Group B meningococcal recombinant vaccine

Abstract

The present disclosure relates to an immunogenic composition comprising a combination of meningococcal antigens including at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisseria adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). The meningococcal antigens may be derived from meningococcal serogroup B. The combination of antigens provided broad coverage of bacterial strains. Additionally, the present disclosure relates to the use of the immunogenic composition in a method for eliciting an immune response.

Description

[Technical field]

[0001] Related Applications This application claims priority to European Patent Application No. 21305211.1, filed February 19, 2021, and U.S. Provisional Patent Application No. 63 / 172,885, filed April 9, 2021, the disclosures of which are incorporated by reference in their entireties herein.

[0002] Technical Field The present disclosure relates to the field of vaccines. The present disclosure relates to immunogenic compositions and vaccines for preventing meningococcal infections, such as Neisseria meningitidis (N. meningitidis or Nm) serogroup B (MenB) infections. [Background technology]

[0003] Neisseria meningitidis is a Gram-negative diplococcus whose only known natural host is humans. Neisseria meningitidis frequently colonizes the human nasopharynx and oropharynx, but is also found in other areas of the body, such as the anal mucosa, conjunctiva, and urogenital tract (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3).

[0004] At least 12 meningococcal serogroups have been classified based on the immunochemical properties of the capsular polysaccharide (PS). Some strains are more likely to cause infection than others. Worldwide, most cases of meningococcal disease are caused by serogroups A, B, C, W, X, and Y. Serogroup B is responsible for endemic disease and some outbreaks (4; 5; 6; 7; 8).

[0005] Meningococcal serogroup B is a respiratory-transmitted bacterium that cannot survive in the environment (via droplets) and requires close and prolonged contact or direct physical contact (e.g., kissing) for effective transmission. Asymptomatic carriers, present in less than 2% of children under 5 years of age and 20-25% of adolescents and young adults, are the major component in the transmission and maintenance of the pathogen in nature, even during epidemics (Non-Patent Document 9; Non-Patent Document 3).

[0006] In general, the age group with the highest carriage rates are adolescents and young adults, who tend to engage in behaviors that are recognized as risk factors for carriage and the eventual emergence of invasive meningococcal disease (IMD) (Non-Patent Document 9; Non-Patent Document 2; Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12). Vaccination of these age groups may therefore affect the incidence of IMD in other age groups, as has been demonstrated in several European countries where vaccination campaigns with serogroup C conjugate vaccines have led to population protection in unvaccinated age groups (Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15).

[0007] Invasive meningococcal disease (IMD) is a severe disease caused by Neisseria meningitidis (including meningococcal serogroup B); symptoms include severe headache, fever, nausea, vomiting, photophobia, neck stiffness, lethargy, myalgia, and a characteristic punctate rash (Non-Patent Document 4). IMD can cause meningococcal meningoencephalitis and meningococcemia. Meningococcemia is perhaps the most rapidly fatal infectious disease in humans, with approximately 90% of deaths reported within the first 2 days after hospitalization. An inflammatory syndrome may develop in 6-15% of IMD patients, which is due mainly to the deposition of antigen-antibody complexes consisting of capsular polysaccharides, specific immunoglobulins, and complement fraction C3. These reactions generally occur 4-12 days after onset and include arthritis, which is mostly monoarticular (7-14% of patients), cutaneous vasculitis, iritis, episcleritis, pleuritis, and pericarditis. Concomitantly, fever, leukocytosis, and increased serum C-reactive protein may occur.Other complications that may occur in patients with IMD include activation of herpes simplex infection, symmetric distal necrosis, extensive ulceration on vasculitis topography, gastrointestinal bleeding, subdural effusion, myocarditis, rhabdomyolysis, adult respiratory distress syndrome, acid-base and water-electrolyte abnormalities, cerebral infarction, and intracranial suppuration.

[0008] IMD survivors may develop sequelae. The risk of developing neurological sequelae is 7-12% (lower than for pneumococcal meningitis) and occurs mainly in infants. Hearing loss (permanent or transient) is the most common complication, occurring in approximately 4% of cases. Other sequelae include visual impairment, hydrocephalus, ataxia, aphasia, motor disorders, developmental delay, arthropathy, spasticity, convulsions, renal failure, osteonecrosis, atrophic scarring, loss of parts of limbs, learning disabilities, and behavioral disorders (Non-Patent Document 3; Non-Patent Document 16; Non-Patent Document 17; Non-Patent Document 18).

[0009] Serogroup B is an important cause of endemic disease and has been responsible for multiple long-term epidemics in several developed countries, including Cuba (Non-Patent Document 19), Norway (Non-Patent Document 20), and New Zealand (Non-Patent Document 21; Non-Patent Document 22) (Non-Patent Document 23). Smaller outbreaks caused by single strains have also been reported in other countries, such as France (2000-2003) (Non-Patent Document 24; Non-Patent Document 25) and the United States (2013-2017), some of which have been associated with universities (Non-Patent Document 26; Non-Patent Document 27).

[0010] Two protein-based vaccines with broad spectrum of protection targeting meningococcal group B have recently been licensed: 1) the 4-component MenB protein vaccine (4 CMeNB; GlaxoSmithKline's [GSK] BEXSERO® vaccine), licensed in the United States (US) as a 2-dose schedule for individuals aged 10-25 years and in Europe, Australia, Canada, and some South American countries for individuals aged 2 months to 50 years; 2) the bivalent recombinant fHBP protein-based (rLP2086) vaccine (Pfizer's TRUMENBA® vaccine), licensed in the US and Europe as a 2- or 3-dose schedule for individuals aged 10-25 years.

[0011] Clinical trials with both of these licensed vaccines have revealed that fever is an adverse event of particular concern for the pediatric population. Fever above 38°C was reported in up to 70% of infants receiving the BEXSERO vaccine along with their regular vaccines (6–12% had fevers above 39°C), leading to the recommendation of prophylactic use of paracetamol at the time of (and within 24 hours after) administration of the BEXSERO vaccine. In a phase I / IIb trial of the TRUMENBA vaccine in the pediatric population, fever (mostly below 39.0°C) was reported in 64% and 90% of participants receiving 20 μg or 60 μg of rLP2086, respectively, leading to early termination of the trial (Non-Patent Document 28).

[0012] Humans immunized with these vaccines generate complement-mediated serum bactericidal antibody (SBA) responses. However, for the TRUMENBA and BEXSERO vaccines, fHBP-associated SBA activity and coverage was shown to be reduced against some MenB strains, especially in young children and infants (Non-Patent Document 29; Non-Patent Document 30). It was shown that binding of host molecules to vaccine antigens reduces immunogenicity by masking important epitopes or reducing vaccine uptake, which leads to reduced antigen processing and presentation (Non-Patent Document 31). Preclinical studies have shown that binding of factor H (fH) to wild-type recombinant fHBP antigen impairs protective serum anti-fHBP antibody responses in human fH transgenic mice and infant rhesus monkeys (Non-Patent Document 32; Non-Patent Document 33). [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Rouphael et al., Methods Mol Biol. 2012;799:1-20 [Non-Patent Document 2] Stephens, Vaccine.2009;27 Suppl 2:B71~7 [Non-Patent Document 3] Batista et al., Asian Pac J Trop Med. 2017;10(11):1019-29 [Non-Patent Document 4] Harrison et al., [eds.] Orenstein WA, Offit PA, Edwards KM Plotkin SA, “Vaccines.7”, Philadelphia (PA): Elsevier; 2018, pp. 619-43. [Non-Patent Document 5] Borrow et al., Expert Rev Vaccines. 2017;16(4):313-28 [Non-Patent Document 6] Harrison et al., Emerg Infect Dis. 2013;19(4):566 - 573 [Non - Patent Document 7] Pollard, Pediatr Infect Dis J. 2004;23(12 Suppl):S274 - S279 [Non - Patent Document 8] Kvalsvig et al., J Clin Pathol. 2003;56(6):417 - 422 [Non - Patent Document 9] Christensen et al., Lancet Infect Dis. 2010;10(12):853 - 861 [Non - Patent Document 10] Bruce et al., JAMA. 2001;286(6):688 - 693 [Non - Patent Document 11] Germinario et al., Hum Vaccin. 2010;6(12):1025 - 1027 [Non - Patent Document 12] MacLennan et al., Emerg Infect Dis. 2006;12(6):950 - 957 [Non - Patent Document 13] Maiden et al., J Infect Dis. 2008;197(5):737 - 743 [Non - Patent Document 14] Trotter et al., Lancet. 2004;364(9431):365 - 367 [Non - Patent Document 15] Bijlsma et al., Clin Infect Dis. 2014;59(9):1216 - 1221 [Non - Patent Document 16] Stephens et al., "Neisseria meningitidis", in J.E. Bennett, R. Dolin and M.J. Blaser (eds.). Philadelphia: Elsevier Saunders; 2015, pp. 2425 - 2445 [Non - Patent Document 17] Campsall et al., Crit Care Clin. 2013;29(3):393 - 409

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[0014] Thus, there appears to be a continuing need for immunogenic compositions, eg, vaccines, against Neisseria meningitidis, eg, MenB, that have a high coverage with respect to bacterial strains.

[0015] There is also a need for immunogenic compositions that provide broader coverage of MenB strains than BEXSERO or TRUMENBA.

[0016] There is also a need for immunogenic compositions that have a good reactogenicity profile.

[0017] There is a need for immunogenic compositions that have good reactogenicity and safety profiles for use in infants and children.

[0018] Additionally, there is a need for immunogenic compositions that have an improved reactivity profile compared to TRUMENBA.

[0019] There is also a need for immunogenic compositions that have an improved reactivity profile compared to BEXSERO.

[0020] There is also a need for immunogenic compositions against MenB that can be readily combined with other antigens, such as meningococcal ACWY antigens, such as ACWY polysaccharide conjugated to diphtheria or tetanus toxoid.

[0021] The present disclosure is intended to meet all or some of these needs. [Means for solving the problem]

[0022] In one aspect, a multicomponent meningococcal immunogenic composition is disclosed, which comprises at least four antigens and is intended to provide broad-spectrum protection against meningococcal infections, such as invasive meningococcal disease (IMD) caused by MenB, with an acceptable safety profile for infant indication. It is composed of three major surface-exposed recombinant Neisseria proteins (Pizza et al., Science. 2000;287(5459):1816-20), selected on the basis of their important role in the pathogenesis of meningococci and their ability to induce serum bactericidal antibodies (SBA) against homologous and heterologous MenB strains, namely two non-lipidated factor H binding proteins (fHBPs) from the A and B subfamilies and the Neisseria adhesin A (NadA) protein. To improve the immunogenicity and potential strain coverage of the multicomponent MenB vaccine, outer membrane vesicles (OMVs) obtained by detergent extraction (also called detergent-extracted OMVs, dOMVs, outer membrane protein complexes (OMPCs)) were added to the formulation. The dOMVs were derived from MenB strains, e.g., strains expressing PorA proteins, e.g., PorA VR2 P1.2. The efficacy of dOMVs is associated with bactericidal antibodies directed primarily against the cognate PorA, which is highly abundant on the bacterial surface.

[0023] Unexpectedly, the inventors have observed that the combination of MenB antigens in the immunogenic compositions disclosed herein confers on the composition broad coverage against MenB strains capable of eliciting an immune response, while inducing low reactogenic and proinflammatory effects.

[0024] Indeed, as shown in the Examples section, the immunogenic compositions, e.g., vaccines, of the present disclosure allow for a broader coverage of MenB strains than TRUMENBA or BXSERO. Notably, the immunogenic compositions disclosed herein elicited protective immune responses against six MenB strains not covered by BXSERO.

[0025] Advantageously, the antigens of the immunogenic compositions disclosed herein provide cross-protection between pandemic IMD-causing MenB strains.

[0026] Advantageously, the immunogenic composition disclosed herein comprises two non-lipidated fHBP recombinant antigens, A05 (Pfizer classification, Novartis nomenclature variant 3.45 or Peptide ID 45 according to PubMLST nomenclature) and B01 (Pfizer classification, Novartis nomenclature variant 1.55 or PubMLST nomenclature peptide ID 55), one variant antigen each from two genetically and immunologically diverse subfamilies of fHBP, ensuring broad protection against all MenB strains and allowing indication in infants from 6 weeks of age onwards.

[0027] As an additional advantage, the immunogenic compositions disclosed herein include dOMVs that can induce specific protective responses against strains expressing homologous PorA, such as PorA VR2 strains, and, for example, PorA VR2 P1.2 strains, as well as protective cross-reactivity against heterologous strains.

[0028] A further advantage of the immunogenic compositions disclosed herein is that they induce an immune response against MenB strains from ST-41 / 44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and the ST-461 clonal complex. Another advantage is that the immunogenic compositions disclosed herein induce an immune response against the ST-11 clonal complex, an emerging highly virulent clonal complex.

[0029] Moreover, as shown in the Examples section, the immunogenic compositions disclosed herein show a reactogenicity profile comparable to that of BEXSERO, which is improved (i.e., less reactogenic) compared to TRUMENBA, and overall less reactogenic. Notably, the disclosed immunogenic compositions show a weaker proinflammatory cytokine response than TRUMENBA. Furthermore, they have only a minimal effect on cell viability.

[0030] According to one of its aspects, the present disclosure relates to an immunogenic composition comprising a combination of meningococcal antigens, said combination comprising at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisseria adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV), wherein the antigens are present in an immunologically effective amount.

[0031] In one embodiment, the meningococcal antigen may be from N. meningitidis serogroup B.

[0032] According to another of its aspects, the present disclosure relates to an immunogenic composition comprising a combination of meningococcal serogroup B antigens comprising at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). The fHBP A protein and / or the fHBP B protein are non-lipidated.

[0033] In another embodiment, the fHBP A protein may be a lipidated or non-lipidated protein, for example, it is a non-lipidated protein.

[0034] In another embodiment, the fHBP B protein may be a lipidated or non-lipidated protein, for example, it is a non-lipidated protein.

[0035] In another embodiment, the fHBP A protein and / or the fHBP B protein are non-lipidated. In one embodiment, both the fHBP A protein and the fHBP B protein are non-lipidated.

[0036] In one embodiment, the fHBP A protein can be a non-naturally occurring fHBP.

[0037] In one embodiment, the fHBP B protein can be a non-naturally occurring fHBP.

[0038] In one embodiment, the fHBP A and / or fHBP B protein can be a non-naturally occurring fHBP.

[0039] In one embodiment, the fHBP A and / or fHBP B protein can be a mutated fHBP. In one embodiment, the fHBP A and / or fHBP B protein can be a mutated, non-lipidated fHBP.

[0040] In one embodiment, the fHBP A protein can be a mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1.

[0041] In another embodiment, the fHBP A protein may comprise at least one amino acid substitution selected from at least one of the following, based on the numbering of SEQ ID NO:6: a) an amino acid substitution of asparagine at amino acid 115 (N115); b) an amino acid substitution of aspartic acid at amino acid 121 (D121); c) an amino acid substitution of serine at amino acid 128 (S128); d) an amino acid substitution of leucine at amino acid 130 (L130); e) an amino acid substitution of valine at position 131 (V131); f) an amino acid substitution of glycine at position 133 (G133); g) an amino acid substitution of lysine at position 219 (K219); and h) an amino acid substitution of glycine at position 220 (G220).

[0042] In one embodiment, the fHBP A protein may be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprising at least one amino acid substitution selected from at least one of the following based on the numbering of SEQ ID NO:6: a) an amino acid substitution of asparagine (N115) at amino acid 115; b) an amino acid substitution of aspartic acid (D121) at amino acid 121; c) an amino acid substitution of serine (S128) at amino acid 128; d) an amino acid substitution of leucine (L130) at amino acid 130; e) an amino acid substitution of valine (V131) at position 131; f) an amino acid substitution of glycine (G133) at position 133; g) an amino acid substitution of lysine (K219) at position 219; and h) an amino acid substitution of glycine (G220) at position 220.

[0043] In one embodiment, the fHBP A protein can include at least the amino acid substitution G220S based on the numbering of SEQ ID NO: 6. The fHBP A protein can be a non-lipidated protein that includes at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO: 1 and includes at least the amino acid substitution G220S based on the numbering of SEQ ID NO: 6.

[0044] In one embodiment, the fHBP A protein may comprise at least three amino acid substitutions selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. In another embodiment, the fHBP A protein may comprise only three amino acid substitutions, G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6. The fHBP A protein may be a mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity with SEQ ID NO: 1 and comprises at least three amino acid substitutions, or only those, selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6.

[0045] In another embodiment, the fHBP A protein may comprise or consist of SEQ ID NO:2.

[0046] In one embodiment, the fHBP B protein can be a mutein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:3.

[0047] In another embodiment, the fHBP B protein may comprise at least one amino acid substitution selected from at least one of: a) an amino acid substitution at amino acid 38 with glutamine (Q38); b) an amino acid substitution at amino acid 92 with glutamic acid (E92); c) an amino acid substitution at amino acid 130 with arginine (R130); d) an amino acid substitution at amino acid 223 with serine (S223); and e) an amino acid substitution at amino acid 248 with histidine (H248), based on the numbering of SEQ ID NO:6.

[0048] In one embodiment, the fHBP B protein may be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:3, and comprising at least one amino acid substitution selected from at least one of: a) an amino acid substitution of glutamine at amino acid 38 (Q38); b) an amino acid substitution of glutamic acid at amino acid 92 (E92); c) an amino acid substitution of arginine at amino acid 130 (R130); d) an amino acid substitution of serine at amino acid 223 (S223); and e) an amino acid substitution of histidine at amino acid 248 (H248), based on the numbering of SEQ ID NO:6.

[0049] In another embodiment, the fHBP B protein may include at least the amino acid substitution H248L based on the numbering of SEQ ID NO: 6. In another embodiment, the fHBP B protein may include only the amino acid substitution H248L based on the numbering of SEQ ID NO: 6. The fHBP B protein may be a mutein that includes at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO: 3 and includes at least, or only, the amino acid substitution H248L based on the numbering of SEQ ID NO: 6.

[0050] In another embodiment, the fHBP B protein may comprise or consist of SEQ ID NO:4.

[0051] In one embodiment, fHBP A protein and / or fHBP B may be present in an amount ranging from about 20 μg / dose to about 200 μg / dose, or from about 25 μg / dose to about 180 μg / dose, or from about 40 μg / dose to about 140 μg / dose, or from about 50 μg / dose to about 120 μg / dose, or from about 75 μg / dose to about 100 μg / dose, or from about 25 μg / dose, or from about 50 μg / dose, or from about 100 μg / dose.

[0052] As used herein, the term "dose" refers to the total amount or volume of a composition administered to an individual. The dose may range from about 0.1 ml to about 1 ml, for example, from about 0.2 ml to about 0.8 ml, from about 0.4 ml to about 0.6 ml, or may be about 0.5 ml.

[0053] In one embodiment, the NadA protein may be a NadA1 protein.

[0054] In another embodiment, the NadA protein may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO: 5. In another embodiment, the NadA protein may comprise or consist of SEQ ID NO: 5.

[0055] In one embodiment, the NadA protein may be present in an amount ranging from about 20 μg / dose to about 200 μg / dose, or from about 25 μg / dose to about 180 μg / dose, or from about 40 μg / dose to about 140 μg / dose, or from about 50 μg / dose to about 120 μg / dose, or from about 75 μg / dose to about 100 μg / dose, or from about 25 μg / dose, or from about 50 μg / dose, or from about 100 μg / dose. In one embodiment, the NadA protein may be present in an amount of about 50 μg / dose.

[0056] In one embodiment, the dOMV may comprise the outer membrane protein Porin A (PorA). In one embodiment, the dOMV may comprise the outer membrane protein Porin B (PorB). In another embodiment, the dOMV may comprise the outer membrane protein Porin A (PorA) and the outer membrane protein Porin B (PorB).

[0057] In another embodiment, the dOMV may comprise the outer membrane protein Porin A (PorA) and / or the outer membrane protein Porin B (PorB). The dOMV may comprise Porin A (serotype PorA VR2 P1.2), Porin B (serotype PorB P2.2a), and optionally, immunotype LOS L3,7. PorA and PorB may represent approximately 50% of the protein of the dOMV.

[0058] PorA may be present in an amount ranging from about 3% to about 15%, or from about 5% to about 9% or about 10% of the total protein present in the dOMV.PorB may be present in an amount ranging from about 30% to about 70%, or from about 35% to about 65%, or from about 38% to about 58% of the total protein present in the dOMV.

[0059] In one embodiment, the dOMV may comprise Porin A (PorA). In one embodiment, the PorA may be a PorA of the VR2 family. In one embodiment, the dOMV may comprise a PorA VR2 P1.2 subtype.

[0060] In one embodiment, the dOMV can be derived from MenB strain 99M expressing PorA VR2, P1.2.

[0061] In one embodiment, the dOMV may comprise PorA VR2 P1.2 and PorB P2.2a.

[0062] In one embodiment, dOMVs can be obtained by a detergent extraction method using at least a deoxycholate treatment step.

[0063] In one embodiment, dOMV may be present in an amount ranging from about 5 μg / dose to about 400 μg / dose, or from about 10 μg / dose to about 300 μg / dose, or from about 25 μg / dose to about 250 μg / dose, or from about 35 μg / dose to about 225 μg / dose, or from about 50 μg / dose to about 200 μg / dose, or from about 75 μg / dose to about 180 μg / dose, or from about 100 μg / dose to about 150 μg / dose, or from about 110 μg / dose to about 125 μg / dose, or about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0064] In another embodiment, the composition disclosed herein may further comprise an adjuvant.In one embodiment, the adjuvant may be an aluminum-based adjuvant.In one embodiment, the aluminum-based adjuvant may be an aluminum-based adjuvant selected from the group comprising aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate salt adjuvant, aluminum sulfate hydroxyphosphate adjuvant, aluminum potassium sulfate adjuvant, aluminum hydroxycarbonate, a combination of aluminum hydroxide and magnesium hydroxide, and mixtures thereof.In one embodiment, the adjuvant may be an aluminum phosphate adjuvant.

[0065] In another embodiment, the compositions disclosed herein may further comprise a pharma- ceutically acceptable excipient.

[0066] In another embodiment, the composition disclosed herein may further comprise a buffer. In one embodiment, the buffer is selected in the group including Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer, or histidine buffer. In one embodiment, the buffer may be an acetate buffer. In one embodiment, the acetate buffer may be a sodium acetate buffer. In one embodiment, the sodium acetate buffer may be present at a concentration in the range of about 10 mM to about 300 mM, or in the range of about 10 mM to about 250 mM, or in the range of about 20 mM to about 250 mM, or in the range of about 20 mM to about 150 mM, or in the range of about 20 mM to about 130 mM, or in the range of about 30 mM to about 120 mM, or in the range of about 40 mM to about 100 mM, or in the range of about 50 mM to about 80 mM, or in the range of about 50 mM to about 60 mM, or at a concentration of, for example, about 50 mM.

[0067] In another embodiment, the compositions disclosed herein may further comprise a salt, for example, a sodium salt, a calcium salt, or a magnesium salt. The sodium salt may be, for example, a sodium salt selected from the group including sodium chloride, sodium phosphate. In one embodiment, the sodium salt may be sodium chloride. The calcium salt may be a calcium chloride salt. The magnesium salt may be a magnesium chloride salt. In one embodiment, the sodium salt may be present at a concentration ranging from about 10 mM to about 300 mM, or from about 30 mM to about 280 mM, or from about 50 mM to about 250 mM, or from about 60 mM to about 220 mM, or from about 80 mM to about 200 mM, or from about 100 mM to about 180 mM, or from about 120 mM to about 160 mM, or may be present at a concentration of, for example, about 150 mM. The calcium salt or magnesium salt may be present in an amount ranging from about 1 mM to about 15 mM, or from about 5 mM to about 10 mM.

[0068] In another embodiment, the compositions disclosed herein may have a pH in the range of about 4.0 to about 9.0. In one embodiment, the pH of the compositions disclosed herein may range from about 4.5 to about 8.5, or about 4.8 to about 8.2, or about 5.0 to about 8.0, or about 5.2 to about 7.5, or about 5.4 to about 7.0, or about 5.5 to about 6.8, or about 5.7 to about 6.5, or about 5.8 to about 6.2, or may be about 6.0. In another embodiment, the compositions disclosed herein may comprise or consist of a non-lipidated fHBP A protein comprising or consisting of SEQ ID NO:2, a non-lipidated fHBP B protein comprising or consisting of SEQ ID NO:4, a NadA protein, and a dOMV derived from MenB expressing a PorA protein. In the composition, NadA may be NadA1, or may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity with SEQ ID NO:5, or may consist of SEQ ID NO:5, and / or dOMV may comprise PorA VR2 subtype or PorA VR2 P1.2, and optionally PorB P2.2a, or may be obtained from MenB strain 99M. The compositions disclosed herein may comprise an aluminum phosphate adjuvant. The compositions disclosed herein may comprise 50 mM acetate buffer and pH 6.0.

[0069] In another embodiment, the compositions disclosed herein may comprise or consist of a non-lipidated mutant fHBP A protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1, a non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least 99%, or at least about 99.5% identity to SEQ ID NO:3, a NadA protein from MenB expressing a PorA protein, and a dOMV. In the composition, NadA may be NadA1, or may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity with SEQ ID NO:5, or may consist of SEQ ID NO:5, and / or dOMV may comprise PorA VR2 subtype or PorA VR2 P1.2, and optionally PorB P2.2a, or may be obtained from MenB strain 99M. The compositions disclosed herein may comprise an aluminum phosphate adjuvant. The compositions disclosed herein may comprise 50 mM acetate buffer and pH 6.0.

[0070] In another embodiment, the composition disclosed herein comprises a non-lipidated mutant fHBP comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprising at least one amino acid substitution selected from at least one of the following, based on the numbering of SEQ ID NO:6: a) an amino acid substitution of asparagine at amino acid 115 (N115); b) an amino acid substitution of aspartic acid at amino acid 121 (D121); c) an amino acid substitution of serine at amino acid 128 (S128); d) an amino acid substitution of leucine at amino acid 130 (L130); e) an amino acid substitution of valine at position 131 (V131); f) an amino acid substitution of glycine at position 133 (G133); g) an amino acid substitution of lysine at position 219 (K219); and h) an amino acid substitution of glycine at position 220 (G220). The fHBP A protein may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:3, and may include at least one amino acid substitution selected from at least one of: a) an amino acid substitution of glutamine at amino acid 38 (Q38); b) an amino acid substitution of glutamic acid at amino acid 92 (E92); c) an amino acid substitution of arginine at amino acid 130 (R130); d) an amino acid substitution of serine at amino acid 223 (S223); and e) an amino acid substitution of histidine at position 248 (H248), based on the numbering of SEQ ID NO:6. In the composition, NadA may be NadA1 or may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5, or may consist of SEQ ID NO:5, and / or dOMV may comprise PorA VR2 subtype or PorA VR2 P1.2, and optionally PorB P2.2a, or may be derived from MenB strain 99M. The compositions disclosed herein may include an aluminum phosphate adjuvant.The compositions disclosed herein can comprise or consist of 50 mM acetate buffer and pH 6.0.

[0071] In another embodiment, the composition disclosed herein may comprise or consist of a non-lipidated mutant fHBP A protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprising at least one amino acid substitution selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO:6, a non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:3 and comprising at least the amino acid substitution H248L based on the numbering of SEQ ID NO:6, a NadA protein, and a dOMV from MenB expressing a PorA protein. In the composition, NadA may be NadA1, or may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity with SEQ ID NO:5, or may consist of SEQ ID NO:5, and / or dOMV may comprise PorA VR2 subtype or PorA VR2 P1.2, and optionally PorB P2.2a, or may be obtained from MenB strain 99M. The composition disclosed herein may comprise an aluminum phosphate adjuvant. The composition disclosed herein may comprise 50 mM acetate buffer and pH 6.0.

[0072] In another embodiment, the composition disclosed herein may comprise or consist of a non-lipidated mutant fHBP A protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprising at least three amino acid substitutions selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO:6, a non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:3 and comprising at least the amino acid substitution H248L based on the numbering of SEQ ID NO:6, a NadA protein, and a dOMV from MenB expressing a PorA protein. In the composition, NadA may be NadA1, or may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity with SEQ ID NO:5, or may consist of SEQ ID NO:5, and / or dOMV may comprise PorA VR2 subtype or PorA VR2 P1.2, and optionally PorB P2.2a, or may be obtained from MenB strain 99M. The composition disclosed herein may comprise an aluminum phosphate adjuvant. The composition disclosed herein may comprise 50 mM acetate buffer and pH 6.0.

[0073] In another embodiment, the composition disclosed herein may comprise or consist of a non-lipidated mutant fHBP A protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprises only three amino acid substitutions selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO:6, a non-lipidated mutant fHBP B protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:3 and comprises only the amino acid substitution H248L based on the numbering of SEQ ID NO:6, a NadA protein, and a dOMV from MenB expressing a PorA protein. In the composition, NadA may be NadA1, or may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity with SEQ ID NO:5, or may consist of SEQ ID NO:5, and / or dOMV may comprise PorA VR2 subtype or PorA VR2 P1.2, and optionally PorB P2.2a, or may be obtained from MenB strain 99M. The compositions disclosed herein may comprise an aluminum phosphate adjuvant. The compositions disclosed herein may comprise 50 mM acetate buffer and pH 6.0.

[0074] In another embodiment, the composition disclosed herein can comprise or consist of non-lipidated fHBP A protein comprising or consisting of SEQ ID NO:2, non-lipidated fHBP B protein comprising or consisting of SEQ ID NO:4, NadA protein comprising or consisting of SEQ ID NO:5, dOMV derived from MenB or obtained from MenB strain 99M expressing PorA VR2 P1.2 and optionally PorB P2.2a. The composition disclosed herein can comprise an aluminum phosphate adjuvant. The composition disclosed herein can comprise 50 mM acetate buffer and pH 6.0.

[0075] In another embodiment, the compositions disclosed herein may comprise or consist of a non-lipidated fHBP A protein consisting of SEQ ID NO:2, a non-lipidated fHBP B protein consisting of SEQ ID NO:4, a NadA protein consisting of SEQ ID NO:5, and a dOMV derived from MenB or obtained from MenB strain 99M expressing PorA VR2 P1.2 and optionally PorB P2.2a. The compositions disclosed herein may include an aluminum phosphate adjuvant.

[0076] In another embodiment, the compositions disclosed herein may comprise or consist of a non-lipidated fHBP A protein consisting of SEQ ID NO:2, a non-lipidated fHBP B protein consisting of SEQ ID NO:4, a NadA protein consisting of SEQ ID NO:5, dOMVs derived from MenB or obtained from MenB strain 99M expressing PorA VR2 P1.2 and optionally PorB P2.2a, an aluminum phosphate adjuvant, 50 mM acetate buffer, and pH 6.0.

[0077] In another embodiment, the compositions disclosed herein can comprise or consist of an mRNA encoding a fHBP A protein that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:2, an mRNA encoding a fHBP B protein that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:4, an mRNA encoding a NadA protein that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5, and a dOMV from MenB expressing PorA. PorA can be PorA VR2 subtype or PorA VR2 P1.2.

[0078] In another embodiment, the compositions disclosed herein can comprise or consist of about 25 to about 100 μg / dose of non-lipidated fHBP A protein comprising SEQ ID NO:2, about 25 to about 100 μg / dose of non-lipidated fHBP B protein comprising SEQ ID NO:4, about 25 to about 100 μg / dose of NadA protein comprising SEQ ID NO:5, about 20 to about 150 μg / dose of dOMV derived from a MenB strain expressing PorA VR2 P1.2, about 100 to about 600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer, pH 6.0.

[0079] In another embodiment, the compositions disclosed herein can comprise or consist of about 25 to about 100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 25 to about 100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 25 to about 100 μg / dose of NadA protein consisting of SEQ ID NO:5, about 20 to about 150 μg / dose of dOMV derived from a MenB strain expressing PorA VR2 P1.2, about 100 to about 600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer, pH 6.0.

[0080] In another aspect, the present disclosure relates to a vaccine comprising the composition disclosed herein.

[0081] In another aspect, the present disclosure relates to the compositions disclosed herein as medicaments, particularly as vaccines.

[0082] In another aspect, the disclosure relates to a composition as disclosed herein for use in protecting against meningococcal infection, and in an exemplary embodiment, against meningococcal serogroup B infection.

[0083] In another aspect, the disclosure relates to a composition as disclosed herein for use in inducing an immune response against Neisseria meningitidis, and in an exemplary embodiment, against Neisseria meningitidis serogroup B bacteria.

[0084] In another aspect, the disclosure relates to a composition as disclosed herein for use in inducing an immune response against meningococcal serogroup B bacteria from ST-41 / 44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and / or the ST-461 clonal complex.

[0085] In another aspect, the disclosure relates to a composition as disclosed herein for use in inducing an immune response against Neisseria meningitidis serogroup B bacteria from the ST-11 clonal complex.

[0086] In another aspect, the disclosure relates to a method for protecting an individual against meningococcal infection, and in an exemplary embodiment, against meningococcal serogroup B infection, comprising at least the step of administering to the individual an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0087] In another aspect, the disclosure relates to a method of reducing the risk of meningococcal infection, and in an exemplary embodiment, the development of invasive meningococcal disease caused by meningococcal serogroup B infection, in an individual, comprising at least the step of administering to the individual an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0088] In another aspect, the disclosure relates to a method for eliciting an immune response in an individual against Neisseria meningitidis, and in an exemplary embodiment against Neisseria meningitidis serogroup B bacteria, comprising at least the step of administering to the individual an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0089] In another aspect, the disclosure relates to a method for eliciting an immune response against meningococcal serogroup B bacteria from ST-41 / 44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and / or the ST-461 clonal complex, comprising at least the step of administering to an individual an immunogenic composition as disclosed herein, or a vaccine as disclosed herein.

[0090] In another aspect, the disclosure relates to a method for eliciting an immune response against meningococcal serogroup B bacteria from the ST-11 clonal complex, the method comprising at least the step of administering to an individual an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0091] In another aspect, the present disclosure relates to a method for preparing an immunogenic composition as disclosed herein or a vaccine as disclosed herein, comprising at least the step of mixing meningococcal antigens, including at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV), and optionally an aluminum salt.

[0092] In one embodiment, the step of mixing may optionally include AlPO 4 of at least one factor H binding protein (fHBP) A protein adsorbed onto the 4 A first mixture of at least one fHBP B protein adsorbed on a salt is optionally combined with AlPO 4 of at least one NadA protein adsorbed onto said mixture and with a second mixture of dOMVs.

[0093] In one embodiment, the step of mixing comprises mixing AlPO 4 of at least one factor H binding protein (fHBP) A protein adsorbed onto AlPO 4 A first mixture of at least one fHBP B protein adsorbed onto a salt is added to AlPO 4 of at least one NadA protein adsorbed onto said mixture and with a second mixture of dOMVs.

[0094] In another embodiment, the antigen and aluminum salt for use in the methods for preparation disclosed herein may be in a buffer.

[0095] In another aspect, the present disclosure relates to a kit of parts comprising a plurality of containers, each of the containers comprising at least one meningococcal antigen or a combination of at least two meningococcal antigens, said antigens being selected from the group comprising fHBP A protein, fHBP B protein, NadA protein, and detergent-extracted outer membrane vesicles (dOMV).

[0096] In one embodiment, at least one of the antigens of the kit of parts may be in a dry form. In one embodiment, at least one of the antigens may be in a lyophilized form or a dry micropellet form. In one embodiment, the kit may optionally include a container that contains a physiologically injectable vehicle. [Brief description of the drawings]

[0097] [Figure 1A] Figure 1A shows specific anti-A05tmN (Figure 1A) or anti-B01smN (Figure 1B) IgG titers measured by ELISA in sera collected on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56 (LLOQ means lower limit of quantification). [Figure 1B] Figure 1A shows specific anti-A05tmN (Figure 1A) or anti-B01smN (Figure 1B) IgG titers measured by ELISA in sera collected on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56 (LLOQ means lower limit of quantification). [Figure 2A] FIG. 1 shows specific anti-NadA IgG titers measured by ELISA in sera collected on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with BEXSERO or with formulations F1-F5 and F1 co-administered with MENQUADFI on days 0 and 28. [Figure 2B] FIG. 1 shows specific anti-dOMV IgG measured by ELISA in sera collected on days 0 and 42 from rabbits immunized with formulations F1-F5 and F1 co-administered with MENQUADFI on days 0, 28 and 56. [Diagram 3] FIG. 1 shows hSBA measured against the closely related MenB B44 strain (strain no. 3) in purified IgG on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA, BEXSERO or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56. [Figure 4] FIG. 1 shows hSBA measured against the heterologous MenB B24 strain (strain no. 4) in purified IgG on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA, BEXSERO or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56. [Diagram 5] FIG. 1 shows hSBA measured against the heterologous MenB B24 strain (strain no. 18) in purified IgG on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA, BEXSERO or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56. [Figure 6] FIG. 1 shows hSBA measured against the closely related MenB A56 strain (strain no. 1) in purified IgG on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA, BEXSERO or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56. [Figure 7]FIG. 1 shows hSBA measured against the heterologous MenB A22 strain (strain no. 2) in purified IgG on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA, BEXSERO or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56. [Figure 8] FIG. 1 shows hSBA measured against MenB NadA1 strain (strain no. 6) in purified IgG on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA, BEXSERO or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56. [Figure 9] FIG. 1 shows hSBA measured against MenB OMV PorA 1.2 strain (strain no. 5) in purified IgG on day 0 (open / white symbols) and day 42 (filled / black symbols) from rabbits immunized with TRUMENBA, BEXSERO or formulations F1-F6 or F1 co-administered with MENQUADFI on days 0, 28 and 56. [Figure 10] Figure 1 shows cytokine production (IL-6, TNFα, MIP-1β and IL-1b) of adult PTE-derived dendritic cells (DCs) treated with different doses of BEXSERO and TRUMENBA: Culture supernatants from PTEs were collected 48 hours after treatment and assessed for cytokine secretion using multiplex arrays. The graph shows the geometric mean values ​​(GMV) of cytokine production of IL-6, TNFα, IL-1β, and MIP-1β in dendritic cells (DCs). Treatment was with 10-fold dilutions of the human dose (n=20 donors). N / A is mock control without antigen. [Figure 11]Figure 1 shows cytokine production (IL-6, TNFα, MIP-1β and IL-1β) of neonatal PTE-derived DCs treated with different doses of BEXSERO and TRUMENBA: Culture supernatants from PTEs were collected 48 hours after treatment and assessed for cytokine secretion using multiplex arrays. The graph shows the GMV of cytokine production of IL-6, TNFα, IL-1β, and MIP-1β in DCs. Treatment was with 10-fold dilutions of human doses (n=20 donors). [Figure 12A] Forest plot of the geometric mean ratio with 95% confidence interval of cytokine secretion induced by BEXSERO in adult and neonatal PTE compared to TRUMENBA at a dilution of 1:10000. The dotted line is set at a value of 1, and confidence of values ​​and intervals below 1 means that BEXSERO is inferior to TRUMENBA, and values ​​and intervals above 1 mean that BEXSERO is superior to TRUMENBA. [Figure 12B] Forest plot of the geometric mean ratio with 95% confidence interval of cytokine secretion induced by BEXSERO in adult and neonatal PTE compared to TRUMENBA at a dilution of 1:10000. The dotted line is set at a value of 1, and confidence of values ​​and intervals below 1 means that BEXSERO is inferior to TRUMENBA, and values ​​and intervals above 1 mean that BEXSERO is superior to TRUMENBA. [Figure 13-1] Figure 1. Cytokine (IL-6, TNFα, MIP-1β and IL-1β) secretion induced by F1-F5 formulations in adult PTE modules. MIMIC adult PTEs were treated with different doses of F1-F5 formulations and control (BEXSERO). Culture supernatants were then harvested and assessed for cytokine secretion by multiplex array. Mean values ​​± 95% CI for IL-6, TNFα, IL-1β, and MIP-1β; n = 16-24. [Figure 13-2] Continued from Figure 13-1. [Figure 13-3] Continued from Figure 13-2. [Figure 13-4]Continued from Figure 13-3. [Figure 14] Figures 14A, 14B and 14C show forest plots of the ratio of geometric means with 95% confidence intervals of cytokine secretion induced by BEXSERO compared to formulations F1, F2, F3, F4 and F5 in adult PTE at a dilution of 1:10000. The dotted line is set at a value of 1, with confidence levels of values ​​and intervals below 1 meaning that BEXSERO is inferior to treatment, and values ​​and intervals above 1 meaning that BEXSERO is superior to treatment. Figure 14A shows the results obtained with formulations F2 and F3, and Figures 14B and 14C show the results obtained with formulations F1, F4 and F5. [Figure 15] FIG. 1 shows immune cytotoxicity (i.e., percentage of viable cells after treatment) following treatment with F1-F5 formulations, and BEXSERO in adult MIMIC PTE. [Figure 16-1] Figure 1. Increased cytokine (IL-6, TNFα, MIP-1β and IL-1β) secretion levels of F1-F5 formulations compared to BEXSERO in neonatal PTE modules. MIMIC neonatal PTEs were treated with different doses of F1-F5 formulations and control (BEXSERO). Culture supernatants were then harvested and assessed for cytokine secretion by multiplex array. Mean ± 95% CI for IL-6, TNFα, IL-1β, and MIP-1β (n=16-24). N / A is mock control without antigen. [Figure 16-2] Continued from Figure 16-1. [Figure 16-3] Continued from Figure 16-2. [Figure 16-4] Continued from Figure 16-3. [Figure 17]Figures 17A and 17B show forest plots of the ratio of geometric means with 95% confidence intervals of cytokine secretion induced by BEXSERO compared to formulations F1, F2, F3, F4 and F5 in neonatal PTE at a dilution of 1:10000. The dotted line is set at a value of 1, and confidence levels of values ​​and intervals below 1 mean that Bexsero® is inferior to treatment, and values ​​and intervals above mean that Bexsero® is superior to treatment. Figure 17A shows the results obtained with the F2 and F3 formulations, and Figure 17B shows the results obtained with the F1, F4 and F5 formulations. [Figure 18] FIG. 1 shows immune cytotoxicity (i.e., percentage of live cells after treatment) following treatment with F1-F5 formulations, and BEXSERO in neonatal MIMIC PTE. [Figure 19] Figure 1 shows a summary of hSBA results illustrating different formulations tested on 18 MenB strains from different clonal complexes: different clonal complexes covered or not covered by F3, TRUMENBA and BEXSERO. Black arc: BEXSERO. Dotted arc: TRUMENBA. Grey arc: F3 formulation. Numbers within the arcs indicate the number of MenB strains covered by the formulation out of the total number of MenB strains tested per clonal complex. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] Array Description SEQ ID NO:1 represents the fHBP A05 wild type sequence without the signal peptide responsible for lipidation.

[0099] CSSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSL INQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

[0100] SEQ ID NO: 2 represents the mutated fHBP A05 sequence without the signal peptide responsible for lipidation and with the mutations G220S, L130R, G133D (numbering is determined with reference to SEQ ID NO: 6 (fHBP B24)).

[0101] CSSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSL INQRSFRVSDLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKSTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

[0102] SEQ ID NO:3 represents the fHBP B01 wild type sequence without the signal peptide responsible for lipidation.

[0103] CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVA KRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHHIGLAAKQ

[0104] SEQ ID NO: 4 represents the mutated fHBP B01 sequence without the signal peptide responsible for lipidation and with the mutation H248L (numbering is determined with reference to SEQ ID NO: 6 (fHBP B24)).

[0105] CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVA KRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHLIGLAAKQ

[0106] SEQ ID NO:5 represents the NadA1 sequence from MenB strain MC58 in which 23 amino acids of the N-terminal signal peptide and the last 55 amino acids of the C-terminus are deleted.

[0107] MTSDDDVKKAATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKID EKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVG

[0108] SEQ ID NO:6 represents the fHBP B24 wild type sequence, and the numbering of the positions of the mutations in A05 and B01 is determined accordingly.

[0109] CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKR QFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ

[0110] SEQ ID NO:7 represents the wild-type NadA1 sequence from MenB strain MC58.

[0111] MKHFPSKVLTTAILATFCSGALAATSDDDVKKAATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEA FNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFRFTENFAAKAGVAVGTSSGSSAAYHVGVNYEW

[0112] definition It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an antigen" includes a plurality of such antigens, a reference to "the protein" includes a reference to one or more proteins, and so forth.

[0113] The term "about" or "approximately" as used herein refers to the usual error range for each value, which is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. In some embodiments, the term "about" refers to ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% of the given value. However, whenever the value in question refers to an indivisible entity, e.g., a molecule or other entity that loses its identity once subdivided, "about" refers to ±1 of the indivisible entity.

[0114] The term "antigen" includes any molecule, e.g., a peptide, protein, polysaccharide, or glycoconjugate, that contains at least one epitope that elicits and / or is the subject of an immune response. For example, an antigen is a molecule that elicits an immune response, optionally after processing, that is specific, e.g., against the antigen or a cell expressing the antigen. After processing, the antigen can be presented by MHC molecules to react specifically with T lymphocytes (T cells). Thus, the antigen or a fragment thereof must be recognizable by a T cell receptor and, in the presence of an appropriate co-stimulatory signal, induce the clonal expansion of T cells bearing a T cell receptor that specifically recognizes the antigen or fragment, resulting in an immune response against the antigen or a cell expressing the antigen. According to the present disclosure, any suitable antigen that is a candidate for an immune response can be envisaged. The antigen may correspond to or be derived from a naturally occurring antigen.

[0115] It will be understood that the aspects and embodiments of the disclosure described herein include "having," "including," "consisting," and "consisting essentially of" aspects and embodiments. It will be understood that the words "having" and "comprising," or variations such as "having," "having," "consisting," or "consisting of," imply the inclusion of the recited elements (e.g., a composition of matter or method step) but not the exclusion of any other elements. The term "consisting of" means including the recited elements and excluding additional elements. The term "consisting essentially of" means including the recited elements and may include other elements if the other elements do not materially affect the basic and novel characteristics of the disclosure. It will be understood that various embodiments of the disclosure that use the term "consisting of" or equivalents include within their scope embodiments in which the term is replaced with "consisting of" or "consisting essentially of."

[0116] The phrase "disease caused by a strain of Neisseria meningitidis" encompasses any clinical symptom or combination of clinical symptoms present in human infection with Neisseria meningitidis, including, but not limited to, colonization of the upper respiratory tract (e.g., mucosa of the nasopharynx and tonsils) with pathogenic strains of Neisseria meningitidis, bacterial penetration of mucosal and submucosal vascular beds, sepsis, septic shock, inflammation, hemorrhagic skin lesions, activation of fibrinolysis and blood coagulation, organ dysfunction, e.g., renal, pulmonary, and cardiac failure, adrenal hemorrhage and muscle infarction, capillary leak, edema, peripheral limb ischemia, respiratory distress syndrome, pericarditis, and meningitis.

[0117] As used herein, the terms "individual" or "subject" or "patient" are used interchangeably and are intended to refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In some exemplary embodiments, the individual or subject is a human.

[0118] In the context of the present disclosure, the phrase "pharmaceutical acceptable carrier" refers to a carrier or vehicle that is physiologically acceptable for administration to a mammal, e.g., a human, while retaining the physiological activity of the immunogenic composition disclosed herein, i.e., its ability to elicit an immune response with low reactogenic effect.

[0119] As used herein, with respect to a disease or disorder, the terms "prevent", "preventing" or "delaying progression" (and their grammatical variations) refer to prophylactic treatment of a disease or disorder, for example, in an individual suspected of having the disease or at risk of developing the disease. Prevention includes, but is not limited to, preventing or delaying the onset or progression of a disease, and / or maintaining one or more symptoms of a disease or disorder at a desired or subpathological level. The term "preventing" does not require 100% elimination of the possibility or likelihood of the occurrence of an event. Instead, it indicates that the likelihood of the occurrence of an event has been reduced in the presence of the compositions or methods described herein.

[0120] The term "protective immunity" means that a vaccine or immunization schedule administered to a mammal induces an immune response that prevents, delays the onset or reduces the severity of disease caused by meningococcus, or attenuates or completely eliminates the symptoms of the disease. Protective immunity may involve the production of bactericidal antibodies. It should be noted that the production of bactericidal antibodies against meningococcus is recognized in the art as predictive of the protective efficacy of a vaccine in humans (Goldschneider et al. (1969) J. Exp. Med. 129:1307).

[0121] In this disclosure, the term "significantly" as used in reference to a change is intended to mean that the change observed is noticeable and / or that it has statistical significance.

[0122] In this disclosure, the term "substantially" as used in connection with a feature of the disclosure is intended to define a set of embodiments related to this feature that are generally similar to this feature, but not completely similar to this feature. The differences between the set of embodiments related to a given feature and the given feature are such that in the set of embodiments, the properties and functions of the given feature are not substantially affected.

[0123] The phrase "in an amount sufficient to induce an immune response" or "immunologically effective amount" used in reference to an antigen or a combination of an antigen and an adjuvant is intended to refer to an amount effective to induce an immune response to the antigen when administered to a subject. This amount may vary depending on various factors, such as the health or physical condition of the subject, its age, the ability of the subject's immune system to produce antibodies, the degree of protection desired, the formulation of the composition containing the antigen, and the evaluation of the medical situation by the treating physician. This amount can be determined by routine methods known to those skilled in the art. Indicators of immune response include, but are not limited to, antibody titers or specificities detected by assays such as enzyme-linked immunoassays (ELISA), bactericidal assays, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays, such as spot, Western blot, or antigen array; cytotoxicity assays, and the like.

[0124] As used herein, in the context of eliciting an immune response, the terms "treat", "treatment", "therapy" and the like refer to the administration or consumption of a composition disclosed herein for the purpose of curing, relieving, alleviating, altering, treating, ameliorating, or affecting the symptoms of a disease or disorder, condition, or for the purpose of preventing or delaying the onset of symptoms, complications, or otherwise halting or inhibiting further progression of a disorder in a statistically significant manner. Also, as used herein, in the context of this disclosure, the terms "treat", "treatment" and the like refer to the alleviation or mitigation of pathological processes mediated by meningococcal infection. In the context of this disclosure, insofar as any of the other conditions described herein are concerned, the terms "treat", "treatment", and the like refer to the alleviation or mitigation of one or more symptoms associated with such condition.

[0125] As used herein, the term "vaccine" is intended to mean an immunogenic composition directed against a pathogen that is administered to a subject to elicit an immune response with the intent of protecting or treating the subject against disease caused by the pathogen. The vaccines disclosed herein are intended for use as prophylactic (preventative) vaccines to be administered to a subject prior to infection with the intent of preventing or reducing the likelihood of occurrence of initial (and / or recurrent) infection.

[0126] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide that codes for at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. An mRNA may include one or more coding and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). The non-coding regions in an mRNA include the 5' cap, 5' untranslated region (UTR), 3'UTR, and poly(A) tail. An mRNA can be purified from a natural source, produced using a recombinant expression system (e.g., in vitro transcription) and optionally purified, or chemically synthesized.

[0127] The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA comprises at least one chemical modification. In some embodiments, the mRNA disclosed herein may comprise one or more modifications that typically enhance RNA stability. Exemplary modifications include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In some embodiments, the disclosed mRNAs may contain modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydro- uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queuosine, β-D-mannosyl-queuosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0128] In some embodiments, the disclosed mRNAs may contain at least one chemical modification, including but not limited to pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0129] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0131] The lists of sources, ingredients, and components set forth below are recited as such, as are combinations and mixtures thereof that are contemplated and are within the scope of this specification.

[0132] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0133] All lists of items, such as, for example, lists of ingredients, are intended to be and should be construed as Markush groups, and therefore all lists should be read and interpreted as items "selected from the group consisting of" a list of items "and combinations and mixtures thereof."

[0134] References herein may be trade names of components that contain various ingredients utilized in this disclosure.The inventors do not intend to be limited herein by any particular trade name material.Equivalent materials (e.g., those obtained from different sources with different names or reference numbers) to those referenced by trade name can be substituted and utilized in the description herein.

[0135] antigen The immunogenic compositions disclosed herein include at least one combination of meningococcal antigens. The combination of antigens may include at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisseria adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

[0136] In one embodiment, the meningococcal antigen may be from N. meningitidis serogroup B.

[0137] fHBP Meningococcal fHBP, also referred to in the art as lipoprotein 2086 (LP2086), ORF2086, genome-derived Neisseria antigen (GNA) 1870, or "741", is a lipoprotein expressed on the bacterial surface of nearly all invasive meningococcal isolates. fHBP is an important virulence factor because it binds to human complement factor H (fH), a negative regulator of the alternative complement pathway (Seib et al., Expert Rev Vaccines. 2015;14(6):841-59). Binding of fHBP to human fH allows the pathogen to survive in human serum and blood, evading alternative pathway-mediated killing by the host's innate immune system.

[0138] Three main genetic and immunological fHBP variants have been described: variant 1, which corresponds to subfamily B, and variants 2 and 3, which are classified in subfamily A (Seib et al., Expert Rev Vaccines. 2015;14(6):841-59). In addition to the nomenclature provided by Pfizer (fHBP A and B) and Novartis (variants 1, 2 and 3), fHBPs are identified by unique ID numbers in the PubMLST database. Although there is significant antigenic diversity between fHBP subfamilies A and B, protein sequences within the subfamily are highly conserved, with sequence identity between different strains exceeding 86%. Each unique fHBP found in N. meningitidis is also assigned a fHBP peptide ID according to neisseria.org or pubmlst.org / neisseria / fHBP / website. Because the length of the variant 2 (v.2) fHBP protein (from strain 8047, fHBP ID 77) and variant 3 (v.3) fHBP (from strain M1239, fHBP ID 28) differs from that of MC58 (fHBP ID 1) by -1 and +7 amino acid residues, respectively, the numbering used to refer to residues in the v.2 and v.3 fHBP proteins differs from the numbering based on the actual amino acid sequences of these proteins. Thus, for example, a reference to a leucine residue (L) at position 166 in the v.2 or v.3 fHBP sequence is referring to residues at position 165 in the v.2 protein and 173 in the v.3 protein. Members of variants 1, 2 and 3 are present in approximately 65%, 25% and 10%, respectively, of MenB clinical isolates that cause invasive disease.The top 10 most frequent fHBP variants in the global population of MenB strains collectively represent approximately 80% of invasive disease-causing strains in the United States and Europe (Bambini et al., Vaccine. 2009;27(21):2794-803; Chang, J Infect 2019;S0163-4453(19):30272-5; Lucidarme, Clin Vaccine Immunol 2010;17(6):919-29; and Murphy et al., The Journal of infectious diseases. 2009;200(3):379-89; Wang et al., Vaccine. 2011;29(29-30):4739-44).

[0139] An fHBP used in accordance with the present disclosure may be a wild-type (or naturally occurring) polypeptide or may be modified by amino acid substitutions, insertions, or deletions (not naturally occurring), provided that the polypeptide is capable of eliciting an immune response.

[0140] The fHBP used according to the present disclosure can be lipidated fHBP or non-lipidated fHBP.Lipidated protein usually contains specific peptide sequence for lipidation in its N-terminal sequence.This sequence is cleaved during the maturation stage of protein.These lipidation signal peptides are specific for each type of protein and the host cell that produces the protein.

[0141] The ORF2086 polypeptide is expressed in N. meningitidis as a precursor protein with a lipoprotein signal motif that is cleaved during processing to leave an N-terminal cysteine ​​residue that is co-translationally modified with a lipid anchor that anchors the protein to the Neisserial outer membrane (McNeil et al., (2013) MMBR 77(2):234-252).

[0142] In order to avoid the lipidation of recombinant protein, various techniques known in the art can be used.For example, it is possible to delete lipidation peptide signal or replace lipidation peptide signal with another peptide signal that is not recognized by the cell in which the protein is produced.US Patent 10,300,122 B2 describes the use of this technique for ORF 2086.

[0143] It is also possible to replace the codon encoding the N-terminal cysteine ​​with a codon encoding another amino acid, or to remove the codon encoding the N-terminal cysteine.For example, with respect to fHBP, US Patent No. 10,300,122 B2 describes fusing an ATG (methionine) codon directly to the second 5'-terminal codon of ORF2086 encoding the mature polypeptide, resulting in the deletion of the cysteine ​​lipidation site (or replacing the cysteine ​​with methionine).Also, for example, US Patent No. 9,724,402 B2 or US Patent No. 11,077,180 B2 disclose obtaining a non-lipidated fHBP in which the N-terminal Cys is replaced with an amino acid that is not a Cys residue.

[0144] The fHBP used according to the present disclosure may be a naturally occurring protein or a non-naturally occurring protein. Non-naturally occurring protein refers to an "artificial protein" and includes fHBPs with heterologous components not found in nature, as opposed to naturally occurring proteins. Non-naturally occurring proteins may be chimeric or mutant proteins. "Chimeric protein" in the context of the present disclosure is intended to refer to a protein that includes two or more different components, each of which is derived from a different fHBP (e.g., variant 1, 2, or 3). Mutations in mutant proteins may include amino acid substitutions, insertions, or deletions. In one embodiment, the mutation is an amino acid substitution.

[0145] A non-naturally occurring fHBP suitable for the immunogenic composition disclosed herein can still induce an immune response against fHBP. In one embodiment, the non-naturally occurring fHBP used according to the present disclosure can be a mutated fHBP. Mutations, e.g., amino acid substitutions, can also be introduced to reduce or inhibit the binding of fHBP antigen to coagulation factor H (fH) normally present in the blood of an individual. Thus, prevention of fH binding to fHBP antigens used in the immunogenic composition of the present disclosure can increase the amount of antigens accessible to the immune system, improving the efficacy and efficiency of the immune response against these antigens. Advantageously, the mutated fHBP can induce an anti-fHBP antibody repertoire that is directed against fHBP epitopes within the fH binding site, resulting in greater protective complement deposition activity than antibodies induced by wild-type (WT) fHBP antigens that target fHBP epitopes outside the fH binding site.

[0146] A non-naturally occurring fHBP contemplated for the immunogenic compositions disclosed herein may exhibit reduced affinity for fH or improved thermal stability compared to the corresponding naturally occurring fHBP. Affinity and thermal stability for fH protein can be measured as disclosed in WO 2016 / 014719 A1 (as in Examples 1 or 3 of this document).

[0147] For convenience and clarity, unless otherwise stated, the native or naturally occurring amino acid sequence of fHBP B24 of SEQ ID NO:6 (or fHBP ID 1 or v.1 fHBP of meningococcal strain MC58) is selected as the reference sequence for all naturally occurring and non-naturally occurring fHBP amino acid sequences herein. Thus, when referring to an amino acid residue position in fHBP, the position number used herein corresponds to the amino acid residue number of SEQ ID NO:6 (fHBP B24). As a result, position number 1 is the first amino acid residue shown in SEQ ID NO:6, which is a cysteine. This also holds true when additional amino acids are added to the N-terminus of SEQ ID NO:6 before this cysteine.

[0148] In one embodiment, the mutations, e.g., amino acid substitutions, introduced into the fHBP A or B antigens used in this disclosure can be as disclosed in WO 2011 / 126863 A1, WO 2015 / 017817 A1, or WO 2016 / 014719 A1.

[0149] The immunogenic compositions disclosed herein can include a non-naturally occurring fHBP that differs in amino acid sequence from wild-type meningococcal fHBP by between 1 and 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), between 10 and 15 amino acids, between 15 and 20 amino acids, between 20 and 30 amino acids, between 30 and 40 amino acids, or between 40 and 50 amino acids.

[0150] In some embodiments, a fHBP may comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to a reference fHBP sequence.

[0151] Identity (e.g., percent homology) can be determined using any homology comparison software that calculates pairwise sequence alignment, including various known sequence comparison tools, such as the Blast software of the National Center of Biotechnology Information (NCBI), for example, by using default parameters. This identity is global identity, i.e., identity over the entire amino acid or nucleic acid sequence, not just a portion. Pairwise global alignment is defined by Needleman et al., Journal of Molecular Biology, vol. 48, 1970, pp. 443-53. For example, starting from a polypeptide sequence and comparing it with another polypeptide sequence, the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available at http: / / emboss.sourceforge.net / apps / cvs / emboss / apps / needle.html) can be used to find the optimal alignment along the entire length of the two sequences ("global alignment").

[0152] The fHBP antigen used in the immunogenic composition disclosed herein is available as disclosed in WO 2016 / 014719 A1. fHBP can be obtained as a recombinant protein from a recombinant expression vector (or construct) transfected into a host cell, such as an E. coli strain, for production. Vectors suitable for transferring and expressing nucleic acid encoding fHBP can vary in composition. Integration vectors can be conditionally replicating or suicide plasmids, bacteriophages, etc.

[0153] The constructs may contain a variety of elements including, for example, promoters, selectable genetic markers (e.g., genes that confer resistance to antibiotics, such as kanamycin, erythromycin, chloramphenicol, or gentamicin), origins of replication (to facilitate replication in host cells, e.g., bacterial host cells), and the like. The choice of vector will depend on a variety of factors, such as the type of cell in which growth is desired and the purpose of growth. Certain vectors are useful for amplifying and producing large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Selection of an appropriate vector is well within the skill of one of ordinary skill in the art. Many such vectors are commercially available.

[0154] In one example, the vector may be an episomal plasmid-based expression vector that contains a selectable drug resistance marker and elements that provide for autonomous replication in different host cells (e.g., both E. coli and N. meningitidis). One example of such a "shuttle vector" is the plasmid pFPIO (Pagotto et al. (2000) Gene 244:13-19). The vector may provide for extrachromosomal maintenance in the host cell or may provide for integration into the host cell genome. Vectors are fully described in numerous publications well known to those skilled in the art, including, for example, "Short Protocols in Molecular Biology" (1999), edited by F. Ausubel et al., Wiley & Sons. The vector may provide for expression of the nucleic acid encoding the subject fHBP, may provide for propagation of the subject nucleic acid, or both.

[0155] Examples of vectors that can be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA. For example, plasmid vectors, such as pBR322, pUC 19 / 18, pUC 118, 119, and M13 mp series vectors can be used. pET21 is also an expression vector that can be used. Bacteriophage vectors include λgtl0, λgtl, λgtl8-23, λzAp / R, and EMBL series bacteriophage vectors. Additional vectors that can be used include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16, and charomid 9 series vectors.

[0156] The recombinant expression vector may comprise a transcriptional control element, such as a nucleotide sequence encoding fHBP, operably linked to a promoter. The promoter may be constitutive or inducible. The promoter may be adapted for use in prokaryotic or eukaryotic host cells.

[0157] The expression vector can provide transcriptional and translational regulatory sequences to provide inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of a transcriptional initiation region and a transcriptional and translational termination region. These regulatory regions can be native to the fHBP from which the subject fHBP is derived, or can be derived from an exogenous source. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. The promoter can be either constitutive or inducible, and can be a strong constitutive promoter (such as, for example, T7).

[0158] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of a nucleic acid sequence encoding a protein of interest. Constructs (recombinant vectors) can be prepared, for example, by inserting a polynucleotide of interest into the backbone of the construct, typically by DNA ligase ligation into a restriction enzyme site cleaved in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous or site-specific recombination. Typically, homologous recombination can be achieved by ligating homologous regions into the vector flanking the desired nucleotide sequence, while site-specific recombination can be achieved through the use of sequences that promote site-specific recombination (e.g., cre-lox, att sites, etc.). Nucleic acids containing such sequences can be added, for example, by ligation of oligonucleotides or by polymerase chain reaction using primers that contain both a homologous region and a portion of the desired nucleotide sequence.

[0159] In addition, expression constructs can contain additional elements.For example, expression vectors can have one or two replication systems, allowing them to be maintained in organisms, for example, in mammalian cells or insect cells for expression, and in prokaryotic hosts for cloning and amplification.In addition, expression constructs can contain selection marker genes to allow the selection of transformed host cells.Selection genes are well known in the art and vary according to the host cells used.

[0160] Amino acid substitutions can be introduced into the fHBP nucleotide sequence by any technique known in the art.For example, amino acid substitutions can be obtained as disclosed in WO 2011 / 126863 A1, WO 2015 / 017817 A1, or WO 2016 / 014719 A1.In other exemplary embodiments, amino acid substitutions can be obtained as disclosed in WO 2015 / 128480, WO 2010 / 046715, WO 2016 / 008960, WO 2020 / 030782, or WO 2011 / 051893.

[0161] Recombinant fHBP can be obtained in purified form from the culture by any purification method known in the art, for example, as described in the Examples section.

[0162] In one embodiment, fHBP A protein and / or fHBP B may be present in the immunogenic compositions disclosed herein in an amount of about 20 μg / dose to about 200 μg / dose, or about 25 μg / dose to about 180 μg / dose, or about 40 μg / dose to about 140 μg / dose, or about 50 μg / dose to about 120 μg / dose, or about 75 μg / dose to about 100 μg / dose. In one embodiment, fHBP A protein and / or fHBP B may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0163] fHBP A In one embodiment, the immunogenic composition disclosed herein may comprise at least one fHBP A variant antigen. At least one fHBP A protein may be a lipidated protein or a non-lipidated protein. In an exemplary embodiment, the fHBP A protein may be a non-lipidated protein.

[0164] In one embodiment, the fHBP A protein may be a naturally occurring fHBP or a non-naturally occurring fHBP. In one embodiment, the fHBP A protein may be a naturally occurring fHBP. In another embodiment, the fHBP A protein may be a non-naturally occurring fHBP.

[0165] In one embodiment, the fHBP A protein can be a protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO: 1. At least one fHBP A protein can be a lipidated or non-lipidated protein and / or can be a naturally occurring or non-naturally occurring fHBP (a non-naturally occurring fHBP A05 protein is not 100% identical to fHBP A05 or SEQ ID NO: 1).

[0166] The non-naturally occurring fHBP A protein may be a chimeric protein as disclosed in WO 2011 / 126863 A1 or WO 2015 / 017817 A1, or a mutant fHBP A protein as disclosed in WO 2016 / 014719 A1, WO 2011 / 051893, WO 2016 / 008960, or WO 2015 / 128480. In an exemplary embodiment, the fHBP A protein may be a mutant protein.

[0167] In an exemplary embodiment, the non-naturally occurring fHBP A protein can be a mutant protein. The non-naturally occurring fHBP A protein can be a non-lipidated protein. In an exemplary embodiment, the fHBP A protein can be a non-naturally occurring, e.g., a mutant, non-lipidated fHBP A protein.

[0168] In one embodiment, the non-naturally occurring fHBP A protein can differ in amino acid sequence from a wild-type meningococcal fHBP A protein, e.g., fHBP A05, by 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 to 15 amino acids, 15 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.

[0169] In one embodiment, the non-naturally occurring fHBP A protein can be a mutein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 1. The non-naturally occurring fHBP A05 protein is not 100% identical to fHBP A05 or SEQ ID NO: 1.

[0170] In another embodiment, the non-naturally occurring or mutated fHBP A protein may comprise at least one amino acid substitution selected from at least one of the following, based on the numbering of SEQ ID NO:6: a) an amino acid substitution of asparagine at amino acid 115 (N115); b) an amino acid substitution of aspartic acid at amino acid 121 (D121); c) an amino acid substitution of serine at amino acid 128 (S128); d) an amino acid substitution of leucine at amino acid 130 (L130); e) an amino acid substitution of valine at position 131 (V131); f) an amino acid substitution of glycine at position 133 (G133); g) an amino acid substitution of lysine at position 219 (K219); and h) an amino acid substitution of glycine at position 220 (G220).

[0171] In another embodiment, the non-naturally occurring or mutated fHBP A protein may contain at least one amino acid deletion or substitution at any one of the following positions disclosed in WO 2011 / 051893 with respect to the numbering of the fHBP sequence identified as SEQ ID NO: 5 in WO 2011 / 051893 (mature lipoprotein form of fHBP A19): D37, K45, T56, E83, E95, E112, S122, I124, R127, T139, F141, N142, Q143, L197, D210, R212, K218, N43, N116, K119, T220 and / or 240. In one embodiment, the amino acid deletion or substitution is as disclosed in WO 2011 / 051893.

[0172] In another embodiment, the non-naturally occurring or mutated fHBP A protein may include at least one amino acid deletion or substitution at any one of the following positions: S32, L126 and / or E243, as disclosed in WO 2016 / 008960 with respect to the numbering of the fHBP sequence (A124 or variant 3.28 or ID28) identified as SEQ ID NO: 17 in WO 2016 / 008960. One, two or three residues may be deleted. Alternatively, they may be replaced by different amino acids. For example, Leu-126 may be replaced by any of the other 19 naturally occurring amino acids. When a substitution is made, the replacement amino acid in some embodiments may be a simple amino acid, for example, glycine or alanine. In other embodiments, the substituted amino acid is a conservative substitution, for example, made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is non-conservative. In one embodiment, the substitution at a particular residue is as follows: S32V; L126R; and / or E243A.

[0173] In another embodiment, the non-naturally occurring or mutated fHBP A protein may contain at least one amino acid deletion or substitution at any one of the following positions: S32, L123 and / or E240, as disclosed in WO 2016 / 008960 with respect to the numbering of the fHBP sequence (mature lipoprotein form) identified in WO 2016 / 008960 as SEQ ID NO: 5. One, two or three residues may be deleted. Alternatively, they may be replaced by different amino acids. For example, Leu-123 may be replaced by any of the other 19 naturally occurring amino acids. When a substitution is made, the replacement amino acid in some embodiments may be a simple amino acid, for example, glycine or alanine. In other examples, the substituted amino acid is a conservative substitution, for example, made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is non-conservative. In one embodiment, the substitution at a particular residue can be as follows: S32V; L123R; and / or E240A.

[0174] In another embodiment, the non-naturally occurring or mutated fHBP A protein may include at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2015 / 128480 with respect to the numbering of the fHBP sequence (fHBP A19 or v2.16 or ID16) identified as SEQ ID NO:5 in WO 2015 / 128480: S32, V33, L39, L41, F69, V100, 1113, F122, L123, V124, S125, G126, L127, G128, S151, H239, and / or E240. In one embodiment, the residues for mutation may be S32, V100, L123, V124, S125, G126, L127, G128, H239, and / or E240. Mutations at these residues result in proteins with better stability compared to wild-type fHBP A. In one embodiment, the residues for mutation can be S32, L123, V124, S125, G126, L127, and / or G128. In one embodiment, the residues for mutation can be S32, L123, V124, S125, G126, L127, and / or G128. In another embodiment, residues S32 and / or L123 can be mutated, e.g., S32V and / or L123. If one or more of V100, S125, and / or G126 are mutated, mutations can be introduced at residues outside the triad.

[0175] The designated residue may be deleted, but preferably it is replaced by a different amino acid. For example, Ser-32 may be replaced by any of the other 19 naturally occurring amino acids. When a substitution is made, the replacement amino acid in some embodiments may be a simple amino acid, for example, glycine or alanine. In other embodiments, the replacement amino acid is a conservative substitution, for example, it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is non-conservative. In some embodiments, the substitution does not use alanine.

[0176] For example, substitutions at the specified residues can be: S32V; V33C; L39C; L41C; F69C; V100T; I113S; F122C; L123R; V124I; S125G or S125T; G126D; L127I; G128A; S151C; H239R; or E240H.

[0177] In another embodiment, the non-naturally occurring or mutated fHBP A protein may include at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2015 / 128480 with respect to the numbering of the fHBP sequence (A124 or variant 3.28 or ID28) identified as SEQ ID NO: 17 in WO 2015 / 128480: S32, V33, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242, and / or E243. In one embodiment, the residues for mutation may be S32, V103, L126, V127, S128, G129, L130, G131, H242, and / or E243. In one embodiment, the residues for mutation can be S32, L126, V127, S128, G129, L130, and / or G131. In another embodiment, residues S32, L126, V127, S128, G129, L130, and / or G131, e.g., residues S32 and / or L126, can be mutated to, e.g., S32V and / or L126R.

[0178] The designated residue may be deleted, but preferably it is replaced by a different amino acid. For example, Ser-32 may be replaced by any of the other 19 naturally occurring amino acids. When a substitution is made, the replacement amino acid in some embodiments may be a simple amino acid such as glycine or alanine. In other embodiments, the replacement amino acid is a conservative substitution, for example, it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitution is non-conservative. In some embodiments, the substitution does not use alanine.

[0179] For example, substitutions at the specified residues can be as follows: S32V; I33C; L39C; L41C; F72C; V103T; T116S; F125C; L126R; V127I; S128G or S128T; G129D; L130I; G131A; S154C; H242R; E243H.

[0180] The amino acid substitution of asparagine (N115) at amino acid 115 can be an N115I substitution (I: isoleucine). Other amino acids with non-polar, positively charged, or aromatic side chains, such as valine, leucine, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, in some cases, fHBP can include an N115V substitution, an N115L substitution, an N115K substitution, an N115R substitution, an N115H substitution, an N115F substitution, an N115Y substitution, or an N115W substitution.

[0181] The amino acid substitution of aspartic acid at amino acid 121 (D121) can be a D121G substitution (G: glycine). Other amino acids with non-polar, positively charged, or aromatic side chains, such as leucine, isoleucine, valine, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, fHBP variants can include a D121L substitution, a D121I substitution, a D121V substitution, a D121K substitution, a D121R substitution, a D121H substitution, a D121F substitution, a D121Y substitution, or a D121W substitution.

[0182] The amino acid substitution of serine (S128) at amino acid 128 can be S128T substitution (T: threonine). Other amino acids with polar, charged or aromatic side chains, such as methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, can also be substituted at this position. Thus, for example, in some cases, fHBP variants can include S128M substitution, S128N substitution, S128D substitution, S128E substitution, S128K substitution, S128R substitution, S128H substitution, S128F substitution, S128Y substitution or S128W substitution.

[0183] fHBP A can include an amino acid substitution of leucine (L130) at amino acid 130. The amino acid substitution of leucine (L130) at amino acid 130 can be an L130R substitution (R: arginine).

[0184] fHBP A may include an amino acid substitution of valine (V131) at amino acid 131. Other amino acids with charged or aromatic side chains, such as glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some cases, fHBP may include a V131E substitution, a V131K substitution, a V131R substitution, a V131H substitution, a V131F substitution, a V131Y substitution, or a V131W substitution.

[0185] fHBP A can include an amino acid substitution of glycine (G133) at amino acid 133. The amino acid substitution of glycine (G133) at amino acid 133 can be a G133D substitution (D: aspartic acid).

[0186] fHBP A may include an amino acid substitution of lysine at position 219 (K219). Other amino acids with polar, negatively charged, or aromatic side chains, such as glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some cases, fHBP may include a K219Q substitution, a K219D substitution, a K219E substitution, a K219F substitution, a K219Y substitution, or a K219W substitution.

[0187] The amino acid substitution of glycine acid (G220) at amino acid 220 can be a G220S substitution (S: serine). Other amino acids with polar, charged, or aromatic side chains, such as asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, can also be substituted at this position. Thus, for example, in some cases, fHBP can include a G220N substitution, a G220Q substitution, a G220D substitution, a G220E substitution, a G220K substitution, a G220R substitution, a G220H substitution, a G220F substitution, a G220Y substitution, or a G220W substitution.

[0188] In an exemplary embodiment, the amino acid substitution for leucine at amino acid 130 (L130) can be an L130R substitution (R: arginine).

[0189] In an exemplary embodiment, the amino acid substitution for glycine at amino acid 133 (G133) can be a G133D substitution (D: aspartic acid).

[0190] In an exemplary embodiment, the amino acid substitution at glycine at position 220 (G220) can be a G220S substitution (S: serine).

[0191] In another embodiment, the non-naturally occurring or mutated fHBP A protein may contain at least one amino acid substitution selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. In another embodiment, the non-naturally occurring fHBP A protein may contain at least three amino acid substitutions selected from the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. In another embodiment, the fHBP A protein may contain only three amino acid substitutions, G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6.

[0192] In one embodiment, the non-naturally occurring (or mutated) fHBP A protein may be a non-lipidated mutated fHBP A protein comprising at least one of the amino acid substitutions selected from the group consisting of: a) an amino acid substitution of asparagine at amino acid 115 (N115); b) an amino acid substitution of aspartic acid at amino acid 121 (D121); c) an amino acid substitution of serine at amino acid 128 (S128); d) an amino acid substitution of leucine at amino acid 130 (L130); e) an amino acid substitution of valine at position 131 (V131); f) an amino acid substitution of glycine at position 133 (G133); g) an amino acid substitution of lysine at position 219 (K219); and h) an amino acid substitution of glycine at position 220 (G220), based on the numbering of SEQ ID NO:6.

[0193] In another embodiment, the non-naturally occurring (or mutated) fHBP A protein is selected from the group consisting of an N115I substitution, an N115V substitution, an N115L substitution, an N115K substitution, an N115R substitution, an N115H substitution, an N115F substitution, an N115Y substitution, an N115W substitution, a D121G substitution, a D121L substitution, a D121I substitution, a D121V substitution, a D121K substitution, a D121R substitution, a D121H substitution, a D121F substitution, a D121Y substitution, a D121W substitution, an S128T substitution, an S128M substitution, an S128N substitution, an S128D substitution, an S128E substitution, an S128K substitution, an S128R substitution, an S128H substitution, an S128F substitution, an S128Y substitution, an S128 The fHBP A protein may be a non-lipidated mutant fHBP A protein comprising at least one amino acid substitution selected from the group consisting of a W substitution, an L130R substitution, a V131E substitution, a V131K substitution, a V131R substitution, a V131H substitution, a V131F substitution, a V131Y substitution, a V131W substitution, a G133D substitution, a K219Q substitution, a K219D substitution, a K219E substitution, a K219F substitution, a K219Y substitution, a K219W substitution, a G220S substitution, a G220N substitution, a G220Q substitution, a G220D substitution, a G220E substitution, a G220K substitution, a G220R substitution, a G220H substitution, a G220F substitution, a G220Y substitution, or a G220W substitution.

[0194] In one exemplary embodiment, the non-naturally occurring (or mutated) fHBP A protein may be a non-lipidated mutant fHBP A protein comprising at least one of the amino acid substitutions selected in the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. In another embodiment, the non-naturally occurring or mutated non-lipidated fHBP A protein may comprise at least three amino acid substitutions selected in the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. In another exemplary embodiment, the non-lipidated mutant fHBP A protein may comprise only three amino acid substitutions, G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6.

[0195] In an exemplary embodiment, the fHBP A protein may be a non-lipidated mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 1 and comprises at least one of the amino acid substitutions selected in the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. The mutated non-lipidated fHBP A protein may comprise at least three amino acid substitutions selected in the group consisting of G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6. In another exemplary embodiment, the non-lipidated mutant fHBP A protein may comprise only the three amino acid substitutions G220S, L130R, and G133D based on the numbering of SEQ ID NO: 6.

[0196] In one embodiment, the fHBP A protein may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO:2.

[0197] In another embodiment, the fHBP A protein may comprise or consist of SEQ ID NO:2.

[0198] Another embodiment is directed to an mRNA encoding an fHBP A protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 2. The mRNA can encode an fHBP A protein that comprises or consists of SEQ ID NO: 1, or encode an fHBP A protein that comprises or consists of SEQ ID NO:2.

[0199] Amino acid substitutions can be introduced into the fHBP A nucleotide sequence by any technique known in the art. For example, amino acid substitutions can be obtained as disclosed in WO 2016 / 014719 A1.

[0200] In one embodiment, fHBP A protein may be present in the immunogenic compositions disclosed herein in an amount of about 20 μg / dose to about 200 μg / dose, or about 25 μg / dose to about 180 μg / dose, or about 40 μg / dose to about 140 μg / dose, or about 50 μg / dose to about 120 μg / dose, or about 75 μg / dose to about 100 μg / dose. In one embodiment, fHBP A protein and / or fHBP B may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0201] fHBP B In one embodiment, the immunogenic composition disclosed herein may comprise at least one fHBP B variant antigen. At least one fHBP B protein may be a lipidated protein or a non-lipidated protein. In an exemplary embodiment, the fHBP B protein may be a non-lipidated protein.

[0202] In one embodiment, the fHBP A and fHBP B proteins may be lipidated. In one embodiment, the fHBP A and fHBP B proteins may be non-lipidated. Alternatively, the fHBP A protein may be lipidated and the fHBP B protein may be non-lipidated. Furthermore, the fHBP A protein may be non-lipidated and the fHBP B protein may be lipidated.

[0203] In one embodiment, the fHBP B protein may be a naturally occurring fHBP or a non-naturally occurring fHBP. In one embodiment, the fHBP B protein may be a naturally occurring fHBP. In another embodiment, the fHBP B protein may be a non-naturally occurring fHBP.

[0204] In one embodiment, the fHBP A and fHBP B proteins can be naturally occurring fHBPs. In one embodiment, the fHBP A and fHBP B proteins can be non-naturally occurring fHBPs. Alternatively, the fHBP A protein can be a naturally occurring fHBP and the fHBP B protein can be a non-naturally occurring fHBP. Furthermore, the fHBP A protein can be a non-naturally occurring fHBP and the fHBP B protein can be a naturally occurring fHBP.

[0205] In one embodiment, the fHBP B protein can be a protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO: 3. At least one fHBP B protein can be a lipidated or non-lipidated protein and / or can be a naturally occurring or non-naturally occurring fHBP (a non-naturally occurring fHBP B01 protein is not 100% identical to fHBP B01 or SEQ ID NO: 3).

[0206] The non-naturally occurring fHBP B protein may be a chimeric protein as disclosed in WO 2011 / 126863 A1 or WO 2015 / 017817 A1, or may be a mutant fHBP B protein as disclosed in WO 2016 / 014719 A1, WO 2011 / 051893, or WO 2020 / 030782. In an exemplary embodiment, the fHBP B protein may be a mutant protein.

[0207] In an exemplary embodiment, the non-naturally occurring fHBP B protein can be a mutant protein. The non-naturally occurring fHBP B protein can be a non-lipidated protein. In an exemplary embodiment, the fHBP B protein can be a non-naturally occurring, e.g., a mutant, non-lipidated fHBP B protein.

[0208] In one embodiment, the non-naturally occurring fHBP B protein can differ in amino acid sequence from a wild-type meningococcal fHBP B protein, e.g., fHBP B01, by 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 to 15 amino acids, 15 to 20 amino acids, 20 to 30 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.

[0209] In one embodiment, the non-naturally occurring fHBP B protein can be a mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 3. The non-naturally occurring fHBP B protein is not 100% identical to fHBP B01 or SEQ ID NO:3.

[0210] In another embodiment, the non-naturally occurring or mutated fHBP B protein may comprise at least one amino acid substitution selected from at least one of: a) an amino acid substitution at amino acid 38 with glutamine (Q38); b) an amino acid substitution at amino acid 92 with glutamic acid (E92); c) an amino acid substitution at amino acid 130 with arginine (R130); d) an amino acid substitution at amino acid 223 with serine (S223); and e) an amino acid substitution at amino acid 248 with histidine (H248), based on the numbering of SEQ ID NO:6.

[0211] In another embodiment, the non-naturally occurring or mutated fHBP B protein can include at least one amino acid deletion or substitution at any one of the following positions disclosed in WO 2011 / 051893 with respect to the numbering of the fHBP sequence identified as SEQ ID NO: 4 in WO 2011 / 051893 (mature lipoprotein form of fHBP B24): D37, K45, T56, E83, E95, E112, K122, V124, R127, T139, F141, D142, K143, I198, S211, L213, K219, N43, D116, H119, S221, and K241.

[0212] In another embodiment, the non-naturally occurring or mutated fHBP B protein can include at least one amino acid substitution at any one of the following positions: E211, S216, or E232, as disclosed in WO 2020 / 030782 with respect to the numbering of the fHBP sequence identified as SEQ ID NO:2 in WO 2020 / 030782 (mature lipoprotein form of fHBP B09).

[0213] In another embodiment, the non-naturally occurring or mutated fHBP B protein may include at least one of the following amino acid substitutions at any one of the following positions as disclosed in WO 2020 / 030782 with respect to the numbering of the fHBP sequence identified as SEQ ID NO:2 in WO 2020 / 030782 (mature lipoprotein form of fHBP B09): E211A, S216R, or E232A.

[0214] In another embodiment, the non-naturally occurring or mutated fHBP B protein can include at least one amino acid substitution at any one of the following positions: E214, S219, or E235, as disclosed in WO 2020 / 030782 with respect to the numbering of the fHBP sequence identified as SEQ ID NO: 6 in WO 2020 / 030782 (fHBP B44 in the mature lipoprotein form).

[0215] In another embodiment, the non-naturally occurring or mutated fHBP B protein can include at least one of the following amino acid substitutions at any one of the following positions as disclosed in WO 2020 / 030782 with respect to the numbering of the fHBP sequence identified as SEQ ID NO:2 in WO 2020 / 030782 (the mature lipoprotein form of fHBP B44): E214A, S219R, or E235A.

[0216] In another embodiment, a naturally occurring or mutated fHBP The B protein may include at least one of the following amino acid substitutions at any one of the following positions as disclosed in WO2010046715 with respect to the numbering of the fHBP sequence (mature lipoprotein form of fHBP24) identified as SEQ ID NO: 1 in WO2010046715: 103, 106, 107, 108, 109, 145, 147, 149, 150, 154, 156, 157, 180, 181, 182, 183, 184, 185, 191, 193, 194, 195, 196, 199, 262, 264, 266, 267, 268, 272, 274, 283, 285, 286, 288, 289, 302, 304, 306, 311 and 313. In one embodiment, the one or more amino acids that may be varied in the factor H binding protein may be selected from the group including amino acid numbers 103, 106, 107, 108, 180, 181, 183, 184, 185, 191, 193, 195, 262, 264, 266, 272, 274, 283, 286, 304 and 306 with respect to the numbering of the fHBP sequence identified as SEQ ID NO:1 in WO2010046715.

[0217] The amino acid substitution of glutamine (Q38) at amino acid 38 can be a Q38R substitution (R: arginine). Other amino acids with positive charge or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, can also be substituted at this position. Thus, in some cases, fHBP can include a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, or a Q38W substitution.

[0218] The amino acid substitution of glutamic acid at amino acid 92 (E92) can be an E92K substitution. Other amino acids with positive charge or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine or tryptophan, can also be substituted at this position. Thus, for example, in some cases, the fHBP variant can include an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution.

[0219] The amino acid substitution for arginine at amino acid 130 (R130) can be an R130G substitution (G: glycine). Other amino acids with negatively charged or aromatic side chains, such as aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, can also be substituted at R130. Thus, for example, in some cases, fHBP variants can include an R130D substitution, an R130E substitution, an R130F substitution, an R130Y substitution, or an R130W substitution.

[0220] The amino acid substitution of the serine at amino acid 223 (S223) can be an S223R substitution (R: arginine). Other amino acids with positive charge or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, can also be substituted at this position. Thus, for example, in some cases, the fHbp variant comprises an S223K substitution, an S223H substitution, an S223F substitution, an S223Y substitution, or an S223W substitution.

[0221] In an exemplary embodiment, the amino acid substitution of histidine (H248) at amino acid 248 can be a H248L substitution (L:leucine). Other amino acids with non-polar, negatively charged, or aromatic side chains, such as isoleucine, valine, aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan, can also be substituted with H248. Thus, for example, in some cases, fHBP can include a H248I substitution, a H248V substitution, a H248D substitution, a H248E substitution, a H248F substitution, a H248Y substitution, or a H248W substitution.

[0222] In another embodiment, the non-naturally occurring or mutated fHBP B protein can include at least the H248L amino acid substitution. In another embodiment, the non-naturally occurring fHBP B protein can include only the H248L amino acid substitution, based on the numbering of SEQ ID NO:6.

[0223] In one embodiment, the non-naturally occurring (or mutated) fHBP B protein may be a non-lipidated mutated fHBP B protein comprising at least one of the amino acid substitutions selected from the group consisting of: a) an amino acid substitution at amino acid 38 with glutamine (Q38); b) an amino acid substitution at amino acid 92 with glutamic acid (E92); c) an amino acid substitution at amino acid 130 with arginine (R130); d) an amino acid substitution at amino acid 223 with serine (S223); and e) an amino acid substitution at amino acid 248 with histidine (H248), based on the numbering of SEQ ID NO:6.

[0224] In another embodiment, a naturally occurring (or mutated) fHBP The B protein may be a non-lipidated mutated fHBP B protein comprising at least one amino acid substitution selected from the group consisting of a Q38R substitution, a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, a Q38W substitution, an E92K substitution, an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, an E92W substitution, an R130G substitution, an R130D substitution, an R130E substitution, an R130F substitution, an R130Y substitution, an R130W substitution, an S223R substitution, an S223K substitution, an S223H substitution, an S223F substitution, an S223Y substitution, an S223W substitution, an H248L substitution, an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution, or an H248W substitution based on the numbering of SEQ ID NO:6.

[0225] In another exemplary embodiment, the non-naturally occurring or mutated fHBP B protein can be a non-lipidated mutant fHBP B protein that includes at least the amino acid substitution H248L based on the numbering of SEQ ID NO: 6. In another exemplary embodiment, the non-lipidated mutant fHBP B protein can be a non-lipidated mutant fHBP B protein that includes only the amino acid substitution H248L based on the numbering of SEQ ID NO: 6.

[0226] In an exemplary embodiment, the fHBP A protein may be a non-lipidated mutant protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to SEQ ID NO: 3 and includes at least the amino acid substitution H248L based on the numbering of SEQ ID NO: 6. The non-lipidated mutant fHBP B protein may include only the amino acid substitution H248L based on the numbering of SEQ ID NO: 6.

[0227] In one embodiment, the fHBP B protein may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO:4.

[0228] In another embodiment, the fHBP B protein may comprise or consist of SEQ ID NO:4.

[0229] Another embodiment is directed to an mRNA encoding an fHBP B protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 4. The mRNA can encode an fHBP B protein that comprises or consists of SEQ ID NO: 3, or encode an fHBP A protein that comprises or consists of SEQ ID NO: 4.

[0230] Amino acid substitutions can be introduced into the fHBP B nucleotide sequence by any technique known in the art. For example, amino acid substitutions can be obtained as disclosed in WO 2016 / 014719 A1.

[0231] In one embodiment, fHBP B protein may be present in the immunogenic compositions disclosed herein in an amount of about 20 μg / dose to about 200 μg / dose, or about 25 μg / dose to about 180 μg / dose, or about 40 μg / dose to about 140 μg / dose, or about 50 μg / dose to about 120 μg / dose, or about 75 μg / dose to about 100 μg / dose. In one embodiment, fHBP A protein and / or fHBP B may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0232] NadA Neisserial adhesin A (NadA, previously known as GNA1994) is a surface-exposed trimeric protein that forms oligomers anchored in the outer membrane via a transmembrane domain and plays an important role in adhesion to and invasion of epithelial cells (Capecchi et al., Mol. Microbiol. 2005;55:(pp. 687-98)). Sequences of NadA antigens from many strains have been published and the activity of the protein as a neisserial adhesin is well documented. The NadA gene is present in approximately 50% of meningococcal isolates. NadA shows growth phase-dependent expression, with highest levels during stationary growth phase.

[0233] The NadA antigen was included in the published genome sequence of N. meningitidis serogroup B strain MC58 as gene NMB1994 (GenBank accession number GI:7227256).

[0234] NadA polypeptides for use in accordance with the present disclosure may be wild-type polypeptides or may be modified by amino acid substitution, insertion or deletion, provided that the polypeptide is capable of eliciting an immune response against NadA.

[0235] In some embodiments, the NadA protein used may be an N-terminally and / or C-terminally truncated NadA or NadA protein containing amino acid deletions or insertions, for example as disclosed in references WO 01 / 64920; WO 01 / 64922; or WO 03 / 020756.

[0236] In one embodiment, the recombinant NadA protein used in the immunogenic compositions disclosed herein may be a NadA1 variant. As shown in the Examples, NadA1 has been shown to induce a strong hSBA response.

[0237] NadA1 can be derived from the NadA sequence of MenB strain MC58.

[0238] In one embodiment the NadA protein may comprise or consist of the sequence SEQ ID NO:7.

[0239] In some embodiments, the NadA protein may comprise at least 190 contiguous amino acids from SEQ ID NO:7, such as 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more contiguous amino acids from SEQ ID NO:7, such as 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, or 360 or more amino acids from SEQ ID NO:7.

[0240] In some embodiments, the NadA protein may, for example, lack 5-10 amino acids, or 10-15 amino acids, or 15-20 amino acids, or 25, or 30, or 35, or 40, or 45, or 50, or 55 amino acids from the C-terminus and / or N-terminus of SEQ ID NO: 7. If N-terminal residues are deleted, such deletions should not remove the ability of NadA to adhere to human epithelial cells.

[0241] In one embodiment, the NadA protein may lack a signal peptide at the N-terminus. For example, the NadA protein may lack the N-terminal 23 amino acids of, for example, SEQ ID NO:7.

[0242] In one embodiment, the NadA protein may lack a membrane anchor peptide at the C-terminus, for example the NadA protein may lack the C-terminal 55 amino acids of, for example, SEQ ID NO:7.

[0243] NadA may be used in monomeric or oligomeric form, for example in trimeric form.

[0244] For example, the NadA protein may not have its C-terminal membrane anchor (e.g., deletion of residues 308-362 for strain MC58 (SEQ ID NO: 7)) and expression of NadA without its membrane anchor domain in E. coli may result in secretion of the protein into the culture supernatant with concomitant removal of the 23 amino acid signal peptide (e.g., deletion of residues 2-24 of SEQ ID NO: 7 leaving a 284 amino acid protein - SEQ ID NO: 5).

[0245] In one embodiment, the NadA protein may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 5. In another embodiment, the NadA protein may comprise or consist of SEQ ID NO: 5.

[0246] Another embodiment is directed to an mRNA encoding a NadA protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO: 5. The mRNA can encode a NadA protein that comprises or consists of SEQ ID NO:5.

[0247] The NadA protein used in the immunogenic compositions disclosed herein can be obtained according to any recombinant technique known in the art, for example as generally disclosed above. The NadA protein can be obtained as a recombinant protein from a recombinant expression vector (or construct) transfected into a host cell, for example an E. coli strain, for production. The recombinant NadA can be obtained in purified form from the culture by any purification method known in the art, for example as described in the Examples section.

[0248] In one embodiment, the NadA protein may be present in an amount ranging from about 20 μg / dose to about 200 μg / dose, or from about 25 μg / dose to about 180 μg / dose, or from about 40 μg / dose to about 140 μg / dose, or from about 50 μg / dose to about 120 μg / dose, or from about 75 μg / dose to about 100 μg / dose. In one embodiment, the NadA protein may be present in an amount of about 50 μg / dose.

[0249] dOMV The immunogenic composition disclosed herein comprises detergent-extracted outer membrane vesicles (dOMV), also called outer membrane protein complexes (OMPC).Usually, detergent-extracted outer membrane vesicles are called dOMV or OMV when used as antigen.When used as protein carrier, they are called OMPC.

[0250] OMPC was used as the carrier protein platform for the polyribosylribitol phosphate (PRP) conjugated vaccines PedvaxHIB (Haemophilus influenzae type b vaccine) (Einhorn et al., Lancet (London, England). 1986;2(8502):299-302; Moro et al., The Journal of pediatrics. 2015;166(4):992-7) and VAXELIS (diphtheria, tetanus, pertussis, polio, hepatitis B and Haemophilus influenzae type b vaccine) (Syed, Paediatric drugs. 2017;19(1):69-80).

[0251] dOMVs are large proteolipid vesicles that contain integral outer membrane proteins and residual lipooligosaccharide (LOS) found in bacterial outer membranes (Helting, Acta Pathol Microbiol Scand C, 1981;89(2):69-78). Over 300 proteins can be identified in dOMVs. 75% of the total protein content of dOMVs is accounted for by 10 highly abundant proteins, including the outer membrane proteins Porin A (PorA) and Porin B (PorB), which account for up to 50% of the total protein.

[0252] Meningococcal porin proteins (Por) are antigenic determinants for serotyping. Two classes of porins, PorA and PorB, and antigenically distinct variants within each class resulting from sequence diversity in the por gene variable regions (VRs) that encode the surface-exposed loops, have been identified.

[0253] Suitable dOMV for the immunogenic composition disclosed herein can be obtained from various MenB strains.dOMV can be isolated from detergent extract of MenB strain.Suitable MenB strain can be wild-type MenB strain or engineered MenB strain to overexpress porin protein, for example PorA or PorB protein, for example PorA protein.

[0254] In one embodiment, the dOMV may be derived from a MenB strain expressing the PorA protein.

[0255] In one embodiment, the dOMV can be obtained from a MenB strain expressing the PorA VR2 subtype, which can be the PorA VR2 type P1.2, P1.4, P1.7, P1.10, or P1.13 protein.

[0256] In one embodiment, the dOMV may be derived from a MenB strain expressing the PorA VR2 type P1.2 protein.

[0257] In one embodiment, the dOMV can be obtained from a MenB strain expressing the PorA VR2 subtype and PorB P2.2a. The dOMV can be obtained from a MenB strain expressing PorA VR2 P1.2 and PorB P2.2a.

[0258] In one embodiment, the dOMV may comprise a PorA VR2 subtype and PorB P2.2a. The dOMV may comprise PorA VR2 P1.2 and PorB P2.2a.

[0259] In another embodiment, the dOMV may comprise PorA VR2 P1.2 and PorB P2.2a and immunotype LOS L3,7. PorA and PorB may account for approximately 50% of the proteins of the dOMV.

[0260] In some embodiments, the dOMV can be derived from a single MenB strain or from multiple different MenB strains, where the MenB strains can express the same subtype of PorA protein, or different PorA protein subtypes, or different types of Por proteins, e.g., PorA and PorB proteins.

[0261] The usable MenB strains from which the dOMVs displaying the desired porin protein are derived can be identified, for example, from the PubMLST database (https: / / pubmlst.org / ). For example, suitable MenB strains can be obtained by selecting MenB strains from epidemic outbreaks in such databases, and then selecting within such subsets MenB strains that have a gene encoding the desired porin protein, for example, the PorA VR2 P1.2 protein. The selected strains can then be evaluated for their efficient expression of the desired porin protein using techniques known in the art.

[0262] Examples of MenB strains suitable for obtaining dOMV according to the present invention include: NG H36, BZ 232, DK 353, B6116 / 77, BZ 163, 0085 / 00, NG P20, 0046 / 02, M11 40123, M12 240069, N5 / 99, 99M, or M07 240677.

[0263] In an exemplary embodiment, the MenB strain can be MenB strain 99M, which expresses the PorA VR2 P1.2 protein subtype.

[0264] In one embodiment, the dOMV may comprise Porin A (PorA) VR2 subtype P1.2.

[0265] In another embodiment, the dOMV may comprise the outer membrane protein Porin A (PorA) and / or the outer membrane protein Porin B (PorB). PorA may be present in an amount ranging from about 3% to about 15%, or from about 5% to about 9% or 10% of the total protein present in the dOMV. PorB may be present in an amount ranging from about 30% to about 70%, or from about 35% to about 65%, or from about 38% to about 58% of the total protein present in the dOMV.

[0266] In one embodiment, dOMVs can be obtained by a detergent extraction method using at least a deoxycholate treatment step.

[0267] Suitable methods for obtaining dOMVs can be as disclosed in Helting et al. (Acta Pathol Microbiol Scand C. April 1981;89(2):69-78) or in Example 2 of U.S. Pat. No. 4,695,624. For example, the bacterial culture can be centrifuged to obtain a pellet, which can then be extracted with a detergent, e.g., deoxycholate or sodium dodecyl sulfate (SDS), under heating, e.g., at about 50° C. to about 60° C., or about 56° C., for a time period ranging from about 10 minutes to about 20 minutes, or for about 15 minutes. The resulting material can then be centrifuged and the pellet further suspended and purified according to any method known in the art.

[0268] In one embodiment, the dOMV may be present in an amount ranging from about 5 μg / dose to about 400 μg / dose, or from about 10 μg / dose to about 300 μg / dose, or from about 25 μg / dose to about 250 μg / dose, or from about 35 μg / dose to about 225 μg / dose, or from about 50 μg / dose to about 200 μg / dose, or from about 75 μg / dose to about 180 μg / dose, or from about 100 μg / dose to about 150 μg / dose, or from about 110 μg / dose to about 125 μg / dose. In one embodiment, the dOMV may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0269] Further antigens In one embodiment, the immunogenic compositions disclosed herein may comprise at least one further antigen compared to the combination of meningococcal antigens disclosed herein.

[0270] In an exemplary embodiment, the further antigen may be a carbohydrate antigen from N. meningitidis serogroups A, C, W135, Y and / or X conjugated to a carrier protein. In one embodiment, the further antigen may be a combination of MenA, MenC, MenW-135 and MenY capsular polysaccharides conjugated to a carrier protein.

[0271] In an exemplary embodiment, the additional antigen may be a carbohydrate antigen from N. meningitidis serogroups A, C, W135, and / or Y conjugated to a carrier protein. In one embodiment, the additional antigen may be a combination of MenA, MenC, MenW-135, and MenY capsular polysaccharides conjugated to a carrier protein.

[0272] The carrier proteins for different capsular polysaccharides may be different or the same. Carrier proteins may include inactivated bacterial toxins such as diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesin protein (PsaA) may also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin (PPD) may also be used as carrier proteins. The carrier protein may be CRM197 protein, tetanus toxoid, or diphtheria toxoid. In one embodiment, the carrier protein is tetanus toxoid.

[0273] The conjugate may be a population comprising molecules having molecular weights in the range of 700 kDa to 1400 kDa or 800 kDa to 1300 kDa.

[0274] Within each dose, the amount of individual saccharide antigens may be 1-50 μg, measured as the mass of saccharide. For example, a total of 40 μg of saccharide may be administered per dose. For example, 10 μg of each polysaccharide and approximately 55 μg of carrier protein, such as tetanus toxoid protein, may be administered.

[0275] In one embodiment, the further antigen may be a combination of MenA, MenC, MenW-135 and MenY capsular polysaccharides, each conjugated to a tetanus toxoid carrier protein, where the MenA polysaccharide is conjugated to the tetanus toxoid carrier via an adipic acid dihydrazide (ADH) linker, while the MenC, MenW-135 and MenY polysaccharides are each directly conjugated to the tetanus toxoid carrier (TT).

[0276] In one embodiment, the additional antigen may be a combination of MenA, MenC, MenW-135 and MenY capsular polysaccharides conjugated to tetanus toxoid carrier protein. In an exemplary embodiment, the conjugated carbohydrate antigens from meningococcal serogroups A, C, W135 and / or Y may be as disclosed in WO 2018 / 045286 A1 or WO 2002 / 058737 A2.

[0277] In one embodiment, the additional antigen is that of the commercially available MenACYW-TT conjugate vaccine MENQUADFI®.

[0278] Adjuvants In one embodiment, the compositions disclosed herein may further comprise an adjuvant.

[0279] In one embodiment, the adjuvant may be an aluminum-based adjuvant (or aluminum salt). Aluminum-based adjuvants include aluminum hydroxide adjuvants (e.g., aluminum oxyhydroxide-AlOOH), aluminum phosphate adjuvants (e.g., aluminum hydroxyphosphate-Al(OH)PO 4- or aluminum orthophosphate - AlPO 4 ), an aluminum sulfate salt adjuvant, an aluminum sulfate hydroxyphosphate adjuvant, an aluminum potassium sulfate adjuvant, aluminum hydroxycarbonate, a combination of aluminum hydroxide and magnesium hydroxide (commercially available as Imject® Alum), or a mixture thereof. In one embodiment, the aluminum-based adjuvant is an aluminum hydroxide adjuvant (e.g., aluminum oxyhydroxide-AlOOH), an aluminum phosphate adjuvant (e.g., aluminum hydroxyphosphate-Al(OH)PO 4 - or aluminum orthophosphate - AlPO 4 ), or a mixture thereof. In one embodiment, the aluminum-based adjuvant is an aluminum phosphate adjuvant (e.g., aluminum hydroxyphosphate (Al(OH)PO 4 ) or aluminum orthophosphate-(AlPO 4 )). The adjuvant may be in any suitable form, e.g., gel, crystalline, amorphous. In one embodiment, the aluminum-based adjuvant may be an amorphous aluminum hydroxyphosphate salt. In one embodiment, the aluminum-based adjuvant may be an aluminum orthophosphate salt. In another embodiment, the aluminum-based adjuvant may be a crystalline aluminum oxyhydroxide salt (boehmite).

[0280] Aluminum hydroxyphosphate or orthophosphate adjuvants can be obtained by precipitation of aluminum oxyhydroxide in the presence of phosphoric acid. The reaction conditions and reactant concentrations during the precipitation reaction affect the degree of substitution of phosphate for hydroxyl in the salt.

[0281] An aluminum hydroxyphosphate or orthophosphate adjuvant may have a PO / Al molar ratio of 0.3 to 0.99, such as a ratio of 0.8 to 0.95 (eg, 0.88+0.05).

[0282] Aluminum hydroxyphosphate [[Al(OH)x(PO 4 )y]adjuvant, where the sum of the valences of each anion and their mole fractions is -3] is formed by the presence of hydroxyl groups into AlPO 4 For example, 3146cm -1 The IR spectrum band of (eg, when heated to 200° C.) indicates the presence of structural hydroxyls.

[0283] Aluminum oxyhydroxide [AIO(OH)] can be detected by IR spectroscopy, especially at 1070 cm -1 and 3090-3100cm -1 It can be distinguished from Al(OH)3 by the presence of a strong shoulder at

[0284] Mixtures of different aluminium-based adjuvants may also be used.

[0285] In one embodiment, the adjuvant may be an aluminum phosphate adjuvant.

[0286] In exemplary embodiments, the immunogenic compositions disclosed herein may include a single aluminum-based adjuvant, i.e., the aluminum adjuvant present in the composition may account for more than 90%, more than 99%, or even more than 99.9%. The aluminum-based adjuvant may be Al 3+ Al concentration is about 0.5mg / mL to about 1.5mg / mL 3+ or about 0.8 to about 1.2 mg / mL Al 3+ , or about 1.00 mg / mL Al 3+ There may exist such that

[0287] Aluminum-based adjuvants, e.g., aluminum phosphate adjuvants, may be present in the compositions disclosed herein in an amount ranging from about 100 μg / dose to about 1000 μg / dose, or from about 150 μg / dose to about 900 μg / dose, or from about 200 μg / dose to about 800 μg / dose, or from about 250 μg / dose to about 700 μg / dose, or from about 300 μg / dose to about 600 μg / dose, or from about 350 μg / dose to about 550 μg / dose, or from about 400 μg / dose to about 500 μg / dose, or from about 400 μg / dose to about 800 μg / dose, or from about 400 μg / dose, or about 800 μg / dose.

[0288] The compositions may include other adjuvants in addition to (or instead of) the aluminium-based adjuvant.

[0289] Suitable adjuvants include, but are not limited to, calcium phosphate adjuvant, MF59 (4.3% w / v squalene, 0.5% w / v Tween 80™, 0.5% w / v Span 85), CpG-containing nucleic acids (cytosines are unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT / CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like. For experimental animals, Freund's adjuvant (incomplete Freund's adjuvant; complete Freund's adjuvant), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, also called nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also called MTP-PE), and RIBI, which is a mixture of three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS), in 2% squalene / Tween, can be used. The effectiveness of an adjuvant can be determined by measuring the amount of antibody directed to the immunogenic antigen or antigenic epitope.

[0290] Further exemplary adjuvants for enhancing the effectiveness of the compositions include, but are not limited to, the following: (1) oil-in-water emulsion formulations (with or without other specific immunostimulants, such as muramyl peptides (see below) or components of bacterial cell walls), such as (a) MF59 (WO 90 / 14837), which contains 5% squalene, 0.5% Tween 80, and 0.5% SPAN 85 (optionally containing MTP-PE) and is formulated into submicron particles using a microfluidizer; (b) SAF, which contains 10% squalane, 0.4% Tween 80, 5% pluronic blocked polymer L121, and thr-MDP, which is either microfluidized to a submicron emulsion or vortexed to produce a larger particle size emulsion; and (c) 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components, e.g., monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), e.g., MPL+CWS (Detox™), the RIBI adjuvant system (RAS) (Ribi Immunochem, Hamilton, Mont.), (2) saponin adjuvants, e.g., QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.), may be used, or particles produced therefrom, e.g., ISCOMs (immunostimulating complexes), where ISCOMs may lack additional surfactants, e.g., WO 00 / 07621; (3) complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (W099 / 44636), etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.;(5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL), e.g. GB-2220221, EP-A-0689454, optionally in the substantial absence of alum when used together with pneumococcal saccharides, e.g. WO 00 / 56358; (6) combinations of 3dMPL with, e.g., QS21 and / or oil-in-water emulsions, e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides containing CpG motifs (see, e.g., WO 98 / 52581), e.g., oligonucleotides containing at least one CG dinucleotide in which the cytosines are unmethylated; (8) polyoxyethylene ethers or polyoxyethylene esters (see, e.g., WO 99 / 52549); (9) a polyoxyethylene sorbitan ester surfactant in combination with octoxynol (WO 01 / 21207) or at least one further non-ionic surfactant, e.g., a polyoxyethylene alkyl ether or ester surfactant in combination with octoxynol (WO 01 / 21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide) (WO 00 / 62800); (11) a microparticle of an immunostimulant and a metal salt, e.g., WO 00 / 23105; (12) a saponin and an oil-in-water emulsion, e.g., WO 99 / 11241; (13) a saponin (e.g., QS21) + 3dMPL + IM2 (optionally + sterol), e.g., WO 98 / 57659; (14) other substances that act as immunostimulants to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutarinyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE, etc.;

[0291] Adjuvants suitable for administration to humans can be aluminum salt adjuvants (eg, aluminum phosphate or aluminum hydroxide).

[0292] immunogenic composition The immunogenic compositions disclosed herein may include a combination of meningococcal antigens comprising at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

[0293] The immunogenic compositions disclosed herein may include a combination of meningococcal serogroup B antigens comprising at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisseria adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). The fHBP A protein and / or the fHBP B protein are non-lipidated.

[0294] The fHBP A protein may be a mutant protein comprising at least about 85% identity to SEQ ID NO:1, and / or the fHBP B protein may be a mutant protein comprising at least about 85% identity to SEQ ID NO:3.

[0295] The fHBP A protein may comprise at least one amino acid substitution selected from at least one of the following, based on the numbering of SEQ ID NO:6: a) an amino acid substitution of asparagine at amino acid 115 (N115); b) an amino acid substitution of aspartic acid at amino acid 121 (D121); c) an amino acid substitution of serine at amino acid 128 (S128); d) an amino acid substitution of leucine at amino acid 130 (L130); e) an amino acid substitution of valine at position 131 (V131); f) an amino acid substitution of glycine at position 133 (G133); g) an amino acid substitution of lysine at position 219 (K219); and h) an amino acid substitution of glycine at position 220 (G220), or may comprise or consist of SEQ ID NO:2, and / or may be a fHBP A protein. The B protein can comprise at least one amino acid substitution selected from at least one of: a) an amino acid substitution of glutamine (Q38) at amino acid 38; b) an amino acid substitution of glutamic acid (E92) at amino acid 92; c) an amino acid substitution of arginine (R130) at amino acid 130; d) an amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248, based on the numbering of SEQ ID NO:6, or comprises or consists of SEQ ID NO:4.

[0296] The fHBP A protein and / or fHBP B may be present in an amount ranging from about 20 μg / dose to about 200 μg / dose, or from about 25 μg / dose to about 180 μg / dose, or from about 40 μg / dose to about 140 μg / dose, or from about 50 μg / dose to about 120 μg / dose, or from about 75 μg / dose to about 100 μg / dose, or from about 25 μg / dose, or from about 50 μg / dose, or from about 100 μg / dose.

[0297] The NadA protein may be a NadA1 protein or may comprise at least about 85% identity to SEQ ID NO:5 or comprise or consist of SEQ ID NO:5.

[0298] The NadA protein may be present in an amount ranging from about 20 μg / dose to about 200 μg / dose, or from about 25 μg / dose to about 180 μg / dose, or from about 40 μg / dose to about 140 μg / dose, or from about 50 μg / dose to about 120 μg / dose, or from about 75 μg / dose to about 100 μg / dose, or about 50 μg / dose.

[0299] The dOMV may comprise Porin A (PorA).

[0300] The dOMV may be present in an amount ranging from about 5 μg / dose to about 400 μg / dose, or from about 10 μg / dose to about 300 μg / dose, or from about 25 μg / dose to about 250 μg / dose, or from about 35 μg / dose to about 225 μg / dose, or from about 50 μg / dose to about 200 μg / dose, or from about 75 μg / dose to about 180 μg / dose, or from about 100 μg / dose to about 150 μg / dose, or from about 110 μg / dose to about 125 μg / dose, or about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0301] The composition may also include an adjuvant, for example an aluminum-based adjuvant selected from the group including aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate salt adjuvant, aluminum sulfate hydroxyphosphate adjuvant, aluminum potassium sulfate adjuvant, aluminum hydroxycarbonate, a combination of aluminum hydroxide and magnesium hydroxide, and mixtures thereof, such as an aluminum phosphate adjuvant.

[0302] The composition can comprise or consist of about 25 to about 100 μg / dose of non-lipidated fHBP A protein comprising or consisting of SEQ ID NO:2, about 25 to about 100 μg / dose of non-lipidated fHBP B protein comprising or consisting of SEQ ID NO:4, about 25 to about 100 μg / dose of NadA protein comprising or consisting of SEQ ID NO:5, about 20 to about 150 μg / dose of dOMV derived from a MenB strain expressing PorA VR2 P1.2, about 100 to about 600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer, pH 6.0.

[0303] The composition may further comprise at least bound capsular saccharides from one or more of meningococcal serogroups A, C, W135 and / or Y.

[0304] Also disclosed are vaccines comprising the compositions described herein.

[0305] The compositions or vaccines disclosed herein may be for use in protecting against meningococcal infection or in inducing an immune response against meningococcal.

[0306] Further disclosed are compositions comprising or consisting of an mRNA encoding a fHBP A protein that comprises at least about 85%, at least about 90%, at least 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:2, an mRNA encoding a fHBP B protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:4, an mRNA encoding a NadA protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5, and a dOMV from MenB expressing PorA VR2 P1.2.

[0307] formulation In another aspect, the present disclosure relates to a vaccine comprising the composition disclosed herein.

[0308] The immunogenic or vaccine compositions disclosed herein can be formulated as preparations in solid, semi-solid, or liquid form, such as tablets, capsules, powders, aerosols, solutions, suspensions, or emulsions. Typical routes of administration of such compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. In some embodiments, the vaccine compositions disclosed herein can be administered by transdermal, subcutaneous, intradermal, or intramuscular routes. The compositions of the present disclosure are formulated based on the mode of delivery, including, for example, compositions formulated for parenteral delivery, such as delivery via intramuscular, intradermal, or subcutaneous injection.

[0309] The immunogenic compositions disclosed herein can be administered via any suitable route, for example, mucosal administration (e.g., intranasal or sublingual), parenteral administration (e.g., intramuscular, subcutaneous, transdermal, or intradermal routes), or oral administration. As will be appreciated by those skilled in the art, the immunogenic compositions can be suitably formulated to be compatible with the intended route of administration. In one embodiment, the immunogenic compositions disclosed herein can be formulated to be administered via an intramuscular, intradermal, or subcutaneous route. In one embodiment, the immunogenic compositions can be formulated to be administered via an intramuscular route.

[0310] The compositions disclosed herein are formulated so as to allow the active ingredients contained therein to be bioavailable when the composition is administered to a subject.

[0311] Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

[0312] The immunogenic compositions disclosed herein can be formulated with any pharma- ceutically acceptable excipient. The compositions can include at least one inert diluent or carrier. One exemplary pharma-ceutically acceptable vehicle is physiological saline buffer. Other physiologically acceptable vehicles are known to those skilled in the art and are described, for example, in "Remington's Pharmaceutical Sciences" (18th ed.), A. Gennaro, ed., 1990, Mack Publishing Company, Easton, Pa. The immunogenic compositions described herein may optionally include pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, non-essential amino acids, L-arginine hydrochloride, saccharose, D-trehalose dehydrate, sorbitol, tris(hydroxymethyl)aminomethane, and / or urea. In addition, the vaccine compositions may optionally include pharma- ceutically acceptable additives, such as diluents, binders, stabilizers, and preservatives.

[0313] In one embodiment, the composition may be in liquid form, for example, solution, emulsion or suspension. The liquid may be for delivery by injection. The composition intended to be administered by injection may include at least one of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents. The liquid composition disclosed herein may include at least one of sterile diluents, for example, distilled water for injection, saline, for example, physiological saline, Ringer's solution, isotonic sodium chloride, fixed oil, for example, synthetic mono- or diglycerides that can function as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents, for example, benzyl alcohol or methylparaben; antioxidants, for example, ascorbic acid or sodium bisulfite; chelating agents, for example, ethylenediaminetetraacetic acid; buffers, for example, acetates, citrates or phosphates, and agents for adjusting tonicity, for example, sodium chloride or dextrose; agents that act as cryoprotectants, for example, sucrose or trehalose.

[0314] In another embodiment, the compositions disclosed herein may have a pH ranging from about 4.0 to about 9.0.

[0315] The pH of the immunogenic compositions disclosed herein may range from about 4.5 to about 8.5, or from about 4.8 to about 8.2, or from about 5.0 to about 8.0, or from about 5.2 to about 7.5, or from about 5.4 to about 7.0, or from about 5.5 to about 6.8, or from about 5.7 to about 6.5, or from about 5.8 to about 6.2. In one embodiment, the pH of the compositions disclosed herein may be about 6.0. A stable pH may be maintained by the use of a buffer.

[0316] In one embodiment, the composition disclosed herein may further comprise a buffer.Possible usable buffers include Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer, or histidine buffer.

[0317] In an exemplary embodiment, the compositions disclosed herein may include a sodium acetate buffer. The sodium acetate buffer may be present at a concentration in the range of about 10 mM to about 300 mM, or in the range of about 10 mM to about 250 mM, or in the range of about 20 mM to about 250 mM, or in the range of about 20 mM to about 150 mM, or in the range of about 20 mM to about 130 mM, or in the range of about 30 mM to about 120 mM, or in the range of about 40 mM to about 100 mM, or in the range of about 50 mM to about 80 mM, or in the range of about 50 mM to about 60 mM, or at a concentration of, for example, about 50 mM.

[0318] The immunogenic composition can be isotonic with respect to a mammal, for example a human.

[0319] The immunogenic composition may also include one or more additional salts, such as a sodium salt, a calcium salt, or a magnesium salt. In one embodiment, the sodium salt may be selected in the group including sodium chloride, sodium phosphate. In one embodiment, the sodium salt may be sodium chloride. The calcium salt may be a calcium chloride salt. The magnesium salt may be a magnesium chloride salt. In one embodiment, the sodium salt may be present at a concentration ranging from about 10 mM to about 300 mM, or from about 30 mM to about 280 mM, or from about 50 mM to about 250 mM, or from about 60 mM to about 220 mM, or from about 80 mM to about 200 mM, or from about 100 mM to about 180 mM, or from about 120 mM to about 160 mM, or may be present at a concentration of, for example, about 150 mM. Calcium or magnesium may be present in an amount ranging from about 1 mM to about 15 mM, or from about 5 mM to about 10 mM.

[0320] The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Injectable compositions, for example, are sterile.

[0321] The immunogenic compositions disclosed herein may be sterilized by conventional sterilization techniques, for example, by UV or gamma radiation, or may be sterile filtered. The resulting composition of the liquid immunogenic compositions disclosed herein after sterile filtration may be packaged and stored in liquid form, or may be lyophilized. Lyophilized compositions may be reconstituted with a sterile aqueous carrier before administration. Dry compositions may contain stabilizers, such as mannitol, sucrose, or dodecyl maltoside, as well as mixtures thereof, such as lactose / sucrose mixtures, sucrose / mannitol mixtures, and the like.

[0322] The compositions disclosed herein are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used; the metabolic stability and length of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject receiving the therapy.

[0323] In one embodiment, the composition disclosed herein can comprise or consist of a non-lipidated fHBP A protein comprising or consisting of SEQ ID NO:2, a non-lipidated fHBP B protein comprising or consisting of SEQ ID NO:4, a NadA protein comprising or consisting of SEQ ID NO:5, a dOMV from MenB expressing PorA VR2 P1.2. The composition can comprise an aluminum phosphate adjuvant. The composition can comprise 50 mM acetate buffer and pH 6.0.

[0324] In one embodiment, the composition disclosed herein can comprise or consist of a non-lipidated fHBP A protein consisting of SEQ ID NO:2, a non-lipidated fHBP B protein consisting of SEQ ID NO:4, a NadA protein consisting of SEQ ID NO:5, a MenB-derived dOMV expressing PorA VR2 P1.2, an aluminum phosphate adjuvant, 50 mM acetate buffer, and pH 6.0.

[0325] In another embodiment, the compositions disclosed herein can comprise or consist of about 25 to about 100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 25 to about 100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 25 to about 100 μg / dose of NadA protein consisting of SEQ ID NO:5, about 20 to about 150 μg / dose of dOMV derived from a MenB strain expressing PorA VR2 P1.2, about 100 to about 800 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer, pH 6.0.

[0326] In another embodiment, the compositions disclosed herein may comprise or consist of about 25 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 25 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 25 μg / dose of NadA protein consisting of SEQ ID NO:5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0327] In another embodiment, the compositions disclosed herein may comprise or consist of about 25 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 25 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 50 μg / dose of NadA protein consisting of SEQ ID NO:5, about 50 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0328] In another embodiment, the compositions disclosed herein may comprise or consist of about 50 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 50 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 50 μg / dose of NadA protein consisting of SEQ ID NO:5, about 25 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0329] In another embodiment, the compositions disclosed herein may comprise or consist of about 50 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 50 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 50 μg / dose of NadA protein consisting of SEQ ID NO:5, about 50 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0330] In another embodiment, the compositions disclosed herein may comprise or consist of about 50 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 50 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 50 μg / dose of NadA protein consisting of SEQ ID NO:5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0331] In another embodiment, the compositions disclosed herein may comprise or consist of about 50 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 50 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 50 μg / dose of NadA protein consisting of SEQ ID NO:5, about 50 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 200 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0332] In another embodiment, the compositions disclosed herein may comprise or consist of about 75 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 75 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 75 μg / dose of NadA protein consisting of SEQ ID NO:5, about 75 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 300 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0333] In another embodiment, the compositions disclosed herein may comprise or consist of about 100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 100 μg / dose of NadA protein consisting of SEQ ID NO:5, about 125 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0334] In another embodiment, the compositions disclosed herein may comprise or consist of about 100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 50 μg / dose of NadA protein consisting of SEQ ID NO:5, about 50 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 400 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0335] In another embodiment, the compositions disclosed herein may comprise or consist of about 100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 50 μg / dose of NadA protein consisting of SEQ ID NO:5, about 50 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0336] In one embodiment, the disclosure relates to a container comprising a composition disclosed herein. The container may contain an immunogenic composition comprising a combination of meningococcal antigens, said combination comprising at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisseria adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

[0337] Further, the container may contain a composition comprising or consisting of about 25-100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2, about 25-100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4, about 25-100 μg / dose of NadA protein consisting of SEQ ID NO:5, about 20-150 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100-800 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0338] The container may be a vial. The vial may be a multi-dose or single-dose vial. A suitable vial may be a small glass or plastic container sealed with a suitable stopper and seal.

[0339] Alternatively, the container can be a pre-filled syringe.The pre-filled syringe can include a syringe barrel that stores the liquid composition disclosed herein.A gasket and a plunger are inserted into the syringe barrel.The gasket seals the syringe barrel in a liquid-tight manner to prevent leakage of liquid drug, and the plunger slides the gasket.Various types of pre-filled syringes are known in the art, for example, as described in US Patent No. 10,625,025 or WO 2013 / 046855.

[0340] When the composition disclosed herein is mixed with another vaccine composition, for example, a tetravalent MenACWY conjugate composition, and injected, both compositions can be packaged in a single vial or container that is a prefilled syringe, or in a dual chamber syringe. A dual chamber syringe, also known as a sequential syringe or bypass syringe, can include a single barrel separated into two compartments, proximal and distal, by a septum. Depression of the plunger of the syringe forces the mixing of the two vaccine compositions in the distal compartment. Various types of dual chamber syringes are known in the art, for example, as described in U.S. Pat. No. 10,695,505. A dual chamber syringe can also be used when the vaccine composition is formulated in a dry form, such as a lyophilized form, and stored with a liquid vehicle for reconstitution. In such a case, the dry vaccine is stored in one chamber, and the liquid for reconstitution and injection is stored in the second chamber.

[0341] Manufacturing method The compositions disclosed herein can be prepared by methodology well known in the pharmaceutical art.

[0342] In one embodiment, the present disclosure relates to a method for preparing an immunogenic composition as disclosed herein or a vaccine as disclosed herein, comprising at least one step of mixing meningococcal antigens and, optionally, an aluminum salt, wherein said antigens comprise at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

[0343] In one embodiment, the step of mixing may optionally include AlPO 4 of at least one factor H binding protein (fHBP) A protein adsorbed onto the 4 A first mixture of at least one fHBP B protein adsorbed on a salt is optionally combined with AlPO 4 combining said at least one NadA protein adsorbed thereon with a second mixture of at least one dOMV.

[0344] In one embodiment, the step of mixing comprises mixing AlPO 4 of at least one factor H binding protein (fHBP) A protein adsorbed onto AlPO 4 A first mixture of at least one fHBP B protein adsorbed onto a salt is added to AlPO 4 combining said at least one NadA protein adsorbed thereon with a second mixture of at least one dOMV.

[0345] The antigens fHBP A and B and NadA are first adjuvanted with an aluminum-based adjuvant, e.g., AlPO 4The fHBP A and B adsorbed on an aluminum-based adjuvant can be mixed together in a suitable buffer. The NadA and dOMV adsorbed on an aluminum-based adjuvant can be mixed together in a suitable buffer. The two mixtures can then be mixed together to obtain the immunogenic composition disclosed herein.

[0346] The additional antigen composition, e.g., a conjugated MenACWY polysaccharide composition, can then be mixed with the compositions disclosed herein. The additional antigen composition, e.g., a conjugated MenACWY polysaccharide composition, can be mixed with the immunogenic composition disclosed herein immediately prior to administration to the patient, optionally via a dual chamber syringe that mixes the two compositions together prior to administration.

[0347] In another embodiment, the immunogenic composition or vaccine disclosed herein is co-administered with a conjugated MenACWY polysaccharide immunogenic composition or vaccine. The conjugated MenACWY polysaccharide immunogenic composition or vaccine that can be co-administered with the immunogenic composition or vaccine disclosed herein can be MenA, MenC, MenW-135 and MenY capsular polysaccharide, each of which is conjugated to a tetanus toxoid carrier protein, and optionally MenA polysaccharide is conjugated to a tetanus toxoid carrier via an adipic acid dihydrazide (ADH) linker, and MenC, MenW-135 and MenY polysaccharide are each directly conjugated to a tetanus toxoid carrier. The conjugated MenACWY polysaccharide immunogenic composition or vaccine that is co-administered can be as disclosed in WO 2018 / 045286 A1 or WO 2002 / 058737 A2. The co-administered conjugated MenACWY polysaccharide immunogenic composition or vaccine may be the commercially available MenACYW-TT conjugate vaccine MENQUADFI®.

[0348] In another embodiment, the antigen and aluminum salt for use in the preparation methods disclosed herein may be in a buffer.

[0349] The compositions disclosed herein can be prepared in liquid or solid, e.g., lyophilized, form.

[0350] In one embodiment, the immunogenic compositions disclosed herein can be packaged and stored in a dry form, e.g., a lyophilized composition, or as a micropellet obtained via a prilling process as described in WO 2009 / 109550. In one embodiment, the different components of the composition, e.g., the MenB antigen, and possibly further antigens, can all be present in the same micropellet. In another embodiment, the components of the immunogenic composition disclosed herein may each be in a separate micropellet, i.e., one component per micropellet, or all or a portion thereof, in which case the other one or more components may each be in a separate micropellet, or may be combined in the same micropellet as a duo or triplet (bivalent or trivalent formulation) (such as fHBP A+B and NadA+dOMV, fHBP A+NadA and fHBP B+dOMV, fHBP B+NadA and fHBP A+dOMV, fHBP A+NadA+fHBP B and dOMV, or fHBP A+dOMV+fHBP B and NadA). In such an embodiment, different micropellets containing different components separately or any combination of at least two of the different components may be mixed before administration to a subject. In one embodiment, they may be mixed before reconstitution in the liquid carrier. In another embodiment, they may be mixed at the time of reconstitution in the liquid carrier by adding them to a volume of the liquid carrier. In another embodiment, they can first be added separately to separate volumes of each liquid carrier, and then the different volumes of the liquid carrier can be mixed together to obtain the final liquid composition that is administered to a subject.

[0351] In one embodiment, four antigens and AlPO 4 are in the same container.

[0352] In another embodiment, the adjuvant and the antigen can be prepared in at least two separate compositions. The separate compositions can then be mixed together immediately prior to administration to the patient, optionally via a dual chamber syringe that mixes the two compositions together prior to administration. In another embodiment, the separate compositions can be administered separately, i.e., administered simultaneously (actually, within seconds or minutes, e.g., less than 5 minutes apart), but via at least two separate administration sites, e.g., at least two separate injection sites. In another embodiment, the separate compositions can be administered sequentially, i.e., at least two separate time points, e.g., at least 5 minutes apart, or several hours or one or two days apart. In such an embodiment, the separate compositions can be administered to the same administration site, e.g., the same injection site, or to different administration sites, e.g., different injection sites. In such an embodiment, at least one of the different components of the compositions disclosed herein can be provided separately as a kit of parts.

[0353] Parts Kit In one embodiment the present disclosure relates to a kit of parts comprising a plurality of containers, each of the containers comprising at least one or a combination of at least two meningococcal antigens, said antigens being selected from the group comprising fHBP A protein, fHBP B protein, NadA protein, and detergent extracted outer membrane vesicles (dOMV).

[0354] In one embodiment, at least one of the antigens of the kit of parts may be in a dry form. In one embodiment, at least one of the antigens may be in a lyophilized or dry micropellet form. In one embodiment, the kit may optionally include a container containing a physiologically injectable vehicle.

[0355] In one embodiment, the different antigens of the immunogenic compositions disclosed herein can be prepared and stored in separate containers or vials, which can then be mixed at the time of administration to an individual.

[0356] In one embodiment, each of the antigens fHBP A, fHBP B, NadA and dOMV can be stored in one container. In another embodiment, the antigens can be combined by pairs or triplets (bivalent or trivalent formulations). Any kind of combination can be envisaged, such as fHBP A+B and NadA+dOMV, fHBP A+NadA and fHBP B+dOMV, fHBP A+NadA and fHBP A+dOMV, fHBP A+NadA+fHBP B and dOMV, or fHBP A+dOMV+fHBP B and NadA.

[0357] In one embodiment, the fHBP A and B antigens can be stored in a first container, the NadA antigen can be stored in a second container, and the dOMV antigen can be stored in a third container. In another embodiment, the fHBP A and B antigens can be stored in a first container, and both the NadA antigen and the dOMV antigen can be stored in a second container.

[0358] The antigens can be stored in liquid formulations or in dry form. When formulated in dry form, an auxiliary container can contain an injectable liquid carrier that is used to resuspend and mix the different antigens. A suitable injectable liquid carrier can include a buffer. In addition, it can include an adjuvant as described above. The adjuvant can be an aluminum-based adjuvant, e.g., AlPO 4 It could be.

[0359] Optionally, the kit of parts may comprise at least one further container or a plurality of further containers for at least one further antigen or a plurality of further antigens. In one embodiment, the further antigen may be a conjugated MenACWY polysaccharide.

[0360] In one embodiment, the kit of parts may comprise a first container comprising an immunogenic composition as disclosed herein, and a second container comprising a conjugated MenACWY polysaccharide.

[0361] Methods of Use and Treatment In one embodiment, the present disclosure relates to the compositions disclosed herein for use as medicaments, in particular as vaccines.

[0362] In another aspect, the disclosure relates to a composition disclosed herein for use in protecting against meningococcal infection, and in one exemplary embodiment, against meningococcal serogroup B infection.

[0363] In another aspect, the disclosure relates to a composition disclosed herein for use in inducing an immune response against Neisseria meningitidis, and in one exemplary embodiment, against Neisseria meningitidis serogroup B bacteria.

[0364] In another aspect, the disclosure relates to a composition disclosed herein for use in inducing an immune response against meningococcal serogroup B bacteria from ST-41 / 44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and / or the ST-461 clonal complex.

[0365] In another aspect, the disclosure relates to a composition disclosed herein for use in inducing an immune response against Neisseria meningitidis serogroup B bacteria from the ST-11 clonal complex.

[0366] In another aspect, the disclosure relates to a method for protecting an individual against meningococcal infection, and in one exemplary embodiment against meningococcal serogroup B infection, comprising at least the step of administering to the individual an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0367] In another aspect, the disclosure relates to a method of reducing the risk of developing invasive meningococcal disease due to meningococcal infection, and in one exemplary embodiment due to meningococcal serogroup B infection, in an individual, comprising at least the step of administering to the individual an immunogenic composition as disclosed herein, or a vaccine as disclosed herein.

[0368] In another aspect, the disclosure relates to a method of eliciting an immune response in an individual against Neisseria meningitidis, and in one exemplary embodiment against Neisseria meningitidis serogroup B bacteria, comprising at least the step of administering to the individual an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0369] In another aspect, the disclosure relates to a method of eliciting an immune response against meningococcal serogroup B bacteria from ST-41 / 44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and / or the ST-461 clonal complex, comprising at least the step of administering to an individual an immunogenic composition as disclosed herein, or a vaccine as disclosed herein.

[0370] In another aspect, the disclosure relates to a method of eliciting an immune response against meningococcal serogroup B bacteria from the ST-11 clonal complex, comprising at least the step of administering to an individual an immunogenic composition disclosed herein, or a vaccine disclosed herein.

[0371] In one embodiment, the immunogenic compositions disclosed herein may be for use in eliciting an immune response against meningococcal serogroup B in an individual. The individual may be a mammal, such as a human, such as an infant, a toddler, a child, a teenager, a young adult, an adult, and an elderly person. In one embodiment, the individual may be 6 weeks or older, 2 months or older, or 10 years or older. As an exemplary embodiment, the individual may be 6 weeks to 55 years or older, such as 2 months to 55 years or older, or such as 10 to 55 years or older.

[0372] The methods generally involve administering an effective amount of a subject immunogenic composition to an individual in need thereof. Amounts effective for therapeutic use will depend, for example, on the antigenic composition, the method of administration, the patient's weight and general state of health, and the judgment of the prescribing physician. Single or multiple administrations of the antigenic composition will be administered depending on the dosage and frequency required and tolerated by the patient, and the route of administration.

[0373] The immunogenic compositions disclosed herein are administered in a 2, 3, 2+1, or 3+1 dose regimen.

[0374] In one embodiment, the immunogenic compositions disclosed herein are administered in two or three doses. Subsequent doses are administered about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, or about 20 months apart from the previous dose. In one embodiment, subsequent doses are administered about 1 month, about 2 months, about 5 months, about 6 months, about 8 months, about 10 months, about 12 months, about 14 months, or about 16 months apart from the previous dose. In one embodiment, subsequent doses are administered about 1 month, about 2 months, about 5 months, about 6 months, or about 8 months apart from the previous dose. In one embodiment, subsequent doses are administered about 30 days, about 60 days, or about 180 days apart from the previous dose.

[0375] In a two-dose regimen, the second dose is administered about 1 month after the first dose, or about 2 months after the first dose, or about 6 months after the first dose. Alternatively, in a two-dose regimen, the second dose is administered about 30 days after the first dose, or about 60 days after the first dose, or about 180 days after the first dose. Such two-dose regimens may be suitable for adults and / or adolescents.

[0376] In a two-dose regimen, the second dose is administered about 2 months after the first dose. Alternatively, in a two-dose regimen, the second dose is administered about 60 days after the first dose. Such a two-dose regimen may be suitable for young children.

[0377] In a three dose regimen, the second dose is administered about 1 month after the first dose and the third dose is administered about 6 months after the first dose. Alternatively, in a three dose regimen, the second dose is administered about 30 days after the first dose and the third dose is administered about 180 days after the first dose. Such a three dose regimen may be suitable for adults and / or adolescents.

[0378] In a three dose regimen, the second dose is administered about 2 months after the first dose and the third dose is administered about 10 months after the first dose. Alternatively, in a three dose regimen, the second dose is administered about 60 days after the first dose and the third dose is administered at about 12 months of age. Such a three dose regimen may be suitable for infants.

[0379] In one embodiment, in addition to the two or three doses, the third or fourth dose is administered.This subsequent dose is administered at least one year after the last dose of the two or three doses, for example 16 months after the last dose.In such a regimen, the first two or three doses are suitable as prime doses, and the subsequent one (+1) dose is suitable as boost dose.

[0380] In one embodiment, infants and toddlers, for example, 6 weeks of age or 2 months to 2 years of age, may receive a 2+1 or 3+1 dose regimen. In another embodiment, children, for example, 2-10 years of age, may receive a 2 dose regimen. In another embodiment, teenagers and adults, for example, 10-55 years of age, may receive a 2+1 dose regimen.

[0381] The immunogenic compositions disclosed herein may be administered by any suitable route. For example, administration by the intramuscular route is contemplated.

[0382] The present invention is further described by the following clauses and embodiments.

[0383] According to embodiment 1, the present invention relates to an immunogenic composition comprising a combination of meningococcal serogroup B antigens, said combination comprising at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisseria adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

[0384] According to embodiment 2, the present invention relates to a composition according to embodiment 1, wherein the fHBP A protein and / or the fHBP B protein is not lipidated.

[0385] According to embodiment 3, the present invention relates to a composition according to embodiment 1 or 2, wherein the fHBP A protein is a mutant protein comprising at least about 85% identity with SEQ ID NO: 1, and / or the fHBP B protein is a mutant protein comprising at least about 85% identity with SEQ ID NO: 3.

[0386] According to embodiment 4, the present invention relates to a fHBP A protein comprising at least one amino acid substitution selected from at least one of the following based on the numbering of SEQ ID NO: 6: a) an amino acid substitution of asparagine at amino acid 115 (Ν115); b) an amino acid substitution of aspartic acid at amino acid 121 (D121); c) an amino acid substitution of serine at amino acid 128 (S128); d) an amino acid substitution of leucine at amino acid 130 (L130); e) an amino acid substitution of valine at position 131 (V131); f) an amino acid substitution of glycine at position 133 (G133); g) an amino acid substitution of lysine at position 219 (K219); and h) an amino acid substitution of glycine at position 220 (G220), or comprising or consisting of SEQ ID NO: 2, and / or comprising or consisting of a fHBP A protein comprising at least one amino acid substitution selected from the following based on the numbering of SEQ ID NO: 6: a) an amino acid substitution of asparagine at amino acid 115 (Ν115); The composition according to any one of embodiments 1 to 3, wherein protein B comprises at least one amino acid substitution selected from at least one of the following, based on the numbering of SEQ ID NO: 6: a) an amino acid substitution of glutamine (Q38) at amino acid 38; b) an amino acid substitution of glutamic acid (E92) at amino acid 92; c) an amino acid substitution of arginine (R130) at amino acid 130; d) an amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248, or comprises or consists of SEQ ID NO: 4.

[0387] According to embodiment 5, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein fHBP A protein and / or fHBP B is present in an amount of about 20 μg / dose to about 200 μg / dose, or about 25 μg / dose to about 180 μg / dose, or about 40 μg / dose to about 140 μg / dose, or about 50 μg / dose to about 120 μg / dose, or about 75 μg / dose to about 100 μg / dose, or about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0388] According to embodiment 6, the present invention relates to a composition according to any one of embodiments 1 to 5, wherein the NadA protein is a NadA1 protein or has at least about 85% identity with SEQ ID NO: 5 or comprises or consists of SEQ ID NO: 5.

[0389] According to embodiment 7, the present invention relates to a composition according to any one of embodiments 1 to 6, wherein the NadA protein is present in an amount of about 20 μg / dose to about 200 μg / dose, or about 25 μg / dose to about 180 μg / dose, or about 40 μg / dose to about 140 μg / dose, or about 50 μg / dose to about 120 μg / dose, or about 75 μg / dose to about 100 μg / dose, or about 50 μg / dose.

[0390] According to embodiment 8, the present invention relates to a composition according to any one of embodiments 1 to 7, wherein the dOMV comprises porin A (PorA).

[0391] According to embodiment 9, the present invention relates to a composition according to any one of embodiments 1 to 8, wherein dOMV is present in an amount of about 5 μg / dose to about 400 μg / dose, or about 10 μg / dose to about 300 μg / dose, or about 25 μg / dose to about 250 μg / dose, or about 35 μg / dose to about 225 μg / dose, or about 50 μg / dose to about 200 μg / dose, or about 75 μg / dose to about 180 μg / dose, or about 100 μg / dose to about 150 μg / dose, or about 110 μg / dose to about 125 μg / dose, or about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0392] According to embodiment 10, the present invention relates to a composition according to any one of embodiments 1 to 9, further comprising an adjuvant, optionally an aluminum-based adjuvant selected from the group consisting of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate salt adjuvant, aluminum sulfate hydroxyphosphate adjuvant, aluminum potassium sulfate adjuvant, aluminum hydroxycarbonate, a combination of aluminum hydroxide and magnesium hydroxide, and mixtures thereof.

[0393] According to an eleventh embodiment, the present invention relates to a composition according to any one of the preceding embodiments, comprising or consisting of 25-100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO: 2, 25-100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO: 4, 25-100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20-150 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, 100-600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0394] According to embodiment 12, the present invention relates to a composition according to any one of embodiments 1 to 11, further comprising at least a conjugated capsular saccharide from one or more of meningococcal serogroups A, C, W135 and / or Y.

[0395] According to embodiment 13, the present invention relates to a vaccine comprising a composition according to any one of embodiments 1-13.

[0396] According to embodiment 14, the present invention relates to a composition according to any one of embodiments 1 to 12 or a vaccine according to embodiment 12 for use in protecting against meningococcal infections or for use in inducing an immune response against meningococci.

[0397] According to embodiment 15, the present invention relates to a composition comprising or consisting of an mRNA encoding a fHBP A protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:2, an mRNA encoding a fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:4, an mRNA encoding a NadA protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5, and a dOMV from MenB expressing PorA VR2 P1.2.

[0398] According to embodiment 16, the present invention relates to an immunogenic composition comprising a combination of meningococcal serogroup B antigens, said combination comprising at least one non-lipidated factor H binding protein (fHBP) A protein comprising at least about 85% identity to SEQ ID NO: 1 and at least the amino acid substitution G220S based on the numbering of SEQ ID NO: 6, at least one non-lipidated fHBP B protein, at least one Neisseria adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

[0399] According to embodiment 17, the present invention relates to the composition according to embodiment 16, wherein the non-lipidated fHBP B protein is a mutein comprising at least about 85% identity with SEQ ID NO:3.

[0400] According to embodiment 18, the present invention relates to a composition according to embodiment 16 or 17, wherein the non-lipidated fHBP B protein comprises the amino acid substitution H248L according to the numbering of SEQ ID NO:6.

[0401] According to embodiment 19, the present invention relates to a composition according to any one of embodiments 16 to 18, wherein the non-lipidated fHBP A protein comprises or consists of SEQ ID NO:4.

[0402] According to embodiment 20, the present invention relates to a composition according to any one of embodiments 16 to 19, wherein the non-lipidated fHBP A protein further comprises the amino acid substitutions L130R and G133D according to the numbering of SEQ ID NO: 6.

[0403] According to embodiment 21, the present invention relates to a composition according to any one of embodiments 16 to 20, wherein the non-lipidated fHBP A protein comprises or consists of SEQ ID NO:2.

[0404] According to embodiment 22, the present invention relates to a composition according to any one of embodiments 16 to 21, wherein the NadA protein is a NadA1 protein or has at least about 85% identity with SEQ ID NO: 5 or comprises or consists of SEQ ID NO: 5.

[0405] According to embodiment 23, the present invention relates to a composition according to any one of embodiments 16 to 22, wherein the dOMV comprises the PorA VR2 subtype.

[0406] According to embodiment 24, the present invention relates to a composition according to any one of embodiments 16 to 23, wherein the dOMV comprises PorA VR2 P1.2.

[0407] According to embodiment 25, the present invention relates to a composition according to any one of embodiments 16 to 24, further comprising an adjuvant, optionally an aluminum-based adjuvant selected from the group consisting of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate salt adjuvant, aluminum sulfate hydroxyphosphate adjuvant, aluminum potassium sulfate adjuvant, aluminum hydroxycarbonate, a combination of aluminum hydroxide and magnesium hydroxide, and mixtures thereof.

[0408] According to embodiment 26, the present invention relates to a method for the preparation of a medicament ... or about 50 μg / dose, and dOMV is present in an amount of about 5 μg / dose to about 400 μg / dose, or about 10 μg / dose to about 300 μg / dose, or about 25 μg / dose to about 250 μg / dose, or about 35 μg / dose to about 225 μg / dose, or about 50 μg / dose to about 200 μg / dose, or about 75 μg / dose to about 180 μg / dose, or about 100 μg / dose to about 150 μg / dose, or about 110 μg / dose to about 125 μg / dose, or about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0409] According to embodiment 27, the present invention relates to a composition according to any one of embodiments 16 to 26, comprising or consisting of 25 to 100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO: 2, 25 to 100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO: 4, 25 to 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20 to 150 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, 100 to 600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.0.

[0410] According to embodiment 28, the present invention relates to a composition according to any one of embodiments 16 to 27, further comprising at least a conjugated capsular saccharide from one or more of N. meningitidis serogroups A, C, W135 and / or Y.

[0411] According to embodiment 29, the present invention relates to a vaccine comprising a composition according to any one of embodiments 16 to 28.

[0412] According to embodiment 30, the present invention relates to a method of treating meningococcal infection or inducing an immune response against meningococcal, comprising administering to an individual in need thereof a composition comprising or consisting of a fHBP A protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:2, a fHBP B protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:4, a NadA protein that comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5, and a dOMV from MenB expressing PorA VR2 P1.2.

[0413] At least one limitation, element, descriptive term of a clause, etc. from at least one of the enumerated claims should be understood to encompass all of the variations, combinations, and permutations that are introduced into another claim relying on the same base claim (or any other claim, if relevant), unless otherwise indicated or unless it is obvious to one skilled in the art that a contradiction or inconsistency would result. When elements are presented as a list, for example in a Markush group or similar format, it should be understood that each subgroup of elements is also disclosed and any element can be removed from the group. In general, when the disclosure, or aspects of the disclosure, are said to include certain elements, features, etc., they should be understood to also encompass embodiments that consist of or consist essentially of such elements. For purposes of brevity, those embodiments have not been explicitly and specifically described in every instance herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether a specific exclusion is described herein. Publications and other reference materials referenced herein to describe the background of the disclosure and to provide additional details regarding its implementation are incorporated herein by reference.

[0414] The sequences disclosed herein serve as references. The same sequences are also shown in the sequence listing, which is formatted according to standard requirements for patent purposes. In the event that any sequence is inconsistent with the standard sequence listing, the sequences described herein are the reference sequences. EXAMPLES

[0415] Without limiting the disclosure, a number of embodiments of the disclosure are described below for purposes of illustration. EXAMPLES

[0416] Antigen Production and Immunogenic Compositions 1. Preparation and purification of MenB antigen 1. Non-lipidated mutant fHBP A05 (A05tmN) To produce non-lipidated A05tmN, three point mutations (G220S, L130R and G133D, numbering relative to SEQ ID NO: 6) were introduced into the wild-type fHBP A05 sequence, and the first cysteine ​​residue that anchors the lipid moiety at the N-terminus was replaced with a methionine (non-lipidated A05tmN: SEQ ID NO: 2) to obtain a non-lipidated protein. The DNA sequence of A05tmN was synthesized and then used to produce a plasmid construct. Briefly, DNA sequences of Xba1 and Xho1 sites were added to both ends of the A05tmN sequence. To generate an expression plasmid, the Xba1 / Xho1-containing plasmid was digested and the resulting DNA fragment was ligated into Xba1 / Xho1-digested pET28a(+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba I / Xho I digestion. The A05tmN plasmid was transformed into E. coli and a cell bank was produced after three rounds of colony purification.

[0417] The E. coli strain transformed with A05tmN was amplified in semi-synthetic medium (pH 6.8, dissolved oxygen: 20%) with stirring at 37°C. Antigen expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0418] The cultures were harvested as raw bulk and the bacterial biomass was separated from the medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was processed through a homogenizer to produce a cell homogenate. The homogenate was then centrifuged to collect the pellet fraction. The homogenate pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5) and subjected to a pH shock treatment (pH 12, 1 h at room temperature with mixing). The pH was returned to 8.5 with 85% phosphoric acid. The supernatant fraction of the pH shocked material was collected after centrifugation and then filtered to obtain the filtered supernatant.

[0419] The supernatant was adjusted to pH 8.5 and <5.0 mS / cm conductivity and loaded onto a capture column, GigaCap Q-650M. The elution pool was adjusted to 0.9 M ammonium sulfate (AmS) and then further purified by medium pressure chromatography, Toyopearl Phenyl 600M. After hydrophobic interaction chromatography, the elution pool was adjusted to pH 8.5 and <8.0 mS / cm conductivity and further purified by Nuvia aPrime 4A chromatography. This was followed by a final ultrafiltration and diafiltration using a 5 kDa regenerated cellulose tangential flow filtration (TFF) membrane, and 0.2 μm filtration.

[0420] A05tmN was dissolved in AlPO4 at 1.00 mg Al / mL in acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0). 4 The mixture was adsorbed onto the

[0421] 2. Non-lipidated mutant fHBP B01 (B01smN) To produce non-lipidated B01smN, a single point mutation was introduced into the wild-type fHBP B01 sequence, replacing the first cysteine ​​residue that anchors the lipid moiety at the N-terminus with a methionine (non-lipidated B01smN: SEQ ID NO: 4) to obtain a non-lipidated protein. The DNA sequence of B01smN was synthesized and then used to produce a plasmid construct. Briefly, DNA sequences for Xba1 and Xho1 sites were added to both ends of the B01smN sequence. To generate an expression plasmid, the Xba1 / Xho1-containing plasmid was digested and the resulting DNA fragment was ligated into Xba1 / Xho1-digested pET28a(+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba I / Xho I digestion. The B01smN plasmid was transformed into E. coli, and a cell bank was produced after three rounds of colony purification.

[0422] The E. coli strain transformed with B01smN was amplified in semi-synthetic medium (pH 6.8, dissolved oxygen: 20%) with stirring at 37°C. Antigen expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0423] The culture was harvested as raw bulk and the bacterial biomass was separated from the medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was processed through a homogenizer to produce a cell homogenate. The homogenate was then centrifuged to collect the supernatant fraction. The supernatant fraction was then filtered.

[0424] The filtered supernatant was adjusted to pH 8.5 and <5.0 mS / cm conductivity, loaded onto a CaptoQ ImpRes chromatography column and purified in bind and elute mode. The CaptoQ ImpRes elution pool was then adjusted to 1.8 M AmS for loading onto a second chromatography column, Phenyl Sepharose HP. After elution, the material was concentrated and diafiltered into acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0) using a 5 kDa Ultracel TFF membrane, then 0.2 μm filtered.

[0425] B01smN was dissolved in AlPO4 at 1.00 mg Al / mL in acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0). 4 The mixture was adsorbed onto the

[0426] 3. NadA A truncated form of NadA was produced from NadA_MC58. The truncated NadA had the leader sequence (residues 1-23) and anchor domain (residues 308-362) of NadA_MC58 removed (truncated NadA: SEQ ID NO: 5). In the truncated sequence of NadA_MC58, the first amino acid after the leader sequence was alanine, and the alanine was replaced with methionine. The DNA sequence of NadA was synthesized and then used to produce a plasmid construct. DNA sequences for Xba1 and Xho1 sites were added to both ends of the NadA sequence. To generate an expression plasmid, the Xba1 / Xho1-containing plasmid was digested and the resulting DNA fragment was ligated into Xba1 / Xho1-digested pET28a(+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba I / Xho I digestion. The NadA plasmid was transformed into E. coli and a cell bank was produced after three rounds of colony purification.

[0427] The NadA1 transformed E. coli strain was amplified in semi-synthetic medium (pH 6.8, dissolved oxygen: 20%) with agitation at 37°C. Antigen expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0428] The culture was harvested as raw bulk and the bacterial biomass was separated from the medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was processed through a homogenizer to produce a cell homogenate. The homogenate was then centrifuged to collect the supernatant fraction. The supernatant fraction was then filtered.

[0429] The supernatant fraction was loaded onto a Capto DEAE column. The Capto DEAE elution fraction was adjusted with AmS powder until a concentration of 500 mM AmS was obtained. The adjusted Capto DEAE elution fraction was loaded onto a Toyopearl Butyl-650M column. The Toyopearl Butyl-650M elution fraction was loaded onto a CHT Type I 40 μm column, and the CHT elution fraction was concentrated using a 30 kDa regenerated cellulose TFF membrane, then diafiltered into 50 mM sodium acetate, 150 mM NaCl, pH 6.0. After TFF, the product was 0.2 μm filtered to produce NadA antigen.

[0430] NadA was prepared by diluting AlPO4 at 1.00 mg Al / mL in acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0). 4 The mixture was adsorbed onto the

[0431] 4.dOMV dOMVs were purified from wild-type meningococcal serotype B, strain 99M, provided by the Walter Reed Army Institute of Research (WRAIR).

[0432] NmB 99M was cultured in the presence of 1 g / L yeast extract and 1M Hepes at 37°C and CO 2 The cells were cultured at 0.5% in a synthetic medium as described by Fu et al. (Biotechnology (NY). February 1995; 13(2):170-4) and in US Pat. No. 5,494,808.

[0433] Culture harvest was performed using low speed centrifugation of the heat-treated suspension (55°C, 2 h) to recover a wet bacterial pellet. Two successive detergent-mediated extraction steps (56°C, 15 min) were performed with an extraction buffer consisting of a detergent (sodium deoxycholate) to extract dOMVs from the bacterial outer membrane and to deplete lipooligosaccharides (as described in Helting et al., Acta Pathol Microbiol Scand C. 1981 Apr;89(2):69-78). Sodium deoxycholate and EDTA solubilize the bacterial outer membrane, which then reorganize itself into dOMVs (vesicles and microparticles). Resuspension was completed by homogenizing the pellet suspended in extraction buffer using an Ultra-Turrax (rotor-stator device). The dOMV supernatants were pooled followed by benzonase treatment in the presence of MgCl2 (37°C, 15 min).

[0434] After dOMV extraction, dOMV enrichment was performed using 300 kDa modified polyethersulfone (mPES) hollow fibers. Several ultracentrifugation steps were used to separate dOMV from "soluble" contents such as nucleic acids, cytoplasmic proteins, extracted lipopolysaccharides or buffer components. The resulting pellet was then resuspended in extraction buffer using an Ultra-Turrax (rotor-stator device) at the lowest speed for a few seconds. After the initial resuspension, high-pressure homogenization was used to thoroughly resuspend dOMV in extraction buffer, increasing detergent accessibility to the dOMV surface.

[0435] Then, after centrifugation, the supernatant was finally filtered through a 0.45 / 0.2 μm cellulose acetate filter.

[0436] 5. Antigen combinations The MenB antigens in the multicomponent immunogenic composition are purified non-lipidated mutant A05 fHBP (A05tmN), non-lipidated mutant fHBP (B01smN), NadA and dOMV. The A05tmN, B01smN and NadA antigens were incubated with aluminum phosphate (AlPO 4 ) was adsorbed onto the

[0437] The solvent was acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0) and AlPO 4 (1.00mg AL 3+ / mL).

[0438] To formulate the final immunogenic composition, the formulation, AlPO 4 , AlPO 4 B01smN, AlPO 4 The A05tmN adsorbed on the target antigen and aluminum were mixed with acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0) to obtain a solution containing the target antigen and aluminum (250 μg / mL for B01smN, 250 μg / mL for A05tmN, and 100 μg / mL for AlPO 4 dOMVs were dissolved in acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0) and AlPO 4 Various NadA-dOMV compositions were formulated by mixing NadA adsorbed to fHBP to obtain different concentrations of antigen (F1-F5) in the various formulations tested below. A formulation of fHBP only was diluted with carrier to obtain formulation F6.

[0439] 2. TRUMENBA (registered trademark) TRUMENBA® is a 10 mM histidine buffer pH 6.0, sodium chloride (NaCl), and aluminum phosphate (AlPO 4 The MenB bivalent recombinant lipidated fHBP (rLP2086) composition is a sterile liquid suspension consisting of fHBP subfamily A (A05) and B (B01) proteins formulated at 60 μg / mL / subfamily in 0.5 mg / mL aluminum as a suction solution, and polysorbate 80 (PS80).

[0440] 3. BEXSERO (registered trademark) BEXSERO® contains three recombinant proteins: rp287-953 (NHBA chimera) at 50 μg / 0.5 mL dose, rp936-741 (fHBP chimera with non-lipidated B24, at 50 μg / 0.5 mL dose), and rp961c (NadA) at 50 μg / 0.5 mL dose; dOMV at 25 μg / 0.5 mL dose, and AlOOH (e.g., Al 0.5 mg) at 1.5 mg / 0.5 mL dose in a buffer containing histidine 0.776 mg / 0.5 mL dose, sodium chloride at 3.125 μg / 0.5 mL dose, sucrose 10 mg / 0.5 mL dose, and water.

[0441] 4. MENQUADFI® MENQUADFI® is a commercially available vaccine containing the obtained ACWY polysaccharide antigen, disclosed in WO 2018 / 045286 A1, conjugated to tetanus toxoid (TT). The formulation contains meningococcal capsular saccharides from serogroups A, C, Y, and W135 separately conjugated to tetanus toxoid protein. The target active ingredient concentration is 10 μg of each polysaccharide and approximately 55 μg of tetanus toxoid protein per 0.5 mL dose. The antigen was formulated in a sterile aqueous solution containing 30 mM sodium acetate buffer (1.23 mg / dose) and sodium chloride (0.67%, 3.35 mg / dose).

[0442] 5. Test Compositions In the following study, six formulations of the MenB multicomponent vaccine disclosed herein (F1-F5, and F1 co-administered with MENQUADFI) were tested and compared to the reference compositions BEXSERO, TRUMENBA and a comparative bivalent (fHBP A and fHBP B) composition (F6). The different amounts of the various antigens in the test compositions are summarized in Table 1 below. F1, F2, F4-F6 and F1 co-administered with MENQUADFI were administered at 0.4 mg AlPO per dose. 4 while F3 contains 0.8 mg AlPO per dose. 4 Includes:

[0443] [Table 1]

[0444] The following volumes per formulation were used for administration of Example 2:

[0445] F1: 400 μL

[0446] F2: 400 μL

[0447] F3: 800 μL

[0448] F4: 400 μL

[0449] F5: 400 μL

[0450] F6: 400 μL

[0451] F1+MENQUADFI: Animals received 400 μL of F1 on days 0, 28, and 56. On day 0, animals also received 500 μl of MENQUADFI in the opposite thigh.

[0452] TRUMENBA: 500μL

[0453] BEXSERO: 500μL EXAMPLES

[0454] Preclinical evaluation of MenB vaccine immunogenicity: Rabbit immunogenicity study 1. Materials and Methods 1. Study Design Nine groups of seven rabbits (New-Zealand KBL - female - 8 weeks old) received three immunizations with seven formulations - designated F1 to F6 and F1+MENQUADFI in Table 1 of Example 1 - given by the IM route on days 0, 28 and 56, or one each of the licensed reference vaccines TRUMENBA and BEXSERO (see Example 1). Formulation F1 was also tested in co-administration with MENQUADFI. The composition was administered by the IM route for the first injection into the right thigh, the second injection into the left thigh and the last injection into the right thigh. For the group receiving F1+MENQUADFI, the animals were co-administered with MENQUADFI given by the IM route on day 0 in the opposite thigh.

[0455] Clinical signs and temperatures were measured 4, 24 and 48 hours after immunization. Blood samples were collected in tubes (BD Vacutainer SST II 8.5mL, ref. 366468A) containing a coagulation activator and serum separator on day 0 and 2 weeks after the second immunization (day 42), and on day 63. To separate the serum from the blood cells, the tubes were centrifuged at 3500 rpm for 15 minutes. The serum was transferred to 4.5mL NUNC tubes and heat inactivated at +56°C for 30 minutes. The serum was stored at -20°C until use in ELISA, purification and bactericidal assays.

[0456] After the second dose a plateau in the antibody response was already reached, therefore immunogenicity was analyzed after the second dose.

[0457] The immunogenicity of the test compositions was measured on day 42 by assessing antigen (Ag)-specific IgG responses measured by ELISA and functional serum bactericidal antibody activity (hSBA) against a panel of 7 or 18 MenB strains. Responders showed at least a 4-fold increase in hSBA between days 0 and 42.

[0458] The positive threshold for a positive response was defined as follows:

[0459] Before (day 0): hSBA<8 - After (day 42): If hSBA is >8, serum is positive

[0460] Before (day 0): hSBA ≧8-After (day 42): If hSBA increases ≧4-fold compared to day 0, the serum is positive.

[0461] The number of immunized animals whose sera were able to induce specific hSBA was greater than 70% of the observed responders (ie, 5 / 7 per formulation).

[0462] 2. Determination of IgG antibody titers by ELISA The specific IgG responses generated against each antigen were measured from rabbit sera on days 0 and 42. ELISA analysis was performed from individual sera collected from each immunization group.

[0463] Briefly, 96-well microplates were coated with 100 μL of specific antigen 1 μg / mL per well in carbonate buffer coating solution and kept overnight at +4°C. The coating solution was removed by inverting the plate, after which the plate was tapped on a paper towel. Free sites were blocked with 150 μL of buffer 1 (PBS / Tween20 0.05% / skim milk 1%) and after a 60 min incubation period at +37°C the plate was emptied. Serum was serially diluted in buffer 1 in a volume of 100 μL (12 times) in the microplate. The plate was incubated for 90 min at +37°C and then washed with buffer 2 (PBS / Tween20 0.05%). 100 μL of diluted anti-rabbit IgG was then added to each well. After 90 min incubation at +37°C the plate was washed with buffer 2. The reaction was developed by adding 100 μL of tetramethylbenzidine substrate solution to each well. After 30 min at room temperature, the reaction was chemically stopped with HCl (1N) and the absorbance was measured at 450–650 nm in a spectrophotometer (Versamax reader p248, Molecular Devices). Results were expressed as arbitrary ELISA units / mL according to the reciprocal of the dilution corresponding to OD = 1 using the CODUNIT program of the robotic ELISA.

[0464] 3. Purification of rabbit serum IgG for hSBA testing Purification of IgG was necessary to avoid non-specific killing induced by rabbit serum on days 0 and 42. Purification of rabbit serum was performed using an RPROTEIN A GRAVTITRAP™ column (GE healthcare GE28-9852-54) and an Ab buffer kit (GE Healthycare ref. 28-9030-59). After an initial step of equilibrating the column with binding buffer (sodium phosphate 20 mM pH=7), IgG binding was performed by adding serum (2 mL) adjusted to neutral pH with binding buffer (V / V) to the column. The column was washed with binding buffer and IgG was collected by elution with elution buffer (glycine HCl 0.1 M pH 2.7). To preserve the activity of IgG, neutralization buffer (Tris-HCl 1 M, pH 9.0) was added to the elution fraction to obtain a final pH of approximately neutral. Dialysis of each sample was performed using a Slide-A-Lyser G2 dialysis cassette (Thermo Scientific 87730) to exchange the sample in PBS buffer. Quantification of IgG concentration was performed by NANODROP.

[0465] 4.hSBA test Measurement of serum bactericidal antibodies, using human complement as the complement source, is widely accepted as a surrogate marker of protection against meningococcal disease (Borrow et al., Vaccine. 2005;23(17-18):2222-7; Borrow et al., Vaccine. 2006;24(24):5093-107; Frasch et al., Vaccine. 2009;27 Suppl 2:B112-6; Goldschneider et al., J Exp Med. 1969;129(6):1307-26).

[0466] The SBA assay refers to the ability of antibodies to lyse and kill bacteria in the presence of complement. The source of complement was human complement (Pel Freez IgG / IgM depleted batch #13441). Briefly, serum was heat inactivated at +56°C for 30 min, followed by IgG purification. The purified IgG was then serially diluted two-fold (9 times) in Dulbecco's PBS buffer containing Ca++ and Mg++) + 0.2% gelatin (dilution buffer) in a 96-well microplate.

[0467] Bacterial precultures were grown on Mueller-Hinton agar (Petri dishes) at +37°C and 5% CO 2 , for 18 h to obtain confluent bacterial growth. Bacteria were then grown in BHI suspension at an initial OD of approximately 0.20 at λ600nm for 2 h 30 min at +37 °C with shaking (100 rpm). After incubation, bacteria were diluted 5-fold to obtain 1.4.104 CFU / mL. 25 μL of working bacterial suspension, 50 μL of prediluted serum, and 25 μL of diluted human complement (15% final concentration) were deposited in a 96-well microplate and incubated at +37 °C for 1 h with shaking (100 rpm). The Zephyr robotic application automatically deposited 40 μL for each well on a square dish (40 × 40) containing Mueller-Hinton agar. The agar plates were incubated at +37 °C and 5% CO2 for 10–12 h. After incubation, the number of colonies per well was counted using Cybele Software from Microvision.

[0468] Bactericidal titers were defined as the serum dilution that resulted in a 50% reduction in colony forming units (CFU) per mL of bacteria compared to complement control wells. SBA titers were calculated by modeling a 4-parameter curve in Soft Max Pro v6.5.1 GxP with a user-defined protocol that fit the plate design. If modeling did not fit the 4-parameter curve, then a trend function around the K50 and manual readings were applied; bactericidal titers were defined as the last reciprocal serum dilution that resulted in at least a 50% reduction.

[0469] The positive threshold for a positive response was defined as follows:

[0470] Before (day 0): hSBA<8 - After (day 42): If hSBA is >8, the serum is positive

[0471] Before (day 0): hSBA ≧8-After (day 42): If hSBA increases ≧4-fold compared to day 0, the serum is positive.

[0472] The number of immunized animals whose sera were able to induce specific hSBA was greater than 70% of the observed responders (ie, 5 / 7 per formulation).

[0473] 5.MenB strains A set of 18 wild-type serogroup B meningococcal isolates (mostly recent clinical isolates) isolated on different isolation dates, from different geographic locations, and representing diverse MLST clonal complexes were selected. MenB strains were selected to evaluate the immunogenicity of each component of the immunogenic composition and to assess breadth of coverage. Immunogenicity was measured by SBA using MenB strains that do not match any of the other antigens of the composition, but are selectively recognized by antibodies against one of the antigens of the composition. Furthermore, the selection was made to ensure that several selected strains represented the diversity and distribution of well-described fHBP sequences of both the A and B subfamilies. Breadth of coverage was assessed using MenB strains that represented epidemiological characteristics such as clonal complex (CC) distribution, geographic origin, antigenic prevalence and diversity.

[0474] The main characteristics of the selected strains are listed in Table 2.

[0475] [Table 2]

[0476] Eighteen strains were selected based on epidemiological characteristics such as clonal complex distribution, prevalence and virulence of clonal complexes, geographic origin, antigen prevalence and diversity, and were characterized with respect to antigenic distribution as follows:

[0477] -11 strains expressed fHBP subfamily A (2 strains close to the fHBP A05 antigen - numbers 1 and 8 - and 9 strains distant from the fHBP A05 antigen - numbers 2, 5, 7, 9, 10, 11, 12, 13, 14),

[0478] -7 strains express fHBP subfamily B (one strain close to the fHBP B01 antigen - number 3 - and six strains distant from the fHBP B01 antigen - numbers 4, 6, 15, 16, 17, 18).

[0479] -14 strains did not express NadA (gene missing or frameshift NadA4 / 5 off), and 4 strains expressed either the homologous NadA1 variant (3 strains) or the heterologous NadA2 / 3 variant (1 strain);

[0480] -2 strains express PorA VR2 P1.2 of the matching dOMV that express low levels of fHBP A (1 strain does not express NadA protein (number 5) and 1 strain expresses a heterologous NadA protein (number 13)).

[0481] 2.Results 1.ELISA results Total IgG antibodies were quantified by ELISA from pooled sera collected from rabbits immunized with the various formulations (F), TRUMENBA or BEXSERO, at day 0 (gray) and from individual sera at day 42 (blue).

[0482] Whatever the ELISA response, the results were homogeneous within the groups (all p-values ​​> 0.061). No outlier animals were detected.

[0483] As shown in Figures 1A and 1B for fHBP-specific IgG responses and Figures 2A and 2B for NadA- and dOMV-specific IgG responses, the ELISA results show that F1-F5 formulations and the F1 formulation coadministered with MENQUADFI all induced antibody responses with Ag-specific IgG titers ranging from 4.05 to 4.99 Log10.

[0484] Furthermore, as shown in Figures 1A and B and Figures 2A and 2B, whatever the formulation used, AlPO 4 Injection of each of the four MenB antigens formulated in combination with fHBP A05 tmN, fHBP B01 smN, NadA protein and dOMV enabled the production of moderate to high titers of antibodies in 100% of animals. Formulations made with 50 μg of A05 tmN and B01 smN fHBP Ags (F1+ / - MENQUADFI, F4, F5 and F6) induced fHBP-specific IgG responses with mean titers of 4.28 Log10 to 4.59 Log10 and 4.05 Log10 to 4.52 Log10, respectively. Formulations made with 50 μg of NadA (F1-F5) induced NadA-specific IgG responses with mean titers of 4.42 Log10 to 4.81 Log10. Formulations made with 50 μg of dOMV (F1–F3) induced dOMV-specific IgG responses with mean titers ranging from 4.77 Log10 to 4.99 Log10. Finally, for various doses of fHBP protein (F2, 25 μg and F3, 100 μg) or dOMV (F4, 25 μg and F5, 125 μg), similar mean titers were measured at all doses.

[0485] TRUMENBA was able to induce specific IgG against fHBP A05 tmN antigen and fHBP B01 smN antigen with mean titers of 5.16 and 5.15 Log10, respectively.

[0486] BEXSERO was able to induce a mean titer of 4.34 Log10 of specific IgG against the NadA variant 1 antigen.

[0487] 2. hSBA results obtained with different compositions F1-F6 or F1+MENQUADFI, TRUMENBA and BEXSERO against seven MenB strains (numbers 1-6 and 18) In a first set of experiments, the different compositions F1 to F6 or F1+MENQUADFI, TRUMENBA and BEXSERO were assayed against seven MenB strains (numbers 1 to 6 and 18).

[0488] hSBA results indicate that the six formulations F1-F5 and F1 co-administered with MENQUADFI were immunogenic in rabbits. The percentage of responders is the percentage of animals that showed a 4-fold increase in hSBA GMT measured on a post-immunization vs. pre-immunization basis, demonstrating a surrogate for protection.

[0489] As shown in Figure 3, whatever the immunization dose used (25 μg F2, 50 μg F1, F4, F5, F6 and 100 μg F3), mutant nonlipidated fHBP B01 smN in combination with mutant nonlipidated fHBP A05 tmN+NadA+dOMV was able to induce bactericidal activity in 100% of animals at day 42 against the closely related fHBP B44 variant strain, with GMTs ranging from 443 to 1147.

[0490] TRUMENBA and BEXSERO were able to induce bactericidal activity in 100% of animals against a MenB strain expressing the closely related fHBP B44 variant, with GMTs of 5773 and 646, respectively.

[0491] As shown in Figures 4 and 5, mutant nonlipidated fHBP B01 smN in combination with mutant nonlipidated fHBP A05tmN+NadA+dOMV was able to induce bactericidal activity in 43–86% of animals against the first MenB strain expressing a different fHBP B24 variant (Figure 4) and in 86–100% of animals against the second different fHBPB24 variant strain tested (Figure 5), with GMTs of 5–18 and 28–219, respectively.

[0492] Interestingly, against the first heterologous B24 variant strain, only the mutated non-lipidated fHBP B01smN in formulation F3 induced a hSBA response in 86% of animals with a GMT of 18.

[0493] TRUMENBA was able to induce bactericidal activity in 100% of animals against two B24 strains, with GMTs of 44 and 143, respectively. BEXSERO was able to induce bactericidal activity in 100% of animals against two B24 strains, with GMTs of 2274 and 2587, respectively.

[0494] Additionally, no significant dose effects were demonstrated for the 25–100 μg B01 smN fHBP doses (all p values ​​≥ 0.056).

[0495] Finally, coadministration of MENQUADFI with formulation F1 demonstrated no significant differences in mutant nonlipidated B01 smN responses (total MenB Ag, 50 μg, all p values ​​≥ 0.339).

[0496] As shown in Figures 6 and 7, whatever the immunization dose used (25 μg F2, 50 μg F1, F4, F5, F6 and 100 μg F3), mutant non-lipidated fHBP A05 tmN in combination with mutant non-lipidated fHBP B01smN+NadA+dOMV was able to induce bactericidal activity in 100% of animals against the closely related fHBP A56 variant expressing strain (Figure 6) and the different fHBP A22 variant expressing strain (Figure 7), with geometric mean titers (GMTs) of 226-382 and 45-108, respectively.

[0497] TRUMENBA was able to induce bactericidal activity in 100% of animals against fHBP A56 and fHBP A22 variant expressing strains, with GMTs of 2058 and 463, respectively. BEXSERO was able to induce bactericidal activity in 57% of animals against fHBP A56 variant expressing strains and in 100% of animals against fHBP A22 variant expressing strains, with GMTs of 7 and 331, respectively.

[0498] Furthermore, no significant dose effect was demonstrated for mutant non-lipidated A05 tmN fHBP doses between 25 and 100 μg (all p values ​​≥ 0.066).

[0499] Finally, when MENQUADFI was coadministered with formulation F1, no significant difference was shown in the mutant non-lipidated A05 tmN response against the A56 strain (p-value=0.281).Regarding hSBA against fHBP A22 expressing strains, titers obtained with F1+MENQUADFI were significantly lower (p=0.020) than those obtained with F1 alone, representing a 2.4-fold reduction with no biological relevance.

[0500] As shown in Figure 8, formulations (F1–F5) produced with 50 μg of NadA were able to induce bactericidal activity in 100% of animals against one homologous NadA variant strain, with GMTs ranging from 217 to 696.

[0501] TRUMENBA and BEXSERO were able to induce bactericidal activity in 86% and 100% of animals against the homologous NadA variant 1 strain, with GMTs of 118 and 1191, respectively.

[0502] Finally, no significant difference in NadA responses was noted when MENQUADFI was co-administered with the multicomponent MenB vaccine (p-value=0.688).

[0503] As shown in Figure 9, whatever the formulations (25 µg F4, 50 µg F1, F2, F3 and 125 µg F5) produced with dOMVs, they were able to induce bactericidal activity in 100% of animals against the homologous PorA_1.2 strain, with GMTs ranging from 350 to 1324.

[0504] TRUMENBA and BEXSERO were unable to induce bactericidal activity against this dOMV strain.

[0505] Furthermore, no significant dOMV dose effect was demonstrated (all p values ​​> 0.637).

[0506] No significant effects of NadA and dOMV antigens on fHBP A or B responses were observed (all p values ​​≥ 0.208).

[0507] The only significant effect of the dose of fHBP antigen in combination with NadA / dOMV antigen was in terms of NadA-specific hSBA responses between F1 (50 μg) and F3 (100 μg) formulations. As shown in FIG. 8A, the titers obtained with the F3 formulation were higher than those obtained with the F1 formulation (p=0.004, 3.2-fold increase). No significant differences were noted between all formulations in terms of porA / dOMV-specific hSBA responses.

[0508] This test is 4 We demonstrated immunogenicity with each of the four selected MenB antigens, the mutant non-lipidated fHBPs A05 tmL and B01 smL, NadA and dOMV in combination, formulated with .

[0509] All antigens included in the new vaccine formulations were able to enhance Ag-specific bactericidal antibody responses measured by hSBA against Ag-specific MenB strains. In the rabbit model, no significant dose effect was observed for fHBP. Similarly, no significant dose effect was observed for dOMV, with the lowest dose (25 μg) of dOMV resulting in a strong hSBA response. Interestingly, formulation F3 (i.e., containing 100 μg / dose of fHBP, 5 μg / dose of NadA and 50 μg / dose of dOMV) tended to result in the highest hSBA response against all strains tested, inducing a positive response in more than 71% of animals. Furthermore, no effect of MenQuadfi coadministration on MenB-specific hSBA responses was observed. fHBP A05tmN vaccine Ag in all test formulations induced hSBA responses in 100% of animals against the closely related A56 variant strain and the heterologous A22 variant strain, with geometric mean titers (GMTs) ranging from 226 to 382 and 45 to 108, respectively. fHBP B01smN vaccine Ag in all test formulations induced hSBA responses in 100% of animals against the closely related B44 variant strain, with GMTs ranging from 443 to 1147. When testing hSBA responses against the first heterologous H44 / 76 B24 variant strain, only fHBP B01smN in formulation F3 induced positive hSBA responses in 86% of animals, with a GMT of 18, whereas all other test formulations induced hSBA responses in only 29 to 57% of animals, with GMTs ranging from 5 to 10. In contrast, when a second heterologous B24 strain, strain 03S-0291, was used, fHBP B01smN in all test formulations induced hSBA in 86–100% of animals with GMTs ranging from 28–219.

[0510] NadA vaccine Ags in all formulations tested induced hSBA against NadA1 variant strains in 100% of animals with GMTs ranging from 217 to 696.

[0511] dOMV in all formulations tested induced hSBA in 100% of animals against the PorA subtype VR2 P1.2 strain, with GMTs ranging from 350 to 1324.

[0512] Overall, the immunogenicity results indicate that the six formulations of the immunogenic composition disclosed herein were immunogenic in rabbits. All antigens included in the new vaccine were able to raise Ag-specific bactericidal antibody responses as measured by hSBA against Ag-specific MenB strains. In the rabbit model, formulations made with 100 μg fHBP tended to produce the highest hSBA responses, and no significant dose effect was observed for fHBP, even though it induced responses in more than 70% of animals against all strains tested. Similarly, no significant dose effect was observed for dOMV, with the lowest dOMV dose (25 μg) being sufficient to induce a strong hSBA response. Interestingly, no effect of MENQUADFI co-administration on MenB-specific hSBA responses was observed.

[0513] Of note, neither hyperthermia nor major clinical signs (such as erythema) were observed in the animals throughout the study.

[0514] In conclusion, the immunogenicity results show that six formulations of the disclosed immunogenic composition were immunogenic in rabbits. All antigens included in the MenB multicomponent vaccine were able to raise Ag-specific bactericidal antibody responses as measured by hSBA against Ag-specific MenB strains.

[0515] 3. hSBA results obtained with compositions F1, F3, TRUMENBA and BEXSERO against 17 MenB strains (numbers 1-17) In a second set of experiments, sera obtained in the preceding experiments after immunization with compositions F1, F3, TRUMENBA and BEXSERO were assayed against 11 additional MenB strains (for a total of 17 MenB strains, including the 6 strains tested above).

[0516] The overall results are summarized in Table 3 below, which shows the efficacy of the various test compositions against a panel of selected MenB strains.

[0517] [Table 3]

[0518] The results showed that strains no. 4 and 5 were poorly (<71% responders) or not covered (0% responders) by TRUMENBA, whereas these strains were largely or completely covered by the F1 and F3 formulations.

[0519] The results also showed that strain numbers 1, 5, 7, 8, 9, and 11 were poorly or not covered by BEXSERO, whereas they tended to be well or completely covered by the F1 and F3 formulations.

[0520] Notably, strain no. 5 is not covered at all by TRUMENBA or BEXSERO.

[0521] Table 3 shows that 16 of 17 and 17 of 17 MenB strains were covered by the F1 and F3 compositions. In contrast, TRUMENBA appeared to cover 15 of 17 MenB strains and BEXSERO appeared to cover only 11 of 17 MenB strains.

[0522] As shown in Figure 19, the test formulation F3 was able to induce protection against all the different MenB strains from the different clonal complexes, ST-41 / 44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and ST-461. In contrast, the coverage induced by TRUMENBA and BEXSERO shows some gaps.

[0523] In conclusion, it is noted that the MenB multi-component immunogenic compositions of the present disclosure provide excellent strain coverage even against low fHBP expressers and different strains: F3 formulation covered 17 / 17 MenB strains with >71% responders, and F1 formulation covered 16 / 17 MenB strains with >71% responders.

[0524] TRUMENBA covered 15 / 17 MenB strains. The uncovered strains (0% responders) had zero fHBP A10 expression and belonged to the ST-11 clonal complex. The least covered strains (71% responders) were B24 expressers.

[0525] BEXSERO covered 11 / 17 MenB strains. Six strains with little or no coverage (0–57% responders) expressed fHBP A variants. They may represent up to 29% of circulating MenB strains and belonged to common or highly virulent clonal complexes: ST-213; ST-11; ST-35; and ST-461.

[0526] The newly developed vaccine composition shows increased breadth of protection against 18 strains representative of current MenB molecular epidemiology; i.e., strains derived from the most common and highly virulent clonal complexes (CCs), and strains derived from the most common antigenic variants. EXAMPLES

[0527] MIMIC PTE assay 1. Materials and Methods 1. Test Composition and Experimental Design In the first study, the commercial vaccines BEXSERO and TRUMENBA were compared in MIMIC adult and neonatal Peripheral Tissue Equivalent (PTE) cultures (see below) using 20 donors each. TRUMENBA and BEXSERO vaccine compositions were as disclosed in Example 1.

[0528] In a second study, MIMIC PTE cultures (see below) were treated with five vaccine candidate formulations - F1, F2, F3; F4 and F5 (formulations were as shown in Table 1 in Example 1) - in a dose-ranging curve of 1:100 to 1:1,000,000 based on 10-fold dilutions of the human dose. F1 was considered the standard formulation, as it contained 50 μg / dose of each component and a standard dose (0.4 mg / dose) of AlPO4; F2 formulation contained 0.4 mg / dose AlPO4. 4 The F1 and F2 formulations were combinations of low and medium doses of fHBP (25 μg / dose) and standard doses of NadA and dOMV (50 μg / dose); the F3 formulation was combination of high dose fHBP (100 μg / dose), standard doses of NadA and dOMV (50 μg / dose), and high dose AlPO4 (0.8 mg / dose); the F4 formulation combined standard doses of fHBP and NadA (50 μg / dose each), low dose dOMV (25 μg / dose), and standard dose AlPO4; and the F5 formulation combined standard doses of fHBP and NadA (50 μg / dose), high dose dOMV (125 μg / dose), and standard dose AlPO4. 4 In this study, a dOMV-based MenB vaccine, BEXSERO, was used as the reference control.

[0529] The mock or control composition was an untreated control containing serum-free culture medium only.

[0530] 2. Adult and Neonatal MIMIC PTE Assays The MIMIC PTE construct (Higbee et al., Altern Lab Anim. September 2009;37 Suppl 1:19-27) was constructed in a robotic line using the methods taught by Ma et al. (Immunology, 2010, 130:374-87).

[0531] Briefly, endothelial cells were grown to confluence on a collagen matrix (Advanced Biomatrix, San Diego, CA). Donor PBMCs (adult MIMIC PTE) or donor umbilical cord blood (neonatal MIMIC PTE) prepared from frozen stocks were then applied to the assay wells. After 90 min (adult version) or 3 h (umbilical cord blood version) of incubation, non-migrating cells were washed away, and then various treatments (Table 1 in Example 1) were added in a final incubation step for 48 h. A mixture of 100 ng / mL LPS (from Pseudomonas aeruginosa, Cat. No. L8643, Millipore Sigma, Burlington, MA) and 10 μg / mL R848 (Cat. No. TLRL-R848, InvivoGen, San Diego, CA) was used as a positive control (assay control) for these assays. Reverse-migrated cells were harvested after the 48-h treatment period and phenotyped for cell viability using flow cytometry, while culture supernatants harvested at the same time points were analyzed for cytokines / chemokines by multiplex assay.

[0532] In a first series of experiments, BEXSERO and TRUMENBA were compared together. In a second series of experiments, immunogenic compositions F1, F2, F3, F4 and F5 of the present disclosure were compared with the present disclosure.

[0533] Culture supernatants were harvested after a 48-h treatment period and analyzed for cytokines / chemokines by multiplex assays. Cells were harvested at the same time and phenotyped for cell viability using flow cytometry.

[0534] 3. Cytokine / Chemokine Analysis The MIMIC culture supernatant was analyzed using the Milliplex Human 12-Plex Multi-Cytokine Detection System (Millipore). The kit included IL-1β, IL-6, MIP-1β, and TNFα. Analyte concentrations were calculated using Bio-Plex Manager software (Luna et al. PloS One vol.13,6 e0197478. June 6, 2018) based on appropriate calibration curves.

[0535] Regarding the criteria for run determination, the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) for each analyte were established based on the recovery rate (observed value / estimated value × 100) at each time point for a 5-parameter logistic (5PL) curve fit of the reference values. Recovery rates in the range of 80% - 120% were considered acceptable such that values falling within this range defined the lower and upper limits of the calibration curve. The raw data files were examined for the number of beads; data points were considered valid if at least 35 beads were counted per region.

[0536] 4. Flow Cytometry Cells derived from MIMIC® PTE were washed with PBS and stained with Live-Dead Aqua (InvitroGen, Carlsbad, CA) on ice for 20 minutes to evaluate cell viability. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). For flow gating, singlets were selected, followed by viable cells.

[0537] 5. Data Analysis and Graph Plotting For graphing, the data was exported to GraphPad Prism (GraphPad Software, San Diego, CA, USA). Cytokine data was exported to an Excel database. Out-of-range high (>OOR) values (values higher than the top of the curve) were replaced with the ULOQ; out-of-range low (<OOR) values were replaced with 1 / 2 of the LLOQ (lower limit of quantification).

[0538] 6. Statistical Analysis Statistical analysis was performed using a non-inferiority model. Parameters including cell viability, CD86, IL-6, IL-1b, TNF-a, and MIP-1b were used as endpoints in this analysis. The geometric means (GM) of the different antigen formulations vs. Bexsero® vaccine were compared, with δ=2 / 3 used as a measure of non-inferiority. The analyses described herein were performed using the SA procedure, PROC TTEST, and presented as dot plot graphs showing GM and 95% CI ranges as recommended in published literature.

[0539] 2.Results 1. BEXSERO AND TRUMENBA IN ADULT AND NEONATAL MIMIC PTE Culture supernatants from untreated and treated adult and neonatal MIMIC PTE cultures were collected after 48 hours and analyzed for cytokine / chemokine secretion using Millipore custom multiplex arrays. The innate chemokines / cytokines IL-6, TNFα, MIP-1β and IL-1β were included in this analysis as they are crucial for innate immune activity and can also drive immune-cytotoxicity.

[0540] The results (Figure 10) showed that TRUMENBA showed a stronger proinflammatory cytokine profile than BEXSERO in adult MIMIC PTE. TRUMENBA also showed a stronger proinflammatory cytokine profile than BEXSERO in neonatal MIMIC PTE (Figure 11).

[0541] The statistical analysis presented in Figure 12 confirmed the observation that for most cytokines, TRUMENBA generates stronger responses than BEXSERO in the adult and neonatal platforms. Indeed, BEXSERO is inferior to TRUMENBA as shown by the forest plot of the ratio of geometric means with 95% confidence intervals of cytokine secretion induced by BEXSERO in adult and neonatal PTEs compared to TRUMENBA.

[0542] 2. F1, F2, F3, F4, F5 and BEXSERO in adult and neonatal MIMIC PTE Culture supernatants from untreated and treated adult and neonatal MIMIC PTE cultures were collected after 48 hours and analyzed for cytokine / chemokine secretion using Millipore custom multiplex arrays. The innate chemokines / cytokines IL-6, TNFα, MIP-1β and IL-1β were included in this analysis as they are crucial for innate immune activity and can also drive immune-cytotoxicity.

[0543] In the adult MIMIC PTE system (see FIG. 13), all test formulations resulted in approximately 1 log higher cytokine secretion than mock controls (i.e., no antigen) at the highest treatment doses (1:100, 1:1000, 1:10000). In nearly all cases, the formulations performed equal to or slightly lower than BEXSERO.

[0544] Statistical analysis in Figures 14A, 14B and 14C showed that in adult MIMIC PTE, F1, F2, F4 and F5 formulations generated less inflammatory responses than Bexsero for all of the reported cytokines. F3 formulation induced secretion of proinflammatory cytokines similar to (TNF-α or IL-1β) or less than (IL6 or MIP-1b) Bexsero in adult MIMIC PTE systems.

[0545] Furthermore, all formulations demonstrated immune cytotoxicity in a dose-dependent manner and to a similar extent as BEXSERO in the adult PTE model. Indeed, as shown in Figure 15, in several tested conditions cell viability was barely affected at the lowest treatment doses (1:100000 and 1:00000 dilutions) and was reduced by up to approximately 50% at the highest dose (1:100 dilution).

[0546] Additionally, in neonatal MIMIC PTE, F1, F2, F3, F4, and F5 formulations exhibited proinflammatory cytokine profiles consistent with BEXSERO (Figure 16). All formulations resulted in approximately 4-fold to 1 log higher cytokine secretion compared to mock conditions. F1, F2, F3, and F4 formulations induced IL-6 and TNF-α secretion similar to or less than BEXSERO. F5 formulation induced slightly higher proinflammatory cytokine secretion than BEXSERO.

[0547] 17A and 17B, statistical analysis showed that overall, F1, F2 and F3 formulations produced inflammatory responses comparable to BEXSERO in neonatal MIMIC PTEs. The F5 formulation tended to produce a slightly higher inflammatory response than BEXSERO in neonatal MIMIC PTEs, while the F4 formulation tended to produce a weaker response than BEXSERO in neonatal MIMIC PTEs.

[0548] As with the adult MIMIC PTE system, all formulations demonstrated a similar immune cytotoxicity profile to BEXSERO. Indeed, as shown in Figure 18, in several tested conditions of the MIMIC neonatal PTE, cell viability was barely affected at the lowest treatment doses (1:100000 and 1:000000 dilutions) and decreased by up to approximately 50% at the highest dose (1:100 dilution).

[0549] 3. Conclusion TRUMENBA induced a stronger proinflammatory cytokine response than BEXSERO in both the adult and neonatal versions of the MIMIC PTE.

[0550] The F1, F2, F3, F4, and F5 formulations produced similar or less proinflammatory cytokine secretion than BEXSERO in adult MIMIC PTE constructs.

[0551] Overall, the F5 formulation induced more proinflammatory cytokines than BEXSERO in neonatal MIMIC PTE, whereas the F1, F2, F3 and F4 formulations induced similar or lower cytokine responses.

[0552] The F1, F2, F3, F4 and F5 formulations had minimal effects on cell viability (except for the 1:100 dilution, where some formulations induced a decrease in cell viability of approximately 40%) and showed a similar trend to BEXSERO. EXAMPLES

[0553] general conclusion From the results presented above, it can be concluded that the disclosed immunogenic compositions comprising a combination of meningococcal antigens including at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV) are capable of inducing an immunogenic protective response as evidenced by the hSBA results, supporting the utility of those compositions as vaccines and for inducing an immunogenic protective response against meningococcal infection.

[0554] Furthermore, the data indicate that the breadth of coverage of common and highly virulent MenB strains is greater than that of TRUMENBA and BEXSERO, allowing it to fill some of the gaps left by those two vaccines.

[0555] Finally, the data show that these compositions exhibit a superior safety profile and a similar or less reactogenicity profile than BEXSERO, which is already less reactogenic than TRUMENBA.

[0556] References Atkinson B, Gandhi A, Balmer P. History of Meningococcal Outbreaks in the United States: Implications for Vaccination and Disease Prevention. Pharmacotherapy. 2016;36(8):880-92. Ausubel FM, Brent R, Kingston RE, Moore DM, Short Protocols in Molecular Biology : A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, ISBN: 047132938X; 4th edition (April 1999) Bambini S, Muzzi A, Olcen P, Rappuoli R, Pizza M, Comanducci M. Distribution and genetic variability of three vaccine components in a panel of strains representative of the diversity of serogroup B meningococcus. Vaccine. 2009;27(21):2794-803. Batista RS, Gomes AP, Dutra Gazineo JL, Balbino Miguel PS, Santana LA, Oliveira L, et al. Meningococcal disease, a clinical and epidemiological review. Asian Pac J Trop Med. 2017;10(11):1019-29. Bijlsma MW, Brouwer MC, Spanjaard L, van de Beek D, van der Ende A. 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Safety and immunogenicity of a meningococcal B bivalent rLP2086 vaccine in healthy toddlers aged 18-36 months: a phase 1 randomized-controlled clinical trial. Pediatr Infect Dis J. 2012;31(10):1061-8. Martin DR, Walker SJ, Baker MG, Lennon DR. New Zealand epidemic of meningococcal disease identified by a strain with phenotype B:4:P1.4. J Infect Dis. 1998;177(2):497-500. Martinon-Torres F, Gimenez-Sanchez F, Bernaola-Iturbe E, Diez-Domingo J, Jiang Q, Perez JL. A randomized, phase 1 / 2 trial of the safety, tolerability, and immunogenicity of bivalent rLP2086 meningococcal B vaccine in healthy infants. Vaccine. 2014;32(40):5206-11. McNeil LK, Zagursky RJ, Lin SL, et al. Role of factor H binding protein in Neisseria meningitidis virulence and its potential as a vaccine candidate to broadly protect against meningococcal disease. Microbiol Mol Biol Rev. 2013;77(2):234-252. doi:10.1128 / MMBR.00056-12 Meri S, Jordens M, Jarva H. Microbial complement inhibitors as vaccines. Vaccine. 2008;26 Suppl 8:I113-7. Moro PL, Jankosky C, Menschik D, Lewis P, Duffy J, Stewart B, et al. Adverse events following Haemophilus influenzae type b vaccines in the Vaccine Adverse Event Reporting System, 1990-2013. The Journal of pediatrics. 2015;166(4):992-7. Murphy E, Andrew L, Lee KL, Dilts DA, Nunez L, Fink PS, et al. Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis. The Journal of infectious diseases. 2009;200(3):379-89. Needleman S.B. and Wunsch C. D., "A general method applicable to the search of similarities in the amino acid sequence of two proteins" Journal of Molecular Biology, 1970, pages 443-53, volume 48 Pace D, Pollard AJ. Meningococcal disease: clinical presentation and sequelae. Vaccine. 2012;30 Suppl 2:B3-9. Pagotto FJ, Salimnia H, Totten PA, Dillon JR. Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication. Gene. 2000 Feb 22;244(1-2):13-9. doi: 10.1016 / s0378-1119(99)00557-0. PMID: 10689182. Pizza M, Scarlato V, Masignani V, Giuliani MM, Arico B, Comanducci M, et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science. 2000;287(5459):1816-20. Pollard AJ. Global epidemiology of meningococcal disease and vaccine efficacy. Pediatr Infect Dis J. 2004;23(12 Suppl):S274-9. Rodriguez AP, Dickinson F, Baly A, Martinez R. The epidemiological impact of antimeningococcal B vaccination in Cuba. Mem Inst Oswaldo Cruz. 1999;94(4):433-40. Rouphael NG, Stephens DS. Neisseria meningitidis: biology, microbiology, and epidemiology. Methods Mol Biol. 2012;799:1-20. Seib KL SM, Comanducci M, Toneatto D, Masignani V. Neisseria meningitidis factor H-binding protein fHBP: a key virulence factor and vaccine antigen. 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US 10,300,122 - Novel immunogenic compositions for the prevention and treatment of meningococcal disease US 10,625,025 - Prefilled syringe US 10,695,505 - Dual-chamber syringe US 11,077,180 B2 - Non-lipidated variants of neisseria meningitidis ORF2086 antigens US 4,695,624 - Covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers, and methods of preparing such polysaccharides and conjugates and of confirming covalency US 5,494,808 - Defined medium OMPC fermentation process US 9,724,402 B2 - Neisseria meningitidis compositions and methods thereof Vu DM, Wong TT, Granoff DM. Cooperative serum bactericidal activity between human antibodies to meningococcal factor H binding protein and neisserial heparin binding antigen. Vaccine. 2011;29(10):1968-73. Vuocolo S, Balmer P, Gruber WC, Jansen KU, Anderson AS, Perez JL, et al. Vaccination strategies for the prevention of meningococcal disease. Hum Vaccin Immunother.2018;14(5):1203-15. Wang X, Cohn A, Comanducci M, Andrew L, Zhao X, MacNeil JR, et al. Prevalence and genetic diversity of candidate vaccine antigens among invasive Neisseria meningitidis isolates in the United States. Vaccine. 2011;29(29-30):4739-44. WO 2001 / 064920 - Hybrid expression of neisserial proteins WO 2001 / 064922 - Heterologous expression of neisserial proteins WO 2002 / 058737 - Multivalent meningococcal polysaccharide - protein conjugate vaccine WO 2003 / 020756 - Hybrid and tandem expression of neisserial proteins WO 2009 / 109550 - Process for stabilizing an adjuvant containing vaccine composition WO 2010 / 046715 - Vaccine compositions comprising a mutated factor h binding protein WO 2011 / 126863 - Factor H binding proteins (FHBP) with altered properties and methods of use thereof WO 2013 / 046855 - Liquid injector WO 2015 / 017817 - Non-naturally occurring factor H binding proteins (fHBP) and methods of use thereof WO 2015 / 128480 - Modified meningococcal fhbp polypeptides WO 2016 / 008960 - Modified meningococcal fhbp polypeptides WO 2016 / 014719 - Factor h binding protein variants and methods of use thereof WO 2018 / 045286 - Neisseria meningitidis vaccine

Claims

1. 1. An immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens, said combination comprising at least one factor H binding protein (fHBP) A protein, at least one fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

2. The composition of claim 1 , wherein the fHBP A protein and / or the fHBP B protein are non-lipidated.

3. 3. The composition of claim 1 or 2, wherein the fHBP A protein and / or the fHBP B protein contain a mutation that reduces its affinity for Factor H compared to the corresponding naturally occurring fHBP.

4. The composition according to any one of claims 1 to 3, wherein the fHBP A protein is a mutant protein comprising at least about 85% identity with SEQ ID NO:1 and / or the fHBP B protein is a mutant protein comprising at least about 85% identity with SEQ ID NO:

3.

5. The fHBP A protein comprises at least one amino acid substitution selected from at least one of the following based on the numbering of SEQ ID NO:6: a) an amino acid substitution of asparagine at amino acid 115 (N115); b) an amino acid substitution of aspartic acid at amino acid 121 (D121); c) an amino acid substitution of serine at amino acid 128 (S128); d) an amino acid substitution of leucine at amino acid 130 (L130); e) an amino acid substitution of valine at position 131 (V131); f) an amino acid substitution of glycine at position 133 (G133); g) an amino acid substitution of lysine at position 219 (K219); and h) an amino acid substitution of glycine at position 220 (G220), or comprises or consists of SEQ ID NO:2, with the proviso that the N-terminal cysteine ​​of SEQ ID NO:2 has been removed or replaced with another amino acid, and / or comprises the amino acid substitution of SEQ ID NO:

2. The B protein is based on the numbering of SEQ ID NO:6: a) an amino acid substitution of glutamine at amino acid 38 (Q38); b) an amino acid substitution of glutamic acid at amino acid 92 (E92); 5. The composition of claim 1, comprising at least one amino acid substitution selected from at least one of: a) an amino acid substitution with an N-terminal cysteine ​​at amino acid 130 (R130); b) an amino acid substitution with an N-terminal cysteine ​​at amino acid 130 (R130); c) an amino acid substitution with an arginine at amino acid 130 (R130); d) an amino acid substitution with an amino acid 223 (S223); and e) an amino acid substitution with an N-terminal histidine at amino acid 248 (H248), or comprising or consisting of SEQ ID NO:4, with the proviso that the N-terminal cysteine ​​of SEQ ID NO:4 has been removed or replaced with another amino acid.

6. 6. The composition of any one of claims 1 to 5, comprising at least one non-lipidated factor H binding protein (fHBP) A protein comprising at least about 85% identity to SEQ ID NO:1, and comprising at least an amino acid substitution G220S based on the numbering of SEQ ID NO:6, at least one non-lipidated fHBP B protein, at least one Neisserial adhesin A (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

7. 7. The composition of claim 6, wherein the non-lipidated fHBP B protein comprises the amino acid substitution H248L based on the numbering of SEQ ID NO:

6.

8. 8. The composition of claim 6 or 7, wherein the non-lipidated fHBP A protein further comprises the amino acid substitutions L130R and G133D based on the numbering of SEQ ID NO:

6.

9. 9. The composition of any one of claims 1-8, wherein the fHBP A protein and / or fHBP B is present in an amount ranging from about 20 μg / dose to about 200 μg / dose, or from about 25 μg / dose to about 180 μg / dose, or from about 40 μg / dose to about 140 μg / dose, or from about 50 μg / dose to about 120 μg / dose, or from about 75 μg / dose to about 100 μg / dose, or about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

10. The composition according to any one of claims 1 to 9, wherein the NadA protein is a NadA1 protein or comprises at least about 85% identity with SEQ ID NO:5 or comprises or consists of SEQ ID NO:

5.

11. 11. The composition of any one of claims 1 to 10, wherein the NadA protein is present in an amount ranging from about 20 μg / dose to about 200 μg / dose, or from about 25 μg / dose to about 180 μg / dose, or from about 40 μg / dose to about 140 μg / dose, or from about 50 μg / dose to about 120 μg / dose, or from about 75 μg / dose to about 100 μg / dose, or about 50 μg / dose.

12. The composition of any one of claims 1 to 11, wherein the dOMV comprises Porin A (PorA).

13. The composition of claim 12 , wherein the dOMV comprises the PorA VR2 subtype.

14. The composition of claim 13 , wherein the dOMV comprises PorA VR2 P1.

2.

15. 15. Any of claims 1-14, wherein dOMV is present in an amount ranging from about 5 μg / dose to about 400 μg / dose, or from about 10 μg / dose to about 300 μg / dose, or from about 25 μg / dose to about 250 μg / dose, or from about 35 μg / dose to about 225 μg / dose, or from about 50 μg / dose to about 200 μg / dose, or from about 75 μg / dose to about 180 μg / dose, or from about 100 μg / dose to about 150 μg / dose, or from about 110 μg / dose to about 125 μg / dose, or about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose. The composition described in claim 1.

16. 16. The composition according to any one of claims 1 to 15, further comprising an adjuvant, in particular an aluminium-based adjuvant, in particular an aluminium-based adjuvant selected in the group comprising aluminium hydroxide adjuvant, aluminium phosphate adjuvant, aluminium sulphate adjuvant, aluminium hydroxyphosphate adjuvant, aluminium potassium sulphate adjuvant, aluminium hydroxycarbonate, a combination of aluminium hydroxide and magnesium hydroxide, and mixtures thereof, in particular an aluminium phosphate adjuvant.

17. 17. The composition of any one of claims 1 to 16, comprising or consisting of 25-100 μg / dose of non-lipidated fHBP A protein consisting of SEQ ID NO:2 with the proviso that the N-terminal cysteine ​​of SEQ ID NO:2 has been removed or replaced with another amino acid, 25-100 μg / dose of non-lipidated fHBP B protein consisting of SEQ ID NO:4 with the proviso that the N-terminal cysteine ​​of SEQ ID NO:4 has been removed or replaced with another amino acid, 25-100 μg / dose of NadA protein consisting of SEQ ID NO:5, 20-150 μg / dose of dOMV derived from a MenB strain expressing PorA VR2 P1.2, 100-600 μg / dose of aluminum phosphate adjuvant, 50 mM acetate buffer and pH 6.

0.

18. 18. The composition of any one of claims 1 to 17, further comprising at least conjugated capsular saccharides from one or more of meningococcal serogroups A, C, W135 and / or Y.

19. 18. The composition of any one of claims 1 to 17, further comprising a combination of MenA, MenC, MenW-135 and MenY capsular polysaccharides each conjugated to a tetanus toxoid carrier protein, wherein the MenA polysaccharide is conjugated to the tetanus toxoid carrier via an adipic acid dihydrazide (ADH) linker, while the MenC, MenW-135 and MenY polysaccharides are each directly conjugated to the tetanus toxoid carrier.

20. A vaccine comprising a composition according to any one of claims 1 to 19.

21. A composition according to any one of claims 1 to 19 or a vaccine according to claim 20 for use in protecting against meningococcal infection or for use in inducing an immune response against meningococcal infection.

22. 20. A composition according to any one of claims 1 to 19 for use in inducing an immune response against meningococcal serogroup B bacteria from ST-41 / 44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and / or the ST-461 clonal complex.

23. A composition according to any one of claims 1 to 19 for use in inducing an immune response against Neisseria meningitidis serogroup B bacteria from the ST-11 clonal complex.

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