Modified meningococcal fHbp polypeptide

Abstract

The present invention provides mutated fHbp polypeptides and fusion proteins comprising the mutated fHbp polypeptides, which can be used as components of immunogenic compositions for immunizing against Neisseria meningitidis infection.

Description

Technical Field

[0001] The present invention is in the field of protein engineering and particularly relates to meningococcal factor H binding protein (fHbp), which is a useful vaccine immunogen.

[0002] background

[0003] Invasive meningococcal disease (IMD) is caused by the bacterial pathogen Neisseria meningitidis ( Neisseria meningitidis ). Of the five serogroups (MenA, B, C, W and Y) that are primarily associated with IMD worldwide, MenB is the predominant serogroup causing IMD in many regions, including Canada, the United States, Australia, New Zealand and Europe. MenB is a serious and often fatal disease that primarily affects infants and young adults. It is easily misdiagnosed, can be fatal within 24 hours of onset, and can cause severe lifelong disability despite treatment.

[0004] Currently, there are two licensed vaccines designed to immunize against serogroup B meningococci: BEXSERO by GSK and TRUMENBA by Pfizer.

[0005] BEXSERO (also commonly referred to as 4CMenB) contains outer membrane vesicles (OMVs) from a prevalent strain of group B meningococcus NZ98 / 254 together with a preparation of 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 exist as fusion proteins (NHBA-GNA1030 fusion protein and GNA2091-fHbp fusion protein). 4CMenB is described in the literature (e.g., 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 herein.

[0006] TRUMENBA contains two lipidated MenB fHbp antigens (v1.55 and v3.45) adsorbed onto aluminum phosphate.

[0007] fHbp (also interchangeably referred to in the art as genome-derived Neisseria 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 control protein (CCP) modules connected by a short linker sequence. hfH circulates in human plasma and regulates the alternative pathway of the complement system. The functional binding of fHbp to hfH depends primarily on the CCP modules (or domains) 6-7 of hfH and enhances the ability of bacteria to resist complement-mediated killing. Therefore, the expression of fHbp enables survival in ex vivo human blood and serum.

[0008] As different fHbp classification schemes have been proposed, a dedicated database with a unified fHbp nomenclature for designating new subvariants is available: (http): / / neisseria.org / nm / typing / fhbp (also as (https): / / pubmlst.org / neisseria / fHbp / ).

[0009] fHbp has been classified into three (major) variants 1, 2 and 3, which are further divided into subvariants fHbp-1.x, fHbp-2.x and fHbp-3.x, where x represents a specific peptide subvariant. In contrast to v2 and v3, fHbp v1 is highly heterogeneous and contains several subvariants. In different naming schemes, subvariants / variants are divided into subfamily A (corresponding to variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity.

[0010] BEXSERO is expected to provide broad coverage against MenB strains circulating worldwide (Medini D et al. , Vaccine 2015; 33:2629–2636; Vogel U et al. Lancet Infect Dis 2013;13:416–425; Křížová 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. Simões MJ et al. PLoS ONE 12(5): e0176177; and Parikh SR et al. Lancet Infect Dis2017; 17:754–62). Furthermore, after the introduction of BEXSERO into the UK National Infant Immunisation Programme in September 2015, data at 10 months showed vaccine efficacy of 83% against all MenB strains after two doses (Parikh SR et al. , Lancet 2016; 388:2775-82).

[0011] However, bactericidal activity is variant-specific; antibodies raised against one variant are not necessarily cross-protective against other variants, although some cross-reactivity between fHbp v2 and v3 has been described (Masignani V et al. , J Exp Med 2003; 197:789–799). Antibodies raised against the subvariant fHbpv1.1 included in the 4CMenB vaccine were highly cross-reactive with the most frequently occurring fHbp v1 subvariant, but had low cross-reactivity with the v1 subvariant that is most distantly related to v1.1. In addition, antibodies raised against the subvariant fHbpv1.1 included in the 4CMenB vaccine had poor cross-reactivity with fHbp v2 and v3 (Brunelli B et al. , Vaccine 2011; 29:1072–1081). This means that 4CMenB coverage cannot be extended to some meningococcal strains carrying fHbp v2, v3 or some v1 subvariants.

[0012] Therefore, despite the efficacy of licensed serogroup B meningococcal vaccines (such as BEXSERO), there remains a need to develop meningococcal vaccines that retain the efficacy of, and are not inferior to, existing licensed vaccines (such as 4CMenB), but have the added value of improved coverage against meningococcal strains carrying fHbp variants that are not well covered by existing vaccines.

[0013] SUMMARY OF THE INVENTION

[0014] A first aspect of the present invention provides a mutant v1.13 meningococcal fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2, wherein the amino acid sequence of the mutant v1.13 meningococcal fHbp polypeptide includes a substitution mutation at E211, S216, E232 or a combination thereof of SEQ ID NO:2.

[0015] A second aspect of the present invention provides a mutant v1.15 meningococcal fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:6, wherein the amino acid sequence of the mutant v1.15 meningococcal fHbp polypeptide includes a substitution mutation at E214, S219, E235 or a combination thereof of SEQ ID NO:6.

[0016] The third aspect of the invention provides a fusion polypeptide comprising all three of the v1, v2 and v3 meningococcal fHbp polypeptides, wherein the variant fHbp sequence is the order v2-v3-v1 from N-terminus to C-terminus, and wherein the v1 polypeptide is a mutant v1.13 fHbp polypeptide according to the first aspect of the invention or a mutant v1.15 fHbp polypeptide according to the second aspect of the invention.

[0017] A fourth aspect of the invention provides an isolated nucleic acid molecule, optionally a plasmid, comprising a nucleotide sequence encoding a mutant v1.13 fHbp polypeptide according to the first aspect of the invention, a mutant v1.15 fHbp polypeptide according to the second aspect of the invention, or a fusion polypeptide according to the third aspect of the invention.

[0018] A fifth aspect of the present invention provides a recombinant host cell transformed with a nucleic acid molecule according to the fourth aspect of the present invention.

[0019] The sixth aspect of the present invention provides an outer membrane vesicle obtained or prepared from the recombinant host cell according to the fifth aspect of the present invention.

[0020] The seventh aspect of the present invention provides an immunogenic composition comprising: a mutant v1.13 fHbp polypeptide according to the first aspect of the present invention, a mutant v1.15 fHbp polypeptide according to the second aspect of the present invention, a fusion polypeptide according to the third aspect of the present invention, or an outer membrane vesicle according to the sixth aspect of the present invention. The immunogenic composition can be used to immunize a mammal, preferably a human, against infection with Neisseria meningitidis.

[0021] Description of the drawings

[0022] Figure 1 A is a graph showing the frequency of meningococcal B strains expressing fHbp v1.x subvariants and indicating which of these v1.x subvariants are covered (black) or not covered (white) by the 4CMenB vaccine. Figure 1 B is a graph showing the frequency of meningococcal B strains carrying fHbp v2 and indicating which of these strains are covered (black) or not covered (white) by the 4CMenB vaccine. Figure 1C is a graph showing the frequency of meningococcal B strains carrying fHbp v3 and indicating which of these strains are covered (black) or not covered (white) by the 4CMenB vaccine.

[0023] Figure 2 Included are four graphs (heat maps) comparing fHbp v1.1 and fHbp v1.13 (wild type) with ( Figure 2 A) fHbpv.1.13 E211A; ( Figure 2 B) fHbp v1.13 S216R; ( Figure 2 C) fHbp v.1.13 E211A / E232A; and ( Figure 2 D) Differential scanning calorimetry (DSC) data of fHbp v.1.13 E211A / S216R.

[0024] Figure 3 Four graphs (sensograms) are included, which compare fHbp v1.1 and fHbp v1.13 (wild type) with ( Figure 3 A)fHbp v.1.13 E211A; ( Figure 3 B) fHbp v1.13 S216R; ( Figure 3 C) fHbp v.1.13 E211A / E232A; and ( Figure 3 D) Binding of fHbp v.1.13 E211A / S216R to factor H domains 6-7.

[0025] Figure 4 (AD) includes four graphs (sensograms) comparing fHbp v1.1 and fHbp v1.13 (wild type) with ( Figure 4 A) fHbp v.1.13 E211A; ( Figure 4 B) fHbp v1.13 S216R; ( Figure 4 C) fHbp v.1.13 E211A / E232A; and ( Figure 4 D) Binding of fHbp v.1.13 E211A / S216R to full-length factor H protein.

[0026] Figure 5 (AD) includes four graphs (sensograms) comparing fHbp v1.1 and fHbp v1.15 (wild type) with ( Figure 5 A) fHbp v.1.15 E214A; ( Figure 5 B) fHbp v1.15 S219R; ( Figure 5 C) fHbp v.1.15 E214A / E235A; and ( Figure 5 D) Binding of fHbp v.1.15 E214A / S219R to factor H domains 6-7.

[0027] Figure 6 (AD) includes four graphs (sensograms) comparing fHbp v1.1 and fHbp v1.15 (wild type) with ( Figure 6 A) fHbp v.1.15 E214A; ( Figure 6 B) fHbp v1.15 S219R; ( Figure 6 C) fHbp v.1.15 E214A / E235A; and ( Figure 6 D) Binding of fHbp v.1.15 E214A / S219R to full-length factor H protein.

[0028] Figure 7 (AB) comprise two graphs (sensograms) showing the binding of two different fHbp fusion proteins according to the invention to Factor H domains 6-7 compared to known fHbp fusions. Figure 7 A Comparison of binding of fHbp 231 wt and fHbp 231S and fHbp 231.13 E211A / S216R fusions. Figure 7 B Comparison of binding of fHbp 231 wt and fHbp 231S and fHbp231.13 E211A / E232A fusion.

[0029] Figure 8 Binding of fHbp 231 wt and fHbp 231S as well as the fHbp 231.15 E214A / E235A fusion according to the invention to factor H domains 6-7 was compared.

[0030] Fig. 9 (AB) include two graphs showing the binding of fHbp fusion proteins, two different fusion proteins according to the present invention, to full-length Factor H protein compared to known fHbp fusions. Fig. 9 A Comparison of binding of fHbp 231 wt and fHbp 231S and fHbp 231.13 E211A / S216R fusions. Fig. 9 B Comparison of binding of fHbp 231 wt and fHbp 231S and fHbp231.13 E211A / E232A fusion.

[0031] Fig.10 Binding of fHbp 231 wt and fHbp 231S as well as the fHbp 231.15 E214A / E235A fusion according to the invention to the full-length Factor H protein was compared.

[0032] Fig.11 (A) rSBA titers (rabbit complement) of each of the six fHbp-related antigens and BEXSERO-like preparations against various meningococcal strains expressing fHbp in v1.x are shown. In the SBA results, each point represents the SBA titer of a single strain analyzed on pooled sera. Fig.11 B shows rSBA titers (human complement) for each of the six fHbp-related antigens and BEXSERO-like preparations tested against the same various strains expressing fHbp in v1.x.

[0033] Fig.12 (A) rSBA titers (rabbit complement) of each of the six fHbp-related antigens and BEXSERO-like preparations against various meningococcal strains expressing fHbp in v2 or v3 are shown. In the SBA results, each point represents the SBA titer of a single strain analyzed on pooled sera. Fig.12 B shows rSBA titers (human complement) for each of the six fHbp-related antigens and BEXSERO-like preparations tested against the same various strains expressing fHbp in either v2 or v3.

[0034] Fig.13 The preparations were shown to be effective for var2 / 3 ( Fig.13 A) and v1.x( Fig.13 B) Pooled hSBA data by strain type. Sera collected from vaccinated mice were tested as a pool against a total of 50 MenB strains, divided into var1 (30 strains) and var2 / 3 strains (20 strains) in the presence of human plasma as a complement source (hSBA). In this figure, fHbp 2-3-1.13 refers to fusions containing wt v1.13, fHbp 2-3-1.13 NB ES refers to 231.13_E211A / S216R fusions, fHbp 2-3-1.13 NB EE refers to 231.13_E211A / E232A fusions, fHbp 2-3-1.15 refers to fusions containing wt v1.15, and fHbp 2-3-1.15 NB EE refers to 231.15_ E214A / E235A fusions.

[0035] Fig.14Shown are the percentage coverage provided by the test vaccine formulations tested in mice against strains expressing fHbp var2 / 3 (A) and fHbp var1 (B). In the figure, fHbp 2-3-1.13 refers to a fusion containing wt v1.13, fHbp 2-3-1.13 NB ES refers to a fusion containing 231.13_E211A / S216R, fHbp 2-3-1.13 NB EE refers to a fusion containing wt v1.15, and fHbp 2-3-1.15 NB EE refers to a fusion containing wt v1.15, and fHbp 2-3-1.15 NB EE refers to a fusion containing 231.15_ E214A / E235A.

[0036] Fig.15 hSBA titers from mouse sera are shown for 11 strains, including the BEXSERO reference strain and fHbp var1.1 and 1.4 strains. In the figure, fHbp 2-3-1.13 refers to a fusion comprising wt v1.13, fHbp2-3-1.13 NB ES refers to a fusion comprising 231.13_E211A / S216R, fHbp 2-3-1.13 NB EE refers to a fusion comprising wt v1.15, and fHbp 2-3-1.15 NB EE refers to a fusion comprising 231.15_ E214A / E235A.

[0037] Fig.16 For a panel of four BEXSERO indicator strains (for fHbp var1.1, M14459 ( Fig.16 A); for PorA P1.4, NZ98 / 254 ( Fig.16 B); for NHBA, M4407( Fig.16 C); and for NadA, 96217 ( Fig.16 D)), comparing the formulation of the present invention comprising BEXSERO + fHbp231.13_E211A / S216R fusion protein (referred to as "BEXSEROPLUS PLUS" in the figure) with the standard BEXSERO formulation.

[0038] Fig.17 The preparations were shown to be effective for var2 / 3 ( Fig.17 A) and v1.x ( Fig.17B) Pooled hSBA data by strain type. Sera collected from vaccinated rabbits were tested as pools against MenB strains (divided into var1 and var2 / 3 strains). In this figure, fHbp 2-3-1.13 refers to a fusion containing wt v1.13, fHbp 2-3-1.13 NB ES refers to a fusion containing wt v1.13, fHbp 2-3-1.13 NB ES refers to a fusion containing wt v1.15, and fHbp 2-3-1.15 NB EE refers to a fusion containing wt v1.15, and fHbp 2-3-1.15 NB EE refers to a fusion containing 231.15_ E214A / E235A.

[0039] Fig.18 Shown are the percentage coverage provided by vaccine formulations tested in rabbits against strains expressing fHbp var2 / 3 (A) and fHbp var1 (B). In the figure, fHbp 2-3-1S refers to the prior art fusion 231.1_R41S, fHbp 2-3-1.13 refers to the fusion containing wt 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 / E232A fusion, fHbp 2-3-1.15 refers to the fusion containing wt v1.15, and fHbp 2-3-1.15 NB EE refers to the 231.15_ E214A / E235A fusion.

[0040] Fig.19 hSBA titers for 11 strains (including BEXSERO reference strains and fHbp var 1.1 and 1.4 strains) from rabbit serum are shown. In the figure, fHbp 2-3-1.13 binding refers to the fusion comprising wt v1.13, fHbp2-3-1.13 non-binding refers to 231.13_E211A / S216R fusion, fHbp 2-3-1.15 binding refers to the fusion comprising wt v1.15, and fHbp 2-3-1.15 non-binding refers to 231.15_ E214A / E235A fusion.

[0041] Fig. 20 Dynamic imaging of bacterial challenge in immunized and non-immunized mice is shown (bioluminescent MC58cc32 / var.1). Mice in group A were immunized with 4CMenB + fHbp 23(S)1.13 wild-type, while mice in group B were immunized with 4CMenB + fHbp 23(S)1.13_E211A / S216R. Non-immunized mice received only phosphate-buffered saline (PBS) as a control.

[0042] Fig.21 (A and B) Quantification and comparison of dynamic imaging signals (bioluminescent MC58 cc32 / var.1) in immunized and non-immunized mice following bacterial challenge. Comparison of crude total signals (number of photons per second and per mouse at each time point) 30 min and 6 h after bacterial challenge was performed using ( Fig.21 A) or by signal ratio ( Fig.21 B) proceed.

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] Lipoprotein factor H binding protein (fHbp) is expressed on the surface of all MenB strains. fHbp binds to human complement regulatory protein factor H (hfH) to form a complex that protects bacteria from complement-mediated killing and provides a survival mechanism for Neisseria meningitidis in the human bloodstream. Antibodies against fHbp have a dual effect: they are bactericidal themselves, and by preventing binding to hfH, they make the strain more susceptible to bacterial killing. Reducing or abolishing the ability of fHbp to bind hfH increases the immunogenicity of the fHbp antigen by preventing the formation of a protective complex between fHbp and hfH, which has the potential to mask the fHbp epitope and prevent antibody binding.

[0045] fHbp exists in three genetically and immunogenic variants (v1, v2 and v3) with many subvariants. Most MenB strains not covered by 4CMenB express v2 or v3 fHbp or v1 subvariants that are distantly related to var1.1.

[0046] like Figure 1 As shown in A, current epidemiological studies using the MATS method (e.g., by Medini et al. Vaccine 2015; 33(23); 2629-36) showed that strains with v1.1 and v1.4 were the most frequent, followed by v1.15, v1.14 and v1.13. Antibodies generated against the subvariant fHbpv1.1 included in the 4CMenB vaccine were highly cross-reactive with these most frequent fHbp v1 subvariants (v1.1 and v1.4), but had low cross-reactivity with the v1 subvariants most distantly related to v1.1 (e.g., v1.15 and v1.13). This is Figure 1 This is exemplified in , because meningococcal B strains expressing fHbp v1.15 and v1.13 are the most frequently occurring strains not covered by the 4CMenB vaccine.

[0047] Furthermore, antibodies generated against the subvariant fHbpv1.1 included in the 4CMenB vaccine had poor cross-reactivity with fHbp v2 and v3 (Brunelli B et al. , Vaccine 2011; 29:1072–1081). Figure 1 B and 1C show gaps in the coverage of 4CMenB against some of the most frequently occurring strains expressing fHbp v2 or v3.

[0048] This means that 4CMenB coverage cannot be extended to some meningococcal strains carrying fHbp v2, v3 or strains carrying some v1 subvariants.

[0049] The present invention provides mutated fHbp variant 1.13 or variant 1.15 (v1.13 or v1.15) polypeptides that are immunogenic and can be combined with existing meningococcal vaccines to provide improved coverage of Neisseria meningitidis strains.

[0050] In particular, the v1 polypeptide of the present invention is a subvariant of fHbp variant 1 which is genetically diverse compared to the fHbpv1.1 antigen included in 4CMenB.

[0051] Furthermore, the v1 polypeptides of the present 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 fHp v1.55 and v3.45 antigens included in TRUMENBA do bind to hfH.

[0052] The V1 polypeptide of the present invention can be provided alone or as a component of a fusion protein with mutant forms of fHbp variants 2 and 3, which have been modified to improve stability and also reduce fHbp binding. By providing a single fusion protein comprising these v2 and v3 antigens together with the v1 antigen of the present invention, the inventors have improved strain coverage relative to existing licensed meningococcal B vaccines. For clarity, neither v2 nor v3 antigens are present in, for example, 4CMenB. The presence of v2 and v3 antigens within the fusion protein of the present invention improves strain coverage compared to, for example, 4CMenB.

[0053] The v1 polypeptides and fusion proteins of the invention can be used alone or in combination with meningococcal NHBA antigens, meningococcal NadA antigens, meningococcal fHbp antigens, and meningococcal outer membrane vesicles (e.g., in combination with a BEXSERO composition), which provides a combined immunogenic composition with increased immunogenicity (due to the addition / inclusion of non-binding forms of fHbp variants) and increased coverage of Neisseria meningitidis strains (due to the addition of new fHbp variants / subvariants) compared to BEXSERO alone.

[0054] Mutant v1.13 meningococcal fHbp polypeptide

[0055] The present inventors have identified residues within 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. The present inventors have also identified combinations of mutations in the v1.13 sequence that are particularly useful for reducing binding to hfH. fHbp v1.13 is also known in the art as fHbp variant B09.

[0056] The mature wild-type fHbp v1.13 lipoprotein from strain M982 (GenBank Accession No. AAR84475.1) has the following amino acid sequence, with the N-terminal poly-glycine signal sequence underlined:

[0057]

[0058] The mature v1.13 lipoprotein differs from the full-length wild-type sequence in that the full-length polypeptide has an additional 19-residue N-terminal leader sequence that is cleaved from the mature polypeptide. Thus, the full-length wild-type fHbp v1.13 has the following amino acid sequence (wherein the N-terminal leader sequence is shown in bold):

[0059]

[0060] The ΔG form of the mature v1.13 lipoprotein lacks the N-terminal poly-glycine 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:

[0061]

[0062] Thus, a first aspect of the present invention provides a mutant v1.13 meningococcal fHbp polypeptide comprising at least k%An amino acid sequence with sequence identity to the mutant v1.13 meningococcal fHbp polypeptide, provided that the amino acid sequence of the mutant v1.13 meningococcal fHbp polypeptide includes a substitution mutation at one or more of residues E211, S216 or E232 of SEQ ID NO:2.

[0063] k The value of may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably 80 (i.e. the mutant fHbp v1.13 amino acid sequence has at least 80% identity with SEQ ID NO: 2) and more preferably 85, more preferably 90, and more preferably 95. Most preferably, the mutant fHbp v1.13 amino acid sequence has at least 97%, at least 98% or at least 99% identity with SEQ ID NO: 2.

[0064] Preferably, the amino acid sequence differs from SEQ ID NO:2 by at least one or more of the substitutions E211A, S216R or E232A. More preferably, the amino acid sequence comprises substitutions at a plurality of residues selected from the group consisting of: (i) E211A and E232A, or (ii) E211A and S216R. More preferably, relative to SEQ ID NO.2, the amino acid sequence comprises substitutions at residues E211A and S216R.

[0065] Without wishing to be bound by theory, the substitution of glutamic acid (E) to alanine (A) at residue 211 of SEQ ID NO. 2 removes a negatively charged residue involved in hfH recruitment, thus contributing to abrogation of fH binding. The substitution of arginine (R) to serine (S) at residue 216 of SEQ ID NO. 2 replaces the wild-type amino acid with the corresponding residue from Neisseria gonorrhoeae, which does not bind hfH.

[0066] In a preferred embodiment, the mutant v1.13 polypeptide of the present invention has the amino acid sequence of SEQ ID NO:3 (v1.13 ΔGE211A / E232A) or SEQ ID NO:4 (v1.13 ΔG (E211A / S216R). More preferably, the mutant v1.13 polypeptide of the present invention has the amino acid sequence of SEQ ID NO:4.

[0067] Upon administration to a host animal, preferably a mammal, and more preferably a human, the mutant v1.13 polypeptides of the invention can elicit antibodies that can recognize the wild-type meningococcal fHbp polypeptide of SEQ ID NO: 1. These antibodies are ideally bactericidal (see below).

[0068] Mutant v1.15 meningococcal fHbp polypeptide

[0069] The inventors have also identified residues within the fHbp v1.15 sequence that can be modified to prevent binding to hfH. Such mutants are referred to herein as non-binding (NB) mutants. The inventors have also identified a combination of mutations in the v1.15 sequence that are particularly useful for preventing binding to hfH. fHbp v1.15 is also known in the art as fHbp variant B44.

[0070] The mature wild-type fHbp v1.15 lipoprotein from strain NM452 (GenBank Accession No. ABL14232.1) has the following amino acid sequence, with the N-terminal poly-glycine signal sequence underlined:

[0071]

[0072] The mature v1.15 lipoprotein differs from the full-length wild-type sequence in that the full-length polypeptide has an additional 19-residue N-terminal leader sequence that is cleaved from the mature polypeptide. Thus, the full-length wild-type fHbp v1.15 has the following amino acid sequence (with the N-terminal leader sequence shown in bold):

[0073]

[0074] The ΔG form of the mature v1.15 lipoprotein lacks the N-terminal poly-glycine 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:31:

[0075]

[0076] Therefore, a second aspect of the present invention provides a mutant v1.15 meningococcal fHbp polypeptide comprising at least k% An amino acid sequence with sequence identity to the mutant v1.15 meningococcal fHbp polypeptide, provided that the amino acid sequence of the mutant v1.15 meningococcal fHbp polypeptide includes a substitution mutation at one or more of residues E214, S219 or E235 of SEQ ID NO:6.

[0077] kThe value of may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably 80 (i.e. the mutant fHbp v1.15 amino acid sequence has at least 80% identity with SEQ ID NO: 6) and more preferably 85, more preferably 90, and more preferably 95. Most preferably, the mutant fHbp v1.15 amino acid sequence has at least 97%, at least 98% or at least 99% identity with SEQ ID NO: 6.

[0078] Preferably, the amino acid sequence differs from SEQ ID NO: 6 by at least one or more of the substitutions E214A, S219R or E235A. More preferably, the amino acid sequence comprises a substitution at a residue selected from the group consisting of: (i) S219R, (ii) E214A and S219R, and (iii) E214A and E235A.

[0079] In a preferred embodiment, the mutant v1.15 polypeptide of the present 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).

[0080] Upon administration to a host animal, preferably a mammal, and more preferably a human, the mutant v1.15 polypeptides of the invention can elicit antibodies that can recognize the wild-type meningococcal fHbp polypeptide of SEQ ID NO: 5. These antibodies are ideally bactericidal (see below).

[0081] Fusion peptide

[0082] The present invention also provides a fusion polypeptide comprising all three of the v1, v2 and v3 meningococcal fHbp polypeptides, wherein the variant fHbp sequence is in the order v2-v3-v1 from the N-terminus to the C-terminus. In a preferred embodiment, the fHbp fusion polypeptide has an amino acid sequence of the formula NH2—A-[-XL]3-B—COOH, wherein each X is a different variant fHbp sequence, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, and B is an optional C-terminal amino acid sequence.

[0083] The v1 fHbp polypeptide component of the fusion of the present invention is a mutant v1.13 fHbp polypeptide or a mutant v1.13 fHbp polypeptide as described above.

[0084] The v2 and v3 fHbp polypeptide components of the fusion of the present invention are preferably mutant v2 and v3 polypeptides having enhanced stability and reduced ability to bind 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 a protective complex between fHbp and hfH, which can mask the fHbp epitope and thereby increase the immunogenicity of the polypeptide antigen.

[0085] The inventors have previously identified residues within the v2 and v3 sequences that can be modified to increase the stability of the polypeptide and reduce binding to hfH. These mutant v2 and v3 sequences are described in detail in WO2015 / 128480.

[0086] Full-length wild-type fHbp v2 from strain 2996 has the following amino acid sequence (the leader sequence is shown in bold and the poly-glycine sequence is underlined):

[0087]

[0088] The mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 10:

[0089]

[0090] The ΔG form of SEQ ID NO: 10 lacks the first 26 amino acids:

[0091]

[0092] In a preferred embodiment, the fusion polypeptide of the present invention comprises a mutant v2 fHbp polypeptide comprising at least k% An amino acid sequence having sequence identity to the v2 fHbp, provided that the v2 fHbp amino acid sequence comprises substitution mutations at residues S32 and L123 of SEQ ID NO: 12. Preferably, the substitutions are S32V and L123R.

[0093] k The value of may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably 80 (i.e., the mutant fHbp v2 amino acid sequence has at least 80% identity with SEQ ID NO: 12) and more preferably 85, more preferably 90, and more preferably 95.

[0094] 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 wild-type mature sequence, SEQ ID NO: 12 has been truncated at the N-terminus up to and including the poly-glycine sequence (compare SEQ ID NO: 11 and 12), but SEQ ID NO: 12 may be truncated at the C-terminus and / or further truncated at the N-terminus.

[0095] In a preferred embodiment, the v2 fHbp polypeptide included in the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO:16.

[0096] Under the same experimental conditions, the v2 fHbp polypeptide included in the fusion protein of the present invention has higher stability than the same polypeptide without sequence differences at 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 by introducing favorable hydrophobic interactions. The L123R mutation eliminates fH binding by introducing conflicts and unfavorable charges with fH.

[0097] Enhanced stability can be assessed using differential scanning calorimetry (DSC), e.g. as described by Johnson (2013) Arch Biochem Biophys 531:100-9 and Bruylants et al. Current Medicinal Chemistry 2005; 12:2011-20. DSC has been used previously to evaluate the stability of v2 fHbp (Johnson et al. PLoS Pathogen 2012;8: e1002981). Suitable conditions for DSC assessment of stability may use 20 µM of the polypeptide in a buffer solution (e.g. 25 mM Tris) having a pH of 6-8 (e.g. 7-7.5) and 100-200 mM NaCl (e.g. 150 mM).

[0098] Increased stability is characterized by an increase in the thermal transition midpoint (Tm) of at least one peak compared to wild-type when evaluated by DSC by at least 5°C, such as at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or more. Wild-type fHbp exhibits two DSC peaks during unfolding (one for the N-terminal domain and one for the C-terminal domain), and in the case where the v2 polypeptide included in the fusion protein of the invention includes two such domains, "increased stability" refers to the Tm of the N-terminal domain. m For the wild-type v2 sequence, the Tm of the N-terminal domain may occur at 40°C or even below 40°C (Johnson et al. (2012) PLoS Pathogen 8:e1002981), while the C-terminal domain may have a Tm of 80°C or higher. Therefore, the mutant fHbp v2 amino acid sequence included in the fusion protein of the present invention preferably has an N-terminal domain that has a Tm of at least 45°C, for example, > 50℃ > 55℃, > 60℃ > 65℃, > 70℃, > 75℃ or even > Tm of 80°C.

[0099] Full-length wild-type fHbp v3 from strain M1239 has the following amino acid sequence (leader sequence is shown in bold and poly-glycine sequence is underlined):

[0100]

[0101] The mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 13:

[0102]

[0103] The ΔG form of SEQ ID NO: 13 lacks the first 31 amino acids (ie lacks the signal sequence and the poly-glycine sequence):

[0104]

[0105] In a preferred embodiment, the fusion polypeptide of the present invention comprises a mutant v3 fHbp polypeptide comprising at least k% An amino acid sequence having sequence identity to the v3 fHbp amino acid sequence, provided that the v3 fHbp amino acid sequence comprises substitution mutations at residues S32 and L126 of SEQ ID NO: 15. Preferably, the substitutions are S32V and L126R.

[0106] k The value of may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably 80 (i.e., the mutant fHbp v2 amino acid sequence has at least 80% identity with SEQ ID NO: 15) and more preferably 85, more preferably 90, and more preferably 95.

[0107] 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 wild-type mature sequence, SEQ ID NO: 15 has been truncated at the N-terminus up to and including the poly-glycine sequence (compare SEQ ID NO: 14 and 15), but SEQ ID NO: 15 may be truncated at the C-terminus and / or further truncated at the N-terminus.

[0108] In a preferred embodiment, the v3 fHbp polypeptide included in the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO:17.

[0109] Under the same experimental conditions, the v3 fHbp polypeptide included in the fusion protein of the present invention has higher stability than the same polypeptide without sequence differences at 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 by introducing favorable hydrophobic interactions. The L126R mutation eliminates fH binding by introducing conflicts and unfavorable charges with fH.

[0110] Enhanced stability can be assessed using differential scanning calorimetry (DSC), e.g. as described by Johnson (2013) Arch Biochem Biophys 531:100-9 and Bruylants et al. (2005) Current Medicinal Chemistry DSC has been previously used to evaluate the stability of v3 fHbp (van der Veen et al. (2014) Infect Immun PMID 24379280). Suitable conditions for DSC assessment of stability may use 20 μM polypeptide in a buffer solution (e.g. 25 mM Tris) having a pH of 6-8 (e.g. 7-7.5) and 100-200 mM NaCl (e.g. 150 mM).

[0111] Increased stability is characterized by an increase in the thermal transition midpoint (Tm) of at least one peak compared to wild-type when evaluated by DSC by at least 5°C, such as at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or more. Wild-type fHbp exhibits two DSC peaks during unfolding (one for the N-terminal domain and one for the C-terminal domain), and in the case where the v3 polypeptide included in the fusion protein of the invention includes two such domains, "increased stability" refers to the Tm of the N-terminal domain. m For the wild-type v3 sequence, the Tm of the N-terminal domain may occur at about 60°C or lower (Johnson et al. (2012) PLoS Pathogen 8:e1002981), while the C-terminal domain may have a Tm of 80°C or higher. Therefore, the mutant fHbp v3 amino acid sequence of the present invention preferably has an N-terminal domain that has a Tm of at least 65°C, for example, > 70℃, > 75℃ or even > Tm of 80°C.

[0112] As described above, in a preferred embodiment, the fHbp fusion polypeptide has an amino acid sequence of the formula NH2—A-[-XL]3-B—COOH, wherein each X is a different variant fHbp sequence, and L is an optional linker amino acid sequence. In a preferred embodiment, the linker amino acid sequence "L" is a glycine polymer or a glycine-serine polymer linker. Exemplary linkers include, but are not limited to, "GGSG", "GGSGG", "GSGSG", "GSGGG", "GGGSG", "GSSSG" and "GSGGGG". Other suitable glycine or glycine-serine polymer linkers will be apparent to the skilled person. In preferred fusion polypeptides according to the present invention, the v2 and v3 sequences and the v3 and v1 sequences are connected by a glycine-serine polymer linker "GSGGGG".

[0113] In a preferred embodiment, the fusion polypeptide of the invention comprises or consists of one of the following amino acid sequences (the glycine-serine linker sequence is underlined and the mutated residues are indicated in bold):

[0114]

[0115]

[0116]

[0117] In a preferred embodiment, the fusion polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO. 19. In an alternative preferred embodiment, the fusion polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO. 18.

[0118] After administration to a host animal, preferably a mammal, and more preferably a human, the fusion polypeptides of the invention can elicit antibodies that can recognize wild-type meningococcal fHbp polypeptides, particularly polypeptides of SEQ ID NO: 31, 32, 10 and / or 13. These antibodies are ideally bactericidal (see below).

[0119] As described above, in a preferred embodiment, the fHbp fusion polypeptide according to the present invention has an amino acid sequence of the formula NH2-A-[-XL]3-B-COOH, wherein each X is a different variant fHbp sequence, and A is an optional N-terminal amino acid sequence. In a preferred embodiment, the fusion protein described herein further comprises the following N-terminal amino acid sequence, which is advantageous for enabling good expression of the fusion protein:

[0120]

[0121] Any of the fusion proteins disclosed herein (e.g., SEQ ID NOs: 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 of SEQ ID NO. 34 is added to the N-terminus of the fHbp v2 component of the fusion polypeptide.

[0122] Bactericidal response

[0123] Preferred v1.13, v1.15 and / or fusion polypeptides of the invention can elicit bactericidal antibody responses against meningococci. Bactericidal antibody responses are conveniently measured in mice and are a standard indicator of vaccine efficacy (e.g., see Pizza et al. (2000) Science 287:1816-1820, end note 14; also WO 2007 / 028408).

[0124] The polypeptides of the first embodiment of the invention may preferably elicit an antibody response that is bactericidal against a strain of Neisseria meningitidis expressing the v1.13 fHbp sequence.

[0125] Preferred polypeptides of the first embodiment of the invention can elicit antibodies in mice that are bactericidal in a serum bactericidal assay against a strain of Neisseria meningitidis expressing the v1.13 fHbp sequence.

[0126] The polypeptides of the second embodiment of the invention may preferably elicit an antibody response that is bactericidal against a strain of Neisseria meningitidis expressing the v1.15 fHbp sequence.

[0127] Preferred polypeptides of the second embodiment of the invention can elicit antibodies in mice that are bactericidal in a serum bactericidal assay against a strain of Neisseria meningitidis expressing the v1.15 fHbp sequence.

[0128] For example, immunogenic compositions comprising these polypeptides can provide >Serum bactericidal titer of 1:4 (using the Goldschneider assay with human complement [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 > Serum bactericidal titer of 1:128 (using baby rabbit complement).

[0129] Peptides

[0130] The polypeptides of the invention can be prepared by various means, e.g., by chemical synthesis (at least in part), by digestion of longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g., from recombinant expression or from Neisseria meningitidis culture), etc. Heterologous expression in an E. coli host is a preferred expression route.

[0131] The polypeptides of the invention are ideally at least 100 amino acids long, e.g., 150aa, 175aa, 200aa, 225aa or longer. They include mutant fHbp v1, v2 and / or v3 amino acid sequences, and the mutant fHbp v1, v2 or v3 amino acid sequences should similarly be at least 100 amino acids long, e.g., 150aa, 175aa, 200aa, 225aa or longer.

[0132] fHbp is the natural lipoprotein of Neisseria meningitidis. It has also been found that it is lipidated when expressed with its natural leader sequence or with a heterologous leader sequence in Escherichia coli. The polypeptide of the present invention may have an N-terminal cysteine ​​residue that can be lipidated, for example, it comprises a palmitoyl group, usually forming tripalmitoyl-S-glyceryl-cysteine. In other embodiments, the polypeptide is not lipidated.

[0133] It is preferred to prepare the polypeptide in substantially pure or substantially isolated form (i.e., substantially free of other Neisseria or host cell polypeptides). Typically, the polypeptide is provided in a non-naturally occurring environment, e.g., separated from its naturally occurring environment. In certain embodiments, the polypeptide is present in a composition that is enriched for the polypeptide compared to the starting material. Purified polypeptides are thus provided, wherein purified means that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, wherein substantially free means that more than 50% (e.g., less than 50%) of the total polypeptides in the composition are present. > 75%, > 80%, > 90%, > 95% or >99%) is the polypeptide of the present invention.

[0134] The polypeptides can be in various forms (eg, native, fusion, glycosylated, non-glycosylated, lipidated, disulfide-linked, etc.).

[0135] If the polypeptide of the present invention is produced by translation in an organism host, an initiation codon is required, which will provide the N-terminal methionine in most hosts. Therefore, the polypeptide of the present invention will include a methionine residue upstream of the SEQ ID NO sequence at least in the nascent stage.

[0136] Cleavage of the nascent sequence means that the mutant fHbp v1, v2 or v3 amino acid sequence may itself provide the N-terminus of the polypeptide. However, in other embodiments, the polypeptide of the present invention may include an N-terminal sequence upstream of the mutant fHbp v1, v2 or v3 amino acid sequence. In some embodiments, the polypeptide has a single methionine at the N-terminus, followed by the mutant fHbp v1, v2 or v3 amino acid sequence; in other embodiments, a longer upstream sequence may be used. Such an upstream sequence may be shorter (e.g., 40 or less amino acids, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid). Examples include leader sequences that direct protein transport, or short peptide sequences that facilitate cloning or purification (e.g., histidine tags, i.e., His n , wherein 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.

[0137] The polypeptides of the invention may also include amino acids downstream of the last amino acid of the mutant fHbp v1, v2 or v3 amino acid sequence. Such C-terminal extensions may be shorter (e.g., 40 or fewer amino acids, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids). Examples include sequences that direct protein transport, short peptide sequences that facilitate cloning or purification (e.g., comprising a histidine tag, i.e., His n , wherein n=4, 5, 6, 7, 8, 9, 10 or more), or a sequence that enhances polypeptide stability. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

[0138] In some embodiments, the invention excludes the inclusion of a histidine tag (see Johnson et al. (2012) PLoS Pathogen 8:e1002981, and Pajon et al. (2012) Infect Immun 80:2667-77) and in particular a C-terminal hexa-histidine-tagged polypeptide.

[0139] The term "polypeptide" refers to an amino acid polymer of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are natural or modified by interventions such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and other modifications known in the art. Polypeptides may exist as single chains or associated chains.

[0140] The polypeptides of the invention may be attached or immobilized to a solid support.

[0141] The polypeptide of the present invention may comprise a detectable label, for example, a radioactive label, a fluorescent label or a biotin label. This is particularly useful in immunoassay techniques.

[0142] The polypeptides of the invention typically consist of an artificial amino acid sequence (ie, a sequence that does not occur in any naturally occurring meningococci).

[0143] The affinity of factor H can be determined using surface plasmon resonance (e.g., as described in Schneider et al. (2009) Nature 458:890-5) is quantitatively evaluated with fixed human fH. Provides at least a 10-fold, and ideally at least a 100-fold reduction in affinity (i.e., dissociation constant K D Mutations that increase the amount of ) are preferred (when measured under the same experimental conditions relative to the same polypeptide but without the mutation).

[0144] Nucleic Acids

[0145] A fourth aspect of the invention provides a plasmid or other nucleic acid comprising a nucleotide sequence encoding a mutant v1.13 fHbp polypeptide, a mutant v1.15 fHbp polypeptide or a fusion polypeptide as defined above.

[0146] Nucleic acids of the invention can be prepared from genomic or cDNA libraries, etc., in a number of ways, for example, by complete or partial chemical synthesis (e.g., phosphoramidite synthesis of DNA), by digestion of longer nucleic acids using nucleases (e.g., restriction enzymes), by ligation of shorter nucleic acids or nucleotides (e.g., using ligases or polymerases).

[0147] The nucleic acid of the present invention can be in various forms, for example, single-stranded, double-stranded, vector, primer, probe, labeled, unlabeled, etc.

[0148] The nucleic acids of the present invention are preferably in isolated or substantially isolated form.

[0149] The term "nucleic acid" includes DNA and RNA, as well as analogs thereof, such as those containing modified backbones, and peptide nucleic acids (PNA) and the like.

[0150] The nucleic acids according to the invention may be labelled, for example, with a radioactive or fluorescent label.

[0151] The present invention also provides vectors (such as plasmids) comprising the nucleotide sequences of the present invention (eg, cloning or expression vectors, such as vectors suitable for nucleic acid immunization) and host cells transformed with such vectors.

[0152] Host cells

[0153] A fifth aspect of the present invention provides a host cell transformed with a plasmid as defined above comprising a nucleotide sequence of the present invention.

[0154] The host cell of the present invention is preferably a bacterium expressing a polypeptide of the present invention. The bacterium may be, for example, a meningococcus or an Escherichia coli. The bacterium may constitutively express the polypeptide, but in some embodiments, expression may be under the control of an inducible promoter. The bacterium may overexpress the polypeptide (see WO2006 / 081259). The expression of the polypeptide is ideally not phase variable.

[0155] The cell may be a meningococcal bacterium, such as a oeLh and optionally also having down-regulation or knockout of: (i) at least one gene involved in making the lipid A portion of LPS toxic, in particular lpxl1 (ii) at least one gene involved in the synthesis or export of capsular polysaccharides, in particular synX and / or ctrA .

[0156] Vesicles

[0157] A sixth aspect of the present invention provides outer membrane vesicles prepared from a host cell of the present invention, in particular from a meningococcal host cell.

[0158] The vesicles prepared from the host cells of the present invention preferably contain the polypeptide according to the present invention, which should be in an immunologically accessible form in the vesicles, that is, antibodies that can bind to the purified polypeptide of the present invention should also be able to bind to the polypeptide present in the vesicles.

[0159] These membrane vesicles include any proteoliposome vesicles formed by disruption of the meningococcal outer membrane or blebbing from the meningococcal outer membrane to form vesicles comprising the protein components of the outer membrane. Thus, the term includes outer membrane vesicles (OMVs; sometimes referred to as 'blebs'), microvesicles (MVs [WO02 / 09643]) and 'native OMVs' ('NOMVs' [Katial et al. (2002) Infect Immun 70:702-707]).

[0160] MV and NOMV are naturally occurring membrane vesicles that are spontaneously formed during bacterial growth and released into the culture medium. MVs can be obtained by culturing Neisseria in broth medium, separating whole cells from smaller MVs in the broth medium (e.g., by filtration or by low-speed centrifugation to precipitate only cells without precipitating smaller vesicles), and then collecting MVs from the cell-depleted culture medium (e.g., by filtration, by differential precipitation or aggregation of MVs, by high-speed centrifugation to precipitate MVs). Strains for producing MVs can generally be selected based on the amount of MVs produced in the culture medium, for example, US 6,180,111 and WO01 / 34642 describe Neisseria with high MV production.

[0161] OMVs are artificially prepared from bacteria and can be prepared using detergent treatment (e.g., with deoxycholate) or by non-detergent means (e.g., see WO2004 / 019977). Techniques for forming OMVs include: using bile salt detergents (e.g., salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., and sodium deoxycholate [see EP0011243 and Fredriksen et al. (1991) NIPH Ann.14(2):67-80] preferably for treating Neisseria) treating the bacteria at a sufficiently high pH that does not precipitate detergents [WO01 / 91788]. Other techniques can be performed essentially in the absence of detergents [WO2004 / 019977] using techniques such as sonication, homogenization, microfluidization, cavitation, osmotic shock, grinding, French press, mixing, etc. Methods without detergents or using low detergents can retain useful antigens such as NspA and fHbp. Thus, the method can use an OMV extraction buffer containing about 0.5% or less, for example about 0.2%, about 0.1%, <0.05% or zero deoxycholate.

[0162] A useful method for OMV preparation is described in WO2005 / 004908 and involves ultrafiltration of crude OMVs rather than replacing high-speed centrifugation. The method may involve a step of ultracentrifugation after ultrafiltration. OMVs may also be purified using a two-stage size filtration method described in WO2011 / 036562.

[0163] The vesicles used in the present invention can be prepared from any meningococcal strain. The vesicles are usually from serogroup B strains, but may also be prepared from serogroups other than B (e.g., WO01 / 91788 describes a method for serogroup A) such as A, C, W135 or Y. The strain can be any serotype (e.g., 1, 2a, 2b, 4, 14, 15, 16, etc.), any serosubtype and any immunotype (e.g., L1; L2; L3; L3,3,7; L10; etc.). The meningococcus can be from any suitable lineage, including highly invasive and highly toxic lineages, such as any of the following seven highly toxic lineages: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37 complex; A4 cluster; lineage 3.

[0164] In addition to encoding a polypeptide of the invention, the bacteria of the invention may have one or more further modifications. For example, they may have a modified fur Gene [WO98 / 56901]. Can be upregulated nspA Expression of expression accompanied by porA and cpsKnockout. Further knockout mutants of Neisseria meningitidis for OMV production are disclosed, for example, in WO2004 / 014417. Claassen et al. (1996) 14(10):1001-8 disclose the construction of vesicles from strains modified to express six different PorA isoforms. Mutant Neisseria with low endotoxin levels achieved by knocking out enzymes involved in LPS biosynthesis can also be used. The present invention can use engineered modifications to reduce or shut down at least one gene involved in conferring toxicity to the lipid A portion of LPS, specifically pxl 1 gene expression mutants of Neisseria [Fisseha et al. (2005) Infect Immun 73:4070–80]. Similarly, the present invention can use mutant Neisseria that have been engineered to reduce or shut down the expression of at least one gene involved in the synthesis or export of capsular polysaccharides, specifically the synX and / or ctrA genes. These or other mutants can be used in the present invention.

[0165] Thus, in some embodiments, the strains used in the present invention may express more than one PorA isoform. 6-valent and 9-valent PorA strains have been previously constructed. The strains may express 2, 3, 4, 5, 6, 7, 8, or 9 PorA isoforms: 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 strains may have downregulated PorA expression, for example, where the amount of PorA has been reduced by at least 20% (e.g., relative to wild-type levels, for example, relative to strain H44 / 76) (e.g., > 30%, > 40%, > 50%, > 60%, > 70%, > 80%, > 90%, > 95%, etc.), or even knocked out.

[0166] In some embodiments, the strain may overexpress certain proteins (relative to the corresponding wild-type strain). For example, the strain may overexpress NspA, protein 287 [WO01 / 52885], fHbp [WO2006 / 081259] (including fHbp of the present invention), TbpA and / or TbpB [WO00 / 25811], Cu, Zn-superoxide dismutase, HmbR, etc.

[0167] The gene encoding the polypeptide of the present invention may be integrated into the bacterial chromosome, or may be present in episomal form, for example, within a plasmid.

[0168] Advantageously, for vesicle production, meningococci can be genetically engineered to ensure that the expression of the polypeptide does not undergo phase transition. Methods for reducing or eliminating phase transitions in gene expression in meningococci are disclosed in WO2004 / 015099. For example, the gene can be placed under the control of a constitutive or inducible promoter, or by removing or replacing the DNA motif responsible for its phase transition.

[0169] In some embodiments, the strain may include one or more of the knockout and / or overexpression mutations disclosed in references WO02 / 09746, WO01 / 09350, WO02 / 062378, and WO2004 / 014417. For example, following the guidance and nomenclature 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, Pho P, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA and / or TbpB; (c) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, Pil C, 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.

[0170] In some embodiments, when a mutant strain is used, it may have one or more or all of the following characteristics: (i) down-regulated or knocked out LgtB and / or GalE to truncate meningococcal LOS; (ii) up-regulated TbpA; (iii) up-regulated NhhA; (iv) up-regulated Omp85; (v) up-regulated LbpA; (vi) up-regulated NspA; (vii) knocked out PorA; (viii) down-regulated or knocked out FrpB; (ix) down-regulated or knocked out Opa; (x) down-regulated or knocked out Opc; (xii) missing cpsGene complex. The truncated LOS may be a truncated LOS that does not include a sialyl-lactose-N-neotetraose epitope, for example, it may be a galactose-deficient LOS. The LOS may not have an α chain.

[0171] Depending on the meningococcal strain used to prepare the vesicles, they may or may not include the strain's native fHbp antigen (WO2004 / 046177).

[0172] In a preferred embodiment, the meningococci do not express a functional MItA protein. As described in WO2006 / 046143 and Adu-Bobie et al. (2004) Infect Immun 72:1914-19, knockout of MItA (membrane-bound lytic transglycosylase, also known as GNA33) in meningococci provides bacteria that spontaneously release large amounts of membrane vesicles into the culture medium from which they can be easily purified. For example, the vesicles can be purified using the two-stage size filtration method of WO2011 / 036562, comprising: (i) a first filtration step, in which vesicles are separated from bacteria based on their different sizes, and the vesicles enter the filtrate; and (ii) a second filtration step, in which the vesicles are retained in the retentate. The MItA mutation (downregulation or knockout) has been used in a 'GMMA' vaccine [Koeberling et al. (2014) Vaccine 32:2688-95] and may be conveniently associated with at least one gene involved in conferring toxicity to the lipid A portion of LPS (particularly lpxl1 ) and / or at least one gene involved in the synthesis or export of capsular polysaccharides (particularly synX and / or ctrA The invention relates to combining further downregulation or knockout of a gene (GMMA). GMMA (generic module for membrane antigens) is a genetically detoxified OMV produced by a meningococcal strain that has been engineered to release GMMA with reduced reactogenicity and increased immunogenicity. When tested in a monocyte activation test (MAT), GMMA induces fewer proinflammatory cytokines than OMV.

[0173] Preferred meningococcal strains for 'GMMA' vaccines using this method express mutant v2 fHbp and / or mutant v3 fHbp of the invention, and expression may be driven by a strong promoter. The vesicles released by this strain include mutant v2 and / or v3 fHbp proteins in an immunogenic form, and administration of the vesicles may provide a bactericidal antibody response as described by Koeberling et al. (2014) Vaccine32:2688-95. The strain may also express v1 fHbp, or may instead provide v1 fHbp as a separate recombinant protein in soluble form (and v1 fHbp may be a wild-type or mutant sequence, e.g., mutated to destroy its ability to bind fH, as discussed above). The invention provides such strains, and also provides vesicles released by these strains, e.g., as purified from culture medium following growth of the strains. Preferred v2 mutants for expression in these strains have mutations at S32 and / or L123 as discussed herein, and preferred v3 mutants for expression in these strains have mutations at S32 and / or L126 as discussed herein. Therefore, vesicles prepared from meningococci expressing these v2 and v3 mutant fHbp sequences are particularly preferred immunogens for use in the vaccines of the invention.

[0174] Useful promoters for use in such strains include those disclosed in WO2013 / 033398 and WO2013 / 113917. For example, the promoter may be: (a) from a porin gene, preferably porA or porB or (b) a rRNA gene promoter (such as the 16S rRNA gene), particularly from Neisseria meningitidis. When a meningococcal porin promoter is used, it is preferably from porA , and even more particularly from meningococci porA -10 region of gene promoter, and / or from meningococci porA The -35 region of a gene promoter (preferably, wherein the -10 region and the -35 region are separated by an intervening sequence of 12-20 nucleotides, and wherein the intervening sequence does not contain a poly-G sequence or comprises a poly-G sequence having no more than eight consecutive G nucleotides). When an rRNA gene promoter is used, it may comprise more specifically (i) the -10 region from a meningococcal rRNA gene promoter and / or (ii) the -35 region from a meningococcal rRNA gene promoter. A hybrid of (a) and (b) may also be used, for example with a promoter having a promoter from porA A promoter comprising the -10 region of a (particularly meningococcal) rRNA gene and the -35 region of a (particularly meningococcal) porA gene, or (ii) the -10 region of a (particularly meningococcal) porA gene and the -35 region of a (particularly meningococcal) rRNA gene.

[0175] If LOS is present in vesicles, the vesicles can be treated to link their LOS and protein components ("intravesicular" conjugation [WO2004 / 014417]).

[0176] Immunogenic composition

[0177] The polypeptides of the invention can be used as one or more active ingredients in an immunogenic composition, and thus the seventh aspect of the invention provides an immunogenic composition comprising a mutant v1.13 fHbp polypeptide according to the first aspect of the invention, a mutant v1.15 fHbp polypeptide according to the second aspect of the invention, a fusion polypeptide according to the third aspect of the invention, or a vesicle according to the sixth aspect of the invention. The immunogenic composition can be used to immunize a mammal, preferably a human, against infection with Neisseria meningitidis.

[0178] In a preferred embodiment of the present invention, in addition to the polypeptide antigen of the first, second or third aspect of the present invention, the immunogenic composition of the present invention further comprises one or more of the antigenic components of 4CMenB.

[0179] As mentioned above, 4CMenB product (BEXSERO) contains the OMV preparation of the popular strain B:4:P1.7b,4 from group B meningococcus NZ98 / 254. The same OMV is found in MeNZB vaccine and is referred to as OMVnz in this article. In addition, 4CMenB comprises five kinds of meningococcal antigens: NHBA (287; Subvariant 1.2), fHbp (741; Subvariant 1.1), NadA (961; Subvariant 3.1), GNA1030 (953) and GNA2091 (936). Four kinds of these antigens exist as fusion proteins (NHBA-GNA1030 fusion protein (287-953) and GNA2091-fHbp (936-741) fusion protein).

[0180] The complete 4CMenB product was formulated to contain 50 μg of each of NHBA-GNA1030, NadA, and GNA2091-fHbp adsorbed onto 1.5 mg of aluminum hydroxide adjuvant, and 25 μg of OMVs from Neisseria meningitidis strain NZ98 / 254. In addition, each 0.5 ml dose of the formulation included 3.125 mg of sodium chloride, 0.776 mg of histidine, and 10 mg of sucrose.

[0181] In a further preferred embodiment, the immunogenic composition of the invention comprises the complete vaccine product 4CMenB, which is sold under the trade name BEXSERO.

[0182] In a further preferred embodiment, the immunogenic composition of the present invention comprises the fHbp fusion polypeptide of SEQ ID NO. 19 (fHbp 23S_1.13_E211A / S216R) and a complete 4CMenB composition.

[0183] Meningococcal serogroups A, C, W135, and Y

[0184] The compositions of the invention may also include capsular saccharide antigens from one or more of meningococcal serogroups Y, W135, C and A, wherein the antigen is conjugated to a carrier protein and / or is an oligosaccharide. Capsular saccharides may be used in oligosaccharide form. These are conveniently formed by fragmenting purified capsular polysaccharides (e.g. by hydrolysis), typically followed by purification of fragments of the desired size.

[0185] Current serogroup C vaccines (MENJUGATE [Costantino et al. (1992) Vaccine 10:691-698,Jones (2001) Curr Opin Investigate Drugs 2:47-49], MENINGITEC and NEISVAC-C) include conjugated sugars. MENJUGATE and MENINGITEC have 197 carrier-conjugated oligosaccharide antigen, while NEISVAC-C uses intact polysaccharide (de-O-acetylated) conjugated to a tetanus toxoid carrier.

[0186] Vaccine products sold under the trade names MENVEO, MENACTRA and NIMENRIX all contain conjugated capsular saccharide antigens from serogroups Y, W135, C and A.

[0187] In MENVEO (also commonly known as meningococcal (groups A, C, Y and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine), the A, C, W135 and Y antigens are each conjugated to CRM 197 Vector conjugation.

[0188] In MENACTRA (also commonly known as Meningococcal (Group A, C, Y and W-135) Polysaccharide Diphtheria Toxoid Conjugate Vaccine), the A, C, W135 and Y antigens are conjugated to a diphtheria toxoid carrier.

[0189] In NIMENRIX (also commonly referred to as meningococcal polysaccharide group A, C, W-135 and Y conjugate vaccine), the A, C, W135 and Y antigens are each conjugated to a tetanus toxoid carrier.

[0190] In a preferred embodiment of the invention, in addition to the polypeptide antigens of the first, second or third aspect of the invention, the immunogenic composition of the invention further comprises one or more conjugated capsular saccharide antigens from Neisseria meningitidis serogroups A, C, W135 and / or Y.

[0191] 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 the complete 4CMenB product, together with one or more conjugated capsular saccharide antigens from Neisseria meningitidis serogroups A, C, W135 and / or Y.

[0192] In a preferred embodiment, in addition to the polypeptide antigens of the first, second or third aspects of the invention, the immunogenic composition of the invention also comprises the intact 4CMenB product, together with conjugated capsular saccharide antigens from each of Neisseria meningitidis serogroups A, C, W135 and / or Y, to form a pentavalent immunogenic composition comprising antigens against each of meningococcal serotypes A, B, C, W135 and Y.

[0193] In a preferred embodiment, the composition includes the A, C, W135, and Y antigen conjugates present in MENVEO, the A, C, W135, and Y antigen conjugates present in MENACTRA, or the A, C, W135, and Y antigen conjugates present in NIMENRIX.

[0194] In a further preferred embodiment, the immunogenic composition of the present invention comprises the fHbp fusion polypeptide of SEQ ID NO. 19 (fHbp 23S_1.13_E211A / S216R), the complete 4CmenB product and the A, C, W135 and Y antigen conjugates present in MENVEO.

[0195] Alternatively, the immunogenic composition of the invention comprising the polypeptide antigen of the first, second or third aspect of the invention may be co-administered with BEXSERO and one or more of MENVEO, MENACTRA or NIMENRIX. Preferably, the immunogenic composition of the invention is co-administered with BEXSERO and MENVEO.

[0196] As used herein, "co-immunization" means that different immunogenic compositions / vaccines can be administered separately or as a combination.

[0197] In the case of separate administration of the vaccines, they will generally be administered at different sites, e.g., one vaccine is administered to the left upper arm, and a second vaccine is administered to the right upper arm. Thus, the two vaccines may be administered to the opposite side (e.g., both arms or both legs or both opposite arms and legs) or to the same side (e.g., the arm and leg on the same side of the body). Although the vaccines are administered separately, they are administered at substantially the same time (e.g., during the same medical consultation or visit to a health care professional or vaccination center), such as within 1 hour of each other.

[0198] However, administration as a combination may be performed rather than separate co-immunizations. Thus, co-immunization may use a combination vaccine, i.e., a single composition in which different immunogens are mixed. Combination vaccines offer the advantage of reducing the number of injections received by the subject, which may result in a clinical advantage of increased compliance.

[0199] Uses of the immunogenic composition of the present invention

[0200] The immunogenic compositions of the present invention are suitable for use in medicine, and in particular can be used to immunize mammals against infection and / or disease caused by Neisseria meningitidis, such that the recipient of the immunogenic composition produces an immune response that provides protection against infection by and / or disease caused by Neisseria meningitidis bacteria.

[0201] In a preferred embodiment, the immunogenic compositions of the invention are useful for immunizing a mammal against meningococcal B infection or disease. However, in embodiments of the invention where meningococcal serogroup B antigens are combined with other meningococcal serogroup antigens (e.g., A, C, W and / or Y antigens), the immunogenic compositions are useful for immunizing a mammal against meningococcal A, B, C, W and / or Y infection or disease.

[0202] Thus, the immunogenic composition according to the invention is used in a prophylactic method for immunizing a subject against infection and / or disease caused by N. meningitidis. The immunogenic composition may also be used in a therapeutic method (ie treating a N. meningitidis infection).

[0203] The invention also provides a method of generating an immune response in vivo against infection by Neisseria meningitidis in a mammal, comprising administering to said mammal an immunogenic composition of the invention. The invention also provides a polypeptide of the invention for use in such a method.

[0204] The immune response is preferably protective and preferably involves antibodies and / or cell-mediated immunity. Preferably, the immune response is a bactericidal antibody response. The method may produce a booster response. By generating an in vivo immune response, mammals may be protected against Neisserial disease, particularly meningococcal infection.

[0205] The invention also provides a method for protecting a mammal against Neisserial (eg, meningococcal) infection comprising administering to the mammal an immunogenic composition of the invention.

[0206] The invention provides polypeptides of the invention for use as a medicament (e.g., as an immunogenic composition or vaccine) or as a diagnostic reagent. It also provides the use of nucleic acids or polypeptides of the invention in the preparation of a medicament for preventing Neisseria (e.g., meningococcal) infection in mammals.

[0207] The immunological composition of the invention is preferably formulated as a vaccine product, which is suitable for therapeutic (ie, treating an infection) or prophylactic (ie, preventing an infection) use. Vaccines are typically prophylactic.

[0208] The mammal is preferably a human. The human may be an adult, a teenager or a child (e.g., a toddler or an infant). Vaccines intended for children may also be administered to adults, e.g., to assess safety, dosage, immunogenicity, etc.

[0209] The uses and methods are particularly useful for preventing / treating diseases, including but not limited to meningitis (particularly bacterial meningitis, such as meningococcal meningitis) and bacteremia. For example, they are suitable for actively immunizing individuals against invasive meningococcal disease caused by Neisseria meningitidis (e.g., in serogroup B).

[0210] Protection against Neisseria meningitidis can be measured epidemiologically, for example, in clinical trials, but it is convenient to use indirect measures to confirm that the immunogenic composition causes serum bactericidal antibodies (SBA) responses in the recipient. In the SBA assay, in the presence of complement (preferably human complement, although often using baby rabbit complement), the serum from the recipient of the composition is incubated with the target bacteria (Neisseria meningitidis in the present invention), and the killing of bacteria is evaluated to measure the SBA activity under various dilutions of the serum. The results observed in the SBA assay can be strengthened by implementing competitive SBA assays to provide further indirect evidence of the immunogenic activity of the target antigen. In the competitive SBA assay, the serum of the recipient of the immunogenic composition containing one or more antigens is pre-incubated with the one or more antigens, and then incubated with the target bacteria in the presence of human complement. Then evaluate the killing of bacteria, and if the killing antibodies in the serum of the recipient bind to the target antigen during the pre-incubation stage and therefore cannot bind to the surface antigens on the bacteria, the killing of bacteria will be reduced or abolished.

[0211] The composition should not necessarily protect against each and every strain of Neisseria meningitidis, nor must it protect each and every receptor of the composition. Such general protection is not a normal standard in the field. Instead, protection is usually evaluated against a panel of reference laboratory strains, often selected on a country-by-country basis, and may vary over time and be measured across populations of receptors.

[0212] Preferred compositions of the present invention can confer antibody titers in patients that are superior to the standards for the seroprotection of each antigen component for an acceptable percentage of human subjects. Antigens with such relevant antibody titers are well known, above which hosts are considered to be seroconverted for the antigens, and such titers are published by organizations such as the WHO. Preferred samples of statistically significant subjects greater than 80% seroconvert, more preferably greater than 90%, still more preferably greater than 93%, most preferably 96-100%.

[0213] Immunogenic compositions contain an immunogen in an immunologically effective amount, and any other specified components as desired.

[0214] By "immunologically effective amount" is meant an amount that is effective for treatment or prevention when administered to an individual in a single dose or as part of a series of doses.

[0215] The term "prevention" means reducing and / or eliminating the progress of the disease, or eliminating the onset of the disease. For example, the immune system of the subject (for example, by vaccination) can be triggered to trigger an immune response and reject infection so as to eliminate the onset of the disease. Therefore, the subject of the vaccination can be infected, but can reject infection better than the control subject. The amount depends on the health and physical condition of the individual to be treated, the age of the individual to be treated, the classification group (for example, non-human primates, primates, etc.), the ability of the individual immune system to synthesize antibodies, the desired degree of protection, the vaccine formulation, the assessment of the medical situation by the treating doctor and other relevant factors. It is expected that the amount will fall within a relatively wide range that can be determined by routine tests. The composition can be used in combination with other immunomodulators.

[0216] Vaccine efficacy

[0217] The immunogenic composition used in the present invention preferably has at least 10%, e.g. > 20%, > 30%, > 40%, > 50%, > 60%, > 70%,> 80%, > 85%, > 90% or higher vaccine efficacy.

[0218] Vaccine efficacy is determined by the reduction in the relative risk of developing meningococcal disease in subjects receiving a composition according to the invention compared to subjects not receiving such a composition (e.g. not immunized or receiving a placebo or negative control). Thus, the incidence of meningococcal disease in a population immunized according to the invention is compared to the incidence in a control population not immunized according to the invention to give a relative risk, and vaccine efficacy is 100% minus this figure.

[0219] Vaccine efficacy is determined for populations rather than individuals. It is therefore a useful epidemiological tool, but cannot predict individual protection. For example, individual subjects may be exposed to very large inocula of infectious pathogens, or may have other risk factors that make them more susceptible to infection, but this does not affect the effectiveness or utility of the efficacy measure. The size of the population immunized according to the present invention and for which the vaccine efficacy is measured is ideally at least 100, and may be higher, such as at least 500 subjects. The size of the control group should also be at least 100, such as at least 500.

[0220] Application

[0221] The compositions of the present invention will generally be directly administered to the patient. Direct delivery can be by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular or to the interstitial space), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, ear, lung or other mucosal administration. Preferably, intramuscular administration is performed in the thigh or upper arm. Injection can be performed via a needle (e.g., a subcutaneous needle), but alternatively, needleless injection can be used. The intramuscular dose is generally about 0.5 ml.

[0222] Neisseria infections affect various areas of the body, so the compositions of the present invention can be prepared in various forms. For example, the composition can be prepared as an injectable, as a liquid solution or suspension. Solid forms suitable for dissolving or suspending in a liquid vehicle prior to injection can also be prepared. The composition can be prepared for topical application, for example, an ointment, cream or powder. The composition can be prepared for oral administration, for example, as a tablet or capsule, or as a syrup (optionally flavored). The composition can be prepared for pulmonary administration using a fine powder or spray, for example, as an inhalant. The composition can be prepared as a suppository or vaginal suppository. The composition can be prepared for nasal, ear or eye administration, for example, as drops. Compositions suitable for parenteral injection are most preferred.

[0223] The present invention can be used to elicit systemic and / or mucosal immunity.

[0224] As used herein, a 'dose' of a composition is a volume of the composition suitable for administration to a subject as a single immunization. Human vaccines are typically administered in a dose volume of about 0.5 ml, although fractional doses may be administered (e.g., to children). The volume of a dose may further vary depending on the concentration of the antigen in the composition.

[0225] The composition may further be provided in a 'multi-dose' kit (ie a single container containing sufficient composition for multiple immunizations).Multi-dose may include a preservative, or the multi-dose container may have a sterile adaptor for removing individual doses of the composition.

[0226] Administration may involve a single dosage regimen, but will typically involve multiple dosage regimens. Preferably, a schedule of at least three doses is given. Suitable intervals between initiating doses may be routinely determined, for example, between 4-16 weeks, such as one or two months. For example, BEXSERO may be administered at 2, 4 & 6 months of age or 2, 3 & 4 months of age, with a fourth optional dose at 12 months.

[0227] The immunized subject is a human being, which can be of any age, such as 0-12 months old, 1-5 years old, 5-18 years old, 18-55 years old, or over 55 years old. Preferably, the immunized subject is an adolescent (e.g., 12-18 years old) or an adult (18 years old or older).

[0228] Optionally, the subject is an adolescent or adult who has been immunized against Neisseria meningitidis during childhood (eg before the age of 12 years) and receives a booster dose of an immunogenic composition according to the invention.

[0229] Where the present invention refers to co-immunization, the different immunogenic compositions / vaccines may be administered separately or as a combination.

[0230] In the case of separate administration of the vaccines, they will generally be administered at different sites, e.g., one vaccine is administered to the left upper arm, and a second vaccine is administered to the right upper arm. Thus, the two vaccines may be administered to the opposite side (e.g., both arms or both legs or both opposite arms and legs) or to the same side (e.g., the arm and leg on the same side of the body). Although the vaccines are administered separately, they are administered at substantially the same time (e.g., during the same medical consultation or visit to a health care professional or vaccination center), such as within 1 hour of each other.

[0231] However, administration as a combination may be performed rather than separate co-immunizations. Thus, co-immunization may use a combination vaccine, i.e., a single composition in which different immunogens are mixed. Combination vaccines offer the advantage of reducing the number of injections received by the subject, which may result in a clinical advantage of increased compliance.

[0232] Non-antigenic components

[0233] The immunogenic compositions of the invention will generally include a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, etc. may also be present in such vehicles. A full discussion of suitable carriers can be found in Gennaro (2000). Remington: The Science and Practice of Pharmacy. 20th edition, ISBN:0683306472.

[0234] The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered, for example between pH 6 and pH 8, typically about pH 7. When the composition comprises an aluminium hydroxide salt, a histidine buffer is preferably used [WO03 / 009869]. The composition of the invention may be isotonic relative to humans.

[0235] Adjuvants that can be used in the compositions of the present invention include, but are not limited to, insoluble metal salts, oil-in-water emulsions (e.g., MF59 or AS03, both of which contain squalene), saponins, non-toxic derivatives of LPS (such as monophosphoryl lipid A or 3-O-deacylated MPL), immunostimulatory oligonucleotides, detoxified bacterial ADP-ribosylating toxins, microparticles, liposomes, imidazoquinolones, or mixtures thereof. Other substances that act as immunostimulants are disclosed in Vaccine Design… (1995) ed. Powell & Newman. ISBN: 030644867X. Chapter 7 of Plenum.

[0236] The use of aluminum hydroxide and / or aluminum phosphate adjuvants is particularly preferred, and polypeptides are typically adsorbed to these salts. These salts include oxyhydroxides and hydroxyphosphates (see, for example, Vaccine Design… (1995) ed. Powell & Newman. ISBN: 030644867X. Plenum, Chapters 8 and 9). The salt may take any suitable form (eg, gel, crystal, amorphous, etc.).

[0237] Additional antigenic components

[0238] The immunogenic compositions of the present invention may include antigens for immunization against other diseases or infections. For example, the composition may include one or more of the following other antigens:

[0239] - From Streptococcus pneumoniae ( Streptococcus pneumoniae ) [e.g., 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].

[0240] - Antigens from hepatitis A virus, such as inactivated virus [e.g., Bell (2000) Pediatr Infect Dis J 19:1187-1188 , Iwarson (1995) APMIS 103:321-326].

[0241] - Antigens from hepatitis B virus, such as surface and / or core antigens [e.g., Gerlich et al. (1990) Vaccine 8 Suppl: S63-68 & 79-80].

[0242] - Diphtheria antigens, such as diphtheria toxoid [e.g. Vaccines (1988) Plotkin & Mortimer. ISBN 0-7216-1946-0, Chapter 3], e.g. CRM 197 Mutants [e.g., Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70].

[0243] - Tetanus antigens, such as tetanus toxoid (e.g. Vaccines (Chapter 4 of Plotkin & Mortimer (1988). ISBN 0-7216-1946-0).

[0244] - From Bordetella pertussis ( Bordetella pertussis ) such as pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) from Bordetella pertussis, optionally in combination with pertactin and / or agglutinogens 2 and 3 (e.g. Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355, and Rappuoli et al. (1991) TIBTECH 9:232-238).

[0245] - From Haemophilus influenzae B ( Haemophilus influenzae B) Carbohydrate antigens [e.g. Costantino et al. (1999) Vaccine17:1251-1263].

[0246] - Poliomyelitis 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 IPV.

[0247] - Measles, mumps and / or rubella antigens (e.g. Vaccines (1988) ed. Plotkin & Mortimer. ISBN 0-7216-1946-0. Chapters 9, 10 and 11).

[0248] - Influenza antigens (eg Vaccines (1988) ed. Plotkin & Mortimer. ISBN 0-7216-1946-0, Chapter 19), such as hemagglutinin and / or neuraminidase surface proteins.

[0249] - From Moraxella catarrhalis ( Moraxella catarrhalis ) antigens [e.g. McMichael (2000) Vaccine 19 Suppl 1: S101-107].

[0250] - From Streptococcus agalactiae ( Streptococcus agalactiae ) (Group B Streptococcus) protein antigens [e.g. Schuchat (1999) Lancet 353(9146):51-6 , WO02 / 34771].

[0251] - From Streptococcus agalactiae ( Streptococcus agalactiae ) (Group B Streptococcus) carbohydrate antigen.

[0252] - From Streptococcus pyogenes ( Streptococcus pyogenes ) (Group A Streptococcus) antigens [e.g. WO02 / 34771, Dale (1999) Infect Dis Clin North Am 13:227-43, Ferretti et al. (2001) PNAS USA 98: 4658-4663].

[0253] - From Staphylococcus aureus ( Staphylococcus aureus ) [e.g. Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages 1218-1219].

[0254] If necessary, the toxic protein antigens can be detoxified (e.g., by chemical and / or genetic detoxification of pertussis toxin [Rappuoli et al. (1991) TIBTECH 9:232-238]).

[0255] When a diphtheria antigen is included in the composition, it is preferred that a tetanus antigen and a pertussis antigen are also included. Similarly, when a tetanus antigen is included, it is preferred that a diphtheria and pertussis antigen are also included. Similarly, when a pertussis antigen is included, it is preferred that a diphtheria and tetanus antigen are also included. The DTP combination is therefore preferred.

[0256] The saccharide antigen is preferably in the form of a conjugate. In general, conjugation enhances the immunogenicity of the saccharide because it converts the saccharide from a T-independent antigen to a T-dependent antigen, thereby eliciting immunological memory. Conjugation is particularly useful for pediatric vaccines and is a well-known technique.

[0257] Typical carrier proteins are bacterial toxins such as diphtheria or tetanus toxins or toxoids or mutants thereof. 197 Diphtheria toxin mutants Research Disclosure , 453077 (Jan 2002)] is useful and is a carrier in the pneumococcal vaccine sold under the trade name PREVNAR. Other suitable carrier proteins include Neisseria meningitidis outer membrane protein complex [EP-A-0372501], synthetic peptides [EP-A-0378881, EP-A-0427347], heat shock proteins [WO93 / 17712, WO94 / 03208], pertussis proteins [WO98 / 58668, EP-A-0471177], cytokines [WO91 / 01146], lymphokines [WO91 / 01146], hormones [WO91 / 01146], growth factors [WO91 / 01146], human CD4+ containing multiple antigens derived from various pathogens. + Artificial proteins of T cell epitopes [Falugi et al. (2001) Eur J Immunol 31:3816-3824] such as N19 [Baraldo et al. (2004) Infect Immun 72(8):4884-7], from Haemophilus influenzae ( H.influenzae ) of protein D [EP-A-0594610, Ruan et al. (1990) J Immunol 145:3379-3384], 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 [WO02 / 091998], ferritin [WO01 / 72337], from Clostridium difficile ( C.difficile ) toxin A or B [WO00 / 61761], recombinant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) Extracellular protein A (rEPA) [WO00 / 33882], etc.

[0258] Any suitable conjugation reaction may be used, using any suitable linker where necessary.

[0259] The sugar will usually be activated or functionalized prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (e.g., 1-cyano-4-dimethylamino pyridinium tetrafluoroborate [Lees et al. (1996) Vaccine 14:190-198, WO95 / 08348]). Other suitable techniques use carbodiimide, hydrazide, active ester, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, etc.

[0260] The linkage can be carried out via a linker group using any known procedure, such as that described in US 4,882,317 and US 4,695,624. One type of linkage involves reductive amination of the polysaccharide, coupling the resulting amino group to one end of an adipic acid linker group, and then coupling the protein to the other end of the adipic acid linker group [Porro et al. (1985) Mol Immunol Other linkers include B-propionamide [WO00 / 10599], nitrophenyl-ethylamine [Gever et al. Med. Microbiol. Immunol, 165: 171-288 (1979)], halogenated acyl halides [US 4,057,685], glycosidic bonds [US 4,673,574; US 4,761,283; US 4,808,700], 6-aminocaproic acid [US 4,459,286], ADH [US 4,965,338], C4 to C 12 Part [US 4,663,160] etc. As an alternative to using a linker, direct linkage can be used. Direct linkage to a protein may include oxidation of the polysaccharide followed by reductive amination with the protein as described, for example, in US 4,761,283 and US 4,356,170.

[0261] Preferred are methods involving the introduction of an amino group into the sugar (e.g. by replacing the terminal =O group with -NH2), followed by derivatization with an adipate diester (e.g. N-hydroxysuccinimidyl adipate), and reaction with a carrier protein. Another preferred reaction uses a CDAP activated protein D carrier, e.g. for MenA or MenC.

[0262] The antigens in the composition are typically each present at a concentration of at least 1 μg / ml. Typically, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.

[0263] The immunogenic compositions of the invention may be used therapeutically (ie, to treat an existing infection) or prophylactically (ie, to prevent future infection).

[0264] As an alternative to using a protein antigen in the immunogenic compositions of the invention, a nucleic acid (which may be RNA, such as self-replicating RNA, or DNA, such as a plasmid) encoding the antigen may be used.

[0265] General concepts

[0266] The term "comprising" encompasses "including" as well as "consisting of," e.g., a composition "comprising" X may consist of X alone or may include additional substances, e.g., X+Y. References to "comprising" (or "comprises," etc.) may optionally be replaced by references to "consisting of" (or "consists of," etc.). The term "consisting essentially of limits the scope of a claim to the specified materials or steps and "those that do not materially affect the basic and novel characteristics of the claimed invention."

[0267] With numerical value x The relative term "about" is optional and means, for example x ±10%.

[0268] The word "substantially" does not exclude "completely", for example a composition "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0269] Where the present disclosure refers to an "epitope", the epitope may be a B-cell epitope and / or a T-cell epitope, but will typically be a B-cell epitope. Such epitopes can be identified empirically (e.g. using PEPSCAN (see, e.g., Geysen et al. (1984) PNAS USA 81:3998-4002 and Carter (1994) Methods Mol Biol36:207-23) or similar methods), or they can be predicted (for example using the Jameson-Wolf Antigenic Index (Jameson, BA et al. 1988, CABIOS 4(1):181-186), matrix-based methods (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), Tsites (Feller & de la Cruz (1991) Nature 349(6311):720-1), hydrophilicity (Hopp (1993) Peptide Research 6:183-190) or antigenicity index (Welling et al. (1985) FEBS Lett. 188:215-218)). Epitopes are the parts of an antigen that are recognized and bound by the antigen binding site of an antibody or T-cell receptor, and they may also be referred to as "antigenic determinants."

[0270] As used herein, references to "percent sequence identity" between a query amino acid sequence and a subject amino acid sequence are understood to refer to identity values ​​calculated using a suitable algorithm or software programs known in the art for pairwise sequence alignments.

[0271] The query amino acid sequence can be described by the amino acid sequence identified in one or more claims herein. The query sequence can have 100% identity with the subject sequence, or it can include up to a specific integer amino acid changes (e.g., point mutations, substitutions, deletions, insertions, etc.) compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity with the subject sequence.

[0272] The preferred alignment tool for performing the alignment and calculating the percentage (%) sequence identity is a local alignment tool, such as the Basic Local Alignment Search Tool (BLAST) algorithm. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Alignment can be determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, and a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981). Adv. Appl. Math. 2: 482-489. Other preferred comparison tools are Water (EMBOSS) and Marcher (EMBOSS). Alternatively, a preferred comparison tool for comparison and calculation of percentage (%) sequence identity is a best fit comparison tool, such as GENEPAST, also referred to as the KERR algorithm.

[0273] To calculate percent identity, the query sequence and the subject sequence can be compared and aligned for maximum correspondence over a specified region (e.g., a region of at least about 40, 45, 50, 55, 60, 65 or more amino acids in length, and can be up to the full length of the subject amino acid sequence). The specified region must include the region of the query sequence that contains any specified point mutations in the amino acid sequence. Alternatively, percent sequence identity can be calculated over the "full length" of the subject sequence. Any N-terminal or C-terminal amino acid extensions that may be present in the query sequence, such as signal peptides or leader peptides or C-terminal or N-terminal tags, should be excluded from the alignment.

[0274] The term "fragment" about a polypeptide sequence means that the polypeptide is a part of a full-length protein. As used herein, a fragment of a mutant polypeptide also comprises one or more mutations. A fragment can have a qualitative biological activity common to the full-length protein, for example, an "immunogenic fragment" contains or encodes one or more epitopes, such as immune epitopes, which allow an immune response identical or similar to the full-length sequence to be produced for the fragment. A polypeptide fragment is typically missing an amino (N) terminal portion and / or a carboxyl (C) terminal portion compared to a native protein, but the remaining amino acid sequence of the fragment is identical to the amino acid sequence of the native protein. 00, 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 integers between 50 and 260, 50 and 255, 50 and 250, 50 and 200, and 50 and 150 of the reference polypeptide sequence. The term fragment specifically excludes the full-length fHbp polypeptide and its mature lipoprotein.

[0275] The classification of meningococcus includes serotype, serosubtype and then immunotype after serogroup, and standard nomenclature lists serogroup, serotype, serosubtype and immunotype, and it is separated by colon separately, for example B:4:P1.15:L3,7,9.In serogroup B, some pedigrees often cause disease (high invasiveness), some pedigrees cause disease (high toxicity) of more serious form than other pedigrees, and other pedigrees rarely cause disease completely.Seven kinds of high toxicity pedigrees are identified, i.e. subgroup I, III and IV-1, ET-5 complex group, ET-37 complex group, A4 cluster and pedigree 3.These are defined by multilocus enzyme electrophoresis (MLEE), but multilocus sequence typing (MLST) has also been used for classification of meningococcus.Four kinds of main high toxicity clusters are ST32, ST44, ST8 and ST11 complex group.

[0276] Reference herein to "enhanced stability" or "higher stability" or "increased stability" means that the mutant polypeptides disclosed herein have a higher relative thermal stability (in kcal / mol) compared to the non-mutant (wild-type) polypeptide under the same experimental conditions. Enhanced stability can be assessed using differential scanning calorimetry (DSC), for example, as described 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 evaluated by differential scanning fluorimetry (DSF). The increase in stability can be characterized as a change in the midpoint of the thermal transition (T m ) increases by at least about 5°C, as assessed by DSC or DSF. See, e.g., 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.

[0277] It will be understood that the present invention has been described above by way of example only, and that modifications may be made while remaining within the scope and spirit of the invention.

[0278] sequence

[0279] SEQ ID NO:1 [v1.13 mature polypeptide from strain M982]

[0280]

[0281] SEQ ID NO:5 [v1.15 mature polypeptide from strain NM452]

[0282]

[0283]

[0284] SEQ ID NO:10 [v2 wt from strain 2996]

[0285]

[0286] SEQ ID NO:11 [v2 mature polypeptide]

[0287]

[0288]

[0289] SEQ ID NO:13 [v3 wt from strain M1239]

[0290]

[0291] SEQ ID NO:14 [v3 mature]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] SEQ ID NO:23 [v1.1 ΔG + His tag]

[0300]

[0301] SEQ ID NO:24 [v1.13 ΔG + His tag]

[0302]

[0303]

[0304] SEQ ID NO:27 [v1.15 ΔG + His tag]

[0305]

[0306] SEQ ID NO:28 [v1.15 ΔG (E214A) + His tag]

[0307]

[0308] SEQ ID NO:29 [fHbp 231 wt fusion polypeptide]

[0309]

[0310] SEQ ID NO:30 [fHbp 231S fusion polypeptide]

[0311]

[0312] SEQ ID NO:31 [v1.13 full length wt sequence]

[0313]

[0314] SEQ ID NO:32 [v1.15 full length wt sequence]

[0315]

[0316] SEQ ID NO:33 [Mature fHbp v1.1]

[0317]

[0318] SEQ ID NO:34 [optional N-terminal amino acid sequence]

[0319]

[0320] Mode for Carrying Out the Invention

[0321] The invention will now be further defined by reference to the following non-limiting examples. Example

[0322] Example 1: Stability analysis of stable fHbp 231 fusions containing variant 1.13 mutants

[0323] As mentioned 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 been used previously to assess the stability of v2 fHbp (Johnson et al. PLoS Pathogen 2012;8:e1002981). Suitable conditions for DSC assessment of stability may use 20 μM polypeptide in a buffer solution (e.g., 25 mM Tris) at a pH of 6 to 8 (e.g., 7-7.5) with 100-200 mM NaCl (e.g., 150 mM).

[0324] The inventors used this technology to study the effect of mutant fHbp variant 1 subvariants on the stability of the 231S fusion protein.

[0325] The stable fHbp 231 fusions used in this example (referred to as "231S") include variant 2 and variant 3 sequences containing stabilizing mutations. Specifically, the v2 component of the 231S fusion has a sequence of SEQ ID NO: 16, which contains S32V and L123R mutations. The v3 component of the 231S fusion has a sequence of SEQ ID NO: 17, which contains S32V and L126R mutations. The inventors changed the v1 component of the 231S fusion to study the effect on stability.

[0326] The fHbp variant polypeptides each comprise an N-terminal and a C-terminal domain, which can be clearly distinguished as two distinct transitions (peaks) in the DSC thermogram. Although a T corresponding to the C-terminal transition of most fHbp variants is seen at around 90°C, m values, but the T of the N-terminal domain between different fHbp variants m The values ​​varied widely, with the lowest values ​​seen in variant 2.1 wild type (42°C) and the highest values ​​seen in var 1.1 (70°C).

[0327] Figure 2 Four different heatmaps comparing DSC data are shown, with the variant 1 group of the fHbp 231S fusion divided into:

[0328] fHbp v1.1 or fHbp v1.13 or (A) fHbp v.1.13 E211A ( Figure 2 A);

[0329] fHbp v1.1 or fHbp v1.13 or (B) fHbp v1.13 S216R ( Figure 2 B);

[0330] fHbp v1.1 or fHbp v1.13 or fHbp v.1.13 E211A / E232A ( Figure 2 C); and

[0331] fHbp v1.1 or fHbp v1.13 or fHbp v.1.13 E211A / S216R ( Figure 2 D).

[0332] From each of these four heat maps, it can be concluded that: (i) the fHbp unit in the fusion construct is correctly folded, and ii) the v1 mutation effectively stabilizes the fusion construct by causing an increase in the C-terminal transition temperature compared to the fusion containing the wild-type v1 sequence.

[0333] Example 2: Binding of fHbp single variant mutants to hfH

[0334] To investigate 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), the following polypeptides shown in Table 1 were generated (with a C-terminal His6 tag added which is not already indicated as part of the reference sequence):

[0335] Table 1

[0336] .

[0337] The inventors studied the binding of fHbp single variant mutants to hfH using surface plasmon resonance (SPR), a technique that enables detailed and quantitative studies of protein-protein interactions and determination of their equilibrium and kinetic parameters (as described, for example, in Karlsson et al. (1994) Methods 6:99-110).

[0338] SPR-based binding methods involve immobilizing ligands on the surface of a sensor chip. Using well-defined chemistry, the target ligand is immobilized on the surface of the sensor chip, solutions with varying concentrations of analyte are allowed to flow through it and their interaction with the immobilized ligand is characterized. The SPR signal originates from changes in the refractive index at the surface of the gold sensor chip.

[0339] Monitoring the SPR signal over time produces a sensorgram, a plot of binding response (RU) versus time, which allows visualization and assessment of the different stages of a binding event.

[0340] During injection of analyte, the increase in binding response is due to the formation of analyte-ligand complexes on the surface, and the sensorgram is dominated by the association phase. After injection, a steady state is reached where bound and dissociated molecules are in equilibrium. The decrease in response after termination of analyte injection is due to the dissociation of the complex, defining the dissociation phase. Fitting the sensorgram data to an appropriate kinetic binding model allows calculation of kinetic parameters such as association (k a ) and dissociation (k d ) rate constants, and the binding affinity of the interactions tested.

[0341] The inventors used the following experimental setup:

[0342] • Chips: CM-5 with ~400 RU of factor H 6-7 domains immobilized via amine chemistry, 10 µg / ml in acetate buffer, pH 4.0; and ~2500 RU of human factor H (MerckMillipore) immobilized via amine chemistry, 20 µg / ml in acetate buffer, pH 4.0

[0343] • Running buffer: HBS-P 1x

[0344] • Antigens analyzed: As shown in Table 1

[0345] • Antigen was applied at a fixed concentration of 250 nM

[0346] • Contact time: 120s, flow rate: 30µl / min, dissociation time: 120s

[0347] • Regeneration buffer: 100 mM glycine-HCl, 3M NaCl pH 2.0.

[0348] Figure 3 The data shown in (AD) compare fHbp v1.1 (fHbp antigen present in BEXSERO®) with fHbpv1.13 (wild type) and fHbp v.1.13 E211A ( Figure 3 A)、fHbp v1.13 S216R ( Figure 3 B), fHbpv.1.13 E211A / E232A ( Figure 3 C) and fHbp v.1.13 E211A / S216R ( Figure 3 D) Binding to factor H (hfH) domains 6-7.

[0349] It has been shown that a fragment of hfH containing only domains 6-7 is sufficient to mimic the fHbp-hfH interaction (Schneider et al. (2009) Nature 458:890-893), thus providing a simplified model system to evaluate the affinity of fHbp mutants and constructs for hfH.

[0350] Although the fHbp v1.13 single mutants showed reduced fH binding compared to v.1.1 and wild-type v1.13 (see Figure 3 A and 3B), but the double mutants v1.13 E211A / E232A and v1.13 E211A / S216R showed greatly reduced binding activity (see Figure 3 C and 3D).

[0351] Figure 4 Data shown in (AD) compare fHbp v1.1 and fHbp v1.13 (wild type) with fHbp v.1.13E211A ( Figure 4 A)、fHbp v1.13 S216R ( Figure 4 B), fHbp v.1.13 E211A / E232A ( Figure 4C) and fHbpv.1.13 E211A / S216R ( Figure 4 D) Binding to full-length factor H protein.

[0352] Although the fHbp v1.13 single mutants showed reduced fH binding compared to v.1.1 and wild-type v1.13 (see Figure 4 A and 4B), but the double mutants v1.13 E211A / E232A and v1.13 E211A / S216R showed greatly reduced binding activity (see Figure 4 C and 4D).

[0353] Figure 5 Data shown in (AD) compare fHbp v1.1 and fHbp v1.15 (wild type) and fHbpv.1.15 E214A ( Figure 5 A)、fHbp v1.15 S219R ( Figure 5 B), fHbp v.1.15 E214A / E235A ( Figure 5 C) and fHbp v.1.15 E214A / S219R ( Figure 5 D) Binding to Factor H domains 6-7.

[0354] Although the fHbp v1.15 single mutant showed reduced fH binding compared to v.1.1 and wild-type v1.15 (see Figure 5 A and 5B), but the double mutants v1.15 E214A / E235A and v1.15 E214A / S219R did not show significant binding to the factor H subdomain fH6-7 (see Figure 5 C and 5D).

[0355] Figure 6 Data shown in (AD) compare fHbp v1.1 and fHbp v1.15 (wild type) and fHbpv.1.15 E214A ( Figure 6 A)、fHbp v1.15 S219R ( Figure 6 B), fHbp v.1.15 E214A / E235A ( Figure 6 C) and fHbp v.1.15 E214A / S219R ( Figure 5 D) Binding to full-length factor H protein.

[0356] Although the fHbp v1.15 single mutant E214A showed reduced fH binding compared to v.1.1 and wild-type v1.15 (see Figure 6A), but the single mutant v1.15 S219R and the double mutant v1.15 E214A / E235A did not significantly bind to the full-length fH protein (see Figure 6 B and C).

[0357] The double mutant v1.15 E214A / S219R appears to show some residual binding activity to the full-length fH protein (see Figure 6 D).

[0358] Thus, in summary, the most promising candidates for reducing or abolishing hfH binding are the fHbp mutants v1.13 E211A / E232A, v1.13 E211A / S216R, v1.15 S219R, and v1.15 E214A / E235A.

[0359] Example 3: Binding of fHbp 23(S)1.13 and 23(S)1.15 fusion mutants to hfH

[0360] To investigate the effect of mutating the var1 component of the fHbp 231 fusion protein on the ability of the fusion to bind human factor H (hfH), the following fusion polypeptides were generated (Table 2) (with the addition of a C-terminal His6 tag that is not already indicated as part of the reference sequence):

[0361] .

[0362] The inventors used SPR to study the binding of fHbp 23(S)1.13 and 23(S)1.15 fusion mutants to hfH as described above in relation to Example 2.

[0363] The fHbp 231S fusion (SEQ ID NO: 30) used in this example includes variant 2 and variant 3 sequences comprising S32V / L123R and S32V / L126R stabilizing mutations, respectively, as described in detail above. Specifically, the v2 component of the 213S fusion has the sequence of SEQ ID NO: 16, and the v3 component of the 213S fusion has the sequence of SEQ ID NO: 17. The v1.1 component of the fHbp 231S fusion includes an fH non-binding point mutation (R→S), as shown in bold in SEQ ID NO: 30 above, and corresponds to the R41S mutation described in WO2011 / 126863.

[0364] The fHbp 231 wt fusion (SEQ ID NO: 29) used in this example corresponds to the fusion of SEQ ID NO: 30, but without the introduction of stabilizing mutations in v2 and v3, and without the non-binding mutations in v1.1.

[0365] fHbp 23S_1.13E211A / S216R fusion (SEQ ID NO: 19) corresponds to the fusion of SEQ ID NO: 30, but the v1 component of the fusion is the v1.13 non-binding E211A / S216R mutant of the present invention.

[0366] fHbp 23S_1.15 E214A / E235A fusion (SEQ ID NO:22) corresponds to the fusion of SEQ ID NO:30, but the v1 component of the fusion is the v1.15 non-binding E214A / E235A mutant of the present invention.

[0367] The fHbp-23S_1.13E211A / E232A fusion (SEQ ID NO: 18) corresponds to the fusion of SEQ ID NO: 30, but the v1 component of the fusion is the v1.13 non-binding E211A / S216R mutant of the present invention.

[0368] fHbp 231 wt (SEQ ID NO: 29) and fHbp 231S (SEQ ID NO: 30) fusion proteins functioned as controls in this experiment.

[0369] Figure 7 The sensorgrams shown in (AB) compare fHbp 231 wt, fHbp 231S, and fHbp 23S_1.13E211A / S216R ( Figure 7 A) and fHbp 23S_1.13E211A / E232A ( Figure 7 B) Binding to factor H (hfH) domains 6-7.

[0370] Figure 8 The sensorgrams shown in compare the binding of fHbp 231 wt fHbp 231S and fHbp 23S_1.15 E214A / E235A to factor H (hfH) domains 6-7.

[0371] It has been shown that a fragment of hfH containing only domains 6-7 is sufficient to mimic the fHbp-hfH interaction (Schneider et al. (2009) Nature 458:890-893), thus providing a simplified model system to evaluate the affinity of fHbp mutants and constructs for hfH.

[0372] from Figure 7 and 8Clearly, all three v1.13 / 1.15 mutants displayed a strong reduction in binding activity to Factor H domains 6-7. With regard to the 2fHbp 31S fusion, the binding activity displayed by the v1.13 and v1.15 mutant fusions was significantly reduced.

[0373] Fig. 9 The sensorgrams shown in (AB) compare fHbp 231 wt, fHbp 231S, and fHbp 23S_1.13E211A / S216R ( Fig. 9 A) and fHbp 23S_1.13E211A / E232A ( Fig. 9 B) Binding to full-length factor H protein.

[0374] Fig.10 The sensorgrams shown in compare the binding of fHbp 231 wt and fHbp 231S as well as fHbp 23S_1.15 E214A / E235A to the full-length factor H protein.

[0375] These data show that all three v1.13 / 1.15 mutants tested displayed strongly reduced binding activity to full-length factor H, comparable to the fHbp 231S fusion.

[0376] Example 4: Study on the effect of the fHbp fusion protein according to the present invention on the expression of various fHbp v1 subvariants Immunogenicity elicited by meningococcal strains (v1.x)

[0377] Main objectives:

[0378] To investigate the immunogenicity elicited by 231.13 fusion proteins containing mutations that reduce or abolish hfH binding against meningococcal strains expressing fHbp in various different fHbp v1 subvariants, in comparison with existing fHbp antigen / fusion proteins and the licensed 4CMenB vaccine. Immunogenicity was determined using rabbit serum bactericidal assays (rSBA) and human serum bactericidal assays (hSBA) against the following fHbp v1 subvariant (v1.x) strains (v1.1, v1.10, v1.13, v1.14, and v1.15).

[0379] These experiments aimed to evaluate whether the fHbp fusion protein according to the invention shows comparable immunogenicity (non-inferiority) against a panel of strains expressing fHbp v1.x compared to existing antigen / fusion compositions and the licensed 4CMenB vaccine.

[0380] Immunization regimen:

[0381] • Seven groups of 10 mice (CD1 females, 6-8 weeks old) received three separate 200µl doses of one of seven different antigen compositions, as detailed in Table 3 below.

[0382] • Mice were immunized intraperitoneally (ip) on days 1, 22, and 36.

[0383] • Mice were bled on days 0, 35, and 50.

[0384] Table 3

[0385]

[0386] *936-741 is the GNA2091-fHbp fusion included in the 4CMenB vaccine

[0387] **BEXSERO-like refers to the complete BEXSERO product but is not necessarily from a batch approved for release.

[0388] end:

[0389] Total IgG elicited against fHbp two weeks after the third immunization.

[0390] rSBA and hSBA analysis was performed on pooled sera from a panel of N. meningitidis strains expressing fHbp v1.1, v1.10, v1.13, v1.14, and v1.15.

[0391] result:

[0392] Fig.11 A shows the rSBA titer (rabbit complement) of each of the 7 antigen compositions tested against various meningococcal strains expressing fHbp in v1.x. In the SBA results, each point represents the SBA titer of a single strain analyzed on pooled sera. Fig.11 B shows the hSBA titer (human complement) of each of the 7 antigen compositions tested against the same various strains expressing fHbp in v1.x.

[0393] The results showed that the fHbp fusion proteins according to the invention (Group 1 and Group 2) showed comparable (non-inferior) immunogenicity to the licensed BEXSERO product and BEXSERO fHbp antigen (936-741) against a panel of v1.x strains.

[0394] Interestingly, these results also show that the fHbp fusion proteins according to the present invention (Group 1 and Group 2) are more immunogenic against a group of v1.x strains than existing fusion proteins known in the art, particularly the 231.1S fusion (herein SEQ ID NO:30), as determined in rSBA and hSBA.

[0395] Example 5: Study on the effect of the fHbp fusion protein according to the present invention on meningococci expressing fHbp v2 / v3 Immunogenicity of the strain

[0396] Main objectives:

[0397] To investigate the immunogenicity elicited by 231.13 fusion proteins containing mutations that reduce or abolish hfH binding against meningococcal strains expressing fHbp in v2 or v3, in comparison with existing fHbp antigen / fusion proteins and the licensed 4CMenB vaccine. These experiments were designed to assess whether fHbp variants containing three different mutations have the potential to increase the breadth of strain coverage compared with existing antigen compositions.

[0398] Immunogenicity was determined using rabbit serum bactericidal assay (rSBA) and human serum bactericidal assay (hSBA) against the following fHbp v2 and v3 strains (v2.16, v3.31 and v3.42).

[0399] Immunization regimen:

[0400] Seven groups of 10 mice (CD1 female, 6-8 weeks old) received three separate 200 µl doses of one of seven different antigen compositions, as detailed in Table 4 below.

[0401] Mice were immunized intraperitoneally (ip) on days 1, 22, and 36.

[0402] Mice were bled on days 0, 35, and 50.

[0403] Table 4

[0404]

[0405] *936-741 is the GNA2091-fHbp fusion included in the 4CMenB vaccine

[0406] **BEXSERO-like refers to the complete BEXSERO product but is not necessarily from a batch approved for release.

[0407] end:

[0408] Total IgG elicited against fHbp two weeks after the third immunization.

[0409] rSBA and hSBA analyses were performed on pooled sera from a panel of N. meningitidis strains expressing fHbp in either variant 2 or variant 3.

[0410] result:

[0411] Fig.12A shows the rSBA titers of each of the 7 antigen compositions tested against various meningococcal strains expressing fHbp in v2 or v3. In the SBA results, each point represents the SBA titer of a single strain analyzed on pooled sera. Fig.12 B shows hSBA titers for each of the 7 antigen compositions tested against the same various strains expressing fHbp in either v2 or v3.

[0412] The results showed that the fusion protein containing all three fHbp variants generated much higher titers in rSBA and hSBA compared to fHbp v1.1 alone, the 936-741 fusion included in BEXSERO, or indeed the BEXSERO product itself. This is evidence that the immune response to strains expressing fHbp in v2 or v3 is significantly improved compared to existing vaccines or vaccine components.

[0413] Example 6: Evaluation of the addition of the fHbp231.x non-binding mutant of the present invention to the current 4CMenB vaccine The value of

[0414] Main objectives:

[0415] a) In two different animal models (mice and rabbits), two weeks after the third dose, the strain coverage (defined as hSBA titer ≥ 64) of 4CMenB + fHbp231.13_E211A / S216R fusion protein of the present invention was evaluated compared with the current 4CMenB vaccine, as measured by hSBA titers against 20 fHbp var 2 and 3 strains of selected serogroup B Neisseria meningitidis.

[0416] b) In two different animal models (mice and rabbits), two weeks after the third dose, the strain coverage (defined as hSBA titer ≥ 64) of 4CMenB + fHbp231.13_E211A / S216R fusion protein of the present invention was evaluated compared with the current 4CMenB vaccine, as measured by hSBA titers against 30 fHbp var 1.x strains of selected serogroup B Neisseria meningitidis.

[0417] c) In two different animal models (mice and rabbits), two weeks after the third dose, the strain coverage of 4CMenB + the fHbp231.13_E211A / S216R fusion protein of the present invention was evaluated (to assess its non-inferiority) compared to the current 4CMenB vaccine, as measured by hSBA titers against 11 serogroup B meningitidis strains (including the 4CMenB reference strain and strains carrying fHbp var 1.1 and 1.4 predicted to be covered by 4CMenB).

[0418] Endpoint: hSBA titers from pooled sera (one pool per group) collected two weeks after the third injection.

[0419] Study design in mice

[0420] CD1 female mice of the 4-6 week old strain received 3 intraperitoneal (IP) injections of 200 μl of 4CMenB adsorbed aluminum hydroxide (Bexsero) or 4CMenB adsorbed aluminum hydroxide (BEXSERO) plus various fHbp 231 fusion proteins (as shown in Table 5 below) on days 1, 22, and 36. Blood samples were collected before the first injection (day 0), and the final bleed was collected 2 weeks after the third injection (day 49).

[0421]

[0422] *fHbp 231S fusion protein includes stabilizing mutations in v2 and v3 components in addition to the fH non-binding point mutation (R→S) in the v1.1 component (as shown in bold in SEQ ID NO:30 herein, which corresponds to the R41S mutation described in WO2011 / 126863).

[0423] Study Design in Rabbits

[0424] 9-week-old female New Zealand rabbits received three intramuscular (IM) injections of 500 μl of 4CMenB (BEXSERO) adsorbed in aluminum hydroxide or 4CMenB-adsorbed aluminum hydroxide (BEXSERO) plus various fHbp 231 fusion proteins (as shown in Table 6 below) on days 1, 21, and 35. Terminal blood was collected two weeks after the third injection.

[0425] Table 6

[0426]

[0427] *fHbp 231S fusion protein includes stabilizing mutations in v2 and v3 components in addition to the fH non-binding point mutation (R→S) in the v1.1 component (as shown in bold in SEQ ID NO:30 herein, which corresponds to the R41S mutation described in WO2011 / 126863).

[0428] Sample size certification

[0429] Ten mice and three rabbits per group were the minimum number of animals that allowed testing of sufficient pooled sera against a large number of strains (approximately 73) in the hSBA, allowing for 20% retesting or further titration.

[0430] A total of 61 fHbp var 1, 2 and 3 strains carrying selected serogroup B N. meningitidis were tested by hSBA.

[0431] Immunological readout

[0432] Functional antibodies were measured by serum bactericidal assay (hSBA) using human complement against 61 N. meningitidis strains carrying fHbp var 1.x, var 2 or var 3 and a panel of reference Bexsero antigenic strains.

[0433] SBA is the only recognized correlate of protection against N. meningitidis in humans.

[0434] result

[0435] Humoral response - functional antibodies measured by rSBA

[0436] To measure the functional antibodies elicited by different BEXSERO formulations capable of triggering complement-mediated killing of N. meningitidis strains, sera collected two weeks after the third vaccination were tested as a pool in a serum bactericidal activity assay (using human rabbit serum as a complement source) (hSBA) against a panel of approximately 60 N. meningitidis strains.

[0437] The total number of selected Neisseria strains was divided into three different strain groups: fHbp var 2 and 3, fHbp var1.x and BEXSERO reference, and fHbp var 1.1 and 1.4 strains.

[0438] • 50 strains were selected to show the added value of the formulation comprising the fHbp231.13_E211A / S216R fusion protein of the present invention compared to BEXSERO:

[0439] o 20 strains carrying fHbp var2 or var3

[0440] • The selection was based on fHbp frequency distribution and included strains carrying fHbp v2.16, v2.19, v2.21, v2.24, v3.116, v3.31, and v3.42.

[0441] o 30 strains carrying fHbp var1.x

[0442] • Selections were performed to address current BEXSERO gaps and sample the entire genetic diversity of fHbp var1 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.

[0443] • 11 strains were selected to show non-inferiority of a formulation comprising the fHbp231.13_E211A / S216R fusion protein of the invention compared to BEXSERO (including the BEXSERO reference strain plus additional strains known to be covered by BEXSERO (including fHbp1.1 and 1.4)).

[0444] Immunogenicity studies in mice

[0445] To measure the added value of formulations comprising the fHbp fusion protein of the invention compared to BEXSERO, sera collected from vaccinated mice were tested as a pool in the presence of human plasma (hSBA) as a source of complement against a total of 50 MenB strains, divided into var1 (30 strains) and var2 / 3 strains (20 strains). It is noteworthy that 50 strains were selected so that they were not covered by BEXSERO and therefore did not match for all BEXSERO antigens. The results are reported in Fig.13 A and 13B.

[0446] from Fig.13 A and 13B It is evident that the formulation comprising the fHbp fusion protein of the present invention performs better than BESXERO against the var 2 / 3 strains, and is also superior to BEXSERO on the var 1.x strain group.

[0447] To help distinguish between covered and uncovered strains, a new threshold of 256 was chosen (four times the initial threshold of 64). The percentage of covered strains was calculated and presented in Fig.14 A and 14B.

[0448] The results shown in these figures again indicate that formulations comprising the fusion protein of the invention presented higher coverage against the var2 and 3 strain groups compared to BEXSERO alone ( Fig.14 A), and elicited higher coverage against most variant 1.x strains compared to BEXSERO ( Fig.14 B).

[0449] Finally, the non-inferiority of formulations comprising the fusion proteins of the invention was also evaluated by testing mouse antisera in mice against a panel of 11 strains, including the BEXSERO reference strain as well as fHbp var1.1 and 1.4 strains ( Fig.15 ). Non-inferiority was evaluated using pooled mouse sera tested in the hSBA against a panel of 11 MenB strains.

[0450] Fig.15 The results shown in show that not only was non-inferiority compared to BEXSERO demonstrated for all formulations comprising the fusion protein of the invention, but also that improved immunogenicity was evident for most strains due to the additional contribution of antibodies against the additional fHbp component.

[0451] Individual mouse sera were also tested against a panel of 4 BEXSERO indicator strains (M14459 for fHbp var1.1; NZ98 / 254 for PorA P1.4; M4407 for NHBA; and 96217 for NadA) using baby rabbit complement (rSBA) as a complement source to confirm that the specific immunogenicity of the BEXSERO antigen was retained after the addition of the new fusion protein, and to evaluate the non-inferiority of the formulation comprising the fHbp231.13_E211A / S216R fusion protein of the present invention compared to BEXSERO. The results are reported in Fig.16 (AD). In these figures, "BEXSERO PLUS PLUS" refers to the formulation BEXSERO + fHbp231.13_ E211A / S216R.

[0452] Immunogenicity studies in rabbits

[0453] The combined hSBA data for the existing BEXSERO formulations and formulations comprising the fusion protein of the present invention are further presented in Fig.17 A (for var2 / 3 strain types) and Fig.17 In B (for var.1.x strain type), the dotted line represents the hSBA threshold of 16 for rabbit serum.

[0454] All formulations comprising fHbp fusion proteins according to the invention provided improved coverage against the var 2 and 3 strain groups compared to BEXSERO alone, and elicited higher hSBA titers against most variant 1.x strains compared to BEXSERO alone.

[0455] As for the mouse studies described above, a value of 256 (four times the initial threshold of 64) was chosen as the new threshold for analysis of the rabbit data.

[0456] A coverage analysis was performed and is summarized in Fig.18 A (for var2 / 3 strains) and Fig.18 These results demonstrate that the formulations comprising the fHbp fusion protein according to the present invention are able to cover a higher percentage of var2 / 3 strains than BEXSERO alone, and are able to cover more than BEXSERO alone and BEXSERO + prior art fHbp fusion 231.1_R41S (in Fig.18 A higher percentage of v1.x strains was found in the samples (referred to as 2-3-1S).

[0457] Finally, the non-inferiority of the formulation comprising the fHbp fusion protein according to the invention compared to BEXSERO was confirmed by testing rabbit antisera in rabbits against a panel of 11 strains, including the BEXSERO reference strain and fHbp var 1.1 and 1.4 strains, as shown in FIG. Fig.19 As reported in.

[0458] These results show that non-inferiority was demonstrated for all formulations comprising the fHbp fusion protein according to the present invention.

[0459] Example 7: Evaluation of 4CMenB + 23(S)1.13 NB mutant comparison in hFH transgenic mouse model Immunogenicity of 4CMenB + 23(S)1.13 wt

[0460] Immunization regimen

[0461] • Two groups of 10 mice (transgenic mice expressing hfH) were immunized intraperitoneally (ip) with one of two formulations (A and B):

[0462] Group A = 4CMenB + fHbp 23(S)1.13 wild type

[0463] Group B = 4CMenB + fHbp 23(S)1.13_E211A / S216R

[0464] • One mouse (non-immunized) received phosphate buffered saline (PBS) only as a control.

[0465] • Administer 3 doses on days 1, 22 and 36.

[0466] • Blood samples were drawn before immunization and after the third dose.

[0467] • For each group pre-immune sera were pooled.

[0468] Bacterial challenge in immunized mice

[0469] Nine mice from each group were challenged (two mice died after sampling and before bacterial challenge; one from group A and one from group B). A bioluminescent variant of the serogroup B strain MC58 (10 7 CFU / mouse in 500 µl saline), and mice were challenged ip.

[0470] result

[0471] Dynamic imaging was performed at 30 min and 6 h after infection, and the total photons per second were scored and expressed as the ratio of the total photons per second and per mouse after 6 h of infection to the total photons per second and per mouse after 0.5 h of infection ( Fig. 20 ).

[0472] The total photons emitted by each mouse were calculated and presented in Fig.21 In both groups, the signal was significantly reduced compared to uninfected mice. Mice in group B showed lower total photons per second compared to group A, but this difference was Fig.21 A did not reach a significant level (p = 0.2). However, if the analysis is expressed as the ratio of signal 6h / 0.5h ( Fig.21 B), the difference was significant (p=0.007) (Mann Whitney test).

[0473] in conclusion

[0474] As from Fig. 20 and 21 It can be seen that mice in both groups immunized with both formulations A and B were protected against MC58 attack, as evidenced by the post-infection clearance seen in mice from both groups at the 6-hour time point (compared to 0.5 hours). In contrast, non-immunized mice were unable to clear the infection by 6 hours after the attack. However, compared with group A mice immunized with 4CMenB + fHbp 23 (S) 1.13 wild-type, post-attack clearance in group B mice immunized with 4CMenB + fHbp 23 (S) 1.13_E211A / S216R was more thorough.

[0475] Thus, these in vivo data support that vaccine compositions comprising mutated non-fH binding fusion polypeptides according to the invention have improved immunogenicity compared to equivalent compositions comprising fusion polypeptides without the non-binding double mutant v1.13 polypeptides of the invention.

Claims

1. A mutant v1.13 meningococcal fHbp polypeptide consisting of the amino acid sequence of SEQ ID NO:

4.

2. A fusion polypeptide consisting of v1, v2 and v3 meningococcal fHbp polypeptides, wherein the fHbp polypeptide sequence is v2-v3-v1 from N-terminus to C-terminus, and wherein the v1 fHbp polypeptide is the mutant v1.13 fHbp polypeptide according to claim 1.

3. The fusion polypeptide of claim 2, wherein: (a) the v2 fHbp polypeptide consists of the amino acid sequence of SEQ ID NO: 16; and / or (b) The v3 fHbp polypeptide consists of the amino acid sequence of SEQ ID NO:

17.

4. The fusion polypeptide of any one of claims 2 to 3, wherein the v2 and v3 sequences and the v3 and v1 sequences are connected by a glycine-serine linker.

5. The fusion polypeptide of claim 4, wherein the glycine-serine linker is "GSGGGG".

6. The fusion polypeptide according to any one of claims 2 to 3 and 5, which consists of the amino acid sequence of SEQ ID NO:

19.

7. The fusion polypeptide according to any one of claims 2 to 3 and 5, further linked to the N-terminal amino acid sequence of SEQ ID NO:

34.

8. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the mutant v1.13 fHbp polypeptide of claim 1, or the fusion polypeptide of any one of claims 2 to 7.

9. A plasmid comprising a nucleotide sequence encoding the mutant v1.13 fHbp polypeptide of claim 1, or the fusion polypeptide of any one of claims 2 to 7.

10. A recombinant host cell transformed with the nucleic acid molecule of claim 8.

11. An immunogenic composition comprising: the mutant v1.13 fHbp polypeptide of claim 1, or the fusion polypeptide of any one of claims 2 to 7.

12. The immunogenic composition of claim 11, further comprising one or more of the following: a meningococcal NHBA antigen, a meningococcal NadA antigen, a meningococcal fHbp antigen, and a meningococcal outer membrane vesicle.

13. The immunogenic composition of claim 12, comprising a 4CMenB composition.

14. The immunogenic composition of any one of claims 11 to 13, further comprising a conjugated capsular saccharide from Neisseria meningitidis serogroups A, C, W135 and / or Y.

15. The immunogenic composition of claim 14, further comprising conjugated capsular saccharides from each of Neisseria meningitidis serogroups A, C, W135 and Y.

16. Use of an immunogenic composition according to any one of claims 11 to 15 in the preparation of a medicament for raising an immune response in a mammal.

17. The immunogenic composition of any one of claims 11-13 and 15 for use in medicine.

18. The immunogenic composition of any one of claims 11-13 and 15 for use as a vaccine.

19. The immunogenic composition of any one of claims 11 to 13 and 15 for use in a method of raising an immune response in a mammal.

20. The immunogenic composition of any one of claims 11 to 13 and 15 for use in immunizing a mammal against infection with Neisseria meningitidis.

21. The use of claim 16, wherein the mammal is a human.

22. The immunogenic composition of claim 19, wherein the mammal is a human.

23. The immunogenic composition of claim 20, wherein the mammal is a human.

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