demonstrate the additional benefits of the composition containing the fHbp231.13_E211A/S216R fusion protein of the invention relative to BEXSERO, 50 strains were selected: 20 strains carrying fHbp variant 2 or variant 3.

Selection was based on the frequency distribution of fHbp and included strains carrying fHbp v2.16, v2.19, v2.21, v2.24, v3.116, v3.31 and v3.42. 30 strains carrying fHbp variants 1.x.

The selection was carried out in such a way as to close the gaps existing in BEXSERO and analyze the entire genetic diversity of fHbp variant 1, and included strains carrying fHbp v1.1, v1.4, v1.13, v1.15, v1.14, v1. 10, v1.260, v1.510, v1.90, v1.275, v1.697, v1.226, v1.110, v1.249, v1.108, v1.227 and v1.215.

To demonstrate that the composition containing the fHbp231.13_E211A/S216R fusion protein of the invention is non-inferior to BEXSERO, 11 strains were selected, including BEXSERO reference strains plus additional strains known to overlap with BEXSERO (including fHbp 1.1 and 1.4).

Immunogenicity study in mice.

To determine the additional benefits of compositions containing the fHbp fusion proteins of the invention relative to BEXSERO, sera obtained from vaccinated mice were pooled in the presence of human plasma as a source of complement (hSBA) on 50 MenB strains grouped as variant 1 strains (30 strains ) and strains of variants 2/3 (20 strains). It should be noted that 50 strains were selected that do not overlap BEXSERO and therefore do not match all BEXSERO antigens. The results are shown in Fig. 13A and B.

As can be seen from FIG. 13A and B, compositions containing the fHbp fusion proteins of the invention are superior to BESXERO with respect to variant 2/3 strains and also superior to BEXSERO with respect to a panel of variant 1.x strains.

A new cutoff value of 256 (four times the original cutoff value of 64) was chosen to distinguish between overlapped and nonoverlap strains. The calculated percentages of overlapping strains are presented in Fig. 14A and B.

The results shown in these graphs again demonstrate that compositions containing the fusion proteins of the invention show greater overlap with respect to the panel of strains of variants 2 and 3 (Fig. 14A) compared to BEXSERO alone, as well as greater overlap with respect to the majority of strains variants 1.x (Fig. 14B) compared to BEXSERO.

Finally, the noninferiority properties of the composition containing the fusion proteins of the invention were also assessed in a mouse study, testing the mouse antiserum against a panel of 11 strains, including the BEXSERO reference strains and fHbp variant 1.1 and 1.4 strains (FIG. 15). Non-inferiority was assessed in the hSBA test using pooled mouse serum to a panel of 11 MenB strains.

The results shown in Fig. 15 confirm not only that all compositions containing the fusion proteins of the invention are non-inferior to BEXSERO, but also demonstrate improved immunogenicity against most strains due to the additional contribution of antibodies directed against additional fHbp components.

The non-inferiority of compositions containing the fHbp231.13_E211A/S216R fusion protein of the invention compared to BEXSERO was also assessed by testing sera from individual mice with rabbit complement as a source of complement (rSBA) on a panel of 4 BEXSERO indicator strains (M14459 for fHbp variant 1.1; NZ98/254 for PorA P1.4; M4407 for NHBA; 96217 for NadA) to confirm that the specific immunogenicity of the BEXSERO antigens is maintained when the new fusion protein is added. The results are shown in Fig. 16A-D. In these graphs, BEXSERO PLUS PLUS refers to the composition BEXSERO + fHbp 231.13_E211A/S216R.

Immunogenicity study in rabbits.

The combined data obtained in the hSBA test for the existing BEXSERO composition and compositions containing the fusion proteins of the invention are further presented in FIG. 17A for var2/3 type strains and FIG. 17B for strains of type var.1.k. The dotted line represents the hSBA test cutoff value of 16 for rabbit serum.

All compositions containing the fHbp fusion proteins of this invention provide improved overlap with respect to the panel of variants 2 and 3 strains compared to BEXSERO from

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Claims (25)

efficiency and lead to higher titers against most 1.x variant strains in the hSBA test compared to BEXSERO alone. As in the mouse study described above, a new cutoff value of 256 (four times the original cutoff value of 64) was chosen for the analysis of the rabbit data. An overlap analysis was performed and the summary data is presented in FIG. 18A (for var2/3 strains) and FIG. 18B (for varJ strains). These results confirm that compositions containing the fHbp fusion proteins of the present invention are capable of covering a greater percentage of var2/3 strains than BEXSERO alone, and are able to cover a greater percentage of v1.x strains than BEXSERO alone and BEXSERO + fHbp 231.1 fusion protein _R41S, known from the prior art (indicated in Fig. 18 as 2-3-1S). Finally, that compositions containing the fHbp fusion proteins of this invention are noninferior to BEXSERO was confirmed in a rabbit study testing the rabbit antiserum against a panel of 11 strains, including BEXSERO reference strains and fHbp strains v1.1 and 1.4, as shown in fig. 19. These results confirm that all compositions containing the fHbp fusion proteins of this invention are non-inferior in their properties. Example 7. Assessment of the immunogenicity of the 44CMenB+23(S)1.13 NB mutant in comparison with wild-type 44CMenB+23(S)1.13 in the hFH transgenic mouse model Immunization schedule. Two groups of 10 mice (transgenic mice expressing hfH) were immunized intraperitoneally (i.p.) with one of two formulations, A and B: group A = 4CMenB + fHbp 23(S)1.13 wild type; group B = 4CMenB + fHbp 23(S)1.13_E211A/S216R. One mouse (non-immunized) served as a control and received phosphate-buffered saline (PBS) only. Immunization was carried out with three doses on days 1, 22 and 36. Blood samples were taken before immunization and after the third dose. In each group, serum obtained before immunization was pooled. Bacterial infection of immunized mice. Nine mice from each group were challenged (two mice died after sampling and before bacterial challenge; one mouse from group A and one from group B). Mice were infected i.p. using a bioluminescent variant of strain MC58 of serogroup B (107 CFU per mouse in 500 μl of saline). Results. Imaging was carried out over time at 30 min and 6 h after infection, the total number of photons per second was calculated and expressed as the total number of photons per second per mouse and as the ratio of the total number of photons per second per mouse after 6 h of infection to the total number of photons in second per mouse after 0.5 h of infection (Fig. 20). The total number of emitted photons per mouse was calculated and shown in FIG. 21. In both groups, signals were significantly reduced compared to uninfected mice. Mice in group B had a lower total number of photons per second compared to group A, but this difference did not reach the level of significance (p=0.2) in Fig. 21A. However, when analyzing the data presented as the ratio of signals after 6 hours/0.5 hours (Fig. 21B), the difference was significant (p=0.007, Mann-Whitney test). Conclusion. As shown in Figs. 20 and 21, mice in both groups immunized with formulations A and B were protected from MC58 challenge, as evidenced by post-challenge clearance observed in mice in both groups at 6 h versus 0.5 h. In contrast, nonimmunized mice were unable to eliminate the infectious agent 6 hours after infection. However, clearance after challenge was more pronounced in group B mice immunized with 4CMenB+fHbp 23(S)1.13_E211A/S216R compared to group A mice immunized with wild-type 4CMenB+fHbp 23(S)1.13. Thus, these in vivo data confirm the improved immunogenicity of a vaccine composition containing the mutated non-fH binding fusion protein of the invention compared to an equivalent composition containing a fusion polypeptide that does not include the non-fH binding double mutant of the v1.13 polypeptide invention. CLAIM 1. Mutant meningococcal polypeptide fHbp v1.13, having a reduced ability to bind to human factor H (hfH), containing the amino acid sequence, - 41 046480 having at least 90% identity to the sequence of SEQ ID NO: 2, where the amino acid sequence of the specified mutant meningococcal polypeptide fHbp v1.13 contains substitutions of residues E211A and S216R of the sequence SEQ ID NO: 2. 2. The polypeptide according to claim 1, containing or consisting of the amino acid sequence SEQ ID NO: 4. 3. A fusion polypeptide containing meningococcal fHbp polypeptides v1, v2 and v3, where the fHbp sequence variants are arranged in the order v2-v3-v1 from N- to C-terminus and where the fHbp v1 polypeptide is a mutant fHbp v1.13 polypeptide according to claim. 1 or 2. 4. The fusion polypeptide according to claim 3, where: (a) an fHbp v2 polypeptide is a mutant fHbp v2 polypeptide containing an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 12, wherein the amino acid sequence of fHbp v2 includes a mutation substituting residues S32 and L123 of SEQ ID NO: 12 and where the replacements are S32V and L123R; and (b) the fHbp v3 polypeptide is a mutant fHbp v3 polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15, wherein the fHbp v3 amino acid sequence includes a mutation substituting residues S32 and L126 of SEQ ID NO: 15 and where the replacements are S32V and L126R. 5. The fusion polypeptide according to claim 4, where: (a) the fHbp v2 polypeptide contains or consists of an amino acid sequence SEQ ID NO: 16 and/or (b) the fHbp v3 polypeptide contains or consists of the amino acid sequence SEQ ID NO: 17. 6. The fusion polypeptide according to any one of claims 3 to 5, wherein the sequences v2 and v3 and the sequences v3 and v1 are connected by a glycine-serine linker. 7. The fusion polypeptide of claim 6, wherein the glycine-serine linker is GSGGGG. 8. A fusion polypeptide according to any one of claims 3 to 7, containing the amino acid sequence of SEQ ID NO: 19. 9. The fusion polypeptide according to any one of claims 3 to 8, further comprising the N-terminal amino acid sequence of SEQ ID NO: 34. 10. An isolated nucleic acid molecule containing a nucleotide sequence encoding the mutant fHbp v1.13 polypeptide according to claim 1 or 2. 11. An isolated nucleic acid molecule containing a nucleotide sequence encoding a fusion polypeptide according to any one of claims 3 to 9. 12. A plasmid containing a nucleotide sequence encoding the mutant fHbp v1.13 polypeptide according to claim 1 or 2 or a fusion polypeptide according to any of claims 3-9. 13. Recombinant host cell transformed with the plasmid according to claim 12. 14. An immunogenic composition containing a mutant fHbp v1.13 polypeptide according to claim 1 or 2 or a fusion polypeptide according to any of claims 3-9. 15. The immunogenic composition of claim 14, further comprising one or more of: meningococcal antigen NHBA, meningococcal antigen NadA, meningococcal antigen fHbp and meningococcal outer membrane vesicles (OMV). 16. Immunogenic composition according to claim 15, containing the composition 44CMenB. 17. Immunogenic composition according to any one of claims 14-16, additionally containing a conjugated capsular saccharide from N. meningitidis serogroups A, C, W135 and/or Y. 18. The immunogenic composition according to claim 17, further containing conjugated capsular saccharides from N. meningitidis of each of serogroups A, C, W135 and Y. 19. A method for producing an immune response in a mammal, comprising administering an immunogenic composition according to any one of claims 14-18. 20. Use of an immunogenic composition according to any one of claims 14-18 in medicine. 21. Use of an immunogenic composition according to any one of claims 14-18 as a vaccine. 22. An immunogenic composition according to any one of claims 14 to 18, where the composition is intended to produce an immune response in a mammal. 23. An immunogenic composition according to any one of claims 14 to 18, wherein the composition is intended to immunize a mammal against infection caused by N. meningitidis. 24. The method of claim 19, wherein the mammal is a human. 25. The immunogenic composition according to claim 22 or 23, wherein the mammal is a human. -