阅读说明：本技术 修饰的脑膜炎球菌fHbp多肽 (Modified meningococcal fHbp polypeptides ) 是由 M·斯卡塞利 D·韦吉 于 2019-08-09 设计创作，主要内容包括：本发明提供了突变的fHbp多肽和包含所述突变的fHbp多肽的融合蛋白,其可用作用于针对脑膜炎奈瑟氏菌感染进行免疫的免疫原性组合物的组分。(The present invention provides mutant fHbp polypeptides and fusion proteins comprising the mutant fHbp polypeptides, which can be used as components of immunogenic compositions for immunization against neisseria meningitidis infection.)

1. A mutant v1.13 meningococcal fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID No.2, wherein the amino acid sequence of the mutant v1.13 meningococcal fHbp polypeptide includes substitution mutations at one or more of residues E211, S216 and E232 of SEQ ID No. 2.

2. The polypeptide of claim 1, wherein the amino acid sequence differs from SEQ ID NO 2 by at least one or more of the substitutions E211A, S216R, and E232A.

3. The polypeptide of claim 2, wherein the amino acid sequence comprises substitutions at a plurality of residues selected from the group consisting of:

(i) E211A and S216R, and

(ii) E211A and E232A.

4. The polypeptide of claim 3, wherein the amino acid sequence comprises substitutions at residues E211A and S216R.

5. The polypeptide of claim 4, which comprises or consists of the amino acid sequence of SEQ ID NO. 4.

6. The polypeptide of claim 3, wherein the amino acid sequence comprises substitutions at residues E211A and E232A.

7. The polypeptide of claim 6, comprising or consisting of the amino acid sequence of SEQ ID NO 3.

8. A mutant v1.15 meningococcal fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID No. 6, wherein the amino acid sequence of the mutant v1.15 meningococcal fHbp polypeptide includes substitution mutations at one or more of residues E214, S219 and E235 of SEQ ID No. 6.

9. The polypeptide of claim 8, wherein the amino acid sequence differs from SEQ ID NO 6 by at least one or more of the substitutions E214A, S219R, and E235A.

10. The polypeptide of claim 9, wherein 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.

11. The polypeptide of any one of claims 8 to 10, comprising or consisting of the amino acid sequence of SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9.

12. A fusion polypeptide comprising v1, v2 and v3 meningococcal fHbp polypeptides, wherein the variant fHbp sequence is a v2-v3-v1 sequence from N-to C-terminus, and wherein the v1 fHbp polypeptide is a mutant v1.13 fHbp polypeptide according to any one of claims 1 to 7 or a mutant v1.15 fHbp polypeptide according to any one of claims 8 to 11.

13. The fusion polypeptide of claim 12, wherein the v1 fHbp polypeptide is a mutant v1.13 fHbp polypeptide according to any one of claims 1 to 7.

14. The fusion polypeptide of claim 12 or 13, wherein:

(a) the v 2fHbp polypeptide is a mutant v 2fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID No. 12, wherein the v 2fHbp amino acid sequence comprises substitution mutations at residues S32 and L123 of SEQ ID No. 12, and wherein the substitutions are S32V and L123R; and is

(b) The v3 fHbp polypeptide is a mutant v3 fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID No. 15, wherein the v3 fHbp amino acid sequence comprises substitution mutations at residues S32 and L126 of SEQ ID No. 15, and wherein the substitutions are S32V and L126R.

15. The fusion polypeptide of claim 14, wherein:

(a) the v 2fHbp polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 16; and/or

(b) The v3 fHbp polypeptide comprises or consists of the amino acid sequence of SEQ ID NO 17.

16. The fusion polypeptide of any one of claims 12 to 15, wherein the v2 and v3 sequences and v3 and v1 sequences are linked by a glycine-serine linker.

17. The fusion polypeptide of claim 16, wherein the glycine-serine linker is "gsggggg".

18. The fusion polypeptide of any one of claims 12 to 17, comprising the amino acid sequence of any one of SEQ ID NOs 18-22.

19. The fusion polypeptide of any one of claims 12 to 18, further comprising the N-terminal amino acid sequence of SEQ ID No. 34.

20. An isolated nucleic acid molecule, optionally a plasmid, comprising a nucleotide sequence encoding: the mutant v1.13 fHbp polypeptide of any one of claims 1 to 7, the mutant v1.15 fHbp polypeptide of any one of claims 8 to 11, or the fusion polypeptide of any one of claims 12 to 19.

21. A recombinant host cell transformed with the nucleic acid molecule of claim 20.

22. An immunogenic composition comprising: the mutant v1.13 fHbp polypeptide of any one of claims 1 to 7, the mutant v1.15 fHbp polypeptide of any one of claims 8 to 11, or the fusion polypeptide of any one of claims 12 to 19.

23. The immunogenic composition of claim 22, further comprising one or more of: meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen, and meningococcal Outer Membrane Vesicles (OMVs).

24. The immunogenic composition of claim 23 comprising a 4CMenB composition.

25. The immunogenic composition of any one of claims 22 to 24 further comprising conjugated capsular saccharide from neisseria meningitidis serogroup A, C, W135 and/or Y.

26. The immunogenic composition of claim 25, further comprising conjugated capsular saccharides from each of neisseria meningitidis serogroups A, C, W135 and Y.

27. A method of generating an immune response in a mammal comprising administering the immunogenic composition of any one of claims 22 to 26.

28. The immunogenic composition of any one of claims 22 to 26 for use in medicine.

29. The immunogenic composition of any one of claims 22 to 26 for use as a vaccine.

30. The immunogenic composition of any one of claims 22 to 26 for use in a method of generating an immune response in a mammal.

31. The immunogenic composition of any one of claims 22 to 26 for use in immunizing a mammal against neisseria meningitidis infection.

32. The method of claim 27 or the immunogenic composition for use according to any one of claims 28 to 31, wherein the mammal is a human.

Technical Field

The present invention is in the field of protein engineering, and in particular relates to meningococcal factor H binding proteins (fhbps), which are useful vaccine immunogens.

Background

Invasive Meningococcal Disease (IMD) by meningitidisTaisui bacteria (A), (B), (C)Neisseria meningitidis) And (4) causing. Of the five serogroups (MenA, B, C, W and Y) that are primarily related to IMD worldwide, MenB is the primary serogroup responsible for IMD in many regions, including canada, the united states, australia, new zealand and europe. MenB is a serious and often fatal disease that affects primarily infants and young adults. It is easily misdiagnosed, can be fatal within 24 hours of onset, and can cause severe lifelong disability despite administration of therapy.

Currently, there are two licensed vaccines that have been designed to immunize against serogroup B meningococcus: BEXSERO for GSK and TRUNEBA for Pfizer.

BEXSERO (also commonly referred to as 4CMenB) contains preparations of Outer Membrane Vesicles (OMVs) from an epidemic strain of group B meningococcus NZ98/254, together with five meningococcal antigens: neisserial heparin binding protein a (nhba), factor H binding protein (fHbp) variant 1.1, neisserial adhesion protein a (nada), and accessory proteins GNA1030 and GNA 2091. Four of these antigens are present as fusion proteins (NHBA-GNA1030 fusion protein and GNA2091-fHbp fusion protein). 4CMenB are described in the literature (see, e.g., Bai et al (2011)Expert Opin Biol Ther. 11:969-85, Su & Snape (2011) Expert Rev Vaccines 10:575-88). The terms "BEXSERO" and "4 CMenB" are used interchangeably herein.

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

fHbp (also interchangeably referred to in the art as genome-derived neisserial antigens (GNA) 1870, LP2086, and protein '741') binds human factor h (hfh), which is a large (180 kDa) multi-domain soluble glycoprotein consisting of 20 Complement Control Protein (CCP) modules connected by short linker sequences. hfH circulates in human plasma and regulates an alternative pathway to the complement system. functional binding of fHbp to hfH is largely dependent on the CCP modules (or domains) 6-7 of hfH and enhances the ability of bacteria to resist complement-mediated killing. Thus, expression of fHbp enables survival in ex vivo human blood and serum.

As different fHbp classification schemes have been proposed, proprietary databases with unified fHbp nomenclature for assigning new sub-variants are available: (http): neissemia. org/nm/typing/fHbp (also as (https):// pubmlst. org/neissemia/fHbp /).

fHbp has been classified into three (major) variants 1,2 and 3, which are further divided into sub-variants fHbp-1.x, fHbp-2.x and fHbp-3.x, where x denotes a particular peptide sub-variant. In contrast to v2 and v3, fHbp v1 is highly heterogeneous and contains several sub-variants. In different nomenclature schemes, the sub-variants/variants are divided into subfamily a (corresponding to variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity.

BEXSERO is expected to provide broad coverage against MenB strains spread worldwide (Medini D et al, Vaccine2015, 33:2629 & 2636, Vogel U et al Lancet Infect Dis2013, 13: 416-, Epidemiol Mikrobiol Imunol2014, 63: 103-106, Tzanakaki G et al BMC Microbiol 2014, 14:111, Wasko I et alVaccine2016, 34: 510-PLoS ONE 12(5) e0176177, and Parikh SR et al Lancet Infect Dis 2017, 17: 754-62). Furthermore, after introduction of BEXSERO into the uk national infant immunization program at 9 months of 2015, data at 10 months showed that the vaccine efficacy of all MenB strains after two doses was 83% (Parikh SR et al), Lancet 2016; 388:2775-82)。

However, the bactericidal activity is variant specific; antibodies raised against one variant are not necessarily cross-protected against the other variants, although some cross-reactivity between fHbp V2 and V3 has been described (Masignani V et al), J Exp Med2003, 197, 789 and 799). Antibodies raised against the sub-variant fHbp vv1.1 included in the 4CMenB vaccine were highly cross-reactive with the most frequently present fHbp v1 sub-variant, but less cross-reactive with the v1 sub-variant that is most correlated with v 1.1. Furthermore, antibodies against the subvariant fHbp pv1.1 included in the 4CMenB vaccine cross-react poorly with fHbp v2 and v3 (Brunelli B et al), Vaccine2011, 29:1072 and 1081). This means that the 4CMenB coverage cannot be extended to some extentA meningococcal strain carrying fHbp v2, v3 or a strain carrying some v1 sub-variant.

Thus, despite the efficacy of licensed serogroup B meningococcal vaccines (such as BEXSERO), there is still a need to develop meningococcal vaccines that retain the efficacy of existing licensed vaccines (such as 4CMenB) and are no inferior to existing licensed vaccines (such as 4CMenB), but with increased value for improved coverage of meningococcal strains carrying fHbp variants that are not well covered by existing vaccines.

Summary of The Invention

In a first aspect, the invention provides a mutant v1.13 meningococcal fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID No.2, wherein the amino acid sequence of the mutant v1.13 meningococcal fHbp polypeptide comprises a substitution mutation at E211, S216, E232, or a combination thereof, of SEQ ID No. 2.

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

A 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 in the order v2-v3-v1 from N-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.

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.

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

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

A seventh aspect of the invention provides an immunogenic composition comprising: a mutant 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 an outer membrane vesicle according to the sixth aspect of the invention. The immunogenic compositions are useful for immunizing a mammal, preferably a human, against neisseria meningitidis infection.

Description of the drawings

Fig. 1A is a graph showing the frequency of meningococcal B strains expressing fHbp v1.x sub-variants and indicating which of these v1.x sub-variants are covered (black) or uncovered (white) by the 4CMenB vaccine. Fig. 1B is a graph showing the frequency of meningococcal B strains carrying fHbp v2, and indicating which of these strains are covered (black) or uncovered (white) by the 4CMenB vaccine. Fig. 1C is a graph showing the frequency of meningococcal B strains carrying fHbp v3, and indicating which of these strains are covered (black) or uncovered (white) by the 4CMenB vaccine.

Fig. 2 includes four graphs (heat maps) comparing fHbp v1.1 and fHbp v1.13 (wild-type) to fHbp v.1.13E 211A (fig. 2A); (fig. 2B) fHbp v 1.13S 216R; (FIG. 2C) fHbp v.1.13E 211A/E232A; and (FIG. 2D) Differential Scanning Calorimetry (DSC) data for fHbp v.1.13E 211A/S216R.

Fig. 3 includes four graphs (sensorgrams) comparing fHbp v1.1 and fHbp v1.13 (wild-type) with fHbp v.1.13E 211A (fig. 3A); (fig. 3B) fHbp v 1.13S 216R; (FIG. 3C) fHbp v.1.13E 211A/E232A; and (FIG. 3D) binding of fHbp v.1.13E 211A/S216R to factor H domains 6-7.

Fig. 4 (a-D) includes four plots (sensorgrams) comparing fHbp v1.1 and fHbp v1.13 (wild-type) with fHbp v.1.13E 211A (fig. 4A); (fig. 4B) fHbp v 1.13S 216R; (FIG. 4C) fHbp v.1.13E 211A/E232A; and (FIG. 4D) binding of fHbp v.1.13E 211A/S216R to full-length factor H protein.

Fig. 5 (a-D) includes four plots (sensorgrams) comparing fHbp v1.1 and fHbp v1.15 (wild-type) to fHbp v.1.15E 214A (fig. 5A); (fig. 5B) fHbp v 1.15S 219R; (FIG. 5C) fHbp v.1.15E 214A/E235A; and (FIG. 5D) binding of fHbp v.1.15E 214A/S219R to factor H domains 6-7.

Fig. 6 (a-D) includes four plots (sensorgrams) comparing fHbp v1.1 and fHbp v1.15 (wild-type) to fHbp v.1.15E 214A (fig. 6A); (fig. 6B) fHbp v 1.15S 219R; (FIG. 6C) fHbp v.1.15E 214A/E235A; and (FIG. 6D) binding of fHbp v.1.15E 214A/S219R to full-length factor H protein.

Fig. 7(a-B) includes two graphs (sensorgrams) showing the binding of two different fHbp fusion proteins according to the invention to factor H domains 6-7, as compared to known fHbp fusions. FIG. 7A compares binding of fHbp231 wt and fHbp 231S and fHbp 231.13E 211A/S216R fusions. FIG. 7B compares binding of fHbp231 wt and fHbp 231S and fHbp 231.13E 211A/E232A fusions.

Figure 8 compares binding of fHbp231 wt and fHbp 231S and fHbp 231.15E 214A/E235A fusions according to the invention to factor H domains 6-7.

Fig. 9(a-B) includes two graphs showing binding of fHbp fusion proteins, two different fusion proteins according to the invention, to full-length factor H proteins compared to known fHbp fusions. FIG. 9A compares binding of fHbp231 wt and fHbp 231S and fHbp 231.13E 211A/S216R fusions. FIG. 9B compares binding of fHbp231 wt and fHbp 231S and fHbp 231.13E 211A/E232A fusions.

Figure 10 compares binding of fHbp231 wt and fHbp 231S and fHbp 231.15E 214A/E235A fusions according to the invention to full-length factor H protein.

Figure 11(a) shows rSBA titers (rabbit complement) for each of the six fHbp-associated antigens and BEXSERO-like preparations 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. Figure 11B shows rSBA titers (human complement) for each of the six fHbp-associated antigens and BEXSERO-like preparations tested against the same various strains expressing fHbp in v1. x.

Figure 12(a) shows rSBA titers (rabbit complement) for each of the six fHbp-associated antigens and BEXSERO-like formulations 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. Figure 12B shows rSBA titers (human complement) for each of the six fHbp-associated antigens and BEXSERO-like preparations tested against the same various strains expressing fHbp in v2 or v3.

FIG. 13 shows the combined hSBA data for the formulations for var2/3 (FIG. 13A) and v1.x (FIG. 13B) strain types. Sera collected from vaccinated mice were tested as a pool against a total of 50 MenB strains, classified as var1(30 strains) and var2/3 strain (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.

Figure 14 shows the percentage of coverage provided by the test vaccine formulations tested in mice against strains expressing fHbp var2/3 (a) and fHbp var1 (B). 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.

Figure 15 shows hSBA titers from mouse sera against 11 strains, including the BEXSERO reference strain and fHbp var1.1 and 1.4 strains. 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.

FIG. 16 compares a preparation of the BEXSERO fusion protein of the invention (referred to in the figure as "BEXSERO PLUS PLUS PLUS") comprising BEXSERO + fHbp231.13_ E211A/S216R fusion protein with a standard BEXSERO preparation against a panel of four BEXSERO indicator strains (M14459 for fHbp var1.1 (FIG. 16A); PorA P1.4, NZ98/254 (FIG. 16B); NHBA, M4407 (FIG. 16C); and NadA 96217 (FIG. 16D)).

FIG. 17 shows the combined hSBA data for the formulations for var2/3 (FIG. 17A) and v1.x (FIG. 17B) strain types. Sera collected from vaccinated rabbits were tested as pools against the MenB strain (divided into var1 and var2/3 strains). 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 2-3-1.15 refers to fusions containing wt v1.15, and fHbp 2-3-1.15 NB EE refers to 231.15_ E214A/E235A fusions.

Figure 18 shows the percentage of coverage provided by vaccine formulations tested in rabbits against strains expressing fHbp var2/3 (a) and fHbp var1 (B). In this 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.

Figure 19 shows hSBA titers from rabbit sera against 11 strains (including the BEXSERO reference strain and fHbp var1.1 and 1.4 strains). In this figure, fHbp 2-3-1.13 binding refers to fusions comprising wt v1.13, fHbp 2-3-1.13 non-binding refers to 231.13_ E211A/S216R fusions, fHbp 2-3-1.15 binding refers to fusions comprising wt v1.15, and fHbp 2-3-1.15 non-binding refers to 231.15_ E214A/E235A fusions.

FIG. 20 shows dynamic imaging of bacterial challenge in immunized and non-immunized mice (bioluminescence MC58 cc 32/var.1). Mice in group A were immunized with 4CMenB + fHbp23 (S)1.13 wild type, while mice in group B were immunized with 4CMenB + fHbp23 (S)1.13_ E211A/S216R. Unimmunized mice received Phosphate Buffered Saline (PBS) alone as a control.

FIG. 21 (A and B) shows quantification and comparison of dynamic imaging signals (bioluminescence MC58 cc32/var.1) of bacterial challenge in immunized and non-immunized mice. The crude total signal (photons per second and per mouse at each time point) after 30 min and 6h challenge with bacteria was compared (fig. 21A) or by signal ratio (fig. 21B).

Detailed Description

Lipoprotein factor H binding protein (fHbp) is expressed on the surface of all MenB strains. fHbp binds human complement regulatory protein factor h (hfh) forming a complex that protects bacteria from complement-mediated killing and provides a survival mechanism for neisseria meningitidis in the human bloodstream. Antibodies to fHbp have a dual role: they are bactericidal in nature and by preventing binding to hfH they make the strain more susceptible to bacterial killing. Reducing or abrogating the ability of fHbp to bind hfH increases the immunogenicity of fHbp antigens by preventing the formation of a protective complex between fHbp and hfH that has the potential to mask fHbp epitopes and prevent antibody binding.

fHbp exists as three distinct genetic and immunogenic variants (v1, v2 and v3), with many sub-variants. Most MenB strains not covered by 4CMenB expressed v2 or v3 fHbp or v1 sub-variants far correlated with var 1.1.

As shown in FIG. 1A, epidemiology currently using the MATS method (as for example by Medini et al)Vaccine2015; 33(23); 2629-36) showed that strains with v1.1 and v1.4 were the most frequently present, followed by v1.15, v1.14 and v 1.13. Antibodies raised against the subvariant fHbp 1.1 included in the 4CMenB vaccine were highly cross-reactive with these most frequently present fHbp v1 subvariants (v1.1 and v1.4), but less cross-reactive with the v1 subvariants most correlated with v1.1 (e.g. v1.15 and v 1.13). This is illustrated in fig. 1, since meningococcal B strains expressing fHbp v1.15 and v1.13 are the most frequently occurring strains not covered by the 4CMenB vaccine.

Furthermore, antibodies raised against the subvariant fHbp pv1.1 included in the 4CMenB vaccine cross-react poorly with fHbp v2 and v3 (Brunelli B et al), Vaccine2011, 29:1072 and 1081). FIGS. 1B and 1C show that 4CMenB is directed to someGaps in coverage of the most frequently occurring strains expressing fHbp v2 or v3.

This means that the 4CMenB coverage cannot be extended to some meningococcal strains carrying fHbp v2, v3 or to strains carrying some v1 sub-variant.

The present invention provides mutant 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 neisseria meningitidis strain coverage.

In particular, the v1 polypeptides of the invention are sub-variants of fHbp variant 1 that are genetically diverse compared to the fHbp v1.1 antigen included in 4 CMenB.

Furthermore, the v1 polypeptide of the invention is mutated to reduce binding to hfH compared to the corresponding wild-type v1 polypeptide. In contrast, fHbp v1.1 antigen included in BEXSERO and fHp v1.55 and v3.45 antigens included in transistor ba did bind hfH.

The V1 polypeptides of the invention can be provided alone or as a component of a fusion protein with mutant forms of fHbp variants 2 and 3 that 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 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, 4 CMenB. The presence of the v2 and v3 antigens within the fusion protein of the invention improves strain coverage compared to, for example, 4 CMenB.

The v1 polypeptides and fusion proteins of the invention can be used alone or in combination with meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen and meningococcal outer membrane vesicles (e.g. in combination with a BEXSERO composition), which provides immunogenic compositions having a combination of increased immunogenicity (due to the addition/inclusion of non-conjugated forms of fHbp variants) and increased neisseria meningitidis strain coverage (due to the addition of new fHbp variants/subvariants) compared to BEXSERO alone.

Mutant v1.13 meningococcal fHbp polypeptides

The 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 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.

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:

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

the Δ G 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:

accordingly, in a first aspect the invention provides a mutant v1.13 meningococcal fHbp polypeptide comprising a sequence having at least one amino acid residue as set forth in SEQ ID No.2k%An amino acid sequence of sequence identity, with the proviso that the amino acid sequence of the mutant v1.13 meningococcal fHbp polypeptide comprises residues E211, S21 of SEQ ID NO:26 or E232.

kMay 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 to 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 to SEQ ID No. 2.

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: (i) E211A and E232A, or (ii) E211A and S216R. More preferably, the amino acid sequence comprises substitutions at residues E211A and S216R relative to SEQ ID No. 2.

Without wishing to be bound by theory, substitution of glutamic acid (E) to alanine (a) at residue 211 of SEQ ID No.2 removes negatively charged residues involved in hfH recruitment, thus contributing to abrogation of fH binding. Substitution of arginine (R) to serine (S) at residue 216 of SEQ ID No.2 the wild type amino acid was replaced with the corresponding residue from neisseria gonorrhoeae which did not bind hfH.

In a preferred embodiment, the mutant v1.13 polypeptide of the invention has the amino acid sequence of SEQ ID NO 3 (v1.13 Δ G E211A/E232A) or SEQ ID NO 4 (v1.13 Δ G (E211A/S216R.) more preferably, the mutant v1.13 polypeptide of the invention has the amino acid sequence of SEQ ID NO 4.

After 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).

Mutant v1.15 meningococcal fHbp polypeptides

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 combinations 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.

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

mature v1.15 lipoproteins differ from the full-length wild-type sequence in that the full-length polypeptide has an additional 19-residue N-terminal leader sequence, which 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 type):

the Δ G 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:

accordingly, in a second aspect the invention provides a mutant v1.15 meningococcal fHbp polypeptide comprising a sequence having at least one amino acid residue as shown in SEQ ID NO 6k%An amino acid sequence of sequence identity, with the proviso 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.

kMay be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably the 80 (i.e., mutant fHbp v1.15 amino acid sequence)Column has at least 80% identity to 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 to SEQ ID No. 6.

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: (i) S219R, (ii) E214A and S219R, and (iii) E214A and E235A.

In a preferred embodiment, the mutant v1.15 polypeptide of the invention has the amino acid sequence of SEQ ID NO 7 (v.1.15_ S219R), SEQ ID NO 8 (v1.15_ E214A/S219R) or SEQ ID NO 9 (v1.15_ E214A/E235A).

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

Fusion polypeptides

The 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 N-terminus to C-terminus. In a preferred embodiment, the fHbp fusion polypeptide has the formula NH₂—A-[-X-L ]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.

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

The v2 and v3 fHbp polypeptide components of the fusions of the 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 binding of fHbp to hfH is advantageous because it prevents the formation of protective complexes between fHbp and hfH, which can mask fHbp epitopes and thereby increase the immunogenicity of the polypeptide antigen.

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

The full length wild-type fHbp v2 from strain 2996 has the following amino acid sequence (leader sequence shown in bold font and poly-glycine sequence underlined):

mature lipoproteins lack the first 19 amino acids of SEQ ID NO 10:

the Δ G form of SEQ ID NO. 10 lacks the first 26 amino acids:

in a preferred embodiment, the fusion polypeptide of the invention comprises a mutant v 2fHbp polypeptide comprising an amino acid sequence having at least the amino acid sequence shown in SEQ ID No. 12k%An amino acid sequence of sequence identity, with the proviso that the v 2fHbp amino acid sequence includes substitution mutations at residues S32 and L123 of SEQ ID NO: 12. Preferably, the substitutions are S32V and L123R.

kMay 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 to SEQ ID NO: 12) and more preferably 85, more preferably 90, and more preferably 95.

In some embodiments, the fHbp v2 polypeptide included in the fusion proteins 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 can be truncated at the C-terminus and/or further truncated at the N-terminus.

In a preferred embodiment, the v 2fHbp polypeptide comprised in the fusion protein of the invention comprises or consists of the amino acid sequence of SEQ ID No. 16.

Under the same experimental conditions, the v 2fHbp polypeptide included in the fusion protein of the invention has greater stability than the same polypeptide, e.g., greater stability than a wild-type meningococcal polypeptide consisting of SEQ ID NO:10, but without sequence differences at residues S32 and L123. The S32V mutation stabilizes the structure by introducing favorable hydrophobic interactions. The L123R mutation abrogated fH binding by introducing collisions and adverse charges with fH.

Stability enhancement can be assessed using Differential Scanning Calorimetry (DSC), e.g., as Johnson (2013)Arch Biochem Biophys531:100-9 and Bruyllants et alCurrent Medicinal Chemistry2005, 12: 2011-20. DSC has previously been used to evaluate the stability of v 2fHbp (Johnson et al) PLoS Pathogen 2012, 8: e 1002981). Suitable conditions for DSC evaluation of stability may use 20 μ M polypeptide in a buffer solution (e.g., 25mM Tris) with pH 6-8 (e.g., 7-7.5) and 100-200mM NaCl (e.g., 150 mM).

The increased stability is characterized by an increase in the thermal transition midpoint (Tm) of at least one peak by at least 5 ℃, e.g., at least 10 ℃,15 ℃, 20 ℃, 25 ℃, 30 ℃,35 ℃ or more, as compared to the wild type, when assessed by DSC. Wild-type fHbp shows two DSC peaks (one N-terminal domain and one C-terminal domain) during unfolding, and where a v2 polypeptide included in a fusion protein of the invention includes two such domains, an "increase in stability" refers to the T of the N-terminal domain_mAt least 5 deg.c. For the wild-type v2 sequence, the Tm of the N-terminal domain can occur at 40 ℃ or even below 40 ℃ (Johnson et al (2012)PLoS Pathogen 8: e1002981), while the C-terminal domain may have a Tm of 80 ℃ or higher.Thus, the mutant fHbp v2 amino acid sequence included in the fusion protein of the invention preferably has an N-terminal domain having a sequence of at least 45 ℃, e.g.,>50℃、>55℃、>60℃、>65℃、>70℃、>75 ℃ or even>Tm of 80 ℃.

The full length wild type fHbp v3 from strain M1239 has the following amino acid sequence (leader sequence shown in bold font and poly-glycine sequence underlined):

mature lipoproteins lack the first 19 amino acids of SEQ ID NO: 13:

the Δ G form of SEQ ID NO:13 lacks the first 31 amino acids (i.e., lacks the signal sequence and poly-glycine sequence):

in a preferred embodiment, the fusion polypeptide of the invention comprises a mutant v3 fHbp polypeptide comprising an amino acid sequence having at least the amino acid sequence shown in SEQ ID No. 15k%Amino acid sequence of sequence identity, with the proviso 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.

kMay 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 to SEQ ID NO: 15) and more preferably 85, more preferably 90, and more preferably 95.

In some embodiments, the fHbp v3 polypeptide included in the fusion proteins 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 can be truncated at the C-terminus and/or further truncated at the N-terminus.

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

Under the same experimental conditions, the v3 fHbp polypeptide included in the fusion protein of the invention has greater stability than the same polypeptide, e.g., greater stability than a wild-type meningococcal polypeptide consisting of SEQ ID NO:13, but without sequence differences at residues S32 and L126. The S32V mutation stabilizes the structure by introducing favorable hydrophobic interactions. The L126R mutation abrogated fH binding by introducing collisions and adverse charges with fH.

Stability enhancement can be assessed using Differential Scanning Calorimetry (DSC), e.g., as Johnson (2013)Arch Biochem Biophys531:100-9 and Bruyllants et al (2005)Current Medicinal Chemistry12: 2011-20. DSC has previously been used to evaluate the stability of v3 fHbp (van der Veen et al (2014)Infect Immun PMID 24379280). Suitable conditions for DSC evaluation of stability may use 20 μ M polypeptide in a buffer solution (e.g., 25mM Tris) with pH 6-8 (e.g., 7-7.5) and 100-200mM NaCl (e.g., 150 mM).

The increased stability is characterized by an increase in the thermal transition midpoint (Tm) of at least one peak by at least 5 ℃, e.g., at least 10 ℃,15 ℃, 20 ℃, 25 ℃, 30 ℃,35 ℃ or more, as compared to the wild type, when assessed by DSC. Wild-type fHbp shows two DSC peaks (one N-terminal domain and one C-terminal domain) during unfolding, and where a v3 polypeptide included in a fusion protein of the invention includes two such domains, an "increase in stability" refers to the T of the N-terminal domain_mAt least 5 deg.c. For the wild-type v3 sequence, the Tm for the N-terminal domain can occur at about 60 ℃ or less (Johnson et al (2012)PLoS Pathogen 8: e1002981), while the C-terminal domain may have a Tm of 80 ℃ or higher. Therefore, the present inventionThe amino acid sequence of the mutant fHbp v3 of the invention preferably has an N-terminal domain having a sequence of at least 65 ℃, e.g.,>70℃、>75 ℃ or even>Tm of 80 ℃.

As noted above, in a preferred embodiment, the fHbp fusion polypeptide has the formula NH₂—A-[-X-L ]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 glycine-serine polymer linker. Exemplary linkers include, but are not limited to, "GGSG," GGSGG, "" GSGSG, "" GSGGG, "" GSSSG, "and" gsggggg. Other suitable glycine or glycine-serine polymer linkers will be apparent to the skilled person. In a preferred fusion polypeptide according to the invention, the v2 and v3 sequences and the v3 and v1 sequences are linked by a glycine-serine polymer linker "gsggggg".

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

in a preferred embodiment, the fusion polypeptide of the invention comprises the amino acid sequence of SEQ ID number 19. In an alternative preferred embodiment, the fusion polypeptide of the invention comprises the amino acid sequence of SEQ ID number 18.

After administration to a host animal, preferably a mammal and more preferably a human, the fusion polypeptide of the invention may elicit antibodies that can recognize wild-type meningococcal fHbp polypeptides, in particular polypeptides of SEQ ID NOs 31, 32, 10 and/or 13. These antibodies are ideally bactericidal (see below).

As mentioned above, in a preferred embodiment, a fHbp fusion polypeptide according to the invention has the formula NH₂—A-[-X-L ]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:

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 number 34 at the N-terminus of the fusion polypeptide, i.e., the amino acid sequence of SEQ ID number 34 is added to the N-terminus of the fHbp v2 component of the fusion polypeptide.

Bactericidal response

Preferred v1.13, v1.15 and/or fusion polypeptides of the invention may elicit an antibody response against meningococcal bactericides. Bactericidal antibody responses are conveniently measured in mice and are a standard indicator of vaccine efficacy (see, e.g., Pizza et al (2000)Science287:1816-1820 tail 14; also WO 2007/028408).

The polypeptide of the first embodiment of the invention may preferably elicit an antibody response which is bactericidal against a neisseria meningitidis strain expressing the v1.13 fHbp sequence.

Preferred polypeptides of the first embodiment of the invention may elicit antibodies in mice that are bactericidal against a neisseria meningitidis strain expressing the v1.13 fHbp sequence in a serum bactericidal assay.

The polypeptide of the second embodiment of the invention may preferably elicit an antibody response which is bactericidal against a neisseria meningitidis strain expressing the v1.15 fHbp sequence.

Preferred polypeptides of the second embodiment of the invention may elicit antibodies in mice that are bactericidal against a neisseria meningitidis strain expressing the v1.15 fHbp sequence in a serum bactericidal assay.

For example, immunogenic compositions comprising these polypeptides can be provided>1:4 (using a 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 1:128 (using baby rabbit complement).

Polypeptides

The polypeptides of the invention can be prepared by various means, such as 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 cultures), and the like. Heterologous expression in an E.coli host is a preferred expression pathway.

The polypeptides of the invention are desirably 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 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.

fHbp is a native lipoprotein of neisseria meningitidis. It has also been found to be lipidated when expressed in E.coli with its native leader sequence or with a heterologous leader sequence. The polypeptides of the invention may have an N-terminal cysteine residue that may be lipidated, e.g., it comprises a palmitoyl group, typically forming tripalmitoyl-S-glyceroyl-cysteine. In other embodiments, the polypeptide is not lipidated.

Preferably, the polypeptide is prepared in substantially pure or substantially isolated form (i.e., substantially free of other neisserial or host cell polypeptides). Typically, the polypeptide is provided in a non-naturally occurring environment, e.g., it is separated from its naturally occurring environment. In certain embodiments, the polypeptide is present in a composition enriched for the polypeptide as compared to the starting material. Thus, a purified polypeptide is provided, wherein purified means that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, wherein substantially free means that more than 50% of the total polypeptide of the composition (e.g., such as>75%、>80%、>90%、>95% or>99%) is a polypeptide of the invention.

The polypeptide may take various forms (e.g., native, fusion, glycosylated, non-glycosylated, lipidated, disulfide, etc.).

If the polypeptide of the invention is produced by translation in a biological host, an initiation codon is required which will provide the N-terminal methionine in most hosts. Thus, the polypeptide of the invention will comprise, at least in the nascent stage, a methionine residue upstream of said SEQ ID NO sequence.

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 invention may comprise 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 immediately by a mutant fHbp v1, v2, or v3 amino acid sequence; in other embodiments, longer upstream sequences may be used. Such upstream sequences 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 acid). Examples include a leader sequence to direct protein transport, or a short peptide sequence (e.g., a histidine tag, His) to facilitate cloning or purification _n Wherein n is 4, 5,6, 7,8. 9, 10, or more). Other suitable N-terminal amino acid sequences will be apparent to those skilled in the art.

The 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 acid). Examples include sequences that direct protein trafficking, short peptide sequences that facilitate cloning or purification (e.g., comprising a histidine tag, His _n Where n is 4, 5,6, 7, 8, 9, 10 or more), or a sequence that enhances the stability of the polypeptide. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

In some embodiments, the present 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 the C-terminal hexahistidine tag.

The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified either naturally or by intervention; the intervention is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation to a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The polypeptide may exist as a single chain or an associated chain.

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

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

The polypeptides of the invention typically consist of artificial amino acid sequences (i.e. sequences that are not found in any naturally occurring meningococcus).

The affinity of factor H can be determined using surface plasmon resonance (e.g., as in Schneider et al (2009)Nature 458: 890-5) was evaluated quantitatively with immobilized human fH. Provide at least a 10-fold, and ideally at least a 100-fold, reduction in affinity (i.e., dissociation constant K)_DIncreased) is preferred (when measured under the same experimental conditions relative to the same polypeptide but without the mutation).

Nucleic acids

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.

The nucleic acids of the invention can be prepared in a number of ways from genomic or cDNA libraries and the like, 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).

The nucleic acids of the invention may take various forms, e.g., single-stranded, double-stranded, vectors, primers, probes, labeled, unlabeled, and the like.

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

The term "nucleic acid" includes DNA and RNA, as well as analogs thereof, such as analogs containing modified backbones, and also Peptide Nucleic Acids (PNAs), and the like.

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

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

Host cell

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

The host cell of the present invention is preferably a bacterium which expresses the polypeptide of the present invention. The bacteria may be, for example, meningococcus or e. 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 WO 2006/081259). The expression of the polypeptide is ideally not phase variable.

The cell may be a meningococcal bacterium, such as a meningococcal bacteriummltAAnd optionally also has a down-regulation or knock-out of a meningococcus of: (i) at least one gene involved in making the lipid A part of LPS toxic, in particularlpxl1(ii) a (ii) At least one gene involved in capsular polysaccharide synthesis or export, in particularsynXAnd/orctrA。

Vesicle

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

The vesicles prepared from the host cells of the invention preferably comprise a polypeptide according to the invention, which should be in an immunologically accessible form in the vesicles, i.e. an antibody that can bind to a purified polypeptide of the invention should also be able to bind to a polypeptide present in the vesicles.

These include any proteoliposomic vesicle formed therefrom by disrupting or blebbing from a meningococcal outer membrane to form a vesicle including the protein component of the outer membrane. Thus, the term includes outer membrane vesicles (OMVs; sometimes referred to as 'blebs)'), microvesicles (MVs [ WO02/09643 ]]) And 'Natural OMVs' ('NOMVs' [ Katial et al (2002)Infect Immun 70:702-707])。

MV and NOMV are naturally occurring membrane vesicles that form spontaneously during bacterial growth and are released into the culture medium. The MV can be obtained by: culturing neisseria in a broth medium, separating the whole cells from the smaller MVs in the broth medium (e.g., by filtration or by low speed centrifugation to pellet only the cells and not the smaller vesicles), and then collecting the MVs from the cell-depleted medium (e.g., by filtration, by differential sedimentation or aggregation of the MVs, by high speed centrifugation to pellet the MVs). Strains for producing MV can generally be selected according to the amount of MV produced in the medium, e.g. US 6,180,111 and WO01/34642 describe neisseria with high MV production.

OMVs are prepared artificially from bacteria and may be prepared using detergent treatment (e.g. with deoxycholate) or by non-detergent means (see e.g. WO 2004/019977). Techniques for forming OMVs include: with bile acid 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 Fredrksen et al (1991))NIPH Ann. 14(2):67-80]Preferably for treating Neisseria) at a sufficiently high pH without precipitating detergents WO01/91788]And (4) treating bacteria. Other techniques may be used in the substantial absence of detergents [ WO2004/019977]Using techniques such as sonication, homogenization, microfluidization, cavitation, osmotic shock, milling, French press crushing, mixing, and the like. Methods that do not use detergent or use low detergent can retain useful antigens such as NspA and fHbp. Thus, the method may use an OMV extraction buffer containing about 0.5% or less, for example about 0.2%, about 0.1%, < 0.05% or zero deoxycholate.

One useful method for OMV preparation is described in WO2005/004908 and involves ultrafiltration of crude OMVs, rather than high speed centrifugation. The method may involve a step of ultracentrifugation after the ultrafiltration is performed. OMVs can also be purified using a two stage size filtration process as described in WO 2011/036562.

The vesicles used in the present invention may be prepared from any meningococcal strain. The vesicles are typically from a serogroup B strain, 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 may be of any serotype (e.g., 1, 2a, 2b, 4, 14, 15, 16, etc.), any serosubtype, and any immunotype (e.g., L1; L2; L3; L3,3, 7; L10; etc.). The meningococcus may be from any suitable lineage, including highly invasive and highly virulent lineages, for example any one of the following seven highly virulent lineages: subgroup I; subgroup III; subgroup IV-1; ET-5 complex group (complex); ET-37 complex group; cluster a 4; lineage 3.

In addition to encoding the polypeptide of the present invention, the bacterium of the present invention may have one or more further modifications. For example, they may have modificationsfurGene [ WO98/56901 ]]. Can be adjusted upwardsnspAExpression is accompanied byporAAndcpsand (4) knocking out. Further knock-out mutants of Neisseria meningitidis for OMV production are disclosed in, for example, WO 2004/014417. Claassen et al (1996) 14(10):1001-8 disclose the construction of vesicles from strains modified to express six different PorA subtypes. Mutant neisseria with low endotoxin levels achieved by knock-out of enzymes involved in LPS biosynthesis may also be used. The present invention may employ at least one gene engineered to reduce or turn off toxicity of the lipid a moiety involved in conferring LPS, in particularlpxl1 expression of the Gene [ Fisseha et al (2005)Infect Immun 73:4070–80]. Similarly, the invention may employ mutant neisseria bacteria engineered to reduce or shut down the expression of at least one gene involved in capsular polysaccharide synthesis or export, in particular the synX and/or ctrA genes. These and other mutants may be used in the present invention.

Thus, in some embodiments, the strains used in the present invention may express more than one PorA subtype. 6-valent and 9-valent PorA strains have been previously constructed. The strain may express 2,3, 4, 5,6, 7, 8 or 9 PorA subtypes: p1.7, 16; p1.5-1, 2-2; p1.19, 15-1; p1.5-2, 10; p1.12-1, 13; p1.7-2, 4; p1.22, 14; p1.7-1,1 and/or P1.18-1,3, 6. In other embodiments, the strain may have down-regulated PorA expression, e.g., wherein the amount of PorA has been reduced by at least 20% (e.g., relative to strain H44/76) relative to wild type levels (e.g., relative to strain H44/76)For example, > 30%、 > 40%、 > 50%、 > 60%、 > 70%、 > 80%、 > 90%、 > 95%, etc.), or even knocked out.

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

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

Advantageously, for vesicle production, meningococci may be genetically engineered to ensure that expression of the polypeptide does not undergo phase transformation. Methods for reducing or eliminating phase shifts in gene expression in meningococcus are disclosed in WO 2004/015099. For example, the gene may be placed under the control of a constitutive or inducible promoter, or by removal or replacement of the DNA motif responsible for its phase transition.

In some embodiments, the strain may comprise one or more of the knockout and/or overexpression mutations disclosed in references WO02/09746, WO01/09350, WO02/062378, and WO 2004/014417. 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, PhoP, PilC, PmrE, PmrF, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB; (c) exbB, exbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB, SiaA, SiaB, SiaC, SiaD, TbpA and/or TbpB; or (d) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorB, SiaD, SynA, SynB, SynX and/or SynC.

In some embodiments, when mutant strains are usedIt may have one or more or all of the following features: (i) down-regulating or knocking-out LgtB and/or GalE to truncate meningococcal LOS; (ii) (ii) an up-regulated TbpA; (iii) up-regulated NhhA; (iv) up-regulated Omp 85; (v) up-regulated LbpA; (vi) up-regulated NspA; (vii) a knock-out PorA; (viii) down-regulated or knock-out FrpB; (ix) down-regulated or knockout Opa; (x) Down-regulated or knockout Opc; (xii) AbsentcpsA gene complex. The truncated LOS may be a truncated LOS that does not include a sialic acid-lacto-N-neotetraose epitope, e.g., it may be a galactose-deficient LOS. The LOS may not have an alpha chain.

Depending on the meningococcal strain used to prepare the vesicles, they may or may not include the strain's native fHbp antigen (WO 2004/046177).

In a preferred embodiment, the meningococcus does not express a functional MltA protein. Such as WO2006/046143 and Adu-Bobie et al (2004)Infect Immun72:1914-19, knock-out of MltA (membrane-bound lytic transglycosylase, also known as GNA33) in meningococcus provides bacteria that spontaneously release large numbers of membrane vesicles into culture media from which they can be easily purified. For example, the vesicles may be purified using the two-stage size filtration process of WO2011/036562, which comprises: (i) a first filtration step, wherein the vesicles are separated from the bacteria based on their different sizes, and the vesicles pass into a filtrate; and (ii) a second filtration step, wherein the vesicles are retained in the retentate. The MltA mutation (down-regulation or knock-out) has been used in 'GMMA' vaccines [ Koeberling et al (2014)Vaccine 32:2688-95]And may conveniently be associated with, in particular, at least one gene involved in conferring toxicity to the lipid A portion of LPS (in particularlpxl1) And/or at least one gene involved in capsular polysaccharide synthesis or export (in particularsynXAnd/orctrAGene) or knock-outs are combined. GMMA (universal module for membrane antigens) is a genetically detoxified OMV, produced by a strain of meningococcus, that has been engineered to release GMMA with reduced reactogenicity and increased immunogenicity. GMMA induces less proinflammatory cytokines than OMV when tested in the Monocyte Activation Test (MAT)。

Preferred meningococcal strains of 'GMMA' vaccines using this method express mutant v 2fHbp and/or mutant v3 fHbp of the invention, and expression may be driven by strong promoters. Vesicles released by the strain include immunogenic forms of mutant v2 and/or v3 fHbp protein, and administration of the vesicles can provide a bactericidal antibody response, such as Koeberling et al (2014)Vaccine 32: 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 wild-type or mutant sequence, e.g. mutated to disrupt 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 the culture medium after growth of the strain. 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. Thus, vesicles prepared from meningococci expressing these v2 and v3 mutant fHbp sequences are particularly preferred immunogens for use in the vaccines of the present invention.

Useful promoters for use in such strains include those disclosed in WO2013/033398 and WO 2013/113917. For example, the promoter may be: (a) from porin genes, preferablyporAOrporBThe promoter of (a), in particular from neisseria meningitidis; 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 derived fromporAAnd even more particularly from meningococcusporARegion-10 of the gene promoter, and/or from meningococcusporAA-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 comprise 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) a promoter derived from a meningococcal rRNA geneRegion-10 and/or (ii) region-35 from the meningococcal rRNA gene promoter. It is also possible to use hybrids of (a) and (b), for example to have a structure derived fromporAThe-10 region of the promoter and the-35 region (which may be the common-35 region) from the rRNA gene promoter. Thus, a useful promoter may be one which comprises (i) a-10 region from a (particularly meningococcal) rRNA gene and a-35 region from a (particularly meningococcal) porA gene, or (ii) a-10 region from a (particularly meningococcal) porA gene and a-35 region from a (particularly meningococcal) rRNA gene.

If LOS is present in the vesicle, the vesicle can be treated to link its LOS and protein components ("intravesicle" conjugation [ WO2004/014417 ]).

Immunogenic compositions

The polypeptide of the invention may 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 compositions are useful for immunizing a mammal, preferably a human, against neisseria meningitidis infection.

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

As described above, the 4CMenB product (BEXSERO) contained OMV preparations of prevalent strain B:4: P1.7b,4 from group B meningococcus NZ 98/254. The same OMV is found in the MeNZB vaccine and is referred to herein as OMVnz. In addition, 4CMenB contains five meningococcal antigens: NHBA (287; subvariant 1.2), fHbp (741; subvariant 1.1), NadA (961; subvariant 3.1), GNA1030 (953) and GNA2091 (936). Four of these antigens were present as fusion proteins (NHBA-GNA1030 fusion protein (287-953) and GNA2091-fHbp (936-741) fusion protein).

A0.5 ml dose of the complete 4CMenB product was formulated to contain 50 μ g of each of NHBA-GNA1030, NadA and GNA2091-fHbp adsorbed to 1.5 mg of aluminium hydroxide adjuvant, and 25 μ g of OMV from Neisseria meningitidis strain NZ 98/254. In addition, 3.125 mg of sodium chloride, 0.776 mg of histidine and 10mg of sucrose were included per 0.5ml dose of the formulation.

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.

In a further preferred embodiment, the immunogenic composition of the invention comprises the fHbp fusion polypeptide of SEQ ID No.19 (fHbp 23S _1.13_ E211A/S216R) and the complete 4CMenB composition.

Meningococcal serogroups A, C, W135 and Y

Compositions of the invention may also include capsular saccharide antigens from one or more of meningococcal serogroups Y, W135, C and a, wherein the antigens are conjugated to a carrier protein and/or are oligosaccharides. Capsular saccharides in the form of oligosaccharides may be used. These are conveniently formed by: the purified capsular polysaccharide is fragmented (e.g., by hydrolysis), usually followed by purification of fragments of the desired size.

Current serogroup C vaccine (MENJUGATE [ Costantino et al (1992)Vaccine 10:691-698, Jones (2001) Curr Opin Investig Drugs 2:47-49]MENINGITEC and NEISVAC-C) include conjugated saccharides. MENJUGATE and MENINGITEC have CRM₁₉₇Carrier conjugated oligosaccharide antigen, and NEISVAC-C used with tetanus toxoid carrier conjugated complete polysaccharide (de-O-acetylated).

Vaccine products sold under the trade names MENVEO, menaca and nimernix all contain conjugated capsular saccharide antigens from serogroups Y, W135, C and a.

In MENVEO (also commonly referred to as meningococcal (A, C, Y and W-135 cluster) oligosaccharide diphtheria CRM197 conjugate vaccine), A, C, W135 and Y antigens were each conjugated to CRM₁₉₇CarrierAnd (6) conjugation.

In menacara, also commonly referred to as meningococcal (group A, C, Y and W-135) polysaccharide diphtheria toxoid conjugate vaccine, both A, C, W135 and Y antigens are conjugated to a diphtheria toxoid carrier.

In nimernix (also commonly referred to as meningococcal polysaccharide A, C, W-135 and group Y conjugate vaccines), each of A, C, W135 and Y antigens is conjugated to a tetanus toxoid carrier.

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

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 intact 4CMenB product together with one or more conjugated capsular saccharide antigens from neisseria meningitidis serogroups A, C, W135 and/or Y.

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 comprises the complete 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.

In a preferred embodiment, the composition comprises A, C, W135 and Y antigen conjugates present in menvo, A, C, W135 and Y antigen conjugates present in menacara, or A, C, W135 and Y antigen conjugates present in nimernix.

In a further preferred embodiment, the immunogenic composition of the 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 menvoo.

Alternatively, an immunogenic composition of the invention comprising a polypeptide antigen of the first, second or third aspect of the invention may be co-administered with BEXSERO and one or more of menvo, menacara or nimernix. Preferably, the immunogenic composition of the invention is co-administered with BEXSERO and menvo eo.

As used herein, "co-immunising" means that the different immunogenic compositions/vaccines can be administered separately or as a combination.

In the case of separate administration of the vaccines, they will typically be administered at different sites, e.g. one vaccine to the left upper arm and a second vaccine to the right upper arm. Thus, the two vaccines can be administered contralaterally (e.g., both arms or legs or contralateral arms and legs) or ipsilaterally (e.g., ipsilateral arms and legs 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.

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 the different immunogens are mixed. Combination vaccines offer the advantage of receiving a reduced number of injections to a subject, which can lead to clinical advantages of increased compliance.

Use of the immunogenic compositions of the invention

The immunogenic compositions of the invention are suitable for use in medicine, and in particular may be used to immunise a mammal against infection and/or disease caused by neisseria meningitidis, such that a recipient of the immunogenic composition generates an immune response that provides protection against infection by and/or disease caused by neisseria meningitidis bacteria.

In a preferred embodiment, the immunogenic compositions of the invention are useful for immunizing mammals against meningococcal B infection or disease. However, in embodiments of the invention in which the meningococcal serogroup B antigen is combined with other meningococcal serogroup antigens (e.g. A, C, W and/or Y antigens), the immunogenic composition may be used to immunise a mammal against meningococcal A, B, C, W and/or Y infection or disease.

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 neisseria meningitidis. The immunogenic compositions may also be used in a method of treatment (i.e., treatment of neisseria meningitidis infection).

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

The immune response is preferably protective and preferably involves antibody and/or cell-mediated immunity. Preferably, the immune response is a bactericidal antibody response. The method may generate a boosted response. Mammals can be protected against neisserial disease (particularly meningococcal infection) by raising an immune response in vivo.

The invention also provides a method for protecting a mammal against neisserial (e.g. meningococcal) infection comprising administering to the mammal an immunogenic composition of the invention.

The invention provides a polypeptide of the invention for use as a medicament (e.g. as an immunogenic composition or vaccine) or as a diagnostic agent. It also provides the use of a nucleic acid or polypeptide of the invention in the manufacture of a medicament for the prevention of neisserial (e.g. meningococcal) infection in a mammal.

The immunological composition of the invention is preferably formulated as a vaccine product, which is suitable for therapeutic (i.e. treatment of infection) or prophylactic (i.e. prevention of infection) use. Vaccines are usually prophylactic.

The mammal is preferably a human. The human may be an adult, adolescent or child (e.g. a toddler or infant). Vaccines intended for children may also be administered to adults, for example, to assess safety, dosage, immunogenicity, and the like.

The uses and methods are particularly useful for the prevention/treatment of 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).

Protection against neisseria meningitidis can be measured epidemiologically, for example in clinical trials, but indirect measures are used to confirm that it is convenient for an immunogenic composition to elicit a Serum Bactericidal Antibody (SBA) response in a recipient. In the SBA assay, serum from the recipient of the composition is incubated with the target bacteria (neisseria meningitidis in the present invention) in the presence of complement (preferably human complement, although young rabbit complement is instead often used), and bacterial kill is assessed at various dilutions of serum to determine SBA activity. The results observed in SBA assays can be potentiated by performing competitive SBA assays to provide further indirect evidence of the immunogenic activity of the antigen of interest. In a competitive SBA assay, sera containing recipients of an immunogenic composition of one or more antigens are pre-incubated with the one or more antigens and subsequently incubated with target bacteria in the presence of human complement. The killing of the bacteria is then assessed and if the bactericidal antibodies in the serum of the recipient bind the target antigen during the pre-incubation period and thus fail to bind the surface antigen on the bacteria, the killing of the bacteria will be reduced or abolished.

The compositions need not be protective against each and every strain of neisseria meningitidis, nor must each and every recipient of the compositions be protected. This universal protection is not a normal standard in the field. In contrast, protection is usually evaluated against a set of reference laboratory strains, often selected on a country-by-country basis (on a county-by-county basis), and possibly varied over time, and measured across populations of recipients.

Preferred compositions of the invention can confer antibody titers in patients that are superior to the criteria for seroprotection of each antigenic component for an acceptable percentage of human subjects. Antigens with such relevant antibody titers above which the host is considered to be seroconverted for the antigen are well known, and such titers are published by tissues such as WHO. Preferably more than 80% of the samples of statistically significant subjects are seroconverted, more preferably more than 90%, still more preferably more than 93%, most preferably 96-100%.

The immunogenic composition comprises an immunologically effective amount of the immunogen, as well as any other specified components, as desired.

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

The term "preventing" means reducing and/or eliminating the progression of the disease, or eliminating the onset of the disease. For example, the immune system of a subject can be primed (e.g., by vaccination) to trigger an immune response and reject infection, such that the onset of disease is eliminated. Thus, the vaccinated subjects can be infected, but are able to reject the infection better than the control subjects. The amount will vary depending on the health and physical condition of the individual to be treated, the age of the individual to be treated, the classification group (e.g., non-human primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. It is expected that the amounts will fall within a relatively wide range that can be determined by routine experimentation. The compositions may be administered in combination with other immunomodulators.

Vaccine efficacy

The immunogenic composition for use in the present invention preferably has at least 10%, e.g. against at least one strain of neisseria meningitidis>20%、>30%、>40%、>50%、>60%、>70%、>80%、>85%、>90% or more vaccine efficacy.

Vaccine efficacy is determined by a reduction in the relative risk of developing meningococcal disease in a subject receiving a composition according to the invention compared to a subject 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 immunised according to the invention is compared to the incidence in a control population not immunised according to the invention to give a relative risk, and the vaccine efficacy is 100% minus this figure.

Vaccine efficacy is determined for a population rather than an individual. Thus, it is a useful epidemiological tool, but does not predict individual protection. For example, individual subjects may be exposed to very large inoculants 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 invention and for which vaccine efficacy is measured is desirably at least 100, and possibly higher, for example at least 500 subjects. The size of the control group should also be at least 100, such as at least 500.

Administration of

The compositions of the invention will generally be administered directly to the patient. Direct delivery can be accomplished by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or to the interstitial space), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, otic, pulmonary, or other mucosal administration. Intramuscular administration is preferably carried out in the thigh or upper arm. The injection may be via a needle (e.g., a hypodermic needle), but alternatively a needle-free injection may be used. The intramuscular dose is usually about 0.5 ml.

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

The invention may be used to elicit systemic and/or mucosal immunity.

As used herein, a 'dose' of a composition is the 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 0.5ml, although partial doses (e.g., to children) may be administered. The volume of the dose may be further varied depending on the concentration of the antigen in the composition.

The compositions may further be provided in a 'multi-dose' kit (i.e., a single container containing sufficient composition for multiple immunizations). Multiple doses may include a preservative, or multiple dose containers may have a sterile adaptor for removing individual doses of the composition.

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 priming 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.

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

Optionally, the subject is a juvenile or adult who has been immunized against neisseria meningitidis during childhood (e.g., before the age of 12 years) and receives a booster dose of an immunogenic composition according to the invention.

Where the invention refers to co-immunisation, the different immunogenic compositions/vaccines may be administered separately or as a combination.

In the case of separate administration of the vaccines, they will typically be administered at different sites, e.g. one vaccine to the left upper arm and a second vaccine to the right upper arm. Thus, the two vaccines can be administered contralaterally (e.g., both arms or legs or contralateral arms and legs) or ipsilaterally (e.g., ipsilateral arms and legs 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.

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 the different immunogens are mixed. Combination vaccines offer the advantage of receiving a reduced number of injections to a subject, which can lead to clinical advantages of increased compliance.

Non-antigenic component

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

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

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

The use of aluminium hydroxide and/or aluminium phosphate adjuvants is particularly preferred, and polypeptides are typically adsorbed to these salts. These salts include oxyhydroxides and hydroxyphosphates (see, e.g., for exampleVaccine Design…(1995) Plaiting Powell&Newman. ISBN: 030644867X. Plenum chapters 8 and 9). The salt may take any suitable form (e.g., gel, crystalline, amorphous, etc.).

Additional antigenic component

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

from Streptococcus pneumoniae: (Streptococcus pneumoniae) Saccharide antigens of [ e.g., Watson (2000)Pediatr Infect Dis J 19:331-332, Rubin (2000) Pediatr Clin North Am 47:269-Microbiol Mol Biol Rev 65:187-207]。

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

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]。

Diphtheria antigens, such as diphtheria toxoid [ e.g. diphtheriaVaccines (1988) Braided Plotkin&Chapter 3 of Mortmier. ISBN 0-7216-]E.g. CRM₁₉₇Mutants [ e.g., Del Guidic et al (1998)Molecular Aspects of Medicine 19:1-70]。

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

From Bordetella pertussis (B.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)。

From Haemophilus influenzae B (B: (B))Haemophilus influenzaeB) Sugar antigens of [ e.g. Costantino et al (1999)Vaccine 17:1251-1263]。

Polio antigen [ e.g. 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.

Measles, mumps and/or rubella antigens (e.g. asVaccines (1988) Braided Plotkin&Chapter 9, 10 and 11 of Mortimer. ISBN 0-7216-.

Influenza antigens (e.g.Vaccines (1988) Braided Plotkin&Chapter 19 of Mortim. ISBN 0-7216-1946-0), such as hemagglutinin and/or neuraminidase surface proteins.

From Moraxella catarrhalis (A)Moraxella catarrhalis) Antigen of (2) [ e.g. McMichael (2000)Vaccine 19 Suppl 1: S101-107]。

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

From Streptococcus agalactiae (Streptococcus agalactiae) Carbohydrate antigen of (group B Streptococcus).

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

From Staphylococcus aureus (S.) (Staphylococcus aureus) Antigen of [ e.g. Ku ]roda et al (2001)Lancet 357(9264) 1225-]。

Where necessary, toxic protein antigens may be detoxified (e.g., pertussis toxin detoxified by chemical and/or genetic means [ Rappuoli et al (1991))TIBTECH 9:232-238])。

When diphtheria antigen is included in the composition, tetanus antigen and pertussis antigen are preferably also included. Similarly, when tetanus antigen is included, diphtheria and pertussis antigens are preferably also included. Similarly, where pertussis antigens are included, diphtheria and tetanus antigens are preferably also included. DTP combinations are therefore preferred.

The saccharide antigen is preferably in the form of a conjugate. Typically, conjugation enhances the immunogenicity of the saccharide, as the conjugation can convert the saccharide from a T-independent antigen to a T-dependent antigen, thus eliciting an immunological memory. Conjugation is particularly useful for pediatric vaccines and is a well known technique.

Typical carrier proteins are bacterial toxins such as diphtheria or tetanus toxins or toxoids or mutants thereof. CRM₁₉₇Diphtheria toxin mutant [ 2 ]Research Disclosure, 453077 (Jan 2002)]Is useful and is a carrier in the streptococcus pneumoniae vaccine marketed under the trade name PREVNAR. Other suitable carrier proteins include the NeisseriｃA meningitidis outer membrane protein complex [ EP-A-0372501]And synthetic peptides [ EP-A-0378881, EP-A-0427347 ]]And heat shock protein [ WO93/17712, WO94/03208 ]]Pertussis proteins [ WO98/58668, EP-A-0471177 ]]Cytokines [ WO91/01146 ]]Lymphokines [ WO91/01146 ]]Hormones [ WO91/01146 ]]Growth factor [ WO91/01146 ]]Human CD4 comprising multiple antigens derived from various pathogens⁺Artificial proteins for 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 (b), (c), (d), (H.influenzae) 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 nontoxic derivatives thereof [ Michon et al (1998)Vaccine. 16:1732-41]Pneumococcus surface protein PspA [ WO02091998]Ferritin [ WO01/72337 ]]From Clostridium difficile (C.difficile) ((C.difficile))C.difficile) Toxin A or B of (1) [ WO00/61761 ]]Recombinant Pseudomonas aeruginosaPseudomonas aeruginosa) Extracellular protein A (rEPA) [ WO00/33882 ]]And the like.

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

The saccharide will typically be activated or functionalized prior to conjugation. Activation may involve, for example, cyanating agents (cyanating agents), such as CDAP (e.g., 1-cyano-4-dimethylaminopyridine tetrafluoroborate) [ Lees et al (1996)Vaccine 14:190-198, WO95/08348]). Other suitable techniques use carbodiimides, hydrazides, active esters, norbornane (norbomane), p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, and the like.

The attachment can be via a linker group using any known procedure, for example, the procedures 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 22:907-919, EP0208375]. Other linkers include B-propionamido [ WO00/10599 ]]Nitrophenyl-ethylamine [ Gever et al Med. Microbiol. Immunol, 165: 171-]Halogenated acyl halides [ US 4,057,685]Glycosidic linkages [ U.S. Pat. No. 4,673,574; U.S. Pat. No. 4,761,283; U.S. Pat. No. 4,808,700 ]]6-aminocaproic acid [ U.S. Pat. No. 4,459,286]、ADH [US 4,965,338]、C₄To C₁₂Section [ US 4,663,160]And the like. As an alternative to using a linker, a direct connection may be used. Direct linkage to the protein may comprise oxidation of the polysaccharide followed by reductive amination with the protein as described in, for example, US 4,761,283 and US 4,356,170.

Preferred is a process involving the steps of: introduction of amino groups into sugars (e.g. by using-NH)₂Replacing the terminal = O group), followed by derivatization with an adipic acid diester (e.g., adipic acid N-hydroxysuccinimidyl diester), and reaction with a carrier protein. Another preferred reaction uses a CDAP-activating protein D vector, for example for MenA or MenC.

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

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

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

General concepts

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, such as X + Y. References to "comprising" (or "comprises", etc.) may optionally be replaced with references to "consisting of" (or "consisting of …", etc.). The term "consisting essentially of …" limits the scope of the claims to the specified materials or steps as well as "those that do not materially affect the basic and novel characteristics of the claimed invention".

And a numerical valuexThe relative term "about" is optional and means, for examplex±10%。

The word "substantially" does not exclude "completely", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the invention, if necessary.

Where the disclosure relates 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-Methods Mol Biol 36:207-23) or the like) or they may be predicted (e.g., using the Jameson-Wolf antigen index (Jameson, BA et al 1988,CABIOS181-& Hammer (2000) Brief Bioinform 1(2) 179-89), MAPIOPE (Bublil et al (2007)Proteins68(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), hydrophilic (Hopp (1993)Peptide Research 183-190) or antigenic index (Welling et al (1985) FEBS Lett. 188:215-218)). Epitopes are portions of antigens 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.

As used herein, reference to "percent sequence identity" between a query amino acid sequence and a subject amino acid sequence is understood to refer to a value of identity calculated using a suitable algorithm or software program known in the art to perform a pairwise sequence alignment.

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

A preferred alignment tool for performing the alignment and calculating percent (%) 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). This can be determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap opening penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62And (6) determining and comparing. The Smith-Waterman homology search algorithm is disclosed in Smith& Waterman (1981) Adv. Appl. Math. 2: 482-489. Other preferred alignment tools are water (emboss) and marcher (emboss). Alternatively, the preferred alignment tool for performing the alignment and calculating percent (%) sequence identity is a best-fit alignment tool, such as genecast, also known as the KERR algorithm.

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 a region of the query sequence that contains any specified point mutation 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 extension that may be present in the query sequence, such as a signal peptide or leader peptide or a C-terminal or N-terminal tag, should be excluded from the alignment.

The term "fragment" with respect to a polypeptide sequence means that the polypeptide is part of a full-length protein. As used herein, a fragment of a mutant polypeptide also comprises one or more mutations. Fragments may have qualitative biological activity in common with the full-length protein, e.g., an "immunogenic fragment" contains or encodes one or more epitopes, such as an immune epitope, that allows the generation of an immune response against the fragment that is the same as or similar to the full-length sequence. A polypeptide fragment typically lacks the amino (N) terminal portion and/or the carboxy (C) terminal portion as compared to the native protein, but wherein the remaining amino acid sequence of the fragment is identical to the amino acid sequence of the native protein. Polypeptide fragments may contain, for example: about 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262 consecutive amino acids, including all integers between two of the reference polypeptide sequences, for example all integers between 50 and 260, 50 and 255, 50 and 250, 50 and 200, 50 and 150 of the reference polypeptide sequence. The term fragment specifically excludes the full-length fHbp polypeptide and its mature lipoprotein.

Serogroup followed by meningococcal classification includes serotype, serosubtype, and then immunotype, and standard nomenclature lists serogroup, serotype, serosubtype, and immunotype, each separated by a colon, e.g., B:4: P1.15: L3,7, 9. Within serogroup B, some lineages often cause disease (highly aggressive), some cause a more severe form of disease than others (highly virulent), and others cause disease very rarely. Seven highly virulent lineages were identified, subpopulations I, III and IV-1, ET-5 complex, ET-37 complex, A4 cluster and lineage 3. These have been defined by multi-site enzyme electrophoresis (MLEE), but multi-site sequence typing (MLST) has also been used to classify meningococci. The four major high virulence clusters are the ST32, ST44, ST8 and ST11 complexes.

Reference herein to "enhanced stability" or "higher stability" or "increased stability" means that the mutant polypeptides disclosed herein have a higher relative thermostability (in kcal/mol) as compared to the non-mutant (wild-type) polypeptide under the same experimental conditions. Stability enhancement can be assessed using Differential Scanning Calorimetry (DSC), e.g., as in Bruyllants et al (R) ((R))Differential Scanning Calorimetry in Life Sciences: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design2005 Current. Med. chem. 12: 2011-Bu 2020) and Calorimetric Sciences Corporation's "Characterising Protein stability by DSC" (Life Sciences Application Note, doc. number 20211021306 February 2006), or by Differential Scanning Fluorometry (DSF). The increase in stability can be characterized as the thermal transition midpoint (T)_m) Increase by at least about 5 ℃ as assessed by DSC or DSF. See, for example, Thomas et al,Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen, 2013 Hum. Vaccin. Immunother. 9(4): 744-752。

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

Sequence of

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

5 [ v1.15 mature polypeptide from strain NM452 ]

10 [ v2 wt from Strain 2996 ]

SEQ ID NO 11 [ v2 mature polypeptide ]

13 [ v3 wt from Strain M1239 ]

14 [ v3 mature ] SEQ ID NO

SEQ ID NO 23 [ v 1.1. delta. G + His tag ]

SEQ ID NO 24 [ v 1.13. delta. G + His tag ]

SEQ ID NO 27 [ v 1.15. delta. G + His tag ]

28 [ v 1.15. delta.G (E214A) + His tag ] SEQ ID NO

29 [ fHbp231 wt fusion polypeptide ]

30 [ fHbp 231S fusion polypeptide ] SEQ ID NO

31 [ v1.13 full Length wt sequence ] of SEQ ID NO

32 [ v1.15 full Length wt sequence ] SEQ ID NO

33 [ mature fHbp v1.1]

34 [ optional N-terminal amino acid sequence ]

Modes for carrying out the invention

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

Examples

Example 1: stability analysis of stable fHbp231 fusions comprising variant 1.13 mutants

As mentioned above, Differential Scanning Calorimetry (DSC) provides information about the thermal stability and domain folding of proteins and is described in the literature, e.g., Johnson (2013)Arch Biochem Biophys531:100-9 and Bruyllants et al Current Medicinal Chemistry2005, 12: 2011-20. DSC has previously been used to assess the stability of v 2fHbp (Johnson et al) PLoS Pathogen 2012, 8: e 1002981). Suitable conditions for DSC evaluation of stability may use 20. mu.M polypeptide in a buffer solution (e.g., 25mM Tris) having 100-200mM NaCl (e.g., 150mM) at a pH of 6 to 8 (e.g., 7-7.5).

The inventors used this technique to investigate the effect of mutant fHbp variant 1 sub-variants on the stability of 231S fusion proteins.

The stable fHbp231 fusions (referred to as "231S") used in this example include variant 2 and variant 3 sequences that include stabilizing mutations. Specifically, the v2 component of the 231S fusion has the sequence of SEQ ID NO 16, which contains the S32V and L123R mutations. The v3 component of the 231S fusion has the sequence of SEQ ID NO 17, which contains the S32V and L126R mutations. The inventors varied the v1 component of the 231S fusion to investigate the effect on stability.

fHbp variant polypeptides each comprise an N-terminal and a C-terminal domain that can be clearly distinguished into two distinct transitions (peaks) in a DSC heatmap. Although T corresponding to the C-terminal transition of most of the fHbp variants was seen around 90 deg.C_mValues, but T of the N-terminal domain between different fHbp variants_mThe values varied widely, with the lowest value seen in variant 2.1 wild type (42 ℃) and the highest value seen in var1.1 (70 ℃).

Figure 2 shows four different heatmaps comparing DSC data, where the variant 1 component of fHbp 231S fusions is:

fHbp v1.1 or fHbp v1.13 or (a) fHbp v.1.13E 211A (fig. 2A);

fHbp v1.1 or fHbp v1.13 or (B) fHbp v 1.13S 216R (fig. 2B);

fHbp v1.1 or fHbp v1.13 or fHbp v.1.13E 211A/E232A (fig. 2C); and

fHbp v1.1 or fHbp v1.13 or fHbp v.1.13E 211A/S216R (FIG. 2D).

From each of these four heat maps the following conclusions can be drawn: (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 a fusion comprising the wild-type v1 sequence.

Example 2: binding of single variant mutant of fHbp to hfH

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 (with the addition of C-terminal His) shown in table 1 were generated₆A tag of the C-terminal His₆The tag has not been indicated as part of the reference sequence):

TABLE 1

。

The inventors investigated binding of fHbp single variant mutants to hfH using Surface Plasmon Resonance (SPR), a technique that enabled detailed and quantitative investigation of protein-protein interactions and determination of equilibrium and kinetic parameters thereof (as described, for example, in Karlsson et al (1994) Methods 6: 99-110).

SPR-based binding methods involve the immobilization of ligands on the surface of a sensor chip. Using well-defined chemistry, target ligands are immobilized on the surface of the sensor chip, allowing solutions with different concentrations of analyte to flow through it and characterizing their interaction with the immobilized ligands. The SPR signal originates from a change in refractive index at the surface of the gold sensor chip.

Monitoring changes in SPR signal over time produces sensorgrams, binding Response (RU) versus time, which allow visualization and assessment of different phases of a binding event.

During injection of analyte, the increase in binding response is due to the formation of analyte-ligand complexes at the surface, and the sensorgram is dominated by the association phase. After injection, a steady state is reached in which bound and dissociated molecules are in equilibrium. The decrease in response after termination of analyte injection is due to dissociation of the complex, defining a dissociation phase. Fitting 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 binding affinities of the interactions tested.

The inventors used the following experimental setup:

chip: CM-5 with-400 RU H factor 6-7 domain chemically immobilized by amine in 10 μ g/ml acetate buffer at pH 4.0; and human factor H (Merck Millipore) with 2500 RU chemically immobilized by amine in acetate buffer at pH 4.0 20 μ g/ml

Run buffer: HBS-P1 x

The antigen analyzed: as shown in table 1

Application of antigen at a fixed concentration of 250nM

Contact time: 120s, flow rate: 30 mul/min, dissociation time: 120s

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

The data shown in FIG. 3 (A-D) compares the binding of fHbp v1.1 (fHbp antigen present in BEXSERO) and fHbp v1.13 (wild type) as well as fHbp v 1.13E211A (FIG. 3A), fHbp v 1.13S 216R (FIG. 3B), fHbp v 1.13E211A/E232A (FIG. 3C) and fHbp v 1.13E211A/S216R (FIG. 3D) to factor H (h) domains 6-7.

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

While the fHbp v1.13 single mutant showed reduced fH binding compared to v.1.1 and wild-type v1.13 (see fig. 3A and 3B), the double mutant v 1.13E211A/E232A and v 1.13E211A/S216R displayed greatly reduced binding activity (see fig. 3C and 3D).

The data shown in fig. 4 (a-D) compares the binding of fHbp v1.1 and fHbp v1.13 (wild-type) to fHbp v.1.13E 211A (fig. 4A), fHbp v 1.13S 216R (fig. 4B), fHbp v.1.13E 211A/E232A (fig. 4C) and fHbp v.1.13E 211A/S216R (fig. 4D) to full-length factor H protein.

While the fHbp v1.13 single mutant showed reduced fH binding compared to v.1.1 and wild-type v1.13 (see fig. 4A and 4B), the double mutant v 1.13E211A/E232A and v 1.13E211A/S216R displayed greatly reduced binding activity (see fig. 4C and 4D).

The data shown in fig. 5 (a-D) compares the binding of fHbp v1.1 and fHbp v1.15 (wild-type) to factor H domains 6-7, as well as fHbp v.1.15E 214A (fig. 5A), fHbp v 1.15S 219R (fig. 5B), fHbp v.1.15E 214A/E235A (fig. 5C), and fHbp v.1.15E 214A/S219R (fig. 5D).

Although the fHbp v1.15 single mutant showed reduced fH binding compared to v.1.1 and wild-type v1.15 (see fig. 5A and 5B), the double mutant v 1.15E 214A/E235A and v 1.15E 214A/S219R did not show significant binding to the factor H subdomain fH6-7 (see fig. 5C and 5D).

The data shown in fig. 6 (a-D) compares the binding of fHbp v1.1 and fHbp v1.15 (wild-type) to full-length factor H protein, as well as fHbp v.1.15E 214A (fig. 6A), fHbp v 1.15S 219R (fig. 6B), fHbp v.1.15E 214A/E235A (fig. 6C), and fHbp v.1.15E 214A/S219R (fig. 5D).

Although fHbp v1.15 single mutant E214A showed reduced fH binding compared to v.1.1 and wild-type v1.15 (see fig. 6A), single mutant v 1.15S 219R and double mutant v 1.15E 214A/E235A did not significantly bind to the full-length fH protein (see fig. 6B and C).

The double mutant v 1.15E 214A/S219R appeared to show some residual binding activity to the full-length fH protein (see fig. 6D).

Thus, in summary, the most promising candidates for reducing or abrogating hfH binding are fHbp mutants v 1.13E211A/E232A, v 1.13E211A/S216R, v 1.15S 219R, and v 1.15E 214A/E235A.

Example 3: binding of fHbp23 (S)1.13 and 23(S)1.15 fusion mutants to hfH

To investigate the effect of the var1 component of the mutant fHbp231 fusion protein on the ability of the fusions to bind human factor h (hfh), the following fusion polypeptides (table 2) were generated (with the addition of a C-terminal His₆A tag of the C-terminal His₆The tag has not been indicated as part of the reference sequence):

。

the inventors investigated binding of fHbp23 (S)1.13 and 23(S)1.15 fusion mutants to hfH using SPR, as described above for example 2.

The fHbp 231S fusion (SEQ ID NO:30) used in this example included variant 2 and variant 3 sequences comprising S32V/L123R and S32V/L126R stabilizing mutations, respectively, as detailed 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 fHbp 231S fusions includes the 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 WO 2011/126863.

The fHbp231 wt fusion used in this example (SEQ ID NO:29) corresponds to the fusion of SEQ ID NO:30, but NO stabilizing mutations were introduced in v2 and v3, and NO non-binding mutations in v 1.1.

The fHbp 23S-1.13E 211A/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 invention.

The fHbp 23S-1.15E 214A/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 invention.

The fHbp-23S-1.13E 211A/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 invention.

The fHbp231 wt (SEQ ID NO:29) and fHbp 231S (SEQ ID NO:30) fusion proteins function as controls in this experiment.

The sensorgrams shown in FIG. 7(A-B) compare the binding of fHbp231 wt fHbp 231S and fHbp 23S _1.13E211A/S216R (FIG. 7A) and fHbp 23S _1.13E211A/E232A (FIG. 7B) to factor H (hfH) domains 6-7.

The sensorgram shown in FIG. 8 compares the binding of fHbp231 wt fHbp 231S and fHbp 23S-1.15E 214A/E235A to factor H (hfH) domains 6-7.

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

From FIGS. 7 and 8 it is clear that all three v1.13/1.15 mutants show a strong reduction of the binding activity to factor H domains 6-7. For the 2fHbp 31S fusion, the binding activity shown by the v1.13 and v1.15 mutant fusions was significantly reduced.

The sensorgrams shown in fig. 9(a-B) compare binding of fHbp231 wt fHbp 231S and fHbp 23S _1.13E211A/S216R (fig. 9A) and fHbp 23S _1.13E211A/E232A (fig. 9B) to full-length factor H protein.

The sensorgrams shown in figure 10 compare binding of fHbp231 wt and fHbp 231S and fHbp 23S — 1.15E 214A/E235A to full-length factor H protein.

These data show that all three v1.13/1.15 mutants tested showed a strong reduction in binding activity to full-length factor H, comparable to fHbp 231S fusions.

Example 4: study of fHbp fusion proteins according to the invention directed to expression of various fHbp v1 sub-variants (v1.x) immunogenicity elicited by meningococcal strains

The main aims are as follows:

to investigate the immunogenicity elicited by 231.13 fusion proteins containing mutations that reduce or abrogate hfH binding against meningococcal strains expressing fHbp in various fHbp v1 sub-variants, comparisons were made with existing fHbp antigens/fusion proteins and licensed 4CMenB vaccines. Immunogenicity was determined using a rabbit serum bactericidal assay (rSBA) and a human serum bactericidal assay (hSBA) against the following fHbp v1 sub-variant (v1.x) strains (v1.1, v1.10, v1.13, v1.14 and v 1.15).

These experiments were aimed at assessing whether fHbp fusion proteins according to the invention show comparable immunogenicity (non-inferiority) against a panel of fHbp v1.x expressing strains compared to existing antigen/fusion compositions and licensed 4CMenB vaccines.

Immunization protocol:

seven groups of 10 mice (CD1 females, 6-8 weeks old) received three separate 200 μ l doses of one of seven different antigen compositions, as detailed in table 3 below.

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

Mice were bled on days 0, 35 and 50.

TABLE 3

936-741 is the GNA2091-fHbp fusion included in the 4CMenB vaccine

BEXSERO-like refers to the complete BEXSERO product, but not necessarily from a batch approved for release.

End point:

two weeks after the third immunization, total IgG elicited against fHbp.

rSBA and hSBA analysis was performed on a pooled serum set of neisseria meningitidis strains expressing fHbp v1.1, v1.10, v1.13, v1.14 and v 1.15.

As a result:

figure 11A shows rSBA titers (rabbit complement) for 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. Figure 11B shows hSBA titers (human complement) for each of the 7 antigen compositions tested against the same various strains expressing fHbp in v1. x.

The results show that fHbp fusion proteins according to the invention (group 1 and group 2) show immunogenicity against a set of v1.x strains comparable (non-inferior) to the licensed BEXSERO product and BEXSERO fHbp antigen (936-741).

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

Example 5: study of fHbp fusion proteins according to the invention against meningococcal bacteria expressing fHbp v2/v3 Immunogenicity elicited by strains

The main aims are as follows:

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, comparisons were made with existing fHbp antigens/fusion proteins and licensed 4CMenB vaccines. These experiments were aimed at assessing whether fHbp variants comprising three different mutations have the potential to increase the breadth of the strain coverage compared to existing antigen compositions.

Immunogenicity was determined using a rabbit serum bactericidal assay (rSBA) and a human serum bactericidal assay (hSBA) against the following fHbp v2 and v3 strains (v2.16, v3.31 and v 3.42).

Immunization protocol:

seven groups of 10 mice (CD1 females, 6-8 weeks old) received three separate 200 μ l doses of one of seven different antigen compositions, as detailed in table 4 below.

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

Mice were bled on days 0, 35 and 50.

TABLE 4

936-741 is the GNA2091-fHbp fusion included in the 4CMenB vaccine

BEXSERO-like refers to the complete BEXSERO product, but not necessarily from a batch approved for release.

End point:

two weeks after the third immunization, total IgG elicited against fHbp.

rSBA and hSBA analysis was performed on a panel of pooled sera from neisseria meningitidis strains expressing fHbp in variant 2 or variant 3.

As a result:

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

The results show that fusion proteins comprising all three fHbp variants generate much higher titers in rSBA and hSBA than fHbp v1.1 alone, the 936-741 fusion included in BEXSERO or indeed the BEXSERO product itself. This is evidence of a significant increase in immune response against strains expressing fHbp in v2 or v3 compared to existing vaccines or vaccine components.

Example 6: evaluation of the increase of the inclusion of the fhbp231.x non-binding mutants of the invention in the current 4CMenB vaccine Value of

The main aims are as follows:

a) in two different animal models (mouse and rabbit), two weeks after the third dose, strain coverage (defined as hSBA titers ≧ 64) of 4CMenB + fHbp231.13_ E211A/S216R fusion proteins of the invention was evaluated, as compared to the current 4CMenB vaccine, as measured by hSBA titers against 20 fHbp var2 and 3 strains of selected serogroup B neisseria meningitidis.

b) In two different animal models (mouse and rabbit), two weeks after the third dose, strain coverage (defined as hSBA titers ≧ 64) of 4CMenB + fHbp231.13_ E211A/S216R fusion proteins of the invention was evaluated as compared to the current 4CMenB vaccine, as measured by hSBA titers against 30 fHbp var1.x strains of selected serogroup B neisseria meningitidis.

c) In two different animal models (mouse and rabbit), two weeks after the third dose, strain coverage of 4CMenB + fHbp231.13_ E211A/S216R fusion proteins of the invention (to evaluate their non-inferiority) was assessed compared to the current 4CMenB vaccine, as measured by hSBA titers against 11 serogroup B neisseria meningitidis (including the 4CMenB reference strain and the strains carrying fHbp var1.1 and 1.4 predicted to be covered by 4 CMenB).

End point: hSBA titers from pooled sera collected two weeks after the third injection (one pool per group).

Study design in mice

CD1 female mice of the 4-6 week old strain received 3 Intraperitoneal (IP) injections with 200 μ l of 4 CMenB-adsorbed aluminum hydroxide (Bexsero) or 4 CMenB-adsorbed aluminum hydroxide (Bexsero) plus various fHbp231 fusion proteins (as shown in table 5 below) on days 1, 22, and 36. Blood samples were collected prior to injection 1 (day 0) and final blood draw was collected 2 weeks after the third injection (day 49).

fHbp 231S fusion proteins include stable mutations in the v2 and v3 components in addition to the fH non-binding point mutation (R → S) in the v1.1 component (shown in bold in SEQ ID NO:30 herein, which corresponds to the R41S mutation described in WO 2011/126863).

Study design in rabbits

New Zealand female rabbits, 9 weeks of age, received 3 Intramuscular (IM) injections of either 4CMenB (BEXSERO) adsorbed in 500. mu.l of aluminum hydroxide, or 4 CMenB-adsorbed aluminum hydroxide (BEXSERO) plus various fHbp231 fusion proteins (as shown in Table 6 below) on days 1, 21 and 35. Final blood was collected two weeks after the third injection.

TABLE 6

fHbp 231S fusion proteins include stable mutations in the v2 and v3 components in addition to the fH non-binding point mutation (R → S) in the v1.1 component (shown in bold in SEQ ID NO:30 herein, which corresponds to the R41S mutation described in WO 2011/126863).

Sample size demonstration

Ten mice and 3 rabbits per group are the minimum number of animals allowed to test sufficient pooled sera against a large set of strains (about 73) in hSBA, considering 20% retesting or further titration.

A total of 61 fHbp var1, 2 and 3 strains carrying the selected serogroup B neisseria meningitidis were tested by hSBA.

Immunological read-out

Functional antibodies were measured by a serum bactericidal assay (hSBA) using human complement on 61 neisseria meningitidis strains carrying fHbp var1.x, var2 or var3 and a panel of reference Bexsero antigen strains.

SBA is the only recognized association in humans for protection against neisseria meningitidis.

Results

Humoral response-functional antibodies measured by rSBA

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

The total number of neisserial strains selected is divided into three distinct groups of strains: fHbp var2 and 3, fHbp var1.x and BEXSERO references, and fHbp var1.1 and 1.4 strains.

Selecting 50 strains to show the increased value of a formulation comprising the fhbp231.13_ E211A/S216R fusion protein of the invention compared to BEXSERO:

o20 strains carrying fHbp var2 or var3

Selecting a strain that is based on fHbp frequency distribution and comprises strains carrying fHbp v2.16, v2.19, v2.21, v2.24, v3.116, v3.31 and v 3.42.

o 30 fHbp-carrying strains of var1.x

Selecting to resolve the current BEXSERO gap and sampling the entire genetic diversity of fHbp var1, and including strains harboring 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 v 1.215.

11 strains were selected to show the 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 fHbp 1.1 and 1.4)).

Immunogenicity studies in mice

To measure the increased value of the formulation comprising the fHbp fusion protein of the invention compared to BEXSERO, 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 strain (20 strains), in the presence of human plasma (hSBA) as a complement source. Notably, 50 strains were selected so as not to be covered by BEXSERO and therefore did not match for all BEXSERO antigens. The results are reported in fig. 13A and 13B.

As is apparent from fig. 13A and 13B, the formulation comprising the fHbp fusion protein of the invention performed better than besjero against var2/3 strain and also outperformed BEXSERO on the var1.x strain group.

To help distinguish between covered and uncovered strains, a new threshold of 256 (four times the initial threshold of 64) was selected. The percentage of the covered strain was calculated and presented in fig. 14A and 14B.

The results shown in these figures again show that the formulation comprising the fusion protein of the invention exhibited higher coverage against the group of var2 and 3 strains compared to BEXSERO alone (fig. 14A) and elicited higher coverage against the majority of variant 1.x strains compared to BEXSERO alone (fig. 14B).

Finally, the non-inferiority of the formulations comprising the fusion proteins of the invention was also assessed by testing mouse antisera against a panel of 11 strains (including the BEXSERO reference strain and fHbp var1.1 and 1.4 strains) in mice (fig. 15). Pooled mouse sera tested against a panel of 11 MenB strains in hSBA were used to evaluate non-inferiority.

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

The non-inferiority of formulations comprising the fp231.13 _ E211A/S216R fusion proteins of the invention compared to BEXSERO was also assessed by testing individual mouse sera against a panel of 4 BEXSERO indicator strains (for fHbp var1.1, M14459; for PorA P1.4, NZ 98/254; for NHBA, M4407; for NadA, 96217) using baby rabbit complement (rSBA) as the complement source to confirm that the specific immunogenicity of the BEXSERO antigen was preserved following addition of the new fusion protein. The results are reported in FIGS. 16 (A-D). In these figures, "BEXSERO PLUS PLUS" refers to the formulation BEXSERO + fHbp 231.13-E211A/S216R.

Immunogenicity Studies in rabbits

The combined hSBA data for the existing BEXSERO formulation and the formulation comprising the fusion protein of the invention are further presented in fig. 17A (for var2/3 strain type) and fig. 17B (for var.1.x strain type). The dashed line represents the hSBA threshold of 16 for rabbit serum.

All formulations comprising fHbp fusion proteins according to the invention provided improved coverage against the group of var2 and 3 strains compared to BEXSERO alone and elicited higher hSBA titers against most variant 1.x strains compared to BEXSERO alone.

As for the mouse study described above, a value of 256 (four times the initial threshold of 64) was selected as a new threshold for analysis of rabbit data.

Coverage analysis was performed and is summarized in FIG. 18A (for var2/3 strain) and FIG. 18B (for var1.x strain). These results demonstrate that formulations comprising fHbp fusion proteins according to the invention are able to cover a higher percentage of var2/3 strain compared to BEXSERO alone and a higher percentage of v1.x strain than BEXSERO alone and BEXSERO + prior art fHbp fusion 231.1_ R41S (referred to as 2-3-1S in fig. 18).

Finally, the non-inferior efficacy of the formulation comprising the fHbp fusion protein according to the invention compared to BEXSERO was confirmed by testing rabbit antisera against a panel of 11 strains (including the BEXSERO reference strain and fHbp var1.1 and 1.4 strains) in rabbits, as reported in fig. 19.

These results show that non-inferiority was demonstrated for all formulations comprising fHbp fusion proteins according to the invention.

Example 7: evaluation of 4CMenB + 23(S)1.13 NB mutant comparison in hFH transgenic mouse model 4CMenB + 23(S)1.13 wt immunogenicity

Immunization protocols

Two groups of 10 mice (hfH expressing transgenic mice) were immunized intraperitoneally (i.p.) with one of the two formulations (a and B):

group a = 4CMenB + fHbp23 (S)1.13 wild type

Group B = 4CMenB + fHbp23 (S)1.13_ E211A/S216R

One mouse (not immunized) received Phosphate Buffered Saline (PBS) only as a control.

3 doses of immunization were performed on day 1, day 22 and day 36.

Blood samples were drawn before immunization and after the third dose.

Pre-immune sera were pooled for each group.

Bacterial challenge in immunized mice

9 mice from each group were challenged (2 mice died after sampling and before bacterial challenge; one from group A and one from group B). Bioluminescent variants of serogroup B strain MC58 (10) were used⁷CFU/mouse in 500 μ l saline), i.p. challenge was performed on the mice.

Results

Dynamic imaging was performed 30 min and 6h post-infection and total photons per second were scored and expressed using the total photons per second and per mouse and the ratio of total photons per second and per mouse 6h post-infection/total photons per second and per mouse 0.5h post-infection (figure 20).

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

Conclusion

As can be seen from figures 20 and 21, mice in both groups immunized with both formulations a and B were protected against MC58 challenge as evidenced by the post-infection clearance seen in mice from both groups at the 6 hour time point (compared to 0.5 hour). In contrast, non-immunized mice failed to clear infection by 6 hours post challenge. However, post-challenge clearance was more complete in group B mice immunized with 4CMenB + fHbp23 (S)1.13_ E211A/S216R compared to group A mice immunized with 4CMenB + fHbp23 (S)1.13 wild-type.

Thus, these in vivo data support an increased immunogenicity of vaccine compositions comprising a mutated non-fH-binding fusion polypeptide according to the invention compared to an equivalent composition comprising a fusion polypeptide not comprising a non-binding double mutant v1.13 polypeptide of the invention.