Immunogenic composition

Abstract

The present invention relates to an immunogenic composition against Neisseria meningitidis serogroup B in liquid form and a reconstituted vaccine against Neisseria meningitidis serogroups A, B, C, W135, and Y comprising this liquid composition. Also provided are kits and methods for the prevention and treatment of Neisseria meningitidis infections and diseases using the immunogenic composition or the reconstituted vaccine.

Description

Technical Field

[0001] The present invention is in the field of vaccines. More specifically, the present invention relates to an immunogenic composition against Neisseria meningitidis serogroup B in liquid form, and a reconstituted vaccine composition against Neisseria meningitidis serogroups A, B, C, W, and Y obtained by reconstituting an immunogenic composition against Neisseria meningitidis serogroups A, C, W, and Y from solid form into a liquid formulation. Kits and methods for immunizing mammals against meningococcal infections and diseases caused by the bacterial pathogen Neisseria meningitidis, particularly infections and diseases caused by serogroups A, B, C, W, and Y of this pathogen, are also provided.

Background Art

[0002] Invasive meningococcal disease (IMD) is caused by the bacterial pathogen Neisseria meningitidis. Of the five serogroups (MenA, B, C, W, Y) that are mainly associated with IMD overall, MenB is the main serogroup causing IMD in many regions including Canada, the United States, Australia, New Zealand, and Europe. MenB is a severe and often fatal disease that mainly affects infants and young adults. It is easily misdiagnosed and can lead to death within 24 hours after onset, and even with treatment, it can cause severe lifelong disabilities.

[0003] Currently, there are two approved vaccines designed for immunization against Neisseria meningitidis serogroup B, namely BEXSERO from GSK and TRUMENBA from Pfizer.

[0004] BEXSERO (also generally referred to herein as 4CMenB) is a preparation of outer membrane vesicles (OMVs) derived from the epidemic strain of Neisseria meningitidis serogroup B NZ98 / 254 and contains five meningococcal antigens: Neisseria heparin-binding protein A (NHBA), factor H-binding protein (fHbp) variant 1.1, Neisseria adhesin A (NadA), and accessory proteins GNA1030 and GNA2091. Four of these antigens exist as fusion proteins (NHBA-GNA1030 fusion protein and GNA2091-fHbp fusion protein). 4CMenB has been described in the literature (see, for example, Bai et al. (2011) Expert Opin Biol Ther. 11:969-85, Su & Snape (2011) Expert Rev Vaccines 10:575-88). The terms “BEXSERO” and “4CMenB” are used interchangeably herein.

[0005] TRUMENBA contains two lipidated MenB fHbp antigens (v1.55 and v3.45) adsorbed to aluminum phosphate.

[0006] fHbp (also known interchangeably in the art as genomic Neisseria antigen (GNA) 1870, LP2086, and protein ‘741’) is a large (180 kDa) multi-domain soluble glycoprotein consisting of 20 complement control protein (CCP) modules connected by short linker sequences that binds to human factor H (hfH). hfH circulates in human plasma and regulates the alternative pathway of the complement system. The functional binding of fHbp to hfH depends primarily on CCP module (or domain) 6-7 of hfH and enhances the ability of bacteria to resist complement-mediated killing. Thus, the expression of fHbp enables survival in ex vivo human blood and serum.

[0007] Since different fHbp classification schemes have been proposed, a dedicated database with a unified fHbp nomenclature for the assignment of new subvariants is available at (http): / / neisseria.org / nm / typing / fhbp (or (https): / / pubmlst.org / neisseria / fHbp / ).

[0008] fHbp is classified into three (major) variants 1, 2, and 3, and further divided into subvariants fHbp-1.x, fHbp-2.x, and fHbp-3.x, where x indicates a specific peptide subvariant. In contrast to v2 and v3, fHbp v1 is highly heterogeneous and contains several subvariants. In different nomenclature schemes, sub / variants are grouped into subfamily A (corresponding to variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity.

[0009] BEXSERO is expected to be effective against a wide range of MenB strains prevalent worldwide (Medini D et al., Vaccine 2015; 33:2629‐2636; Vogel U et al. Lancet Infect Dis 2013;13:416‐425; Krizova et al., Epidemiol Mikrobiol Imunol 2014; 63:103‐106; Tzanakaki G et al. BMC Microbiol 2014;14:111; Wasko I et al. Vaccine 2016;34:510‐515; 6.Simoes MJ et al. PLoS ONE 12(5): e0176177; and Parikh SR et al. Lancet Infect Dis 2017; 17:754‐62). Furthermore, after BEXSERO was introduced into the UK national childhood vaccination program in September 2015, data at 10 months showed 83% vaccine efficacy against all MenB strains after two doses (Parikh SR et al., Lancet 2016; 388:2775-82).

[0010] Recently, WO2020 / 030782 disclosed improved immunogenic compositions against Neisseria meningitidis serogroup B, including mutant fHbp polypeptides and fusion proteins containing these mutant polypeptides. These immunogenic compositions maintain the efficacy of BEXSERO while also improving the coverage against meningococcal strains with fHbp variants, particularly fHbp v2 and v3 variants, and some v1 sub-variants.

[0011] Indeed, the bactericidal activity is variant-specific; although some cross-reactivity has been reported between fHbp v2 and v3, antibodies produced against one variant are not necessarily cross-protective against other variants (Masignani V et al., J Exp Med 2003; 197:789-799). Antibodies produced against the sub-variant fHbp v1.1 included in the 4CMenB vaccine have high cross-reactivity with the most frequently occurring fHbp v1 sub-variants but low cross-reactivity with v1 sub-variants that are least related to v1.1. Furthermore, antibodies produced against the sub-variant fHbp v1.1 included in the 4CMenB vaccine have low cross-reactivity with fHbp v2 and v3 (Brunelli B et al., Vaccine 2011;29:1072-1081), but improved vaccines containing mutated fHbp polypeptides also provide coverage against variants.

[0012] There are also vaccines designed to confer immunity against Neisseria meningitidis serogroups A, C, W135, and Y, such as GSK's MENVEO.

[0013] MENVEO contains meningococcal serogroup A, C, W135, and Y capsular saccharides conjugated to a non-toxic mutant of diphtheria toxin as the carrier protein CRM197. The marketed product consists of a liquid formulation of CRM197-conjugated MenCWY capsular saccharides for reconstituting solid freeze-dried MenA capsular saccharide conjugates.

[0014] The vaccine products sold under the trade names MENACTRA, MENQUADFI, and NIMENRIX also contain conjugate capsular saccharide antigens derived from each of serogroups Y, W135, C, and A.

[0015] To date, no vaccine that covers meningococcal infections and diseases caused by all major serogroups A, B, C, W135, and Y of Neisseria meningitidis with a single pharmaceutical product has been on the market. Such co-immunization with a single vaccine composition containing different immunogens offers the advantage of reducing the number of injections for vaccine recipients and may lead to the clinical benefit of improved compliance.

[0016] In addition to this unmet need for such a product to be available, there is also a need for this product to provide an effective formulation that maintains the integrity of the single antigen and the immunogenicity of the components, while at the same time ensuring the required safety, stability, and ease of use.

Summary of the Invention

[0017] The inventors have found a liquid formulation containing one or more tonicity modifiers and adsorbents, such as aluminum-containing compounds, for an immunogenic composition against Neisseria meningitidis serogroup B, which enhances the integrity and immunogenicity of the antigen and at the same time maintains all relevant quality requirements of the vaccine product, particularly pH and osmotic pressure, at optimal levels. The surprising result that the adsorption of the MenB antigen to the adsorbent in the formulation is enhanced was also observed by the inventors after reconstituting the solid immunogenic composition of the MenACW135Y antigen with this liquid formulation to obtain a MenABCW135Y vaccine product.

[0018] Accordingly, a first aspect of the present invention provides an immunogenic composition against Neisseria meningitidis serogroup B, comprising a Neisseria meningitidis NHBA antigen, a Neisseria meningitidis NadA antigen, a Neisseria meningitidis fHbp antigen, Neisseria meningitidis outer membrane vesicles (OMV), and a fusion polypeptide of a Neisseria meningitidis fHbp polypeptide in a liquid formulation comprising an adsorbent for antigens and polypeptides and one or more pharmaceutically acceptable tonicity modifiers.

[0019] A second aspect of the invention provides a reconstituted vaccine composition against Neisseria meningitidis serogroups A, B, C, W135, and Y, obtained by reconstituting an immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y in solid form (e.g., lyophilized) with the immunogenic composition against Neisseria meningitidis serogroup B in liquid form.

[0020] A third aspect of the invention provides a kit comprising (i) a first container containing the above-described immunogenic composition against Neisseria meningitidis serogroup B in a liquid formulation, and (ii) a second container containing an immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y in solid form.

[0021] A fourth aspect of the invention provides a method for preparing a vaccine composition against Neisseria meningitidis serogroups A, B, C, W135, and Y, comprising the step of reconstituting an immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y in solid form with the above-described immunogenic composition against Neisseria meningitidis serogroup B in liquid form.

[0022] A fifth aspect of the invention is the above-described immunogenic composition against Neisseria meningitidis serogroup B in liquid form, or the reconstituted vaccine composition, for use in immunizing a mammal, preferably a human, against Neisseria meningitidis infections and diseases caused by the bacterial pathogen Neisseria meningitidis.

[0023] A sixth aspect of the invention provides a method for preventing or treating Neisseria meningitidis infections or diseases caused by Neisseria meningitidis, comprising administering to a subject in need thereof a liquid immunogenic composition against Neisseria meningitidis serogroup B or the reconstituted vaccine of the invention.

[0024] A seventh aspect of the invention provides the use of the above-described liquid immunogenic composition against Neisseria meningitidis serogroup B or the reconstituted vaccine composition in the manufacture of a medicament for preventing or treating Neisseria meningitidis infections or diseases caused by Neisseria meningitidis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0026] General The term "comprising" includes not only "comprising" but also "consisting of". For example, a composition "comprising" X may consist of only X or may include additional things such as X + Y. The expression "containing" (or "comprising", etc.) can optionally be replaced with the expression "consisting of" (or "consisting of", etc.). The term "consisting essentially of" limits the scope of the claim to those that "substantially do not affect the basic and novel characteristics" of the specific materials or steps of the invention according to the claim.

[0027] The term "about" with respect to the numerical value x is optional and means, for example, x ± 10%.

[0028] The term "substantially" does not exclude "completely". For example, a composition "substantially free of" Y may in some cases be completely free of Y. The term "substantially" can be omitted from the definition of the invention if necessary.

[0029] When the disclosure relates to an "epitope", this epitope may be a B-cell epitope and / or a T-cell epitope, but is usually a B-cell epitope. Such epitopes can be identified experimentally (e.g., using PEPSCAN (see, e.g., Geysen et al. (1984) PNAS USA 81:3998-4002 and Carter (1994) Methods Mol Biol 36:207-23) or similar methods), or predicted (e.g., Jameson-Wolf antigen index (Jameson, BA et al. 1988, CABIOS 4(1):181-186), matrix-based approach (Raddrizzani & Hammer (2000) Brief Bioinform 1(2):179-89), MAPITOPE (Bublil et al. (2007) Proteins 68(1):294-304), TEPITOPE (De Lalla et al. (1999) J. Immunol. 163:1725-29 and Kwok et al. (2001) Trends Immunol 22:583-88), neural network (Brusic et al. (1998) Bioinformatics 14(2):121-30), OptiMer & EpiMer (Meister et al. (1995) Vaccine 13(6):581-91 and Roberts et al. (1996) AIDS Res Hum Retroviruses 12(7):593-610), ADEPT (Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-7), Tsites (Feller & de la Cruz (1991) Nature 349(6311):720-1), hydrophilicity (Hopp (1993) Peptide Research 6:183-190), or antigen index (Welling et al. (1985) FEBS Lett. 188:215-218)). An epitope is the part of an antigen that is recognized and bound by the antigen-binding site of an antibody or a T-cell receptor, and may also be referred to as an "antigenic determinant".

[0030] The term "suspension" means a mixture in which solid particles are dispersed in the entire liquid, including the entire liquid composition.

[0031] As used herein, reference to the "percent sequence identity" between a query amino acid sequence and a target amino acid sequence is understood to refer to the value of identity calculated using an appropriate algorithm or software program known in the art to perform pairwise sequence alignment. The query amino acid sequence may be described by the amino acid sequence specified in one or more claims of this specification. The query sequence may be 100% identical to the target sequence, or may contain a maximum of a specific integer number of amino acid changes (e.g., point mutations, substitutions, deletions, insertions, etc.) compared to the target sequence such that the % identity is less than 100%. For example, the query sequence is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to the target sequence.

[0032] Preferred alignment tools for performing alignment and calculating the percent sequence identity are local alignment tools such as the Basic Local Alignment Search Tool (BLAST) algorithm. Software for performing BLAST analysis can be obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). The alignment can be determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489. Other preferred alignment tools are Water (EMBOSS) and Marcher (EMBOSS). Alternatively, preferred alignment tools for performing alignment and calculating the percent sequence identity are best fit alignment tools such as GENEPAST (also known as the Kerr algorithm).

[0033] To calculate the percent identity, the query sequence and the target sequence are compared and aligned over a specified region (e.g., a region at least about 40, 45, 50, 55, 60, 65 or more amino acids in length, which can also be the full length of the target amino acid sequence) to obtain the maximum match. The specified region must not include regions of the query sequence that contain specific point mutations in the amino acid sequence. Alternatively, the percent sequence identity can be calculated over the "full length" of the target sequence. N-terminal or C-terminal amino acid extensions that may be present in the query sequence, such as signal peptides, leader peptides, C-terminal tags, or N-terminal tags, need to be excluded from the alignment.

[0034] The term "fragment" with respect to a polypeptide sequence means that the polypeptide is part of the full-length protein. As used herein, fragments of variant polypeptides also include the variant. Fragments can have qualitative biological activities common to the full-length protein. For example, an "immunogenic fragment" contains or encodes one or more epitopes that can elicit the same or a similar immune response against the fragment as against the full-length sequence, such as an immunodominant epitope.

[0035] Polypeptide fragments generally have deletions in the amino (N)-terminal portion and / or the carboxy (C)-terminal portion compared to the native protein, but the remaining amino acid sequence of the fragment is identical to the amino acid sequence of the native protein. Polypeptide fragments include, 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 of a reference polypeptide sequence, including all integral numbers in between, such as a reference polypeptide sequence having 50 to 260, 50 to 255, 50 to 250, 50 to 200, 50 to 150 consecutive amino acids. The term fragment specifically excludes the full-length fHbp polypeptide and its mature lipoprotein.

[0036] Following the serogroup, the classification of Neisseria meningitidis includes serotype, serosubtype, and immunotype. In the standard nomenclature, the serogroup, serotype, serosubtype, and immunotype are described separated by colons, for example, as B:4:P1.15:L3,7,9. Within serogroup B, some lineages frequently cause disease (highly invasive), some lineages cause more severe disease than others (highly virulent), and other lineages cause little disease. Seven highly virulent lineages are recognized, specifically subgroup I, III, IV-1, ET-5 complex, ET-37 complex, A4 cluster, and lineage 3. These are defined by multilocus enzyme electrophoresis (MLEE), although multilocus sequence typing (MLST) is also used for the classification of Neisseria meningitidis. The four major highly virulent clusters are ST32, ST44, ST8, and ST11 complex.

[0037] References to "improved stability" or "higher stability" or "increased stability" in this specification mean that the mutant polypeptides disclosed herein have a higher relative thermal stability (kcal / mol) compared to non-mutant (wild-type) polypeptides under the same experimental conditions. The improvement in stability can be evaluated using differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF), as described, for example, in Bruylants et al. (Differential Scanning Calorimetry in Life Sciences: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design, 2005 Curr. Med. Chem. 12: 2011-2020) and Calorimetry Sciences Corporation's "Characterizing Protein stability by DSC" (Life Sciences Application Note, Doc. No. 20211021306 February 2006). The increase in stability can be characterized as at least about 5 °C increase in the thermal transition midpoint (Tm) upon evaluation 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.

[0038] Meningococcal serogroup B The first subject of the present invention is an immunogenic composition against Neisseria meningitidis serogroup B, which comprises two or more antigens selected from a Neisseria meningitidis NHBA antigen, a Neisseria meningitidis NadA antigen, a Neisseria meningitidis fHbp antigen, Neisseria meningitidis outer membrane vesicles (OMV), and a fusion polypeptide of a Neisseria meningitidis fHbp polypeptide, and is in the form of a liquid preparation containing an adsorbent for the antigens and polypeptides and one or more pharmaceutically acceptable tonicity regulators.

[0039] The MenB composition in the liquid preparation can comprise one or more antigens, as disclosed, for example, in WO2004 / 032958, WO2016 / 008960, and WO2020 / 030782, all of which are incorporated herein by reference.

[0040] The MenB composition in this liquid preparation can comprise, for example, two or more antigens selected from a Neisseria meningitidis NHBA antigen, a Neisseria meningitidis NadA antigen, a Neisseria meningitidis fHbp antigen, and Neisseria meningitidis outer membrane vesicles (OMV). The MenB composition in the liquid preparation can comprise, for example, a Neisseria meningitidis NadA antigen, a Neisseria meningitidis fHbp antigen, and Neisseria meningitidis outer membrane vesicles (OMV). The MenB composition in the liquid preparation can comprise, for example, a Neisseria meningitidis NHBA antigen, a Neisseria meningitidis NadA antigen, a Neisseria meningitidis fHbp antigen, and Neisseria meningitidis outer membrane vesicles (OMV).

[0041] The MenB composition in the liquid preparation can comprise, for example, two or more fHbp antigens. The MenB composition in the liquid preparation can comprise, for example, two or more Neisseria meningitidis fHbp antigens. The MenB composition in the liquid preparation can comprise, for example, a Neisseria meningitidis NadA antigen, two or more Neisseria meningitidis fHbp antigens, and Neisseria meningitidis outer membrane vesicles (OMV). The MenB composition in the liquid preparation can comprise, for example, a Neisseria meningitidis NHBA antigen, a Neisseria meningitidis NadA antigen, two or more Neisseria meningitidis fHbp antigens, and Neisseria meningitidis outer membrane vesicles (OMV).

[0042] In one aspect of the present invention, the liquid formulation of the MenB antigen may further comprise a fusion polypeptide of the meningococcal fHbp polypeptide. The MenB composition in the liquid formulation may include, for example, a meningococcal NHBA antigen, a meningococcal NadA antigen, a fusion polypeptide of the meningococcal fHbp polypeptide, and meningococcal outer membrane vesicles (OMV).

[0043] In a preferred embodiment, the meningococcal NHBA antigen and the meningococcal fHbp antigen of this immunogenic composition in liquid form are fusion proteins with meningococcal accessory proteins, such as GNA1030 and GNA2091. Most preferably, the meningococcal NHBA antigen and the meningococcal fHbp antigen of this immunogenic composition are an NHBA - GNA1030 fusion protein and a GNA2091 - fHbp fusion protein, respectively.

[0044] When the immunogenic composition of the present invention against Neisseria meningitidis serogroup B contains OMV, these OMV can be any proteoliposome vesicles obtained by disruption or blebbing of the outer membrane of Neisseria meningitidis, resulting in the formation of vesicles that retain antigens from the outer membrane. Thus, this term includes, for example, OMV (sometimes called "blebs"), microvesicles (MV), "native OMV" ("NOMV") extracted from cells using a method without using surfactants, and surfactant - extracted OMV (dOMV) such as OMV extracted from cells using deoxycholate treatment.

[0045] The mass of the OMV is measured as the amount of total protein.

[0046] Preferred meningococcal OMV contain PorA serotype 1.4. Preferably, the OMV contain PorA variable region epitope 1.7 - 2 (VR1) and / or 1.4 (VR2). OMV containing both of these epitopes (i.e., P1.7 - 2,4) are even more preferred. OMV obtained from strain NZ98 / 254 are particularly preferred.

[0047] In the most preferred embodiment, the immunogenic composition against Neisseria meningitidis serogroup B in liquid form contains, as OMV antigen, an OMV preparation from the epidemic strain of Neisseria meningitidis group B NZ98 / 254, B:4:P1.7b,4.

[0048] In a further preferred embodiment, the immunogenic composition against Neisseria meningitidis serogroup B of the present invention contains, in addition to the OMV antigen and the fusion fHbp polypeptide, five Neisseria meningitidis antigens: NHBA (287; subvariant 1.2), fHbp (741; subvariant 1.1), NadA (961; subvariant 3.1), GNA1030 (953), and GNA2091 (936). Four of these antigens are present as fusion proteins (NHBA-GNA1030 fusion protein (287-953) and GNA2091-fHbp (936-741) fusion protein).

[0049] In one embodiment, the immunogenic composition of the present invention against Neisseria meningitidis serogroup B contains the complete vaccine product 4CMenB sold under the trade name BEXSERO.

[0050] The polypeptides (or subsets thereof, e.g., non-OMV or soluble polypeptides) in the composition can be present in substantially equal masses, i.e., each mass can be within ±5% of the average mass of all the polypeptides in the composition (or the average mass of a selected subset of polypeptides). For example, if NHBA, fHbp, and NadA are included in the composition, they can be in substantially equal masses, e.g., in a mass ratio a:b:c, where each of a, b, and c can be between 0.95 and 1.05.

[0051] NHBA (Neisseria heparin-binding antigen) NHBA was included as gene NMB2132 (GenBank accession number GI:7227388, herein SEQ ID NO:4) in the publicly available genomic sequence of Neisseria meningitidis serogroup B strain MC58 (Tettelin et al. (2000) Science 287:1809-1815).

[0052] References to NHBA in this specification include truncated variants of NHBA in which the N-terminus of the wild-type NHBA polypeptide sequence is deleted up to the polyglycine sequence (i.e., residues 1 to 24 of meningitis strain MC58 (SEQ ID NO: 4) are deleted). The resulting truncated variants may be distinguished in this specification using the prefix "ΔG". This deletion may enhance expression. The "ΔG" variant of Neisseria meningitidis NHBA is referred to herein as SEQ ID NO: 8.

[0053] Preferred NHBA antigens for use in the present invention include the following amino acid sequences: (a) an amino acid sequence having at least 70% identity (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 4, and / or (b) an amino acid sequence comprising at least "n" consecutive amino acids of SEQ ID NO: 4 where "n" is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) include epitopes from SEQ ID NO: 4. Particularly preferred NHBA antigens for use in the present invention include the amino acid sequence of SEQ ID NO: 8.

[0054] A polypeptide comprising a Neisseria NHBA antigen sequence can comprise only that sequence or can be a fusion protein. One useful fusion partner for the NHBA sequence is typically the GNA1030(953) polypeptide which is downstream of the NHBA sequence. Thus, the NHBA antigen can be present in the compositions of the invention as an NHBA-GNA1030 fusion (e.g., SEQ ID NO: 9).

[0055] NadA (Neisseria adhesin A) The NadA antigen was included as gene NMB1994 (GenBank accession number GI:7227256, SEQ ID NO:5 herein) in the publicly available genomic sequence of Neisseria meningitidis serogroup B strain MC58 (Tettelin et al. (2000) Science 287:1809-1815).

[0056] Preferred NadA antigens for use in the present invention include the following amino acid sequences: (a) an amino acid sequence having at least 70% identity (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO:5; and / or (b) an amino acid sequence comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO:5, where "n" is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) contain epitopes from SEQ ID NO:5. SEQ ID NO:10 is one such fragment. Particularly preferred NadA antigens for use in the present invention include the amino acid sequence of SEQ ID NO:10.

[0057] fHbp (Factor H-binding protein) The fHbp antigen has been characterized in detail. It is also known as protein "741" (SEQ ID NOs:2535 and 2536 of WO99 / 57280), "NMB1870", "GNA1870" (e.g., Masignani V. et al. (2003) J. Exp. Med. 197:789-799), "P2086", "LP2086" or "ORF2086" (e.g., WO03 / 063766). It is a lipoprotein and is expressed in many Neisseria meningitidis serogroups.

[0058] The fHbp antigen is classified into three different variants (see WO2004 / 048404), and in the case of Neisseria meningitidis, sera produced against a particular family have been found to be bactericidal within the same family but inactive against strains expressing either of the other two families. That is, there is cross-protection within the family but not between families. The present invention can use a single fHbp variant, but in order to cover a wider range, the composition can usefully contain fHbp from two or three variants.

[0059] When the composition contains a single fHbp antigen, the composition may contain any of the following: (a) (i) A first polypeptide comprising an amino acid sequence having at least a% sequence identity with SEQ ID NO: 1 (MC58 strain), and / or (ii) an amino acid sequence consisting of a fragment of at least x consecutive amino acids of SEQ ID NO: 1. (b) (i) A second polypeptide comprising an amino acid sequence having at least b% sequence identity with SEQ ID NO: 2 (strain 961-5945), and / or (ii) an amino acid sequence consisting of a fragment of at least y consecutive amino acids of SEQ ID NO: 2. (c) (i) A third polypeptide comprising an amino acid sequence having at least c% sequence identity with SEQ ID NO: 3 (strain M1239), and / or (ii) an amino acid sequence consisting of a fragment of at least z consecutive amino acids of SEQ ID NO: 3.

[0060] When the composition contains two different Neisseria meningitidis fHbp antigens, it can contain a combination of (i) the first and second polypeptides defined above, (ii) the first and third polypeptides defined above, or (iii) the second and third polypeptides defined above. A combination of the first and third polypeptides is preferred. When using a single fHbp antigen, it is preferably the first or third polypeptide as described above.

[0061] In other embodiments, the composition comprises three different Neisseria meningitidis fHbp antigens of the first, second, and third polypeptides defined above. When the composition comprises two or three different Neisseria meningitidis fHbp antigens, these may share some sequences in common, but the first, second, and third polypeptides have different fHbp amino acid sequences.

[0062] In some embodiments, a fragment of at least x consecutive amino acids from SEQ ID NO: 1 is not present within SEQ ID NO: 2 or within SEQ ID NO: 3. Similarly, a fragment of at least y consecutive amino acids from SEQ ID NO: 2 may not be present within SEQ ID NO: 1 or within SEQ ID NO: 3. Similarly, a fragment of at least z consecutive amino acids from SEQ ID NO: 3 may not be present within SEQ ID NO: 1 or within SEQ ID NO: 2. In some embodiments, when the fragment from any of SEQ ID NOs: 1 to 3 is aligned as a contiguous sequence relative to the other two SEQ ID NOs, the identity between the fragment and each of the other two SEQ ID NOs is less than 75%, such as less than 70%, less than 65%, less than 60%, etc.

[0063] The value of a is at least 80, such as 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of b is at least 80, such as 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The value of c is at least 80, such as 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The values of a, b, and c may be the same or different. In some embodiments, a, b, and c are identical.

[0064] The value of x is at least 7, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250. The value of y is at least 7, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250. The value of z is at least 7, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250. The values of x, y, and z may be the same or different. In some embodiments, x, y, and z are identical.

[0065] The fragments preferably contain epitopes from the sequences of the respective SEQ ID NOs. Other useful fragments lack one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the respective SEQ ID NOs and retain at least one epitope.

[0066] The amino acid sequences used in the present invention may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid substitutions compared to SEQ ID NO: 1, 2, or 3, i.e., substitutions of one amino acid with another amino acid having a cognate side chain. Genetically encoded amino acids are generally classified into four families: (1) acidic, i.e., aspartic acid, glutamic acid, (2) basic, i.e., lysine, arginine, histidine, (3) nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine may sometimes be classified together as aromatic amino acids. Substitutions of single amino acids within these families generally do not have a major impact on biological activity. The polypeptide may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to the reference sequence. The polypeptide may also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g., 1, 2, 3, 4, or 5 respective amino acids) relative to the reference sequence.

[0067] Useful first amino acid sequences have at least 85% identity (e.g., >90%, 95% or 100%) with SEQ ID NO: 1. Other useful first amino acid sequences have at least 95% identity (e.g., >98% or 100%) with SEQ ID NO: 12. Preferred fHbp sequences for use according to the present invention include SEQ ID NO: 6. Useful third amino acid sequences have at least 85% identity (e.g., >90%, 95% or 100%) with SEQ ID NO: 3. Other useful third amino acid sequences have at least 95% identity (e.g., >98% or 100%) with SEQ ID NO: 11.

[0068] Combinations comprising a mixture of the first and third sequences based on Accession Nos. 11 and 12 (or approximate variants thereof) are particularly useful. Thus, the composition can include a polypeptide comprising the amino acid sequence of Accession No. 11, and a further polypeptide comprising the amino acid sequence of Accession No. 12.

[0069] The fHbp antigens used in the present invention can be lipidated, for example, with an N-terminal cysteine residue. In other embodiments, they are not lipidated and can include an amino acid sequence upstream of the native mature N-terminal cysteine. Accession Nos. 1 to 3 and 11 to 12 begin with the cysteine at the native N-terminus of the relevant mature fHbp polypeptide. In the case of lipidated fHBP, the lipid attached to the cysteine usually contains palmitoyl residues such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys), dipalmitoyl-S-glycerylcysteine (Pam2Cys), N-acetyl(dipalmitoyl-S-glycerylcysteine), etc.

[0070] The polypeptide comprising the fHbp antigen sequence can contain only that sequence or can be a fusion polypeptide. One useful fusion partner for the fHbp sequence is the GNA2091 polypeptide, which is usually upstream of the fHbp sequence. Thus, the fHbp antigen can be present in the compositions of the present invention as a GNA2091-fHbp fusion (e.g., Accession No. 7).

[0071] The compositions used in the present invention can also include fHbp fusion proteins comprising two or three of the first, second, and third amino acid sequences defined in (a) to (c) above.

[0072] The compositions used in the present invention can also include fHbp proteins that are mutated relative to Accession No. 1, 2, or 3 (fHbp variant 1, 2, or 3, respectively) and that have a reduced binding to human factor H (fH). Suitable mutations are disclosed in Rossi et al. (2013) Vaccine 31:5451-7.

[0073] GNA1030 antigen The "GNA1030" protein derived from Neisseria meningitidis serogroup B is disclosed as "953" (SEQ ID NOs: 2917 and 2918 in this document) in WO99 / 57280 and as "NMB1030" in Tettelin et al. (2000) Science 287:1809-1815 (see also GenBank accession number GI:7226269). The corresponding protein of serogroup A (see Parkhill et al. (2000) Nature 404:502-506) has GenBank accession number 7380108.

[0074] When used according to the present invention, the GNA1030 protein can take various forms. Preferred forms of GNA1030 are cleavage or deletion variants as disclosed in WO01 / 64920, WO01 / 64922, and WO03 / 020756. In particular, the N-terminal leader peptide of GNA1030 is deleted (i.e., deletion of residues 1 to 19 of the MC58 strain [SEQ ID NO: 13]) to obtain GNA1030 (NL) can be obtained.

[0075] Preferred GNA1030 sequences have at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) identity to SEQ ID NO: 13. This includes GNA1030 variants (e.g., allelic variants, homologs, orthologs, paralogs, mutants, etc.).

[0076] Other preferred GNA1030 sequences contain at least n consecutive amino acids from SEQ ID NO: 13, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments contain epitopes from GNA1030, in which case detection of the epitope in the target pathogen can be carried out using monoclonal antibodies against the epitope. Other preferred fragments lack one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or N-terminus of SEQ ID NO: 13.

[0077] GNA2091 antigen The "GNA2091" protein from Neisseria meningitidis serogroup B is disclosed as protein 936 in WO99 / 57280 (SEQ ID NOs: 2883 and 2884) and as "NMB2091" in Tettelin et al. (2000) Science 287:1809-1815 (see also GenBank accession number GI: 7227353). The corresponding gene for serogroup A (see Parkhill et al (2000) Nature 404:502-506) has GenBank accession number 7379093.

[0078] When used according to the present invention, the GNA2091 protein can take various forms. Preferred forms of GNA2091 are cleavage or deletion variants such as those disclosed in WO01 / 64920, WO01 / 64922, and WO03 / 020756. In particular, the N-terminal leader peptide of GNA2091 is deleted (i.e., deletion of residues 1 to 23 of the MC58 strain [SEQ ID NO: 14]) to obtain GNA2091 (NL) can be obtained.

[0079] Preferred GNA2091 sequences have at least 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) identity with SEQ ID NO: 14. This includes variants (e.g., allelic variants, homologs, orthologs, paralogs, mutants, etc.).

[0080] Other preferred GNA2091 sequences contain at least n consecutive amino acids from SEQ ID NO: 14, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments contain epitopes from GNA2091, in which case detection of the epitope in the target pathogen can be performed using monoclonal antibodies against the epitope. Other preferred fragments lack one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or N-terminus of SEQ ID NO: 14.

[0081] Mutant fHbp polypeptide In another embodiment of the present invention, the immunogenic composition against Neisseria meningitidis serogroup B in a liquid formulation contains the antigens in the immunogenic composition disclosed in WO2020 / 030782 incorporated herein by reference, and contains a fusion polypeptide containing a mutant fHbp polypeptide. It has been found that these immunogenic compositions not only maintain the efficacy of the 4CMenB composition (BEXSERO), but also improve the coverage efficiency against meningitis strains carrying fHbp variants due to the inclusion of the fusion fHbp polypeptide.

[0082] Lipoprotein H factor-binding protein (fHbp) is expressed on the surface of all MenB strains. fHbp binds to human complement regulatory protein H factor (hfH), forms a complex that protects bacteria from complement-mediated killing, and provides a mechanism for the survival of meningococci in the human bloodstream. Antibodies against fHbp have two roles. It has a bactericidal effect on its own, and by preventing binding to hfH, it makes the strain more susceptible to bactericidal action. When the ability of fHbp to bind to hfH is reduced or lost, the formation of the protective complex between fHbp and hfH, where the fHbp epitope is hidden and antibody binding is hindered, is prevented, and the immunogenicity of the fHbp antigen is enhanced.

[0083] fHbp exists in three different genetic and immunogenic variants (v1, v2, v3), with many subvariants. Most MenB strains not covered by BEXSERO express fHbp of v2, v3, or v1 subvariants that are poorly related to var1.1 (the fHbp antigen included in BEXSERO).

[0084] W02020 / 030782 discloses mutant fHbp variant 1 (v1) polypeptides that are immunogenic and can improve the coverage of meningococcal strains in combination with existing meningococcal vaccines. In particular, these v1 polypeptides are subvariants of fHbp variant 1 that are genetically diverse compared to the fHbp v1.1 antigen included in BEXSERO.

[0085] Furthermore, the v1 polypeptides disclosed in W02020 / 030782 have been mutated to reduce binding to hfH compared to the corresponding wild-type v1 polypeptides. In contrast, the fHbp v1.1 antigen included in BEXSERO, and the fHp v1.55 and v3.45 antigens included in TRUMENBA, bind to hfH.

[0086] The v1 polypeptide disclosed in WO2020 / 030782 can be provided alone or as a component of a fusion protein together with variants of fHbp variants 2 and 3, which have been modified to improve stability and reduce fHbp binding. By providing a single fusion protein containing these v2 and v3 antigens together with the v1 antigen of the present invention, the inventors improve the strain coverage. Specifically, neither the v2 antigen nor the v3 antigen is present in, for example, BEXSERO. The presence of the v2 antigen and the v3 antigen in the fusion protein of the present invention improves the strain coverage efficiency compared to, for example, BEXSERO.

[0087] The v1 polypeptide and the fusion protein are preferably used in combination with a Neisseria meningitidis NHBA antigen, a Neisseria meningitidis NadA antigen, a Neisseria meningitidis fHbp antigen, and Neisseria meningitidis outer membrane vesicles (for example, in combination with the above-mentioned BEXSERO composition), and compared to BEXSERO alone, the immunogenicity is increased (by the addition / inclusion of unbound fHbp variants), and a combined immunogenic composition with an increased coverage of Neisseria meningitidis serogroup B strains (by the addition of new fHbp variants / sub-variants) is provided.

[0088] The inventors of W02020 / 030782 identified residues within the fHbp v1.13 sequence that can be modified to reduce binding to hfH. Such mutant v1.13 Neisseria meningitidis fHbp polypeptides are referred to herein as non-binding (NB) mutants. The inventors of W02020 / 030782 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.

[0089] The mature wild-type fHbp v1.13 lipoprotein from the M982 strain (GenBank accession number AAR84475.1) has the following amino acid sequence, with the N-terminal polyglycine signal sequence underlined. TIFF2025518168000001.tif28170

[0090] The mature v1.13 lipoprotein has an additional 19-residue N-terminal leader sequence in the full-length polypeptide, which is cleaved in the mature polypeptide, and thus differs from the full-length wild-type sequence. Therefore, the full-length wild-type fHbp v1.13 has the amino acid sequence of SEQ ID NO: 45 below (the N-terminal leader sequence is shown in bold). TIFF2025518168000002.tif14169TIFF2025518168000003.tif15168

[0091] The ΔG form of the mature v1.13 lipoprotein lacks the N-terminal polyglycine sequence of the mature polypeptide, i.e., lacks the first 7 amino acids of SEQ ID NO: 15 and lacks the first 26 amino acids of SEQ ID NO: 45. TIFF2025518168000004.tif28168

[0092] In one embodiment, the serogroup B antigen component of the immunogenic composition of the present invention comprises a v1.13 meningococcal fHbp polypeptide variant comprising an amino acid sequence having at least k% sequence identity with SEQ ID NO: 16. However, the amino acid sequence of the mutant v1.13 meningococcal fHbp polypeptide comprises substitution mutations, i.e., point mutations, at one or more residues of residues E211, S216, or E232 of SEQ ID NO: 16.

[0093] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. k% is preferably 80% (i.e., the mutant fHbp v1.13 amino acid sequence has at least 80% identity with SEQ ID NO: 16), more preferably 85%, more preferably 90%, more preferably 95%. Most preferably, the mutant fHbp v1.13 amino acid sequence has at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 16.

[0094] Accordingly, the present invention provides a recombinant vaccine comprising a mutant v1.13 Neisseria meningitidis fHbp polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 16 and comprising substitution mutations, i.e., point mutations, in one or more of residues S216, E211 or E232 of SEQ ID NO: 16.

[0095] Preferably, the amino acid sequence differs from SEQ ID NO: 16 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 of residues E211A and S216R relative to SEQ ID NO: 16.

[0096] Without wishing to be bound by theory, the substitution of glutamic acid (E) to alanine (A) at residue 211 of SEQ ID NO: 16 removes a negatively charged residue involved in hfH recruitment and thus contributes to the inactivation of fH binding. The substitution of serine (S) to arginine (R) at residue 216 of SEQ ID NO: 16 replaces the wild-type amino acid with the corresponding residue from Neisseria gonorrhoeae (N. gonorrhoeae) that does not bind hfH.

[0097] In a preferred embodiment, the mutant v1.13 polypeptide of the present invention has the amino acid sequence of SEQ ID NO: 17 (v1.13ΔG E211A / E232A) or SEQ ID NO: 18 (v1.13ΔG E211A / S216R). More preferably, the mutant v1.13 polypeptide of the present invention has the amino acid sequence of SEQ ID NO: 18.

[0098] The mutant v1.13 polypeptide of the present invention is capable of inducing antibodies that recognize the wild-type Neisseria meningitidis fHbp polypeptide of SEQ ID NO: 15 after administration to a host animal, preferably a mammal, more preferably a human. These antibodies are ideally bactericidal.

[0099] The inventors of W02020 / 030782 also identified residues within the fHbp v1.15 sequence that can be modified to prevent binding to hfH. Such variants are referred to herein as non-binding (NB) variants. The inventors of W02020 / 030782 identified combinations of mutations in the v1.15 sequence that are particularly useful for blocking binding to hfH. fHbp v1.15 is also known in the art as fHbp variant B44.

[0100] The mature wild-type fHbp v1.15 lipoprotein from the NM452 strain (GenBank accession number ABL14232.1) has the following amino acid sequence, with the N-terminal polyglycine signal sequence underlined. TIFF2025518168000005.tif28170

[0101] The mature v1.15 lipoprotein differs from the full-length wild-type sequence in that the full-length polypeptide has an additional 19-residue N-terminal leader sequence, which is cleaved from the mature polypeptide. Thus, the full-length wild-type fHbp v1.15 has the following amino acid sequence (the N-terminal leader sequence is shown in bold). TIFF2025518168000006.tif28170

[0102] The ΔG form of the mature v1.15 lipoprotein lacks the N-terminal polyglycine sequence, i.e., the first 12 amino acids of SEQ ID NO: 19 are deleted and the first 31 amino acids of SEQ ID NO: 46 are deleted. TIFF2025518168000007.tif27170

[0103] In one embodiment, the serogroup B antigen component of the immunogenic composition of the invention comprises an amino acid sequence having at least k% sequence identity with SEQ ID NO: 20. However, the amino acid sequence of the mutant v1.15 meningococcal fHbp polypeptide comprises substitution mutations, i.e., point mutations, in one or more of residues E214, S219, or E235 of SEQ ID NO: 20.

[0104] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. k% is preferably 80% (i.e., the mutant fHbpv1.15 amino acid sequence has at least 80% identity with SEQ ID NO: 20), more preferably 85%, more preferably 90%, and even more preferably 95%. Most preferably, the mutant fHbpv1.15 amino acid sequence has at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 20.

[0105] Preferably, the amino acid sequence differs from SEQ ID NO: 20 by at least one or more of the substitutions E214A, S219R, or E235A. More preferably, the amino acid sequence contains substitutions at residues selected from (i) S219R, (ii) E214A and S219R, and (iii) E214A and E235A.

[0106] In a preferred embodiment, the mutant v1.15 polypeptide has the amino acid sequence of SEQ ID NO: 21 (v.1.15_S219R), SEQ ID NO: 22 (v1.15_E214A / S219R), or SEQ ID NO: 23 (v1.15_E214A / E235A).

[0107] After administration to a host animal, preferably a mammal, more preferably a human, the mutant v1.15 polypeptide can induce antibodies that recognize the wild-type Neisseria meningitidis fHbp polypeptide of SEQ ID NO: 19. These antibodies are ideally bactericidal.

[0108] The disclosure of W02020 / 030782 also provides a fusion polypeptide comprising all three of the v1, v2, and v3 Neisseria meningitidis fHbp polypeptides, where the variant fHbp sequence is in the order v2-v3-v1 from the N-terminus to the C-terminus. In a preferred embodiment, the serogroup B antigen component of the immunogenic composition of the invention comprises such an fHbp fusion polypeptide.

[0109] Preferably, the fHbp fusion polypeptide has the amino acid sequence of the formula NH 2 -A-[-X-L] 3 -B-COOH, where each X is a different mutant fHbp sequence, L is an arbitrary linker amino acid sequence, A is an arbitrary N-terminal amino acid sequence, and B is an arbitrary C-terminal amino acid sequence.

[0110] The v1 fHbp polypeptide component of the fusion is either the above-mentioned mutant v1.13fHbp polypeptide or the mutant v1.13fHbp polypeptide.

[0111] The v2 and v3 fHbp polypeptide components of the fusion included in the immunogenic composition against Neisseria meningitidis serogroup B of the present invention are preferably mutant v2 and v3 polypeptides with improved stability and reduced binding ability to hfH compared to the wild-type v2 and v3 polypeptides. As described above, without wishing to be bound by theory, reducing the binding of fHbp to hfH is advantageous because it prevents the formation of a protective complex between fHbp and hfH that may mask the fHbp epitope, thereby enhancing the immunogenicity of the polypeptide antigen.

[0112] Residues within the v2 and v3 sequences that can be modified to enhance polypeptide stability and reduce binding to hfH have been identified and are described in detail in WO2015 / 128480.

[0113] The full-length wild-type fHbpv2 derived from strain 2996 has the following amino acid sequence (the leader sequence is shown in bold and the polyglycine sequence is underlined): TIFF2025518168000008.tif28169

[0114] The mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 24. TIFF2025518168000009.tif28170

[0115] The ΔG form of SEQ ID NO: 24 lacks the first 26 amino acids. TIFF2025518168000010.tif28170

[0116] In a preferred embodiment, the fusion polypeptide of the present invention comprises a variant v2fHbp polypeptide comprising an amino acid sequence having at least k% sequence identity to SEQ ID NO: 26, provided that the v2fHbp amino acid sequence comprises substitution mutations, i.e., point mutations, at residues S32 and L123 of SEQ ID NO: 26. Preferably, the substitutions are S32V and L123R.

[0117] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. k% is preferably 80% (i.e., the variant fHbpv2 amino acid sequence has at least 80% identity to SEQ ID NO: 26), more preferably 85%, even more preferably 90%, and even more preferably 95%.

[0118] In some embodiments, the fHbp v2 polypeptide contained in the fusion protein of the present invention is truncated with respect to SEQ ID NO: 26. Compared to the wild-type mature sequence, SEQ ID NO: 26 is already truncated at the N-terminus up to and including the polyglycine sequence (compare SEQ ID NOs: 25 and 26), but SEQ ID NO: 26 may be truncated at the C-terminus and / or further truncated at the N-terminus.

[0119] In a preferred embodiment, the v2 fHbp polypeptide contained in the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 30. The v2 fHbp polypeptide contained in the fusion protein has higher stability than the same polypeptide consisting of, for example, the wild-type Neisseria meningitidis polypeptide of SEQ ID NO: 24, which has no sequence differences at residues S32 and L123 under the same experimental conditions. The S32V mutation stabilizes the structure by introducing favorable hydrophobic interactions. Without wishing to be bound by theory, the L123R mutation abrogates fH binding by introducing a collision with fH and an unfavorable charge.

[0120] Improved stability can be evaluated using differential scanning calorimetry (DSC), as discussed, for example, in Johnson (2013) Arch Biochem Biophys 531:100-9 and Bruylants et al. Current Medicinal Chemistry 2005; 12:2011-20. DSC has been used previously to evaluate the stability of v2 fHbp (Johnson et al. PLoS Pathogen 2012; 8: e1002981). Suitable conditions for DSC to evaluate stability can use 20 pM of polypeptide in a buffer solution (e.g., 25 mM Tris) at pH 6 to 8 (e.g., 7 to 7.5) containing 100 to 200 mM NaCl (e.g., 150 mM).

[0121] Improved stability, when evaluated by DSC, is demonstrated by at least a 5°C, e.g., at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or more increase in the thermal transition midpoint (Tm) of at least one peak compared to the wild type. Wild type fHbp shows two DSC peaks during unfolding (one for the N-terminal domain and the other for the C-terminal domain), and when the v2 polypeptide included in the fusion protein of the present invention contains both such domains, "improved stability" means at least a 5°C increase in the Tm of the N-terminal domain. The Tm of the N-terminal domain can occur at 40°C or less for the wild type v2 sequence (Johnson et al. (2012) PLoS Pathogen 8: e1002981), while the Tm of the C-terminal domain can be 80°C or more. Thus, the mutant fHbp v2 amino acid sequence included in the fusion protein of the present invention preferably has an N-terminal domain with a Tm of at least 45°C, e.g., >50°C, >55°C, >60°C, >65°C, >70°C, >75°C, and even >80°C.

[0122] The full-length wild-type fHbp v3 derived from the M1239 strain has the following amino acid sequence (the leader sequence is shown in bold and the polyglycine sequence is underlined). TIFF2025518168000011.tif28170

[0123] The mature lipoprotein lacks the first 19 amino acids of SEQ ID NO: 27. TIFF2025518168000012.tif28170

[0124] The ΔG form of SEQ ID NO: 27 lacks the first 31 amino acids (i.e., lacks the signal sequence and the polyglycine sequence). TIFF2025518168000013.tif28170

[0125] In a preferred embodiment, the fusion polypeptide of the present invention comprises a mutant v3fHbp polypeptide comprising an amino acid sequence having at least k% sequence identity with SEQ ID NO: 29, provided that the v3fHbp amino acid sequence comprises substitution mutations, i.e., point mutations, at residues S32 and L126 of SEQ ID NO: 29. Preferably, the substitutions are S32V and L126R.

[0126] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. k% is preferably 80% (i.e., the mutant fHbp v2 amino acid sequence has at least 80% identity with SEQ ID NO: 29), more preferably 85%, more preferably 90%, and more preferably 95%.

[0127] In some embodiments, the fHbp v3 polypeptide contained in the fusion protein of the present invention is truncated at the termini relative to SEQ ID NO: 29. Compared to the wild-type mature sequence, SEQ ID NO: 29 is already truncated at the N-terminus up to and including the polyglycine sequence (when comparing SEQ ID NOs: 28 and 29), but SEQ ID NO: 29 may be truncated at the C-terminus and / or further truncated at the N-terminus.

[0128] In a preferred embodiment, the v3 fHbp polypeptide contained in the fusion protein of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 31. The v3 fHbp polypeptide contained in the fusion protein has higher stability than the same polypeptide without differences in the sequences at residues S32 and L126 under the same experimental conditions, for example, higher stability than the wild-type meningococcal polypeptide consisting of SEQ ID NO: 27. The S32V mutation stabilizes the structure by introducing preferred hydrophobic interactions. Without wishing to be bound by theory, the L126R mutation invalidates fH binding by introducing a collision with fH and an unfavorable charge.

[0129] The improved stability can be evaluated using differential scanning calorimetry (DSC), as discussed, for example, in Johnson (2013) Arch Biochem Biophys 531:100-9 and Bruylants et al. (2005) Current Medicinal Chemistry 12:2011-20. DSC has previously been used to evaluate the stability of v3 fHbp (van der Veen et al. (2014) Infect Immun PMID 24379280). Conditions suitable for DSC to evaluate stability can use 20 pM of the polypeptide in a buffer solution (e.g., 25 mM Tris) at pH 6 to 8 (e.g., 7 to 7.5) containing 100 to 200 mM NaCl (e.g., 150 mM).

[0130] Improved stability is demonstrated by an increase in the midpoint of the thermal transition (Tm) of at least one peak of at least 5 °C, such as at least 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C or more, compared to the wild type when evaluated by DSC. Wild-type fHbp shows two DSC peaks during unfolding (one in the N-terminal domain and one in the C-terminal domain), and when the v3 polypeptide included in the fusion protein of the present invention contains both such domains, "improved stability" means an increase in the Tm of the N-terminal domain of at least 5 °C. The Tm of the N-terminal domain can occur at about 60 °C or less for the wild-type v3 sequence (Johnson et al. (2012) PLoS Pathogen 8: e1002981), while the Tm of the C-terminal domain can be 80 °C or more. Thus, the mutant fHbp v3 amino acid sequence of the present invention preferably has an N-terminal domain with a Tm of at least 65 °C, such as >70 °C, >75 °C, or >80 °C.

[0131] As described above, in a preferred embodiment, the fHbp fusion polypeptide has the amino acid sequence of the formula NH 2 -A-[-X-L] 3 -B-COOH, where each X is a different mutant fHbp sequence and L is any linker amino acid sequence. In a preferred embodiment, the linker amino acid sequence "L" is a glycine polymer or a glycine-serine polymer linker.

[0132] Exemplary linkers include, but are not limited to, GGSG (SEQ ID NO: 50), GGSGG (SEQ ID NO: 51), GSGSG (SEQ ID NO: 52), GSGGG (SEQ ID NO: 53), GGGSG (SEQ ID NO: 54), GSSSG (SEQ ID NO: 55), and GSGGGG (SEQ ID NO: 56). Other suitable glycine or glycine-serine polymer linkers will be apparent to those skilled in the art. In the preferred fusion polypeptides of the present invention, the v2 and v3 sequences, and the v3 and v1 sequences are linked by the glycine-serine polymer linker GSGGGG (SEQ ID NO: 56).

[0133] In a preferred embodiment, the fusion polypeptide of the present invention comprises or consists of one of the following amino acid sequences (the glycine-serine linker sequence is underlined and the mutated residues are shown in bold). fHbp23S_1.13_E211A / E232A (SEQ ID NO: 32) TIFF2025518168000014.tif66169

[0134] fHbp23S_1.13_E211A / S216R (SEQ ID NO: 33) TIFF2025518168000015.tif14169TIFF2025518168000016.tif53168

[0135] fHbp_23S_1.15_S219R (SEQ ID NO: 34) TIFF2025518168000017.tif66169

[0136] fHbp_23S_1.15_E214A / S219R (SEQ ID NO: 35) TIFF2025518168000018.tif66170

[0137] fHbp_23S_1.15_E214A / E235A (SEQ ID NO: 36) TIFF2025518168000019.tif20168TIFF2025518168000020.tif47169

[0138] In a preferred embodiment, the fusion polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 33. In another preferred embodiment, the fusion polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 32. After administration of the fusion polypeptide of the present invention to a host animal, preferably a mammal, more preferably a human, antibodies can be induced that recognize wild-type meningococcal fHbp polypeptides, particularly the polypeptides of SEQ ID NO: 45, 46, 24 and / or 27. These antibodies are ideally bactericidal.

[0139] As described above, in a preferred embodiment, for the fHbp fusion polypeptide of the present invention, each X is a different mutant fHbp sequence, and A is any N-terminal amino acid sequence, and has the amino acid sequence of the formula NH 2 -A-[ -X-L] 3 -B-COOH. In a preferred embodiment, the fusion proteins described herein further include the following N-terminal amino acid sequences that are advantageous for enabling good expression of the fusion protein. MGPDSDRLQQRR (SEQ ID NO: 48)

[0140] Any of the fusion proteins disclosed herein (e.g., SEQ ID NOs: 32 to 36, 43, and 44) can be modified to include the amino acid sequence of SEQ ID NO: 48 at the N-terminus of the fusion polypeptide. That is, the amino acid sequence of SEQ ID NO: 48 is added to the N-terminus of the fHbpv2 component of the fusion polypeptide.

[0141] In a preferred embodiment, the serogroup B antigen component of the immunogenic composition of the present invention includes the complete BEXSERO vaccine product together with the fHbp fusion polypeptide defined above. Most preferably, the fHbp fusion polypeptide is fHbp23S_1.13_E211A / S216R. Preferably, the serogroup B antigen component is provided in a single complete liquid formulation.

[0142] The above-mentioned preferred v1.13, v1.15 and / or fusion polypeptides can induce a bactericidal antibody response against Neisseria meningitidis. The bactericidal antibody response can be easily measured in mice and is a standard indicator of the effectiveness of the vaccine (see, for example, Note 14 at the end of Pizza et al. (2000) Science 287: 1816-1820. See also WO2007 / 028408).

[0143] The above-mentioned polypeptide can induce a bactericidal antibody response against Neisseria meningitidis serogroup B strains expressing the v1.13 fHbp sequence.

[0144] The above-mentioned preferred polypeptide can induce bactericidal antibodies against meningitis strains expressing the v1.13fHbp sequence in a mouse serum bactericidal assay.

[0145] The above-mentioned polypeptide can preferably induce a bactericidal antibody response against Neisseria meningitidis serogroup B strains expressing the v1.15fHbp sequence.

[0146] The above-mentioned preferred polypeptide can induce bactericidal antibodies against meningitis strains expressing the v1.15fHbp sequence in a mouse serum bactericidal assay.

[0147] For example, an immunogenic composition containing these polypeptides can provide a serum bactericidal titer exceeding 1:4 using the Goldschneider assay with human complement [Goldschneider et al. (1969) J. Exp. Med. 129:1307-26, Santos et al. (2001) Clinical and Diagnostic Laboratory Immunology 8:616-23, and Frasch et al. (2009) Vaccine 27S:B112-6], and / or can provide a serum bactericidal titer exceeding 1:128 using rabbit complement.

[0148] The above-mentioned polypeptide can be prepared by various means, such as chemical synthesis (at least in part), digestion of long polypeptides using proteases, translation from RNA, purification from cell culture (e.g., recombinant expression or from Neisseria meningitidis culture), etc.

[0149] Heterologous expression in an Escherichia coli (E. coli) host is a preferred expression route.

[0150] The polypeptide is ideally at least 100 amino acids in length, for example 150aa, 175aa, 200aa, 225aa, or more. These include the mutant fHbp v1, v2, and / or v3 amino acid sequences, and the mutant fHbp v1, v2, or v3 amino acid sequences must likewise be at least 100 amino acids in length, for example 150aa, 175aa, 200aa, 225aa, or more.

[0151] fHbp is naturally a lipoprotein of Neisseria meningitidis. It has also been found that when expressed in E. coli using its native leader sequence or a heterologous leader sequence, it is lipidated. The polypeptide of the present invention may have an N-terminal cysteine residue, which may be lipidated, for example, containing a palmitoyl group and usually forming tripalmitoyl-S-glyceryl-cysteine. In other embodiments, the polypeptide is not lipidated.

[0152] The polypeptide is preferably prepared in a substantially pure or substantially isolated form (i.e., substantially free of other Neisseria-derived or host cell polypeptides). Generally, the polypeptide is provided in a non-natural environment separated, for example, from its natural environment. In certain embodiments, the polypeptide is present in a composition that is enriched compared to the starting material. Thus, a purified polypeptide is provided. Purified means that the polypeptide is present in a composition substantially free of other expressed polypeptides, and substantially free means that more than 50% (e.g., >75%, >80%, >90%, >95%, or >99%) of the total polypeptides in the composition are the polypeptide of the present invention.

[0153] The polypeptide can take various forms (e.g., native, fusion, glycosylated, non-glycosylated, lipidated, disulfide-bridged, etc.).

[0154] When a polypeptide is produced by translation in a biological host, an initiation codon that provides an N-terminal methionine is required in most hosts. Thus, at least in the nascent stage, the polypeptide contains a methionine residue upstream of the sequence of the SEQ ID NO.

[0155] Cleavage of the nascent sequence means that the mutant fHbp v1, v2, or v3 amino acid sequence itself may provide the N-terminus of the polypeptide. However, in other embodiments, the polypeptide may contain 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 the mutant fHbp v1, v2, or v3 amino acid sequence. In other embodiments, longer upstream sequences may be used. Such upstream sequences may be short (e.g., 40 amino acids or less, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include a leader sequence that directs protein transport, or a short peptide sequence that facilitates cloning or purification (e.g., a histidine tag, i.e., His n (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.

[0156] The polypeptide 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 short (e.g., 40 amino acids or less, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include a sequence that directs protein transport, a short peptide sequence that facilitates cloning or purification (e.g., a histidine tag, i.e., His n(where n = 4, 5, 6, 7, 8, 9, 10 or more), or sequences that enhance the stability of the polypeptide. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

[0157] In some embodiments, the invention excludes polypeptides comprising a histidine tag (see Johnson et al. (2012) PLoS Pathogen 8:e1002981, and Pajon et al. (2012) Infect Immun 80:2667-77), particularly polypeptides comprising a hexahistidine tag at the C-terminus.

[0158] The term "polypeptide" refers to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified naturally or by intervention. For example, other operations or modifications such as formation of disulfide bonds, glycosylation, lipid addition, acetylation, phosphorylation, or conjugation with a labeling component. Also included in this definition are polypeptides comprising, for example, one or more analogs of amino acids (including, for example, non-natural amino acids), and other modifications known in the art. A polypeptide may exist as a single chain or as related chains.

[0159] A polypeptide can be adhered or immobilized to a solid support.

[0160] A polypeptide can contain a detectable label such as a radioactive label, a fluorescent label, or a biotin label. This is particularly useful in immunoassay techniques.

[0161] A polypeptide usually consists of an artificial amino acid sequence, i.e., a sequence that does not exist in natural meningococcus.

[0162] The affinity for factor H can be quantitatively evaluated using surface plasmon resonance with immobilized human fH (as disclosed, for example, in Schneider et al. (2009) Nature 458:890-5). Mutations that result in at least a 10-fold, ideally at least a 100-fold, decrease in affinity (i.e., an increase in the dissociation constant KD) are preferred (measured under the same experimental conditions compared to the same polypeptide without the mutation).

[0163] Meningococcal serogroups A, C, W135, and Y The immunogenic composition of the present invention in liquid form against Neisseria meningitidis serogroup B can be used for the reconstitution of an immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y in solid form (such as lyophilized) containing capsular saccharides of Neisseria meningitidis serogroups A, C, W135, and Y conjugated to a carrier protein. The capsular saccharides of these four serogroups ACWY of Neisseria meningitidis are well characterized.

[0164] The capsular saccharide of Neisseria meningitidis serogroup A is a homopolymer of (α1→6)-linked N-acetyl-D-mannosamine-1-phosphate, with the C3 and C4 positions being partially O-acetylated. The acetyl groups can be replaced with blocking groups to prevent hydrolysis (see, for example, WO03 / 080678, which is incorporated herein by reference), and such modified saccharides remain serogroup A capsular saccharides as disclosed herein.

[0165] The capsular saccharide of serogroup C is a homopolymer of (α2→9)-linked sialic acid (N-acetylneuraminic acid (「NeuNAc」)). Most serogroup C strains have O-acetyl groups at the C-7 and / or C-8 of the sialic acid residue, but approximately 15% of clinical isolates lack these O-acetyl groups. The sugar structure is denoted as →9)-NeupNAc7 / 8OAc-(α2→.

[0166] Serogroup W135 polysaccharide is a polymer of sialic acid-galactose disaccharide units. Similar to serogroup C polysaccharide, it has variable O-acetylation at the 7- and 9-positions of sialic acid. The structure is represented as →4)-D-Neup5Ac(7 / 9OAc)-α-(2→6)-D-Gal-α-(1→.

[0167] Serogroup Y polysaccharide is similar to serogroup W135 polysaccharide, except that it contains glucose instead of galactose in the disaccharide repeating unit. Similar to serogroup W135, it is variably O-acetylated at the 7- and 9-positions of sialic acid. The serogroup Y structure is represented as follows: →4)-D-Neup5Ac(7 / 9OAc)-α-(2→6)-D-Glc-α-(1→.

[0168] The capsular polysaccharide can be a native capsular polysaccharide obtained from meningococci of the relevant serogroups. The native capsular polysaccharide may be modified by any method available to those skilled in the art, as long as the capsular polysaccharide retains at least one epitope that induces serum bactericidal antibodies. Exemplary modifications are described below. In addition to native and modified capsular polysaccharides, the capsular polysaccharide may be chemically synthesized as long as the synthetic compound (such as a sugar, sugar analog, etc.) contains at least one epitope that induces serum bactericidal antibodies that bind to the capsular polysaccharide. All such native, modified, and chemically synthesized capsular polysaccharides are within the scope of the meningococcal capsular polysaccharides disclosed herein. Exemplary modifications and chemical syntheses are described below.

[0169] The capsular polysaccharide in the vaccine may be O-acetylated as described above (e.g., in the same O-acetylation pattern found in native capsular polysaccharides), or may be partially or completely de-O-acetylated at one or more positions of the sugar ring, or may be over-O-acetylated compared to native capsular polysaccharides.

[0170] The capsular polysaccharides in the vaccine may be shorter than the native capsular polysaccharides found in bacteria. Therefore, the polysaccharide may be partially depolymerized, which usually occurs after purification and before conjugation. Depolymerization shortens the chain length of the polysaccharide and forms polysaccharides of the desired size. In the depolymerization method, hydrogen peroxide is added to the polysaccharide (e.g., final H2 O 2 (Adjust the concentration to 1%), and then incubate the mixture (e.g., at about 55 °C) until the desired shortening of the chain length is achieved. In another depolymerization method, hydrolysis with an acid is carried out (see, for example, WO03 / 007985, which is incorporated herein by reference). Other depolymerization methods are known to those skilled in the art. The capsular saccharides used in vaccines are obtained by any of these depolymerization methods. Depolymerization can be used to provide an optimal chain length for immunogenicity and / or to shorten the chain length for ease of physical handling of the saccharides. Native capsular saccharides are usually called capsular polysaccharides, and depolymerized capsular saccharides are usually called capsular oligosaccharides.

[0171] In this composition, the capsular saccharides may be used in the form of oligosaccharides. These are conveniently formed by fragmentation (e.g., hydrolysis) of the purified capsular polysaccharides, and then usually the fragments of the desired size are purified.

[0172] The vaccine products sold under the trade names MENVEO, MENACTRA, and NIMENRIX all contain conjugate capsular saccharide antigens derived from serogroups Y, W135, C, and A, respectively.

[0173] In MENVEO (also commonly known as meningococcal (groups A, C, Y, and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine), each of the MenA, C, W135, and Y antigens is conjugated to the CRM 197 carrier.

[0174] In MENACTRA (also commonly known as meningococcal (groups A, C, Y, and W-135) polysaccharide diphtheria toxoid conjugate vaccine), each of the A, C, W135, and Y antigens is conjugated to the diphtheria toxoid carrier.

[0175] In MENQUADFI (also generally known as meningococcal (groups A, C, Y, and W-135) polysaccharide conjugate vaccine), each of the A, C, W135, and Y antigens is conjugated to a tetanus toxoid carrier.

[0176] In NIMENRIX (also generally known as meningococcal polysaccharide groups A, C, W135, and Y conjugate vaccine), each of the MenA, C, W135, and Y antigens is conjugated to a tetanus toxoid carrier.

[0177] In a preferred embodiment, the solid-form immunogenic composition reconstituted with the MenB liquid composition of the present invention comprises the MenA, C, W135 and Y antigen conjugates present in MENVEO, the MenA, C, W135 and Y antigen conjugates present in MENACTRA, the MenA, C, W135 and Y antigen conjugates present in MENQUADFI, or the MenA, C, W135 and Y antigen conjugates present in NIMENRIX. In one embodiment, the solid-form immunogenic composition of the present invention comprises MenA, C, W135 and Y antigen conjugates, and their capsular saccharides and / or oligosaccharides are recombinant CRM 197 carrier (rCRM 197 ) comprising CRM 197 carrier, conjugated to a tetanus toxoid carrier (TT) or a diphtheria toxoid carrier (DT). In one embodiment, the solid-form immunogenic composition of the present invention comprises MenA, C, W135, and Y antigen conjugates, and their capsular saccharides and / or oligosaccharides are recombinant CRM 197 carrier (rCRM 197 ) comprising CRM 197 carrier, or conjugated to a tetanus toxoid carrier (TT).

[0178] Preferably, the solid-form immunogenic composition of the present invention comprises MenA, C, W135, and Y antigen conjugates, and their capsular saccharides and / or oligosaccharides are recombinant CRM 197 carrier (rCRM197 )-containing CRM 197 is conjugated to a carrier.

[0179] Additional antigenic components The immunogenic composition of the present invention may contain antigens for immunization against other diseases or infections. For example, the composition may contain one or more of the following additional antigens: - a carbohydrate antigen derived from Streptococcus pneumoniae [e.g., Watson (2000) Pediatr Infect Dis J 19:331-332, Rubin (2000) Pediatr Clin North Am 47:269-285, and Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207]. - an antigen derived from hepatitis A virus, such as inactivated virus [e.g., Bell (2000) Pediatr Infect Dis J 19:1187-1188, Iwarson (1995) APMIS 103:321-326]. - an antigen derived from hepatitis B virus, such as surface antigen and / or core antigen [e.g., Gerlich et al. (1990) Vaccine 8 Suppl: S63-68 & 79-80]. - a diphtheria antigen such as diphtheria toxoid [e.g., Chapter 3 of Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0], e.g., CRM 197 variant [e.g., Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70]. - an antigen derived from tetanus, such as tetanus toxoid [e.g., Chapter 4 of Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0]. - Antigens derived from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) derived 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]. - Carbohydrate antigens derived from Haemophilus influenzae type B [e.g., Costantino et al. (1999) Vaccine 17:1251-1263]. - Polio antigens [e.g., Sutter et al. (2000) Pediatr Clin North Am 47:287-308, Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126], IPV, etc. - Measles, mumps, and / or rubella antigens [e.g., Chapters 9, 10, 11 of Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0]. - Influenza antigens [e.g., Chapter 19 of Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0], such as hemagglutinin and / or neuraminidase surface proteins. - Antigens derived from Moraxella catarrhalis [e.g., McMichael (2000) Vaccine 19 Suppl 1: S101-107]. - Protein antigens derived from Streptococcus agalactiae (group B streptococcus) [e.g., Schuchat (1999) Lancet 353(9146):51-6, WO02 / 34771]. - Carbohydrate antigens derived from Streptococcus agalactiae (group B streptococcus). - Antigens derived from Streptococcus pyogenes (Group A streptococcus) [e.g., WO02 / 34771, Dale (1999) Infect Dis Clin North Am 13:227-43, Ferretti et al. (2001) PNAS USA 98:4658-4663]. - Antigens derived from Staphylococcus aureus [see also Kuroda et al. (2001) Lancet 357(9264):1225-1240, pages 1218-1219].

[0180] In one embodiment, the immunogenic composition of the present invention does not contain antigens against Neisseria meningitidis serogroup X.

[0181] Toxic protein antigens can be detoxified if necessary (e.g., detoxification of pertussis toxin by chemical and / or genetic means [Rappuoli et al. (1991) TIBTECH9:232-238]).

[0182] When the composition contains diphtheria antigen, it is preferably also contains tetanus antigen and pertussis antigen. Similarly, when the composition contains tetanus antigen, it is preferably also contains diphtheria antigen and pertussis antigen. Similarly, when the composition contains pertussis antigen, it is preferably also contains diphtheria antigen and tetanus antigen. Therefore, the DTP combination is preferred.

[0183] Carbohydrate antigens are preferably in conjugate form. Generally, conjugation converts carbohydrates from T-independent antigens to T-dependent antigens, enhancing the immunogenicity of carbohydrates to enable priming of immune memory. Conjugation is particularly useful for pediatric vaccines and is a well-known technique.

[0184] Typical carrier proteins are bacterial toxins such as diphtheria toxin and tetanus toxin, or their toxoids or variants. CRM 197The diphtheria toxin mutant [Research Disclosure, 453077 (January 2002)] is useful and is the carrier of the pneumococcal vaccine sold under the trade name PREVNAR. Also, recombinant CRM 197 (rCRM 197 ) obtained from or derived from Pseudomonas fluorescens or Escherichia coli is also useful as a carrier protein. Other suitable carrier proteins include meningococcal outer membrane protein complex [EP-A-0372501], synthetic peptides [EP-A-0378881, EP-A-0427347], heat shock proteins [WO93 / 17712, WO94 / 03208], pertussis proteins [WO98 / 58668, EP-A-0471177], cytokines [WO91 / 01146], lymphokines [WO91 / 01146], hormones [WO91 / 01146], growth factors [WO91 / 01146], artificial proteins containing multiple human CD4+ T cell epitopes from antigens derived from various pathogens [Falugi et al. (2001) Eur J Immunol 31:3816-3824], such as N19 [Baraldo et al. (2004) Infect Immun 72(8):4884-7], protein D derived from Haemophilus influenzae [EP-A-0594610, Ruan et al. (1990) J Immunol 145:3379-3384], pneumolysin [Kuo et al. (1995) Infect Immun 63:2706-13] or its non-toxic derivative [Michon et al. (1998) Vaccine. 16:1732-41], pneumococcal surface protein PspA [WO02 / 091998], iron uptake proteins [WO01 / 72337], toxin A or B derived from C. difficile [WO00 / 61761], recombinant Pseudomonas aeruginosa exoprotein A (rEPA) [WO00 / 33882], and the like.

[0185] If necessary, appropriate conjugation reactions can be used using appropriate linkers.

[0186] Carbohydrates are usually activated or functionalized before conjugation. Activation may involve, for example, cyanation reagents such as CDAP (e.g., 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [Lees et al. (1996) Vaccine 14:190-198, WO95 / 08348]). Other suitable techniques include using carbodiimide, hydrazide, active ester, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, etc.

[0187] The attachment via a linker group can be carried out using known procedures, such as those described in US4,882,317 and US4,695,624. One type of attachment involves reducing aminating the polysaccharide, attaching the resulting amino group to one end of an adipic acid linker group, and then attaching 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 β-propionamide [WO00 / 10599], nitrophenylethylamine [Gever et al. Med. Microbiol. Immunol, 165: 171-288 (1979)], haloacyl halide [US 4,057,685], glycosidic bond [US 4,673,574; US 4,761,283; US 4,808,700], 6-aminocaproic acid [US 4,459,286], ADH [US 4,965,338], C 4 from C 12 moieties [US 4,663,160], etc. Instead of using a linker, a direct attachment can also be used. The direct attachment to a protein can involve, for example, oxidation of the polysaccharide followed by reductive amination with the protein, as described in US4,761,283 and US4,356,170.

[0188] Introduce an amino group into the carbohydrate (e.g., convert the terminal =O group to NH 2(by replacing with), and then derivatizing with an adipic acid diester (e.g., N-hydroxysuccinimide diester of adipic acid) and reacting with a carrier protein is a preferred process. Another preferred reaction uses CDAP activation with a protein D carrier for, e.g., MenA or MenC.

[0189] The antigens in the composition are usually present at a concentration of at least 1 μg / ml each. Generally, the concentration of any antigen is sufficient to induce an immune response against that antigen.

[0190] The immunogenic composition of the present invention can be used therapeutically (i.e., to treat existing infections) or prophylactically (i.e., to prevent future infections).

[0191] Instead of using a protein antigen in the immunogenic composition of the present invention, a nucleic acid encoding the antigen (which may be RNA such as self-replicating RNA or DNA such as a plasmid) can be used.

[0192] Non-antigenic components and formulation of the composition The immunogenic composition of the present invention generally contains excipients which can be any substance that is pharmaceutically acceptable, does not induce the production of antibodies harmful to the patient to whom the composition is administered, and can be administered without undue toxicity. Depending on the formulation of the immunogenic composition described in more detail below, pharmaceutically acceptable excipients can include liquids such as water, physiological saline, glycerol, ethanol, etc. Auxiliary substances such as wetting agents or emulsifying agents, pH buffering substances can also be present in the above formulations. A detailed description of suitable excipients is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

[0193] The immunogenic composition is preferably sterile.

[0194] The immunogenic composition against Neisseria meningitidis serogroup B of the present invention is formulated as a liquid "adsorbed" vaccine in which the protein antigen is adsorbed to an adsorbent, such as a compound containing aluminum, and contains one or more pharmaceutically acceptable tonicity modifiers.

[0195] A "pharmaceutically acceptable tonicity modifier" is a compound that is physiologically acceptable, imparts an appropriate tonicity to the formulation, and prevents the net flow of water through the cell membrane in contact with the formulation. In some embodiments, the tonicity modifier used in the composition is preferably a salt (or mixture of salts) selected from sodium chloride, sugars such as sucrose and sorbitol, and mixtures thereof. More preferably, the tonicity modifier in these compositions is a mixture of sodium chloride and sucrose, and most preferably an aqueous solution of sodium chloride and sucrose.

[0196] In a preferred embodiment, the immunogenic composition against Neisseria meningitidis serogroup B of the present invention is a liquid formulation such as an aqueous solution having a sodium chloride concentration of up to about 3.8 mg / ml, preferably less than about 3.0 mg / ml, more preferably about 2.8 mg / ml. Intermediate values of the above sodium chloride concentrations (e.g., 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, and 2.9 mg / ml), as well as values less than 2.8 mg / ml, are also intended to be part of the present invention. In a further aspect of the present invention, the concentration of sodium chloride in the MenB liquid formulation may range from 1.0 mg / ml to 3.8 mg / ml, from 1.5 mg / ml to 3.5 mg / ml, from 2.0 to 3.5 mg / ml, from 2.5 to 3.5 mg / ml, or from 2.5 mg / ml to 3.0 mg / ml.

[0197] In a more preferred embodiment, the immunogenic composition against Neisseria meningitidis serogroup B of the present invention comprises an aqueous solution having a sucrose concentration in the range of about 2 to 3% by weight (hereinafter referred to as "w / v"), preferably about 3% w / v, based on the total volume of the solution. In a further aspect of the present invention, the concentration of sucrose in the immunogenic composition of the MenB antigen may be in the range of 2.0 mg / ml to 3.5 mg / ml, 2.5 mg / ml to 3.5 mg / ml, or 2.5 mg / ml to 3.0 mg / ml.

[0198] In an aspect of the present invention, an adjuvant may be added to the immunogenic composition against Neisseria meningitidis serogroup B of the present invention. Adjuvants that can be used in this composition include, but are not limited to, insoluble metal salts, oil-in-water emulsions (e.g., MF59 or AS03 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, or mixtures thereof. Other substances that act as immunostimulants are disclosed in Chapter 7 of Vaccine Design (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum.

[0199] It is preferred to use a compound containing aluminum as an adsorbent, and all MenB antigens in this immunogenic composition containing the fusion fHbp polypeptide are usually adsorbed to this compound. Particularly preferred as an adsorbent in the liquid composition of the present invention are aluminum hydroxide (alum), aluminum phosphate, aluminum potassium sulfate, oxyhydroxide and hydroxyphosphate (see, for example, Chapter 8 and Chapter 9 of Vaccine Design… (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum), and compounds selected from aluminum salts and mixtures thereof. The salt can take any suitable form (e.g., gel, crystal, amorphous, etc.). In the most preferred embodiment, this composition of the liquid formulation is adjuvanted with alum as an adsorbent for the protein antigen. In one aspect, the adsorbent, preferably alum, is present at a concentration of 2 to 5% by weight, for example 3%, based on the total amount of the MenB antigen immunogenic composition of the present invention in liquid form. Further, adsorbent concentrations in the range of, for example, 2 to 4% or 2.5 to 3.5% are also intended to be part of the present invention.

[0200] This immunogenic composition against Neisseria meningitidis serogroup B in the liquid formulation is preferably buffered, for example, between pH 6 and pH 8, preferably around pH 6.5. Suitable buffers can be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and Tris. When the composition contains an aluminum salt, it is preferred to use a histidine buffer [WO03 / 009869]. Histidine can be added to the composition in the form of the amino acid itself, preferably L-histidine, or in the form of a salt. The concentration of histidine in the composition can typically range from at least 1 μM to a maximum of 1 M. In one embodiment, the concentration is from at least 1 mM (e.g., at least 2 mM, 3 mM, 4 mM, 5 mM, etc.) to a maximum of 250 mM (e.g., a maximum of 200 mM, 150 mM, 100 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, etc.). Preferably, the concentration of histidine is between 2 mM and 20 mM (e.g., 5 mM to 15 mM), and most preferably about 10 mM.

[0201] In one embodiment, the reconstituted vaccine composition of the present invention has a pharmaceutically acceptable osmotic pressure in order to avoid cell deformation or lysis. Pharmaceutically acceptable osmotic pressure generally means that the solution has an approximately isotonic or slightly hypertonic osmotic pressure. In one embodiment, the reconstituted vaccine composition has an osmotic pressure in the range of 250 to 750 mOsm / kg, for example, the osmotic pressure is in the range of 250 to 550 mOsm / kg, such as in the range of 280 to 500 mOsm / kg. The liquid formulation of the MenB antigen of the present invention is preferably slightly hypotonic so that the reconstituted vaccine reaches the desired isotonicity or mild hypertonicity as described above, for example, the osmotic pressure is about 210 mOsm / Kg. The osmotic pressure can be measured according to techniques known in the art using a commercially available osmometer, such as Advanced® Model 2020 available from Advanced Instruments Inc. (USA).

[0202] In a preferred embodiment, the immunogenic composition against Neisseria meningitidis serogroups A, C, W, Y in the reconstituted vaccine of the present invention is formulated as a solid, such as a freeze-dried product, and contains a pharmaceutically acceptable bulking agent, such as sugar.

[0203] The term "bulking agent" as used herein refers to an excipient compound or mixture of compounds that, when added to a solution intended for lyophilization, constitutes the bulk lyophilized product. Non-limiting examples of bulking agents include sucrose, mannitol, trehalose, and mixtures thereof. A preferred bulking agent is sucrose. In one embodiment, the bulking agent, preferably sucrose, is added to the capsular polysaccharide conjugate at a concentration of less than about 6% weight / volume (hereinafter "w / v"), based on the total volume of the formulation of MenA, C, W, Y antigens prior to lyophilization. In one embodiment, the bulking agent, preferably sucrose, is added to the capsular polysaccharide conjugate at a concentration of less than about 5% w / v, based on the total volume of the formulation of MenA, C, W, Y antigens prior to lyophilization. In one embodiment, the bulking agent, preferably sucrose, is added to the formulation prior to lyophilization, hereinafter also referred to as the pre-lyophilized bulk formulation, at a concentration of about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, preferably about 3% w / v. In a further aspect, the concentration of the bulking agent, preferably sucrose, in the pre-frozen bulk formulation of MenA, C, W, Y antigens may range from 2% w / v to 5.5% w / v, from 2% w / v to 5% w / v, from 2% w / v to 4% w / v, or from 2.5% w / v to 3.5% w / v. In a further aspect, the concentration of the bulking agent, preferably sucrose, in the pre-frozen bulk formulation of MenA, C, W, Y antigens is 2% w / v or 3% w / v.

[0204] In a more preferred embodiment, the immunogenic composition against Neisseria meningitidis serogroups A, C, W, Y in the reconstituted vaccine of the invention comprises a buffer, such as phosphate buffer at a pH of about 7.2. This phosphate buffer contains potassium dihydrogen phosphate, to which dipotassium phosphate may be added.

[0205] Preferably, the bulking agent and / or buffer is added to the composition and mixed with the antigen prior to lyophilization. In one embodiment, a phosphate buffer at a concentration of at least about 6 mM, at least 10 mM, or at least 40 mM is added to the formulation prior to lyophilization.

[0206] As shown in the following experimental section, the liquid formulation of the MenB antigen and the solid formulations of the MenA, C, W135, and Y antigens described herein are safe, stable, and easy to use, and not only obtain effective vaccine products, but also make it possible to provide vaccines that can be stored for a long time and are effective formulations. The characteristics of the present formulation include, but are not limited to, the chemical stability of the immunogenic composition (e.g., proteolysis or fragmentation of proteins), the physical / thermal stability of the immunogenic composition (aggregation, precipitation, adsorption, etc.), the compatibility of the immunogenic composition with the container / sealing system, the interaction of the immunogenic composition with inert components (buffers, salts, excipients, cryoprotectants, etc.), the manufacturing process, the dosage form (lyophilization, liquid, etc.), the environmental conditions (temperature, humidity, shear force, etc.) encountered during transportation, storage, and handling, and the length of time from manufacture to use.

[0207] The immunogenic composition contains an immunologically effective amount of the protein or conjugate of the present invention and other components. "Immunologically effective amount" means that when administered to an individual as a single dose or as part of a multiple dose, it is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual being treated, age, desired degree of protection, the formulation method of the vaccine, and other relevant factors. It is expected to fall within a relatively wide range that can be determined through routine testing.

[0208] In one embodiment, the immunogenic composition of the present invention in the liquid formulation against Neisseria meningitidis serogroup B infection contains 40 to 60 μg / ml, for example 50 μg / ml, of the OMV antigen. In one embodiment, this immunogenic composition contains 50 to 150 μg / ml, for example 100 μg / ml, of each protein antigen NHBA, fHbp, and NadA. In one embodiment, this immunogenic composition contains 100 to 400 μg / ml, for example 100, 200, 300, or 400 μg / ml, of the fusion fHbp polypeptide. In another embodiment, this immunogenic composition contains 100 to 200 μg / ml, for example, of the fusion fHbp polypeptide, such as the mutant v1.13fHbp polypeptide.

[0209] In one embodiment, the solid - form immunogenic composition against Neisseria meningitidis serogroups ACWY infection in the recombinant vaccine of the present invention contains 5 to 15 μg / ml, for example 10 μg of MenC, MenW, and MenY saccharides respectively. In one embodiment, this immunogenic composition contains 5 to 15 μg, for example 20 μg / ml of MenA saccharide.

[0210] As described above, the solid - form immunogenic composition of the present invention preferably contains MenA, C, W135, and Y antigens in the form of conjugates, and MenA, C, W135, and Y capsular saccharides and / or their oligosaccharides are conjugated to a recombinant CRM 197 carrier (rCRM 197 ) and is included in the CRM 197 carrier conjugated thereto. In one embodiment, a 0.5 - mL unit dose of the recombinant vaccine of the present invention contains 10 μg of MenA saccharide conjugated to 12.5 to 33.3 μg of CRM 197 , 5 μg of MenC saccharide conjugated to 6.3 to 12.5 μg of CRM 197 , 5 μg of MenW135 saccharide conjugated to 3.3 to 10.0 μg of CRM 197 , and 5 μg of MenY saccharide conjugated to 3.3 to 10.0 μg of CRM 197 .

[0211] In one embodiment, the recombinant vaccine composition against Neisseria meningitidis serogroups A, B, C, W135, and Y of the present invention contains 40 to 60 μg / ml, for example 50 μg / ml of OMV, 50 to 150 μg / ml, for example 100 μg / ml of NHBA antigen, NadA antigen, and fHbp antigen respectively, 100 to 400 μg / ml, for example 100, 200, 300, or 400 μg / ml of the fusion fHbp polypeptide; 5 to 15 μg / ml, for example 10 μg / ml of MenC, MenW135, and MenY respectively, 5 to 15 μg / ml, for example 10 μg / ml of MenA; 2 to 5 μg / ml, for example 3 μg / ml of an adsorbent such as alum, and 2.0 to 3.5 μg / ml, for example 2.8 μg / ml of a tonicity regulator such as sodium chloride.

[0212] In one embodiment, a 0.5 ml dose of the recombinant vaccine of the present invention contains 10 μg of Men saccharide, 5 μg each of MenC, MenW135, and MenY saccharide, 25 μg of OMV, 50 μg each of rp287-953, rp936-741, and rp961c antigens, 50 μg of fHbp231.13, 1.4 mg of sodium chloride, 27.5 mg of sucrose, 0.54 mg of potassium phosphate salt, 0.776 mg of histidine, 1.54 mg of aluminum hydroxide, and water for injection up to a 0.5 ml dose. In other embodiments, the same 0.5 dose contains a greater amount of 100 μg or 200 μg of fHbp231.13 instead of 50 μg, and 0.54 to 0.90 mg of potassium phosphate salt.

[0213] Kit and reconstituted vaccine composition The subject of the present invention is a kit comprising (i) a first container containing the immunogenic composition against Neisseria meningitidis serogroup B as described above in a liquid formulation, and (ii) a second container containing the immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y as described above in a solid form. In this kit, the first container and the second container may be separate containers, or may together form a multi-container having separate parts for the liquid and solid components.

[0214] In the present invention, the liquid immunogenic composition in the first container is used to reconstitute the solid immunogenic composition in the second container to form a vaccine composition containing all the antigens of both immunogenic compositions before administration. In one embodiment, the reconstituted vaccine composition is a suspension. In one embodiment, the reconstituted vaccine composition comprises a suspension of the solid immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y as described herein in the liquid immunogenic composition against Neisseria meningitidis serogroup B as described herein.

[0215] In a preferred embodiment, the kit of the present invention comprises (i) a pre-filled syringe as the first container, and (ii) a vial as the second container.

[0216] In certain embodiments, the containers of the kit are silicone-treated to improve the stability of the vaccine upon removal during reconstitution. When the immunogenic composition of the invention is provided in vials, these are preferably made of glass or plastic material, more preferably of silicone-treated glass or plastic material.

[0217] The vials are preferably sterilized before adding the composition. The vial may contain a single dose of the vaccine or may contain multiple doses (a "multi-dose" vial), such as 10 doses. When using a multi-dose vial, each dose should be removed with a sterile needle and syringe under strict aseptic conditions, taking care to avoid contamination of the contents of the vial. Preferred vials are made of colorless glass. The vial can have a cap or stopper (e.g., Luer-Lok™) suitable for inserting a pre-filled syringe with a needle into the cap, discharging the contents of the syringe into the vial (e.g., to reconstitute a lyophilized substance therein), and then removing the contents of the vial back into the syringe. After removing the syringe from the vial, the composition can be administered to the patient. The cap is preferably placed inside a seal or cover such that the seal or cover needs to be removed before accessing the cap.

[0218] In one embodiment, the same needle is used for both reconstitution and parenteral administration. In another embodiment, the needle used for reconstitution is replaced with a different needle for administration of the reconstituted vaccine.

[0219] As the results of the following experimental section show, the liquid formulation of the MenB antigen composition ensures that all relevant quality requirements of the final vaccine are met, particularly with respect to the pH and osmotic pressure of the vaccine composition, the integrity and immunogenicity of each antigen and all antigens. Furthermore, the liquid formulation of the invention of the MenB antigen shows that the adsorption rate of all antigens to the adsorbent is optimal even after reconstituting the solid formulation of the MenACWY antigen.

[0220] Administration The vaccine composition of the present invention is usually directly administered to a patient by a suitable route, such as parenteral injection like intramuscular injection. Intramuscular administration to the thigh or upper arm is preferred. The injection may be performed through a needle (e.g., a hypodermic needle), but alternatively, needle-free injection may be used.

[0221] The reconstituted vaccine composition of the present invention can be administered to a patient in a unit dose ranging from 0.1 to 1 ml, for example, 0.5 ml. A typical intramuscular dose is about 0.5 ml.

[0222] Since Neisseria infections affect various parts of the body, the compositions of the present invention can be prepared in various forms. Compositions suitable for parenteral injection are most preferred.

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

[0224] The "dose" of the composition as used herein is the volume of the composition suitable for administration to a subject as a single immunization. Human vaccines are usually administered in a dose of about 0.5 ml, but may also be administered in divided doses (e.g., in children).

[0225] The composition may further be provided in a "multi-dose" kit, i.e., a single container containing a composition sufficient for multiple immunizations. The multi-dose may contain a preservative, or the multi-dose container may be fitted with a sterile adapter for removing individual doses of the composition.

[0226] The administration may include a single-dose schedule or a multiple-dose schedule. In the latter case, the appropriate interval between priming administrations can be conventionally determined, for example, from 4 to 16 weeks, 1 month or 2 months, etc.

[0227] The subject to be immunized is a human and can be of any age (e.g., from 0 to 12 months, 1 to 5 years, 5 to 18 years, 18 to 55 years, or 55 years or older). Preferably, the subject to be immunized is an adolescent (e.g., 12 to 18 years) or an adult (18 years or older).

[0228] Optionally, the subject is an adolescent or adult who received immunization against Neisseria meningitidis in childhood (e.g., less than 12 years of age) and receives an additional dose of the immunogenic composition according to the invention.

[0229] Use of the immunogenic composition and reconstituted vaccine of the present invention The MenB immunogenic composition of the invention is suitable for medical use, in particular for immunizing mammals against infections and / or diseases caused by Neisseria meningitidis serogroup B such that the recipient of the immunogenic composition initiates an immune response and provides protection against Neisseria meningitidis infections and / or diseases.

[0230] The above recombinant vaccine composition is useful for immunizing mammals against infections or diseases of Neisseria meningitidis serogroups A, B, C, W, and / or Y.

[0231] Accordingly, the immunogenic compositions and recombinant vaccines of the invention are used in a prophylactic method for immunizing a subject against infections and / or diseases caused by Neisseria meningitidis. The immunogenic compositions and recombinant vaccines can also be used in a therapeutic method (i.e., treatment of Neisseria meningitidis infections).

[0232] The invention also provides a method for eliciting an in vivo immune response against Neisseria meningitidis infection in a mammal, comprising administering to the mammal an immunogenic composition or recombinant vaccine of the invention.

[0233] The immune response is preferably protective and preferably involves antibodies and / or cellular immunity. Preferably, the immune response is a bactericidal antibody response. This method can elicit a booster response. By eliciting an immune response in vivo, the mammal can be protected from Neisseria diseases (especially Neisseria meningitidis infections).

[0234] The present invention also provides a method for protecting a mammal from Neisseria bacteria (e.g., meningococcus) infection, which comprises administering to the mammal an immunogenic composition or a reconstituted vaccine of the present invention.

[0235] The immunogenic composition of the present invention is preferably formulated as a vaccine product suitable for therapeutic (i.e., treating an infectious disease) or prophylactic (i.e., preventing an infectious disease) use. Vaccines are typically prophylactic.

[0236] The mammal is preferably a human. The human can be an adult, an adolescent, or a child (e.g., an infant or a baby). A vaccine for children may be administered to adults, for example, to evaluate safety, dosage, immunogenicity, etc.

[0237] The above uses and methods are particularly useful for the prevention / treatment of diseases including but not limited to meningitis (especially bacterial, e.g., meningococcal meningitis) and bacteremia. For example, these are suitable for the active immunization of individuals against invasive meningococcal diseases caused by Neisseria meningitidis, preferably caused by Neisseria meningitidis serogroups A, B, C, W, or Y.

[0238] Protection against meningococcus can be measured epidemiologically, such as in clinical trials, but it is convenient to use an indirect method of measurement to confirm that the immunogenic composition elicits a serum bactericidal antibody (SBA) response in the recipient. In the SBA assay, serum from the recipient of the composition is incubated with the target bacteria (meningococcus in the present invention) in the presence of complement (preferably human complement, although rabbit complement is often used instead), and bactericidal activity of the bacteria is evaluated at various dilutions of the serum to determine SBA activity. The results observed in the SBA assay can be reinforced by performing a competitive SBA assay to provide additional indirect evidence of the immunogenic activity of the target antigen. In the competitive SBA assay, serum from the recipient of the immunogenic composition containing the antigen is pre-incubated with the antigen, followed by incubation with the target bacteria in the presence of human complement. Bactericidal activity of the bacteria is then evaluated, and if the bactericidal antibodies in the recipient's serum bind to the target antigen during the pre-incubation step and thus cannot bind to the surface antigen of the bacteria, the bactericidal antibodies are reduced or absent.

[0239] The composition need not protect against all individual strains of meningococcus, nor must all individual recipients of the composition be protected. Such universal protection is not the standard in the art. Rather, protection is typically evaluated against a panel of reference laboratory strains that are selected country-by-country and likely change over time, and is measured across a population of recipients.

[0240] Preferred compositions of the invention can confer on a permissible percentage of human subjects antibody titers that exceed the criteria for serum protection against each antigenic component in the patient. Antigens with antibody titers at which a host seroconversion to the antigen is recognized are well known, and such antibody titers have been published by organizations such as the WHO. It is preferred that more than 80% of a statistically significant sample of subjects seroconvert, more preferably more than 90%, even more preferably more than 93%, and most preferably 96 - 100%.

[0241] An immunogenic composition comprises an immunologically effective amount of an immunogen and optionally any other specified components.

[0242] "An immunologically effective amount" means that when that amount is administered to an individual as a single dose or as part of a series of doses, it is effective for treatment or prevention.

[0243] The term "prevention" means that the progression of a disease is reduced and / or eliminated, or the onset of a disease is eliminated. For example, the immune system of a subject is primed (e.g., by vaccination) to mount an immune response to fend off an infection, and the onset of the disease is eliminated.

[0244] A vaccinated subject may still become infected, but has a better ability to fend off the infection than an unvaccinated control subject. This amount varies depending on the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (e.g., non-human primate, primate), the antibody synthesis ability of the individual's immune system, the degree of protection desired, the vaccine formulation, the medical situation assessment by the treating physician, and other relevant factors. This amount is expected to fall within a relatively wide range that can be determined through routine testing.

[0245] This composition can be administered in combination with other immunomodulatory agents.

[0246] Vaccine efficacy The recombinant vaccine composition used in the present invention preferably has a vaccine efficacy of at least 10%, such as ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥85%, ≥90%, or more against each strain of Neisseria meningitidis.

[0247] The efficacy of the vaccine is determined by a reduction in the relative risk of developing a meningococcal disease in a subject administered the composition of the invention compared to a subject not administered such a composition (e.g., a non-immunized subject, or a subject administered a placebo or negative control). Thus, the incidence rate of meningococcal disease in a population immunized according to the invention is compared to the incidence rate in a control population not immunized according to the invention to calculate the relative risk, and the efficacy of the vaccine is the value obtained by subtracting this number from 100%.

[0248] The efficacy of the vaccine is determined for a population, not an individual. Thus, while it is a useful epidemiological tool, it does not predict individual protection. For example, an individual subject may be exposed to a very large inoculum of the infectious agent or have other risk factors that make them more susceptible to infection, but this does not negate the validity or usefulness of the efficacy measurement. The size of the population immunized according to the invention and in which the efficacy of the vaccine is measured is ideally at least 100 subjects, probably more, e.g., at least 500 subjects. The size of the control group should also be at least 100 subjects, e.g., at least 500 subjects.

[0249] All references or patent applications cited within this specification are hereby incorporated by reference into this specification. Aspects of the invention are summarized in the numbered items below: 1. An immunogenic composition against Neisseria meningitidis serogroup B, which is a liquid formulation comprising a fusion polypeptide of a Neisseria meningitidis NHBA antigen, a Neisseria meningitidis NadA antigen, a Neisseria meningitidis fHbp antigen, Neisseria meningitidis outer membrane vesicles (OMV), and a Neisseria meningitidis fHbp polypeptide, an adsorbent for said antigen and polypeptide, and one or more pharmaceutically acceptable tonicity modifiers. 2. The immunogenic composition of item 1, wherein the tonicity modifier is selected from sodium chloride, sorbitol, sucrose, and mixtures thereof. 3. The immunogenic composition according to either item 1 or 2, wherein the liquid formulation comprises an aqueous solution of sodium chloride and sucrose. 4. The immunogenic composition according to any one of items 1 to 3, wherein the sodium chloride concentration of the liquid preparation is ≦ 3.8 mg / ml. 5. The immunogenic composition according to any one of items 1 to 4, wherein the sodium chloride concentration of the liquid preparation is in the range of 1 mg / ml to 3.5 mg / ml, for example 2.8 mg / ml.

[0250] 6. The immunogenic composition according to any one of items 1 to 5, wherein the concentration of the fusion polypeptide of the meningococcal fHbp polypeptide is 100 to 400 μg / ml. 7. The immunogenic composition according to any one of items 1 to 6, wherein the sucrose concentration of the liquid preparation is between 2.5 and 3.5% w / v, for example 3% w / v. 8. The immunogenic composition according to any one of items 1 to 7, wherein the fusion polypeptide of the meningococcal fHbp polypeptide contains v1, v2 and v3 meningococcal fHbp polypeptides, and the sequence of the variant fHbp is in the order of v2-v3-v1 from the N-terminus to the C-terminus. 9. The immunogenic composition according to item 8, wherein the v1 fHbp polypeptide is a mutant v1.13 fHbp polypeptide. 10. The immunogenic composition according to item 9, wherein the mutant v1.13 fHbp polypeptide has an amino acid sequence different from SEQ ID NO: 16 by at least one of the substitutions E211A, S216R and E232A.

[0251] 11. The immunogenic composition according to any one of items 1 to 10, wherein the meningococcal NHBA antigen and the meningococcal fHbp antigen are fusion proteins with meningococcal accessory proteins, such as NHBA-GNA1030 fusion protein and GNA2091-fHbp fusion protein. 12. The immunogenic composition according to any one of items 1 to 11, wherein the adsorbent contains an aluminum-containing compound selected from aluminum hydroxide (alum), aluminum salts and mixtures thereof. 13. The immunogenic composition according to any one of items 1 to 12, wherein the adsorbent is alum. 14. An immunogenic composition according to any one of items 1 to 13, further comprising a buffer, preferably a histidine buffer. 15. The immunogenic composition according to any one of items 1 to 14, wherein the OMV is present at 50 μg / ml, the NHBA antigen, NadA antigen, and fHbp antigen are each present at 100 μg / ml, the fusion polypeptide is present at 100 or 200 μg / ml, the sucrose is present at 2.5 to 3.5% w / v, for example 3% w / v, the buffer solution is prepared at pH 6.1 to 6.3, the alum is present at 2 to 5 mg / ml, for example 3 mg / ml, and the sodium chloride is present at 2.0 to 3.5 mg / ml, for example, 2.8 mg / ml.

[0252] 16. A reconstituted vaccine composition comprising an immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y in solid form (e.g., lyophilized) conjugated to a carrier protein and reconstituted with an immunogenic composition against Neisseria meningitidis serogroup B in liquid form as defined in items 1 to 15, comprising capsular polysaccharide antigens of Neisseria meningitidis serogroups A, C, W135, and Y. 17. The reconstituted vaccine according to item 16, wherein the immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y comprises a bulking agent optionally selected from the group consisting of sucrose, mannitol, trehalose, and mixtures thereof, preferably sucrose. 18. The reconstituted vaccine according to any one of items 16 or 17, wherein the immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y further comprises a buffer. 19. The reconstituted vaccine according to item 18, wherein the buffer solution is a phosphate buffer containing potassium dihydrogen phosphate, to which dipotassium phosphate is added. 20. The reconstituted vaccine according to any one of items 16 to 19, wherein the capsular polysaccharide antigens of Neisseria meningitidis serogroups A, C, W135, and Y are conjugated to CRM197 as a carrier protein.

[0253] 21. The reconstituted vaccine according to any one of items 16 to 20, comprising sodium chloride at a concentration of 2 to 3.5 mg / ml (e.g., 2.8 mg / ml). 22. A reconstituted vaccine composition comprising a suspension of an immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y, which contains capsular saccharide antigens of Neisseria meningitidis serogroups A, C, W135, and Y conjugated to a carrier protein in solid form (e.g., freeze-dried), in a liquid immunogenic composition against Neisseria meningitidis serogroup B as defined in items 1 to 15. 23. The immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y is the reconstituted vaccine composition of item 22 as defined in items 16 to 21. 24. A kit comprising: (i) a first container containing an immunogenic composition against Neisseria meningitidis serogroup B in liquid form as described in any of items 1 to 15; and (ii) a second container containing an immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y, which contains capsular saccharide antigens of Neisseria meningitidis serogroups A, C, W135, and Y conjugated to a carrier protein in solid form (e.g., freeze-dried), wherein the first container and the second container are separate containers or together form a multi-container. 25. The kit of item 24, wherein the first container is a prefilled syringe and the second container is a vial.

[0254] 26. The kit according to any of items 24 or 25, wherein the container or the multi-container is silicone-treated. 27. An immunogenic composition as described in any of items 1 to 15, or a reconstituted vaccine as described in any of items 16 to 21, for use as a vaccine. 28. An immunogenic composition as described in any of items 1 to 15, or a reconstituted vaccine as described in any of items 16 to 21, for use in a method for preventing or treating Neisseria meningitidis infection in a mammal, such as a human. 29. A method for preparing a reconstituted vaccine against Neisseria meningitidis serogroups A, B, C, W135, and Y, comprising the step of reconstituting a solid-form immunogenic composition against Neisseria meningitidis serogroups A, C, W135, and Y according to any one of items 16 to 21 with an immunogenic composition against Neisseria meningitidis serogroup B in liquid form as defined in any one of items 1 to 15.

[0255] 30. A method for treating or preventing Neisseria meningitidis infection or a disease caused by Neisseria meningitidis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immunogenic composition according to any one of items 1 to 15 or the reconstituted vaccine according to any one of items 16 to 21. 31. A method for inducing an immune response against Neisseria meningitidis in a subject, comprising administering a therapeutically or prophylactically effective amount of the immunogenic composition according to any one of items 1 to 15 or the reconstituted vaccine according to any one of items 16 to 21. 32. Use of the immunogenic composition according to any one of items 1 to 15 or the reconstituted vaccine according to any one of items 16 to 21 in the manufacture of a medicament for the treatment or prevention of Neisseria meningitidis infection or a disease caused by Neisseria meningitidis.

[0256] It is understood that the present invention has been described for illustrative purposes only above, and modifications can be made without departing from the scope and spirit of the present invention.

[0257] Description of the sequence listing SEQ ID NO: 1 [fHbp protein derived from MC58 strain] TIFF2025518168000021.tif27170 SEQ ID NO: 2 [fHbp protein derived from 961-5945 strain] TIFF2025518168000022.tif27170 SEQ ID NO: 3 [fHbp derived from M1239 strain] TIFF2025518168000023.tif27170 SEQ ID NO: 4 [NHBA wild type derived from MC58 strain] TIFF2025518168000024.tif34170 Sequence number 5 [NadA from strain MC58] TIFF2025518168000025.tif33170 Sequence number 6 [fHbp fusion protein] TIFF2025518168000026.tif27170 Sequence number 7 [GNA2091-fHbp fusion protein] TIFF2025518168000027.tif29170 Sequence number 8 [DG variant of NHBA] TIFF2025518168000028.tif46169 Sequence number 9 [NHBA-GNA1030 fusion] TIFF2025518168000029.tif59169 Sequence number 10 [NadA fragment] TIFF2025518168000030.tif25170 Sequence number 11 [fHbp sequence] TIFF2025518168000031.tif27170 Sequence number 12 [fHbp sequence] TIFF2025518168000032.tif27170 Sequence number 13 [GNA1030 NL TIFF2025518168000033.tif21169 Sequence number 14 [GNA2091 NL TIFF2025518168000034.tif21169 Sequence number 15 [v1.13 mature polypeptide from strain M982] TIFF2025518168000035.tif27169 Sequence number 16 [v1.13ΔG] TIFF2025518168000036.tif26169 Sequence number 17 [v1.13ΔG(E211A / E232A)] TIFF2025518168000037.tif27169 Sequence number 18 [v1.13ΔG(E211A / S216R)] TIFF2025518168000038.tif27169 Sequence number 19 [v1.15 mature polypeptide from strain NM452] ​​TIFF2025518168000039.tif14167TIFF2025518168000040.tif14168Sequence number 20 [v1.15 ΔG] TIFF2025518168000041.tif27169Sequence number 21 [v1.15 ΔG(S219R)] TIFF2025518168000042.tif27170Sequence number 22 [v1.15 ΔG(E214A / S219R)] TIFF2025518168000043.tif26170Sequence number 23 [v1.15 ΔG(E214A / E235A)] TIFF2025518168000044.tif26169Sequence number 24 [v2 wt from strain 2996] TIFF2025518168000045.tif27170Sequence number 25 [v2 mature polypeptide] TIFF2025518168000046.tif13170TIFF2025518168000047.tif13170Sequence number 26 [v2 ΔG] TIFF2025518168000048.tif27170Sequence number 27 [v3 wt from strain M1239] TIFF2025518168000049.tif27170Sequence number 28 [v3 mature] TIFF2025518168000050.tif27170Sequence number 29 [v3 ΔG] TIFF2025518168000051.tif27170Sequence number 30 [v2 ΔG S32V / L123R] TIFF2025518168000052.tif27170Sequence number 31 [v3 ΔG S32V / L126R] TIFF2025518168000053.tif13170TIFF2025518168000054.tif14168Sequence number 32 [(23S_1.13_E211A / E232A)] TIFF2025518168000055.tif66170Sequence number 33 [23S_1.13_E211A / S216R] TIFF2025518168000056.tif66170 Sequence number 34 [23S_1.15_S219R] TIFF2025518168000057.tif53169 TIFF2025518168000058.tif14170 Sequence number 35 [23S_1.15_E214A / S219R] TIFF2025518168000059.tif65169 Sequence number 36 [23S_1.15_E214A / E235A] TIFF2025518168000060.tif66169 Sequence number 37 [v1.1 ΔG+His tag] TIFF2025518168000061.tif27170 Sequence number 38 [v1.13 ΔG+His tag] TIFF2025518168000062.tif14169 TIFF2025518168000063.tif13169 Sequence number 39 [v1.13 ΔG(E211A)] TIFF2025518168000064.tif27169 Sequence number 40 [v1.13 ΔG(S216R)] TIFF2025518168000065.tif27170 Sequence number 41 [v1.15 ΔG+His tag] TIFF2025518168000066.tif27170 Sequence number 42 [v1.15 ΔG(E214A)+His tag] TIFF2025518168000067.tif27170 Sequence number 43 [fHbp231wt fusion polypeptide] TIFF2025518168000068.tif53169 TIFF2025518168000069.tif14169 Sequence number 44 [fHbp231S fusion polypeptide] TIFF2025518168000070.tif66170 Sequence number 45 [v1.13 full-length wt sequence] TIFF2025518168000071.tif27170 Sequence number 46 [v1.15 full-length wt sequence] TIFF2025518168000072.tif27169 Sequence number 47 [mature fHbp v1.1] TIFF2025518168000073.tif Sequence number 48 [Any N-terminal amino acid sequence] TIFF2025518168000074.tif Sequence number 49 [Sequence number 48 + sequence number 33; 23S_1.13_E211A / S216R with additional N-terminal amino acid sequence] TIFF2025518168000075.tif Sequence number 50 [Linker] GGSG Sequence number 51 [Linker] GGSGG Sequence number 52 [Linker] GSGSG Sequence number 53 [Linker] GSGGG Sequence number 54 [Linker] GGGSG Sequence number 55 [Linker] GSSSG Sequence number 56 [Linker] GSGGGG

Examples

[0258] The present invention is further illustrated with reference to the following non-limiting examples. Example 1: Freeze-drying of the MenACWY antigen composition - Selection of excipients By lyophilization, a vaccine component of the title suitable for reconstitution with an aqueous solution containing other vaccine components before administration in a stable form over long-term storage can be obtained.

[0259] In the lyophilization process of the MenACWY antigen composition, the addition of bulking agents was tested to evaluate the potential impact of the lyophilization process and the addition of excipients on the composition itself.

[0260] As shown below, by adding a significantly reduced amount of bulking agent, particularly sucrose, to the bulk formulation before lyophilization, compared to the inventors' experience with similar vaccines, surprising results were obtained.

[0261] As described in detail below, when verified under accelerated stability conditions, by combining a reduced bulking agent of less than 6% w / v in the pre-lyophilized bulk solution with an appropriate phosphate buffer, good results were obtained regarding antigen aggregation and residual moisture content in the lyophilized product.

[0262] For this purpose, three types of lyophilized products with sucrose concentrations of 2%, 3%, and 5% by weight relative to the total amount of the formulation, differing only in the sucrose concentration in the pre-lyophilized bulk formulation of the MenACWY antigen, were prepared.

[0263] A 10 mM phosphate buffer concentration was applied to the three types of tested pre-lyophilized bulk formulations whose compositions are summarized in Table 1.

[0264]

Table 1

[0265] The pH of the three types of formulations was adjusted by adding KOH to the buffer so that the final pH value became 7.2.

[0266] All three batches were freeze-dried with the same lyophilization cycle, then stored at 2 to 8 °C and incubated at 37 °C and 25 °C, 60% RH approximately three months after the manufacturing date.

[0267] The RM% and aggregation% of the lyophilized products were measured at 37 °C and 25 °C / 60% RH, respectively, at time zero and at the stable time (up to 2 months, 6 months). Using the Karl Fischer method, under the following conditions: Oven temperature: 110 °C Nitrogen flow rate: 60 ml / min Stop drift value: 10 μg / min The RM% of the lyophilized cake was measured using a colorimetric Karl Fischer titrator and an oven metrometer 831.

[0268] All lyophilized products manufactured from pre-lyophilized bulk formulations with different sucrose concentrations remained within the 3% RM specification limit until the end of the accelerated stability tests at two different temperatures (37 °C and 25 °C), and the absolute moisture content was similar among the three different formulations.

[0269] The percentage of aggregation was measured by SE-UPLC, and the results obtained are summarized in Table 2 below. No significant change in the aggregation rate was observed among the three different formulations tested, and no tendency for aggregation to increase over time was observed in the pre-lyophilized formulations of the present invention even under stability conditions.

[0270]

Table 2

[0271] Example 2: Preparation of the MenB antigen composition of the liquid formulation containing fHbp231.13 - Pre-formulation experiments and selection of excipients For the MenB antigen composition of the present invention containing the BEXSERO antigen, pre-formulation experiments were carried out as detailed below. To verify the behavior of the new antigen composition when formulating with excipients and buffers and adding alum as an adsorbent for the protein antigen, KPi buffer mimicking the presence of fHbp231.13 was also added at three different concentrations (100 μg / ml, 200 μg / ml, and 400 μg / ml).

[0272] In these experiments, the formulations were stored at 2 to 8 °C and 25 °C for up to 3 months, and then the adsorption rate of the MenB antigen to alum at specific time points was verified by RP-UPLC.

[0273] The results were surprising in that the fHbp-GNA2091 antigen of the BEXSERO formulation showed increased adsorption to alum at all temperatures tested, particularly at the high temperature of 25°C. In fact, in the BEXSERO composition, the fHbp-GNA2091 antigen was not completely adsorbed to alum, and it was expected that adsorption would further decrease due to the possible competitive effect among the adsorbed proteins when another antigen and additional phosphate buffer were added to the MenB composition. In this experiment, it was demonstrated that further addition of phosphate buffer in relation to the addition of the additional antigen fHbp231.13, in order to obtain a heptavalent composition containing seven MenB antigens, had no adverse effect on the overall adsorption of the antigens to alum.

[0274] Furthermore, as summarized in Table 3 below, by simultaneously varying formulation factors using a DoE (Design of Experiments) approach, different concentrations of buffer / excipient containing 3 mg / ml aluminum hydroxide, 50 μg / ml OMV antigen, and 100 μg / ml other protein antigens were evaluated for the MenB antigen liquid formulation of the present invention.

[0275]

Table 3

[0276] The experimental results showed that the NaCl concentration had a significant effect on the amount of fHbp-GNA2091 antigen adsorbed to alum. The antigen was adsorbed better at lower NaCl concentrations, and in particular, at the lowest concentration tested of 2.8 mg / ml, it was observed that the amount of antigen adsorbed was high at all different pH values tested. The sucrose concentration had less of an impact on the adsorption of the antigen to alum compared to the NaCl concentration.

[0277] To optimize the amount of excipient, the following formulations in Table 4 were prepared and tested with the maximum amount of the fHbp231.13 antigen.

[0278]

Table 4

[0279] In the tests conducted on these formulations, formulations No. 2 and 6 with a low NaCl concentration showed better results in that all MenB antigens were well adsorbed to alum, and formulation No. 6 had an optimal 3% w / v sucrose concentration in terms of the stability of the drug substance against the fHbp231.13 antigen.

[0280] The adsorption rate of the MenB antigen to alum was analyzed by RP-UPLC for each antigen according to the following method. 1 ml of the sample was centrifuged at 2100 xg for 20 minutes at 20°C, 500 μl of the supernatant was collected and mixed with 15 μl of an amphoteric ion aqueous solution. The sample was injected into the UPLC system in duplicate, and the chromatogram was recorded. Figure 1 shows a chromatogram of the overlapping profiles of formulations 2, 10, and BEXSERO compared to the standard points at a concentration of 8.0 μg / ml.

[0281] For all formulations, only the peak of fHbp-GNA2091 was detected in the chromatography profile, which is the only antigen that was not completely adsorbed to alum, indicating that adding the fHbp231.13 polypeptide to the formulations of the present invention does not promote the desorption of other MenB components. Furthermore, from the chromatogram in Figure 1, a lower NaCl concentration (formulation number 2) assisted the adsorption of fHbp-GNA2091, and it is clear that the amount of unadsorbed antigen was less compared to other formulations with a high NaCl concentration (formulation number 10, also referred to herein as "BEXSERO-like"). To avoid doubt, the expression "BEXSERO-like formulation" in this specification means a formulation of the MenB antigen of the present invention but with a NaCl concentration of 6.25 mg / ml, which is the same as the BEXSERO formulation and higher than the NaCl concentration in the excipients of the formulations of the present invention.

[0282] After reconstituting the MenACWY lyophilized composition of the present invention with a sucrose content of 2% or 3%, a similar adsorption tendency was observed. Also, similar results were observed in formulations with a low content of the fHbp231.13 antigen, particularly in formulations of 100 μg / ml and 200 μg / ml.

[0283] During this study, the influence of the buffer used to prepare the liquid formulation of the Men antigen was also evaluated, and it was found that the pH value of the histidine buffer affected the pH of the final formulation of the MenB antigen as it was and after reconstitution in the final vaccine composition. When targeting a final pH of approximately 6.5, it was found that a histidine buffer with a pH of 6.1 to 6.3 was most suitable for combination with fHbp231.13 in KPi at pH 7.2.

[0284] In this study, different concentrations of alum, such as 2.8 mg / ml and 3.0 mg / ml, were tested, and a concentration of 3.0 mg / ml was found to be more suitable and was selected for subsequent experiments.

[0285] Antigenicity evaluation - For the following formulations A, B, and D summarized in Table 5:

Table 5

[0286] Flow cytometry analysis was performed for the detection of antigens adsorbed on the surface of the alum suspension, enabling the observation of the conformational behavior of the antigens over time. Antigens adsorbed on the alum can be recognized by specific monoclonal antibodies and then detected by secondary labeled antibodies. Information regarding the conformational behavior of the antigens and their orientation on the alum was obtained according to the signal intensity and polydispersity.

[0287] Using eight different mAbs specific for each MenB antigen in the formulation of the present invention, the presence of important epitopes of the antigens was demonstrated when formulated according to the present invention in the form adsorbed on alum.

[0288] The mAbs used in the FACS analysis are described in Table 6 below, together with their main characteristics:

Table 6

[0289] The results of Formulations A and B and the results of Reference Formulation D are shown in Figures 2, 3, and 4, respectively. These demonstrate that the antigenicity is maintained in these formulations and show that all mAbs recognize their respective antigens similarly in all formulations when the antigens are present at the same concentration.

[0290] Example 4: Preparation of the MenACWY antigen freeze-dried pharmaceutical and the liquid component of the MenB antigen Based on the above results, the following compositions having the components shown in Tables 7 and 8 below were prepared.

Table 7

[0291]

Table 8

[0292] Example 5: Reconstitution of the freeze-dried MenACWY antigen composition with the liquid MenB antigen composition The composition of the vaccine in which the lyophilized MenACWY vaccine was reconstituted with the liquid component of the MenB antigen, where each dose administered corresponded to a volume of 0.5 ml, is shown in Table 9 below.

[0293]

Table 9

[0294] Appropriate procedures are defined to ensure that the lyophilized product is reconstituted with the amount of liquid formulation necessary to obtain the target dose for injection of 0.5 ml.

[0295] The reconstitution and injection procedures are designed to ensure injection of the dosage in all configurations defined for the target dosage using the materials summarized in Table 10 below.

[0296] This procedure means using the same single needle for both the reconstitution / withdrawal step and injection into the subject.

[0297]

Table 10

[0298] To prevent product contamination during use, it is necessary to follow standard hygienic practices regarding contamination prevention during reconstitution and during the recovery procedure.

[0299] For the reconstituted vaccine composition, which is an opalescent suspension, stability tests were carried out, particularly to confirm the adsorption of the MenB protein antigen to aluminum hydroxide after reconstitution. As detailed above, experiments by reverse-phase UPLC were carried out for the purpose of evaluating over time the amount of MenB antigen not adsorbed to aluminum hydroxide, particularly fHbp-GNA2091. Table 11 below summarizes the amount (μg / ml) of fHbp-GNA2091 not adsorbed to aluminum hydroxide before and after reconstitution with the MenACWY lyophilized pharmaceutical for the formulations whose composition was reported in Table 4 above.

[0300]

Table 11

[0301] At time point 0, the non-adsorbed fHbp-GNA2091 protein antigen in the BEXSERO-like formulation (Form10) containing a higher concentration of NaCl in the liquid formulation of the MenB antigen was greater than 10%, but this amount decreased over time up to 3 months, especially significantly in the samples at 25 °C. Furthermore, in the liquid formulations of the MenB antigen with less NaCl content (Form2, 4, 6, 8), the amount of non-adsorbed fHbp-GNA2091 protein antigen was less than 10% in all cases, which is considered the target limit to ensure the integrity and immunogenicity of the optimal antigen.

[0302] When reconstituted with the freeze-dried MenACWY product, the amount of non-adsorbed MenB antigen being evaluated increased in all the formulations tested, but was substantially the same regardless of the amount of sucrose tested corresponding to 2% or 3%.

[0303] Figure 5 shows, in the form of a histogram, the results obtained for all the formulations tested at time point 0 before reconstitution and after storage at 2 to 8 °C for 2 weeks and 6 weeks. Figures 6 and 7 show, in the form of a histogram, the results collected over time for all the formulations at time point 0 after reconstitution and after storage at 2 to 8 °C for 2 weeks. The reported data represent the average of three replicates. From these Figures 6 and 7, it is clear that the percentage of the fHbp-GNA2091 antigen that was not adsorbed exceeded 10% at all time points evaluated for formulation Form10, while in the formulations with less NaCl, the same antigen was adsorbed by alum.

[0304] Conclusion As shown by the results of the above experimental part, the liquid formulations of the MenB antigen composition guarantee that all relevant quality requirements of the final vaccine are met, especially with regard to the pH and osmotic pressure of the vaccine composition, the integrity and immunogenicity of each antigen and all antigens.

[0305] The fHbp231.13 polypeptide added to the MenB antigen of the BEXSERO composition was stable and proven to be compatible with other antigens in the composition when added as an adsorbent to the liquid formulation of the MenB antigen. For the liquid formulation of the MenB antigen and the reconstituted vaccine composition containing all MenABCWY antigens, optimal adsorption rates of all antigens to alum were observed.

Claims

1. An immunogenic composition against meningococcal serogroup B, comprising meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen, meningococcal outer membrane vesicles (OMV), and a fusion polypeptide of meningococcal fHbp polypeptide, and comprising an adsorbent for the antigens and polypeptides, and one or more pharmaceutically acceptable tonicity modifiers.

2. The immunogenic composition according to claim 1, wherein the tonicity regulator is selected from sodium chloride, sorbitol, sucrose, and mixtures thereof.

3. The immunogenic composition according to claim 1, wherein the liquid formulation comprises an aqueous solution of sodium chloride and sucrose.

4. The immunogenic composition according to claim 3, wherein the sodium chloride concentration of the liquid formulation is ≤3.8 mg / ml.

5. The immunogenic composition according to claim 4, wherein the sodium chloride concentration of the liquid formulation is between 1 mg / ml and 3.5 mg / ml, for example, 2.8 mg / ml.

6. The immunogenic composition according to claim 1, wherein the concentration of the fusion polypeptide of the meningococcal fHbp polypeptide is 100 to 400 μg / ml.

7. The immunogenic composition according to claim 3, wherein the sucrose concentration of the liquid formulation is between 2.5 and 3.5% w / v, for example, 3% w / v.

8. The immunogenic composition according to claim 1, wherein the fusion polypeptide of the meningococcal fHbp polypeptide comprises v1, v2, and v3 meningococcal fHbp polypeptides, and the mutant fHbp sequence is in the order v2-v3-v1 from the N-terminus to the C-terminus.

9. The immunogenic composition according to claim 8, wherein the v1fHbp polypeptide is a mutated v1.13fHbp polypeptide and optionally has an amino acid sequence different from SEQ ID NO: 16 by at least one substitution E211A, S216R and E232A.

10. The immunogenic composition according to claim 1, wherein the meningococcal NHBA antigen and the meningococcal fHbp antigen are fusion proteins with meningococcal accessory proteins, for example, NHBA-GNA1030 fusion protein and GNA2091-fHbp fusion protein.

11. The immunogenic composition according to claim 1, wherein the adsorbent comprises an aluminum-containing compound selected from aluminum hydroxide (alum), aluminum salts, and mixtures thereof, and is preferably alum.

12. The immunogenic composition according to claim 1, further comprising a buffer, preferably a histidine buffer.

13. The immunogenic composition according to claim 1, wherein the OMV is present at 50 μg / ml, the NHBA antigen, NadA antigen, and fHbp antigen are each present at 100 μg / ml, the fusion polypeptide is present at 100 or 200 μg / ml, the sucrose is present at 2.5 to 3.5% w / v, for example 3% w / v, the buffer is prepared at pH 6.1 to 6.3, alum is present at 2 to 5 mg / ml, for example 3 mg / ml, and sodium chloride is present at 2.0 to 3.5 mg / ml, for example 2.8 mg / ml.

14. A reconstituted vaccine composition comprising an immunogenic composition against meningococcal serogroup B in liquid form as defined in claim 1, comprising an immunogenic composition against meningococcal serogroups A, C, W135, and Y, and comprising capsular sugar antigens of meningococcal serogroups A, C, W135, and Y in solid form (e.g., lyophilized) conjugated to a carrier protein.

15. The reconstituted vaccine composition according to claim 14, wherein the immunogenic composition against meningococcal serogroups A, C, W135, and Y comprises a volume extender which is optionally selected from the group consisting of sucrose, mannitol, trehalose, and mixtures thereof, preferably sucrose.

16. The reconstituted vaccine composition according to any one of claims 14 or 15, wherein the immunogenic composition against meningococcal serogroups A, C, W135, and Y further comprises a buffer.

17. The reconstituted vaccine composition according to claim 16, wherein the buffer is a phosphate buffer containing monopotassium phosphate, to which dipotassium phosphate is added.

18. The reconstituted vaccine composition according to claim 14, wherein the capsular sugar antigens of meningococcal serogroups A, C, W135, and Y are conjugated to CRM197 as a carrier protein.

19. The reconstituted vaccine composition according to claim 14, comprising sodium chloride at a concentration of 2 to 3.5 mg / ml (e.g., 2.8 mg / ml).

20. (i) a first container (e.g., a pre-filled syringe) comprising an immunogenic composition against meningococcal serogroup B in liquid form as defined in claim 1; and (ii) a second container (e.g., a vial) comprising an immunogenic composition against meningococcal serogroups A, C, W135, and Y, and containing capsular sugar antigens of meningococcal serogroups A, C, W135, and Y conjugated to a solid carrier protein, wherein the first and second containers are either separate containers or together form a multi-container.

21. The kit according to claim 20, wherein the container or the multi-container is silicone-treated.

22. An immunogenic composition according to claim 1, for use as a vaccine.

23. The reconstituted vaccine composition according to claim 14 for use as a vaccine.

24. An immunogenic composition for use in a method for preventing or treating meningococcal infection in a mammal, such as a human, according to Claim 22.

25. A reconstituted vaccine composition for use in a method for preventing or treating meningococcal infection in a mammal, such as a human, according to claim 23.

26. A method for preparing a reconstituted vaccine composition for meningococcal serogroups A, B, C, W135, and Y according to claim 14, comprising the step of reconstituting an immunogenic composition for meningococcal serogroups A, C, W135, and Y in solid form according to claim 1 with an immunogenic composition for meningococcal serogroup B in liquid form as defined in claim 1.

Images