Adjuvanted immunogenic compositions against neisseria meningitidis b

Abstract

The present disclosure relates to an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens and an aluminum hydroxyphosphate (AlPO4) adjuvant, said combination comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, the AlPO4 adjuvant selected having a zero electric point (PZC) of less than 5.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 370,333, filed on August 3, 2022, which is incorporated herein by reference in its entirety for any purpose. Sequence Listing

[0002] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file entitled "PR95421_WO_S17_SANOFI_PASTEUR", which was created on July 21, 2023 and is 14,094 bytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to AlPO4 adjuvanted immunogenic compositions against Neisseria meningitidis B, and methods and uses for stabilizing antigens and enhancing immunogenic responses against self and heterologous antigens by adjuvants. Background Art

[0004] Neisseria meningitidis is a Gram-negative diplococcus for which humans are the only known natural host. Neisseria meningitidis frequently colonizes the human nasopharynx and oropharynx, but can also occur in other parts of the body, such as the anal mucosa, conjunctiva, and urogenital tract (Rouphael et al., Methods Mol Biol. 2012; 799: 1-20; Stephens, Vaccine. 2009; 27 Suppl 2: B71-7; Batista et al., Asian Pac J Trop Med. 2017; 10(11): 1019-29).

[0005] At least 12 different meningococcal serogroups have been classified based on the immunochemistry 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 the cause of endemic disease and some outbreaks (Harrison et al. [eds.] Orenstein WA, Offit PA, Edwards KM Plotkin SA. Vaccines. 7. Philadelphia (PA): Elsevier; 2018. pp. 619-43; Borrow et al. Expert Rev Vaccines. 2017; 16(4): 313-28; Harrison et al. Emerg Infect Dis. 2013; 19(4): 566-73; Pollard, Pediatr Infect Dis J. 2004; 23(12 Suppl): S274-9; Kvalsvig et al. J Clin Pathol. 2003; 56(6): 417-22).

[0006] Neisseria meningitidis serogroup B is a respiratory-transmitted bacterium (via droplets), cannot survive in the environment, and requires close and prolonged contact, or direct physical contact (e.g., kissing) for efficient transmission. Asymptomatic carriers are present in less than 2% of children under 5 years of age and 20%-25% of adolescents and young adults and are a major factor in the transmission pathways of the pathogen and its maintenance in nature, even during epidemics (Christensen et al., Lancet Infect Dis. 2010; 10(12):853-61; Batista et al., Asian Pac J Trop Med. 2017; 10(11):1019-29).

[0007] In general, the age group with the highest incidence of carriage is adolescents and young adults, who are prone to behaviors that are considered risk factors for carriage and ultimately develop invasive meningococcal disease (IMD) (Christensen et al., Lancet Infect Dis. 2010;10(12):853-61; Stephens, Vaccine. 2009;27 Suppl 2:B71-7; Bruce et al., JAMA. 2001;286(6):688-93; Germinario et al., Hum Vaccin. 2010;6(12):1025-7; MacLennan et al., Emerg Infect Dis. 2006;12(6):950-7). Therefore, vaccination of these age groups may affect the incidence of IMD in other age groups, as has been demonstrated in many European countries, where vaccination measures with serogroup C conjugate vaccines have resulted in herd protection in unvaccinated age groups (Maiden et al., J Infect Dis. 2008;197(5):737-43; Trotter et al., Lancet. 2004;364(9431):365-7; Bijlsma et al., Clin Infect Dis. 2014;59(9):1216-21).

[0008] Invasive meningococcal disease (IMD) is a serious illness caused by Neisseria meningitidis (including Neisseria meningitidis serogroup B), and symptoms can include severe headache, fever, nausea, vomiting, photophobia, neck stiffness, lethargy, myalgia, and a characteristic petechial rash (Harrison et al., [eds.] Orenstein WA, Offit PA, Edwards KM Plotkin SA. Vaccines. 7. Philadelphia (PA): Elsevier; 2018. pp. 619-43). IMD can cause meningococcal meningoencephalitis and meningococcal sepsis. Meningococcal sepsis can be the most rapidly fatal infectious condition in humans, with approximately 90% of deaths reported within the first 2 days of hospitalization. 6%–15% of patients with IMD may develop an inflammatory syndrome due to the deposition of antigen-antibody complexes, which are primarily composed of capsular polysaccharides, specific immunoglobulins, and complement component C3. These reactions usually occur 4 to 12 days after the onset of the disease and include arthritis (mostly monoarticular (7%–14% of patients)), cutaneous vasculitis, iritis, episcleritis, pleurisy, and pericarditis. Fever, leukocytosis, and elevated serum C-reactive protein may also recur. Other complications that may occur in patients with IMD include reactivation of herpes simplex infection, symmetrical distal necrosis, extensive ulcers on vasculitis scans, gastrointestinal bleeding, subdural effusions, myocarditis, rhabdomyolysis, adult-onset respiratory distress syndrome, acid-base and water-electrolyte disorders, cerebral infarction, and intracranial suppuration.

[0009] IMD survivors may experience sequelae. The risk of neurologic sequelae is 7%-12% (lower than the rate of pneumococcal meningitis), primarily 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, speech disorders, motor deficits, developmental delay, arthritis, spasticity, convulsions, renal failure, osteonecrosis, atrophic scars, partial limb loss, learning disabilities, and behavioral disorders (Batista et al., Asian Pac J Trop Med. 2017;10(11):1019-29; Stephens et al., Neisseria meningitidis. [Eds.] JEBennett, R. Dolin and MJ Blaser. Philadelphia: Saunders [Elsevier Publishers]; 2015. pp. 2425-45; Campsall et al., Crit Care Clin. 2013;29(3):393-409; Pace et al., Vaccine. 2012;30 Suppl 2:B3-9).

[0010] Serogroup B is an important cause of endemic disease and has caused multiple long-lasting epidemics in several industrialized countries, including Cuba (Rodriguez et al., Mem Inst Oswaldo Cruz. [Memories of the Oswaldo Cruz Institute] 1999;94(4):433-40), Norway (Fredriksen et al., NIPH Ann. [Annals of the Norwegian National Institute of Health] 1991;14(2):67-79; discussion-80), and New Zealand (Martin et al., J Infect Dis. [Journal of Infectious Diseases] 1998;177(2):497-500; Dyet et al., Epidemiol Infect. [Epidemiology and Infection] 2006;134(2):377-83) (Vuocolo et al., Hum Vaccin Immunother. [Human Vaccines and Immunother. [Human Vaccines and Immunother.] 2018;14(5):1203-15). Smaller outbreaks caused by single strains have also been reported in other countries, such as France (2000–2003) (Grodet et al., Microbiol Infect. 2004;10(9):845-8; Caron et al., Lancet Infect Dis. 2011;11(6):455-63) and the United States (2013–2017), some of which have been associated with colleges and universities (Folaranmi et al., 2015. MMWR Morb Mortal Wkly Rep. 2015;64(22):608-12; Atkinson et al., Pharmacotherapy 2016;36(8):880-92).

[0011] Two vaccines targeting N. meningitidis serogroup B have recently been licensed: the 4-component MenB protein vaccine (4CMenB) from GlaxoSmithKline, Inc. [GSK] and vaccines based on the bivalent recombinant fHBP protein (rLP2086) from Pfizer ). Those vaccines were added with the following adjuvants: aluminum hydroxide adjuvant and aluminum hydroxyphosphate adjuvant, respectively.

[0012] Adjuvants are agents incorporated into vaccine formulations to enhance the immunogenicity of vaccine antigens. Aluminum salts (e.g., aluminum phosphate and aluminum hydroxide) are the most commonly used adjuvants in current human and veterinary vaccines. Although many aluminum-containing adjuvants are available, for any particular vaccine formulation to have immunogenicity and long-term stability, an appropriate choice of adjuvant / antigen combination is required.

[0013] Hydroxyaluminum phosphate (AlPO4) adjuvants are amorphous, and most commercially available AlPO4 adjuvants have a zero point of charge (PZC) of 5 to 7 and are neutral or negatively charged at a neutral pH of 7.0 (Hem SL, 2007). The zero point of charge is equivalent to the pI or isoelectric pH of a protein or biomolecule and is defined as the pH at which the net charge on the surface of the molecule is zero. The zero point of charge depends on the ratio of hydroxyl and phosphate ions on the AlPO4 surface. At neutral pH, AlPO4 can strongly adsorb proteins with alkaline pI.

[0014] Aluminum hydroxide in dehydrogenated crystalline form is chemically aluminum oxyhydroxide [AlO(OH)], and in its aqueous phase, by acquiring additional water molecules, it becomes aluminum trihydroxide [Al(OH)3] (Stanley L Hem, 2007). The PZC of aluminum oxyhydroxide is 11, so it is positively charged at a neutral pH of 7.0. This positive charge makes aluminum oxyhydroxide a good adsorbent for negatively charged antigens (e.g., acidic pI proteins). The PZC of aluminum hydroxide can be reduced by titration with phosphate ions. This is caused by ligand exchange between hydroxyl ions and phosphate ions on the surface of aluminum hydroxide.

[0015] The adsorption of antigen on aluminum adjuvant depends on the physical and chemical characteristics of the antigen, the type of aluminum adjuvant used and the adsorption conditions. Factors that may affect the adsorption of antigen on aluminum adjuvant include electrostatic force, hydrophobic interaction, van der Waals force, hydrogen bonding, pH, temperature and the size of adjuvant particles. In general, antigens are adsorbed on aluminum adjuvants by electrostatic attraction (i.e., adjuvant and antigen have opposite charges) and / or by ligand exchange (i.e., the phosphate group on the antigen replaces the hydroxyl group on the adjuvant surface) (Seeber SJ, 1991; Iyer S, 2004). The electrostatic interaction between aluminum adjuvant and antigen protein may be affected by the pH of the formulation, the zero point of charge (PZC) of aluminum and the isoelectric point (pI) of the protein. Usually, the appropriate balance of electrostatic interaction is sought, because proteins that are too strongly bound to aluminum may trigger a poor immune response, while weak interactions may lead to poor adsorption and lower product stability.

[0016] The maximum amount of antigen that can be adsorbed as a monolayer onto an adjuvant is called the "adsorption capacity", and the strength of adsorption is expressed by the "adsorption coefficient" (Jendrek, 2003).

[0017] Adsorption can affect the structure and stability of proteins. Studies on the adsorption effects of aluminum-containing adjuvants have not been entirely consistent. In one study, three proteins (bovine serum albumin [BSA], lysozyme, and ovalbumin) were found to have a significant effect on the adsorption of aluminum. or Become unstable after adsorption. In another study, the structure of BSA and beta-lactoglobulin (BLG) was stabilized by adsorption on aluminum hydroxide (Jones, 2005; Zheng, 2007). Methods for stabilizing liquid formulations of vaccines with aluminum salt adjuvants include lyophilization, freezing, and freeze drying, but these methods generally result in adjuvant aggregation, reduced immunogen concentration, and loss of immunogenicity (Maa, 2003; Diminsky, 1999; Alving, 1993; Warren, 1986). Even for those formulations maintained under refrigerated conditions (e.g., 2°C to 8°C), the adsorbed antigens may be chemically unstable and, therefore, may undergo hydrolysis and fragmentation over time.

[0018] WO 2010 / 109323 discloses an immunogenic composition comprising a factor H binding protein (fHBP) antigen adsorbed on an aluminum hydroxyphosphate adjuvant. The aluminum hydroxyphosphate adjuvant is selected to have a PZC in the range of 5.0 to 7.0 to ensure effective adsorption of the antigen. In addition, the pH of the composition is selected to be within 1.2 pH units of the PZC.

[0019] There remains a need to provide immunogenic compositions comprising aluminum adjuvants that can reduce instability or improve stability of protein antigens.

[0020] There is a need to provide immunogenic compositions comprising an aluminum adjuvant and capable of inducing and enhancing an immune response.

[0021] There is a need to provide immunogenic compositions comprising aluminum adjuvants that provide a good safety profile.

[0022] There is a need to provide immunogenic compositions comprising a combination of Neisseria meningitidis serogroup B antigens and capable of inducing an enhanced immune response.

[0023] There is a need to provide immunogenic compositions comprising a combination of Neisseria meningitidis serogroup B antigens and capable of inducing an enhanced immune response against heterologous antigens.

[0024] There is a need to provide an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens including at least one fHBP A antigen and capable of inducing an enhanced immune response against fHBP A antigens that are heterologous and / or homologous to the fHBP A antigens of the composition.

[0025] There is a need to provide immunogenic compositions comprising a combination of Neisseria meningitidis serogroup B antigens including at least one fHBP B antigen and capable of inducing an enhanced immune response against fHBP B antigens that are heterologous and / or homologous to the fHBP B antigen of the composition.

[0026] There is a need to provide immunogenic compositions comprising a combination of Neisseria meningitidis serogroup B antigens and which improve the stability of the antigens of the composition.

[0027] There is a need to provide immunogenic compositions comprising a combination of Neisseria meningitidis serogroup B antigens including at least one fHBP A and / or Neisseria adhesin A (NadA) antigen and capable of inducing enhanced antigen stability.

[0028] The present disclosure is intended to satisfy all or part of these needs. Summary of the invention

[0029] According to one of its objects, the present disclosure relates to an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens and an aluminum hydroxyphosphate (AlPO4) adjuvant, wherein the combination includes at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and the AlPO4 adjuvant is selected to have a zero charge (PZC) of less than 5.

[0030] The present disclosure relates to an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens and an aluminum hydroxyphosphate (AlPO4) adjuvant, wherein the combination includes at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and the AlPO4 adjuvant has a PZC of less than 5 prior to introduction into the composition.

[0031] The composition of the present disclosure comprises a combination of Neisseria meningitidis serogroup B antigens. The combination includes at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and the composition comprises aluminum hydroxyphosphate (AlPO4) adjuvant. An AlPO4 adjuvant with a "zero point of charge" (PZC) lower than 5 is selected. The isoelectric point (pI) of fHBP A and B is higher than the PZC of the adjuvant. The pH of the composition is at least 1.2 units greater than the PZC of the AlPO4 adjuvant. The pH of the composition is from about 5.5 to about 7.0. About 50% to about 85% of fHBP A and / or B are adsorbed on the AlPO4 adjuvant. The composition may further comprise at least one of NadA or dOMV antigens.

[0032] In the present disclosure, the AlPO4 adjuvant has a PZC of less than 5 before introduction into the immunogenic composition.

[0033] Surprisingly, the inventors observed that by selecting an aluminum hydroxyphosphate (AlPO4) adjuvant with a PZC below 5 (contrary to the suggestion of WO 2010 / 109323) to formulate an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens including an fHBP antigen, the immune response to the antigens can be enhanced, the coverage of Neisseria meningitidis B strains can be expanded, and the antigens can be further stabilized.

[0034] Surprisingly, a beneficial effect was observed when the pH of the composition was 1.2 units above the PZC of the aluminum hydroxyphosphate.

[0035] Surprisingly, a beneficial effect was observed when the adsorption of fHBP antigen was less than 85%.

[0036] As shown in the Examples section, an AlPO4 adjuvant having a PZC lower than 5 is selected to prepare an immunogenic composition comprising at least one factor H binding protein (fHBP) B, such that the composition induces an enhanced immune response (measured by the geometric mean titer - GMT of antibodies) against fHBP B homologous to the fHBP B of the composition, compared to a composition prepared with AlPO4 having a PZC higher than 5.

[0037] In addition, as shown in the Examples section, an AlPO4 adjuvant having a PZC below 5 is selected to prepare an immunogenic composition comprising at least one factor H binding protein (fHBP) B, such that the composition induces an enhanced immune response (measured by GMT and responder percentage) against fHBP B that is heterologous to the fHBP B of the composition, compared to a composition prepared with AlPO4 having a PZC above 5.

[0038] In addition, as shown in the Examples section, an AlPO4 adjuvant having a PZC below 5 is selected to prepare an immunogenic composition comprising at least one factor H binding protein (fHBP) A, such that the composition induces an enhanced immune response (measured by GMT and responder percentage) against fHBP A homologous to the fHBP A antigen of the composition, compared to a composition prepared with AlPO4 having a PZC above 5.

[0039] In addition, as shown in the Examples section, an AlPO4 adjuvant having a PZC below 5 is selected to prepare an immunogenic composition comprising at least one factor H binding protein (fHBP) A, such that the composition induces an enhanced immune response (measured by GMT and responder percentage) against fHBP A that is heterologous to the fHBP A of the composition, compared to a composition prepared with AlPO4 having a PZC above 5.

[0040] Compared to the geometric mean titer (GMT) of functional serum bactericidal antibody activity (hSBA) of human complement obtained with a composition containing AlPO 4 having a PZC greater than 5, the hSBA GMT obtained using the composition according to the present disclosure was increased, thereby observing an enhanced immune response.

[0041] Additionally, the % responders obtained with the composition according to the present disclosure were increased compared to the % responders obtained with the composition containing AlPO 4 having a PZC above 5, thereby observing an enhanced immune response.

[0042] An enhanced immune response may be observed by an increased hSBA GMT fHBP-specific response and / or an increased % responders.

[0043] Additionally, as shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens including at least one fHBPA and / or at least one fHBP B advantageously does not compromise the stability of the fHBP.

[0044] As shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of Neisseria meningitidis serogroup B antigens including at least one fHBP A enhances the stability of fHBP A compared to compositions prepared with an AlPO4 adjuvant with a PZC above 5.

[0045] An AlPO4 adjuvant may be selected with a PZC ranging from about 4.1 to less than 5, or ranging from about 4.2 to about 4.9, or ranging from about 4.3 to about 4.8, or about 4.5.

[0046] An AlPO4 adjuvant may be selected with a PZC of about 4.5.

[0047] The difference between the PZC of the AlPO4 adjuvant of the present disclosure and the pH of the composition ranges from about 0.6 to about 2.9.

[0048] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 0.6 to 2.9 units, or from the PZC of the adjuvant by 1.2 to 2.9 units.

[0049] The pH of the immunogenic composition may range from about 5.5 to about 7.0, or its pH may be about 6.0.

[0050] The immunogenic compositions of the present disclosure may further comprise at least one detergent extracted outer membrane vesicle (dOMV) and / or at least one Neisseria adhesin A (NadA) protein.

[0051] As shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of Neisseria meningitidis serogroup B antigens including at least one NadA protein enhances the stability of the NadA protein compared to compositions prepared with AlPO4 with a PZC above 5.

[0052] As shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of Neisseria meningitidis serogroup B antigens including fHBP A and NadA proteins enhances the stability of fHBP A and NadA proteins compared to compositions prepared with AlPO4 with a PZC above 5.

[0053] Additionally, as shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of Neisseria meningitidis serogroup B antigens including at least one NadA protein advantageously does not impair the immune response induced by the NadA protein.

[0054] In addition, as shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of Neisseria meningitidis serogroup B antigens including at least one detergent-extracted outer membrane vesicle (dOMV) advantageously does not compromise the stability of the dOMV or the immune response induced by the dOMV.

[0055] Additionally, as shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of Neisseria meningitidis serogroup B antigens including at least one detergent extracted outer membrane vesicle (dOMV) advantageously does not increase the pyrogenicity of the composition.

[0056] Additionally, as shown in the Examples section, selecting an AlPO4 adjuvant with a PZC below 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of N. meningitidis serogroup B antigens including at least fHBP A, fHBP B, NadA and dOMV antigens advantageously does not increase the pyrogenicity of the composition.

[0057] In the immunogenic compositions of the present disclosure, the amount of fHBP B that can be adsorbed on the AlPO4 adjuvant is about 85% or less of the total amount of fHBP B present in the composition, or the amount ranges from about 50% to less than 85% of the total amount of fHBP B present in the composition.

[0058] The isoelectric point (pI) of fHBP B may be higher than the PZC of the AlPO4 adjuvant.

[0059] The isoelectric point (pi) of fHBP B may range from about 5.0 to about 7.0, or from 5.2 to about 6.5, or from about 5.3 to about 6.0, or may be about 5.5 or 5.46.

[0060] The isoelectric point of fHBP can be determined empirically by techniques such as isoelectric focusing. However, more conveniently, the isoelectric point is a theoretical isoelectric point. This can be calculated using the pKa values ​​of the amino acids described in Bjellqvist et al. (1993) Electrophoresis [electrophoresis] 14:1023-31 using the relevant ExPASy tools (Gasteiger et al. (2005) Protein Identification and Analysis Tools on the ExPASy Server in The Proteomics Protocols Handbook [protein identification and analysis tools on the ExPASy server in the proteomics protocol manual] (editor John M. Walker), Humana Press [Humana Press] (2005)).

[0061] fHBP B may be non-lipidated.

[0062] fHBP B may be a mutant fHBP B comprising at least one mutation that reduces or inhibits the binding of fHBP B to human factor H (fH).

[0063] The fHBP B may be a mutated fHBP B comprising at least about 85% identity to SEQ ID NO:3.

[0064] fHBP B may 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 comprising or consisting of SEQ ID NO: 4 or SEQ ID NO: 9.

[0065] In some embodiments, fHBP B may comprise or consist of SEQ ID NO:9.

[0066] In the immunogenic compositions of the present disclosure, the amount of fHBP A that can be adsorbed on the AlPO4 adjuvant is about 85% or less of the total amount of fHBP A present in the composition, or the amount ranges from about 50% to less than 85% of the total amount of fHBP A present in the composition.

[0067] The isoelectric point (pi) of fHBP A may range from about 5 to about 7, or from 5.2 to about 6.5, or from about 5.4 to about 6, or about 5.9 or 5.86.

[0068] fHBP A may be non-lipidated.

[0069] The fHBP A may be a mutant fHBP A comprising at least one mutation that reduces or inhibits the binding of the fHBP A to human factor H (fH).

[0070] fHBP A may be a mutein comprising at least about 85% identity to SEQ ID NO:1.

[0071] fHBP A comprises at least one amino acid substitution selected from at least one of the following: 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 phenylalanine (F129) at amino acid 129; e) an amino acid substitution of leucine (L130) at amino acid 130; f) an amino acid substitution of valine (V131) at position 131; g) an amino acid substitution of glycine (G133) at position 133; h) an amino acid substitution of lysine (K219) at position 219; and i) an amino acid substitution of glycine (G220) at position 220, based on the numbering of SEQ ID NO: 6, or comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 8.

[0072] In some embodiments, fHBP A may comprise or consist of SEQ ID NO:8.

[0073] In the immunogenic compositions of the present disclosure, each of fHBP A 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 in an amount of about 50 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0074] The dose may range from about 0.1 mL to about 1 mL, such as about 0.2 mL to about 0.8 mL, about 0.4 mL to about 0.6 mL, or the dose may be about 0.5 mL.

[0075] In the immunogenic compositions of the present disclosure, fHBP A 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 may be present in an amount of about 50 μg / dose, or about 100 μg / dose.

[0076] 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 may be present in an amount of about 50 μg / dose, or about 100 μg / dose.

[0077] In the immunogenic compositions of the present disclosure, 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.

[0078] 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 may be present in an amount of about 50 μg / dose.

[0079] In the immunogenic compositions of the present disclosure, the dOMV may comprise Porin A (PorA).

[0080] Porin A (PorA) may be selected from PorA VR2 isoform or PorA VR2 P1.2.

[0081] dOMV can be present in an amount ranging from 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 can be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0082] The immunogenic compositions of the present disclosure may further comprise a buffer.

[0083] The buffer may be selected from Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer or histidine buffer.

[0084] The buffer may be a sodium acetate buffer.

[0085] The immunogenic composition of the present disclosure may comprise or consist of: 25 to 100 μg / dose of non-lipidated fHBP A consisting of SEQ ID NO: 2, 25 to 100 μg / dose of non-lipidated fHBPB consisting of SEQ ID NO: 4, 25 to 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20 to 250 μg / dose of dOMV from a MenB strain expressing PorAVR2 P1.2, 100 to 800 μg / dose of AlPO4 adjuvant with a PZC of about 4.5, and 50 mM acetate buffer at a pH of 6.0.

[0086] The immunogenic composition of the present disclosure may comprise or consist of: 25 to 100 μg / dose of non-lipidated fHBP A consisting of SEQ ID NO: 8, 25 to 100 μg / dose of non-lipidated fHBPB consisting of SEQ ID NO: 9, 25 to 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20 to 250 μg / dose of dOMV from a MenB strain expressing PorAVR2 P1.2, 100 to 800 μg / dose of AlPO4 adjuvant with a PZC of about 4.5, and 50 mM acetate buffer at a pH of 6.0.

[0087] The immunogenic compositions of the disclosure may further comprise at least one capsular saccharide from one or more of Neisseria meningitidis serogroups A, C, W135 and / or Y conjugated to a carrier protein.

[0088] Conjugated capsular saccharides may be conjugated to a tetanus toxoid carrier.

[0089] As shown in the Examples section, selecting an AlPO4 adjuvant with a PZC of less than 5 to prepare an immunogenic composition according to the present disclosure comprising a combination of Neisseria meningitidis serogroup B antigens and at least one conjugated capsular saccharide from one or more of Neisseria meningitidis serogroups A, C, W135 and / or Y advantageously does not impair the stability of the conjugated capsular saccharide or the immune response induced by the conjugated capsular saccharide.

[0090] The precipitation start time (T onset ) can range from about 3.5 min to about 10 min.

[0091] The initial settling time can be measured by using static multiple light scattering to detect particle migration and size changes in liquid dispersions as disclosed in the Examples section. Two detectors can be used - transmission and backscattering - whose signals are related to particle size and concentration, and their changes are signs of instability occurring. This parameter is a measure of the physical stability of the composition and is related to the flocculation characteristics of the suspension. Other methods known in the art can be used to determine the initial settling time.

[0092] Advantageously, an onset sedimentation time of at least or higher than 3.5 min allows to ensure that the components of the composition will remain suspended during the manufacturing operations and therefore to better control the manufacturing.

[0093] The immunogenic compositions of the present disclosure can enhance immune responses against serogroup B strains of Neisseria meningitidis that express an fHBP B that is heterologous to the fHBP B of the composition.

[0094] The immunogenic compositions of the present disclosure can enhance immune responses against Neisseria meningitidis serogroup B strains that express fHBP B homologous to the fHBP B of the composition.

[0095] The immunogenic compositions of the present disclosure can enhance immune responses against serogroup B strains of Neisseria meningitidis that express an fHBP A that is heterologous to the fHBP A of the composition.

[0096] The immunogenic compositions of the present disclosure can enhance immune responses against serogroup B strains of Neisseria meningitidis that express fHBP A homologous to the fHBP A of the composition.

[0097] The immunogenic compositions of the present disclosure can enhance the stability of fHBP A.

[0098] The immunogenic compositions of the present disclosure can enhance the stability of the NadA protein.

[0099] According to another of its objects, the present disclosure relates to a vaccine comprising the immunogenic composition of the present disclosure.

[0100] According to some embodiments, the immunogenic compositions or vaccines of the present disclosure may be used in methods for inducing an immune response against Neisseria meningitidis B strain.

[0101] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP B heterologous to said fHBP B antigen of said composition.

[0102] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP B homologous to said fHBP B antigen of said composition.

[0103] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP A antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP A heterologous to said fHBP A antigen of said composition.

[0104] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP A antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP A homologous to said fHBP A antigen of said composition.

[0105] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for stabilizing at least one fHBP A in an immunogenic composition.

[0106] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for stabilizing at least one Neisseria adhesin A (NadA) protein in an immunogenic composition.

[0107] According to another object thereof, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for increasing the sedimentation start time (T onset ) is stable within the range of about 3.5 min to about 10 min, wherein the combination includes at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

[0108] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for adjuvanting an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens, the combination comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

[0109] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for the manufacture of an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens, said combination comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

[0110] According to another of its objects, the present disclosure relates to a method for manufacturing an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens and an AlPO4 adjuvant, the combination comprising at least one factor H binding protein (fHBP) A and one factor H binding protein (fHBP) B, the method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, the combining being performed in any order.

[0111] The combination of AlPO4 adjuvant with fHBP A and fHBP B can be performed in any order. For example, AlPO4 adjuvant can be combined with fHBP A and then fHBP B can be added, or AlPO4 adjuvant can be combined with fHBP B and then fHBP A can be added, or AlPO4 adjuvant can be combined with fHBP A and fHBP B at the same time.

[0112] According to another of its objects, the present disclosure relates to a method for stabilizing at least one of fHBP A and NadA proteins in an immunogenic composition, the method comprising at least the steps of: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with fHBP A or NadA protein, and c) obtaining an immunogenic composition wherein the fHBP A or NadA protein is stabilized.

[0113] According to another of its objects, the present disclosure relates to a method for preparing an immunogenic composition comprising a Neisseria meningitidis fHBP B antigen, said composition inducing an enhanced immune response against a Neisseria meningitidis serogroup B strain expressing an fHBP B heterologous to said fHBP B antigen of said composition, said method comprising at least the steps of: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with the fHBP B antigen, and c) obtaining the immunogenic composition.

[0114] According to another of its objects, the present disclosure relates to a method for preparing an immunogenic composition comprising a Neisseria meningitidis fHBP B antigen, said composition inducing an enhanced immune response against a Neisseria meningitidis serogroup B strain expressing a fHBP B homologous to said fHBP B antigen of said composition, said method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with the fHBP B antigen, and c) obtaining the immunogenic composition.

[0115] The composition may further comprise at least one of fHBP A, NadA protein or dOMV.

[0116] According to another of its objects, the present disclosure relates to a method for preparing an immunogenic composition comprising a fHBP A antigen of Neisseria meningitidis, said composition inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP A heterologous to said fHBP A antigen of said composition, said method comprising at least the steps of: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with the fHBP A antigen, and c) obtaining the immunogenic composition.

[0117] According to another of its objects, the present disclosure relates to a method for preparing an immunogenic composition comprising a fHBP A antigen of Neisseria meningitidis, said composition inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP A homologous to said fHBP A antigen of said composition, said method comprising at least the steps of: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with the fHBP A antigen, and c) obtaining the immunogenic composition.

[0118] The composition may further comprise at least one of fHBP B, NadA protein or dOMV.

[0119] According to another of its objects, the present disclosure relates to a method for inducing an immune response against a serogroup B strain of Neisseria meningitidis in an individual in need thereof, the method comprising at least the steps of administering to said individual an immunogenic composition or vaccine according to the present disclosure, wherein said administering step induces an immune response against said serogroup B strain of Neisseria meningitidis.

[0120] A method for enhancing an immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a serogroup B strain of Neisseria meningitidis expressing an fHBP B heterologous to the fHBP B antigen of the composition in an individual in need thereof, the method comprising at least the steps of: administering to the individual an immunogenic composition or vaccine according to the present disclosure, wherein the administering step induces an enhanced immune response against the Neisseria meningitidis serogroup B strain expressing the heterologous fHBP B.

[0121] A method for enhancing an immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a serogroup B strain of Neisseria meningitidis expressing an fHBP B homologous to the fHBP B antigen of the composition in an individual in need thereof, the method comprising at least the steps of: administering to the individual an immunogenic composition or vaccine according to the present disclosure, wherein the administering step induces an enhanced immune response against the Neisseria meningitidis serogroup B strain expressing the homologous fHBP B.

[0122] A method for enhancing an immune response induced by a composition comprising a Neisseria meningitidis fHBP A antigen against a serogroup B strain of Neisseria meningitidis expressing an fHBP A heterologous to the fHBP A antigen of the composition in an individual in need thereof, the method comprising at least the steps of administering to the individual an immunogenic composition or vaccine according to the present disclosure, wherein the administering step induces an enhanced immune response against the Neisseria meningitidis serogroup B strain expressing the heterologous fHBP A.

[0123] A method for enhancing an immune response induced by a composition comprising a Neisseria meningitidis fHBP A antigen against a serogroup B strain of Neisseria meningitidis expressing an fHBP A homologous to the fHBP A antigen of the composition in an individual in need thereof, the method comprising at least the steps of administering to the individual an immunogenic composition or vaccine according to the present disclosure, wherein the administering step induces an enhanced immune response against the Neisseria meningitidis serogroup B strain expressing the homologous fHBP A. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 : Shown are the results of hSBA measured at D0 (grey) and D42 (black) in sera (purified IgG) collected from rabbits immunized at D0 and D28 with a MenB immunogenic composition formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC 4.5) against a strain of Neisseria meningitidis expressing the closely related (also called homologous) fHBP A56 to A05.

[0125] Figure 2 : Shown are the results of hSBA measured at D0 (grey) and D42 (black) in sera (purified IgG) collected from rabbits immunized at D0 and D28 with MenB immunogenic compositions formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC 4.5) against a strain of Neisseria meningitidis expressing the fHBP B44 closely related to B01.

[0126] Figure 3 : Shown are the results of hSBA measured at D0 (grey) and D42 (black) in sera (purified IgG) collected from rabbits immunized at D0 and D28 with MenB immunogenic compositions formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC 4.5) against N. meningitidis strains expressing fHBP A22 heterologous to A05.

[0127] Figure 4 : Shown are the results of hSBA measured at D0 (grey) and D42 (black) in sera (purified IgG) collected from rabbits immunized at D0 and D28 with MenB immunogenic compositions formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC 4.5) against N. meningitidis strains expressing B01 heterologous fHBP B24.

[0128] Figure 5: Shown are the results of hSBA measured against VR2-P1.2-PorA expressing Neisseria meningitidis strains in sera (purified IgG) collected at D0 (grey) and D42 (black) from rabbits immunized at D0 and D28 with MenB immunogenic compositions formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC 4.5).

[0129] Figure 6 : Shown are the results of hSBA measured against NadA-expressing Neisseria meningitidis strains in sera (purified IgG) collected at D0 (grey) and D42 (black) from rabbits immunized at D0 and D28 with a MenB immunogenic composition formulated with AlPO4 adjuvant (PZC5.2) or mod-AlPO4 adjuvant (PZC 4.5).

[0130] Figure 7 : Shown are the results of hSBA measured against Neisseria meningitidis A strain in sera (purified IgG) collected at D0, D28 and D42 from rabbits immunized at D0 and D28 with MenACWY immunogenic composition formulated without AlPO4 adjuvant (white) or with mod-AlPO4 adjuvant (PZC 4.5) (patterned) or with MenB antigen and mod-AlPO4 adjuvant (PZC 4.5) (black).

[0131] Figure 8 : Shown are the results of hSBA measured against Neisseria meningitidis C strain in sera (purified IgG) collected at D0, D28 and D42 from rabbits immunized at D0 and D28 with MenACWY immunogenic composition without AlPO4 adjuvant (white) or with mod-AlPO4 adjuvant (PZC 4.5) (patterned) or with MenB antigen and mod-AlPO4 adjuvant (PZC 4.5) (black).

[0132] Fig. 9 : Shows the results of hSBA measured against Neisseria meningitidis W135 strain in sera (purified IgG) collected at D0, D28 and D42 from rabbits immunized at D0 and D28 with MenACWY immunogenic composition formulated without AlPO4 adjuvant (white) or with mod-AlPO4 adjuvant (PZC 4.5) (patterned) or with MenB antigen and mod-AlPO4 adjuvant (PZC 4.5) (black).

[0133] Fig.10: Shown are the results of hSBA measured against Neisseria meningitidis Y strain in sera (purified IgG) collected at D0, D28 and D42 from rabbits immunized at D0 and D28 with the MenACWY immunogenic composition formulated without AlPO4 adjuvant (white) or with mod-AlPO4 adjuvant (PZC 4.5) (patterned) or with MenB antigen and mod-AlPO4 adjuvant (PZC 4.5) (black).

[0134] Figure 11A-11C : Shown are the results of relative antigenicity (RA) of A05tmN, B01smN, NadA and dOMV formulated in MenPenta immunogenic composition with AlPO4 adjuvant (PZC 5.2) or mod-AlPO4 adjuvant (PZC 4.5) and subjected to heat stress at 45°C (or 37°C for NadA) for 20 days.

[0135] Figures 12A-12D : Shown is the percentage change in free polysaccharide of serogroups A, C, W-135 and Y formulated in MenPenta immunogenic compositions with AlPO4 adjuvant (PZC 5.2) or mod-AlPO4 adjuvant (PZC 4.5) and subjected to heat stress at a temperature of 5°C or 45°C for 28 days.

[0136] Figures 13A-13D : The stability of A05tmN, B01smN, NadA and dOMV in AlPO4 adjuvants with different zero charge points (PZC), AlPO4 adjuvant (PZC 5.2) or mod-AlPO4 adjuvant (PZC of 4.3, 4.5 or 4.8) at 45°C (for B01, A05 and dOMV) or 37°C (for NadA) for 30 days is shown, expressed as the relative antigenicity (RA) of A05tmN, B01smN, NadA or dOMV. Sequence Description

[0137] SEQ ID NO: 1 represents the fHBPA05 wild-type sequence without the first 19 N-terminal amino acids corresponding to the signal peptide responsible for lipidation. CSSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQN GTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSG KAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

[0138] SEQ ID NO: 2 represents the fHBP A05 sequence without the signal peptide responsible for lipidation and with the mutations G220S, L130R, G133D (numbering determined relative to the sequence SEQ ID NO: 6 (fHBP B24)). CSSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSL INQRSFRVSDLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKSTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

[0139] SEQ ID NO: 3 represents the fHBP B01 wild-type sequence without the signal peptide responsible for lipidation. CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVA KRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHHIGLAAKQ

[0140] SEQ ID NO: 4 represents the fHBP B01 sequence without the signal peptide responsible for lipidation and with the mutation H248L (numbering determined relative to the sequence SEQ ID NO: 6 (fHBP B24)). CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVA KRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHLIGLAAKQ

[0141] SEQ ID NO: 5 represents the NadA1 sequence from the MenB MC58 strain, wherein the 23 amino acids of the N-terminal signal peptide and the last 55 amino acids of the C-terminus have been deleted. MTSDDDVKKAATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKID EKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVG

[0142] SEQ ID NO: 6 represents the fHBP B24 wild-type sequence, based on which the numbering of the mutation positions in A05 and B01 was determined. CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKR QFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ

[0143] SEQ ID NO: 7 represents the wild-type NadA1 sequence from the MenB MC58 strain. MKHFPSKVLTTAILATFCSGALAATSDDDVKKAATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEA FNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFRFTENFAAKAGVAVGTSSGSSAAYHVGVNYEW

[0144] SEQ ID NO:8 represents the fHBP A05 sequence with mutations G220S, L130R, G133D (numbering determined relative to the sequence SEQ ID NO:6 (fHBPB24)) and a non-lipidated mutant replacing the N-terminal cysteine ​​with methionine by directly fusing the ATG start codon to the second 5' terminal codon in the DNA sequence encoding the recombinant protein. MSSGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQ NGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFRVSDLGGEHTAFNQLPS GKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKSTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

[0145] SEQ ID NO:9 represents a non-lipidated mutant fHBP B01 sequence having mutation H248L (numbering determined relative to sequence SEQ ID NO:6 (fHBP B24)) and replacing the N-terminal cysteine ​​with methionine by directly fusing the ATG start codon to the second 5' terminal codon in the DNA sequence encoding the recombinant protein. MSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVA KRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHLIGLAAKQ DETAILED DESCRIPTION definition

[0146] Unless otherwise specified herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press can provide a general dictionary of many of the terms used in the present disclosure to those of skill. Although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, exemplary methods and materials are described below. In the event of a conflict, the present specification, including the definitions, shall prevail. Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein, and the techniques thereof, are those well known and commonly used in the art. The methods are performed according to the instructions of the kit manufacturer, as commonly accomplished in the art or as described herein. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0147] Units, prefixes and symbols are all expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers that limit the range. Unless otherwise indicated, amino acid sequences are written from left to right in the direction of amino (N-) to carboxyl (-C). The headings provided herein are not limitations on the various aspects of the present disclosure. Therefore, the terms of the definitions immediately below are more fully defined by reference to the specification (in its entirety).

[0148] All publications and other references mentioned herein are incorporated by reference in their entirety.Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0149] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a plurality of such antigens and reference to "a protein" includes reference to one or more proteins, etc.

[0150] It will be understood that aspects and embodiments of the present disclosure described herein include "having" aspects and embodiments, "comprising" aspects and embodiments, "consisting of" and "consisting essentially of". The words "have" and "comprise" or variations such as "has / having", "comprises / comprising" will be understood to imply the inclusion of the stated elements (such as compositions of matter or method steps) but not the exclusion of any other elements. The term "consisting of" implies the inclusion of the stated elements to the exclusion of any additional elements. The term "consisting essentially of" implies the inclusion of the stated elements, as well as possible other elements, wherein the other elements do not materially affect the basic and novel features of the present disclosure. It will be understood that various embodiments of the present disclosure using the term "comprising" or equivalent terms encompass embodiments in which this term is replaced with "consisting of" or "consisting essentially of".

[0151] In addition, "and / or" where used herein should be considered as a specific disclosure of each of two specified features or components with or without the other specified feature or component. Therefore, the term "and / or" as used in phrases such as "A and / or B" as used herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0152] The term "approximately" or "about" is used herein to mean approximately, roughly, approximately, or about. When the term "approximately" is used in conjunction with a numerical range, it defines the range by extending the boundary above and below the numerical value described. In general, the term "approximately" can limit the numerical value to a change of, for example, 10% above and below (higher or lower) above and below the specified value. In some embodiments, the term represents a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05% or ±0.01% from the specified numerical value. In some embodiments, "approximately" represents a deviation of ±10% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±5% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±4% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±3% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±2% from the indicated numerical value. In some embodiments, "about" means a deviation of ±1% from the indicated value. In some embodiments, "about" means a deviation of ±0.9% from the indicated value. In some embodiments, "about" means a deviation of ±0.8% from the indicated value. In some embodiments, "about" means a deviation of ±0.7% from the indicated value. In some embodiments, "about" means a deviation of ±0.6% from the indicated value. In some embodiments, "about" means a deviation of ±0.5% from the indicated value. In some embodiments, "about" means a deviation of ±0.4% from the indicated value. In some embodiments, "about" means a deviation of ±0.3% from the indicated value. In some embodiments, "about" means a deviation of ±0.1% from the indicated value. In some embodiments, "about" means a deviation of ±0.05% from the indicated value. In some embodiments, "about" means a deviation of ±0.01% from the indicated value.

[0153] In this disclosure, the term "significant" as applied to a change is intended to mean that the observed change is evident and / or statistically significant.

[0154] In this disclosure, the term "substantially" used in conjunction with a feature of the disclosure is intended to define a set of embodiments related to the feature that are largely, but not entirely, similar to the feature.

[0155] In the present disclosure, an "immunogenic composition" is intended to mean a composition comprising at least one antigen in a sufficient amount and in a suitable formulation for inducing an immune response to said antigen in an individual to which the composition is administered. The immune response may be a humoral and / or a cellular response.

[0156] In this disclosure, "PZC" is intended to mean point of zero charge, and is intended to refer to the pH at which the net surface charge of the adsorbent is zero.

[0157] In this disclosure, "pi" is intended to mean isoelectric point, and is intended to refer to the pH at which a particular molecule carries no net charge.

[0158] In the present disclosure, the term "antigen" includes any molecule, such as a peptide or protein, which contains at least one epitope that will cause an immune response and / or an immune response against it. For example, an antigen is a molecule that, optionally after processing, induces an immune response that is, for example, specific to the antigen or a cell expressing the antigen. After processing, the antigen can be presented by an MHC molecule and specifically respond to T lymphocytes (T cells). According to the present disclosure, any suitable antigen that is a candidate for an immune response can be envisioned. The antigen can correspond to or can be derived from a naturally occurring antigen.

[0159] In the present disclosure, the term "adjuvant" is intended to refer to a compound that is capable of enhancing the immune response to an antigen, including enhancing the magnitude and / or duration of the immune response generated by the antigen.

[0160] In this disclosure, the term "buffer" is intended to refer to an aqueous solution containing a weak acid and its salts or a weak base and its salts, and which is resistant to changes in pH. Buffers are used to maintain a stable pH in solution because they can neutralize small amounts of additional acid or base. Buffers suitable for use in immunogenic compositions are known in the art.

[0161] Herein, the expression "immune response" is intended to refer to a biological reaction that occurs in a subject in which the body recognizes and defends itself against antigens, ie, bacteria, viruses, and seemingly foreign harmful substances. An immune response can have either a humoral (ie, antibodies) or a cellular component.

[0162] In this article, the expression "enhanced immune response" is intended to refer to an immune response induced by a first immunogenic composition measured in its body fluids and / or cellular components that is greater than a similarly measured immune response induced by a second immunogenic composition, the first and second compositions differing in one parameter. The difference may be a trend or statistically significant. In the present disclosure, to determine the enhancement of the immune response, the composition of the present disclosure is compared with a composition containing an AlPO4 adjuvant different from the AlPO4 of the present disclosure, the other parameters of the composition being otherwise identical.

[0163] In this article, the terms "stable", "stabilization" or "stability" used with respect to an antigen are intended to refer to maintaining the immunogenic properties of the antigen at a given or non-fluctuating level over a certain period of time. The stabilization of a formulation for an antigen can typically be shown by a reduction or absence of the immunogenicity or efficacy of the antigen during physical, chemical or mechanical stress (e.g., pH changes, temperature changes, surface interactions, foreign impurities, mixing, etc.) in the presence of the formulation, compared to the immunogenicity loss of the antigen in the absence of the formulation or in the presence of another formulation. Immunogenicity or efficacy can be measured at a time point or repeatedly measured over a period of time, such as 2, 3, 4, 6 or 8 times over a period of 3, 6, 9 or 12 months. The immunogenicity (or efficacy) of an antigen is its ability to induce an immune response, and can be measured by any method known in the art.

[0164] Instability of protein antigens can be caused by chemical degradation or aggregation of the molecules to form higher order polymers, dissociation of heterodimers into monomers, deglycosylation, glycosylation modification, or any other structural modification that reduces at least one biological activity of the antigen.

[0165] As used herein, the term "vaccine" is intended to mean an immunogenic composition against a pathogenic agent that is administered to a subject to induce an immune response, with the goal of protecting the subject from a condition caused by the pathogenic agent (i.e., conferring protective immunity) or treating a condition caused by the pathogenic agent. The vaccines disclosed herein can be used as prophylactic (preventive) vaccines for administration to a subject prior to infection, with the goal of preventing a primary (and / or recurrent) infection or reducing the likelihood of a primary (and / or recurrent) infection.

[0166] In the context of the present disclosure, the term "protective immunity" means that a vaccine or vaccination program administered to a mammal induces an immune response that prevents, delays the development of, or reduces the severity of, a disease caused by Neisseria meningitidis, or alleviates or completely eliminates the symptoms of the disease. Protective immunity can be accompanied by the production of bactericidal antibodies. It should be noted that the production of bactericidal antibodies against Neisseria meningitidis is considered in the art to be predictive of the protective effect of a vaccine on a human. (Goldschneider et al. (1969) J. Exp. Med. [Journal of Experimental Medicine] 129: 1307).

[0167] An "isolated" protein or fragment, variant or derivative thereof is one that is not in its native environment. No particular level of purification is required. For example, an isolated protein may simply be removed from its native or natural environment. For purposes of this disclosure, recombinantly produced proteins expressed in host cells are considered isolated, as are native or recombinant polypeptides that are separated, fractionated, or partially or substantially purified by any suitable technique.

[0168] As used herein, the terms "individual" or "subject" or "patient" are used interchangeably and are intended to refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some exemplary embodiments, the individual or subject is a human.

[0169] It should be understood that, for clarity, certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, multiple features of the present invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

[0170] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0171] A list of sources, ingredients, and components as described below is listed such that combinations and mixtures thereof are also contemplated and within the scope herein.

[0172] It should be understood that each maximum numerical limit given throughout this specification includes each lower numerical limit, as if such lower numerical limits were expressly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range that falls within such wider numerical range, as if such narrower numerical ranges were all expressly written herein.

[0173] All item lists (such as, for example, ingredient lists) are intended and should be interpreted as Markush groups. Therefore, all lists can be read and interpreted as items "selected from the group consisting of" the item list "and combinations and mixtures thereof."

[0174] Reference herein may be to the trade names of components including the various ingredients used in the present disclosure. The inventors herein have no intention to be limited by the materials under any particular trade name. Materials equivalent to the materials mentioned by trade names (e.g., materials obtained from different sources with different names or reference numbers) may be substituted and used in the description herein. Aluminum Hydroxyphosphate (AlPO4) Adjuvant

[0175] The compositions of the present disclosure include an aluminum hydroxyphosphate adjuvant.

[0176] Hydroxyaluminum phosphate adjuvant represented by the chemical formula Al(OH)PO4 and referred to as AlPO4 adjuvant in the specification is not a stoichiometric compound, and the amount of hydroxyl and phosphate moieties depends on the preparation conditions. The relative ratio of hydroxyl and phosphate moieties affects the zero charge point (PZC) of the adjuvant.

[0177] The PZC corresponds to the pH at which there is no net charge on the surface. The PZC is negatively correlated with the degree of substitution of hydroxyl groups by phosphate (P / Al molar ratio). Substituting hydroxyl anions with phosphate anions will reduce the PZC. The PZC can be altered by changing the concentration of free phosphate ions in the solution (more phosphate = more acidic or lower PZC) or by adding a buffer such as a histidine buffer (which makes the PZC more basic or higher).

[0178] The AlPO4 adjuvant used in the present disclosure is selected to have a PZC of less than 5. The PZC is measured before the AlPO4 adjuvant is introduced into the composition. Thus, the AlPO4 adjuvant suitable for use in the present disclosure is selected based on its PZC being less than 5 before introduction into the immunogenic composition. Thus, before the selected AlPO4 is introduced into the composition, its PZC is less than 5.

[0179] An AlPO4 adjuvant may be selected with a PZC ranging from about 4.1 to less than 5, or ranging from about 4.2 to about 4.9, or ranging from about 4.3 to about 4.8, or ranging from about 4.4 to about 4.6, or about 4.5.

[0180] The AlPO4 adjuvant may be selected to have a PZC of about 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8 or 4.9.

[0181] An AlPO4 adjuvant may be selected with a PZC of about 4.3, 4.5 or 4.8.

[0182] An AlPO4 adjuvant may be selected with a PZC of about 4.5.

[0183] An AlPO4 adjuvant with a PZC of 4.5 can be selected.

[0184] Different approaches can be used to obtain AlPO4 adjuvants with target PZC.

[0185] To prepare an AlPO4 adjuvant with a target PZC, an aluminum hydroxide (Al(OH)3) adjuvant or an aluminum hydroxyphosphate adjuvant having a PZC higher than the target PZC can be used. Such AlPO4 adjuvants are commercially available.

[0186] In one approach, phosphate buffer (pH 5.8) prepared from a combination of 0.5 M sodium dihydrogen phosphate and 0.5 M sodium hydrogen phosphate can be added to aluminum hydroxide or aluminum hydroxyphosphate adjuvants with higher PZC to prepare an AlPO4 adjuvant with a target PZC.

[0187] In another approach, the PZC of an AlPO4 adjuvant can be altered by titrating the AlPO4 adjuvant with a higher PZC with a stock solution of 0.5 M sodium dihydrogen phosphate salt.

[0188] The AlPO4 adjuvant can be prepared by a batch precipitation method with the following three reactants: aluminum chloride (or other aluminum source), trisodium phosphate, and sodium hydroxide.

[0189] The P / Al molar ratio of the aluminum hydroxyphosphate adjuvant is generally between 0.3 and 1.2, preferably between 0.8 and 1.2, or between 0.85 and 1.0, and more preferably about 0.9. A P / Al molar ratio of at least 0.5 can provide an adjuvant with better immunogenicity and stability properties.

[0190] AlPO4 adjuvants are generally amorphous (i.e., amorphous to X-rays). They are generally granular (e.g., they exhibit a plate-like morphology as seen in transmission electron micrographs). The typical diameter of the plates may be 10-100 nm, and these form aggregates of 0.5-20 μm (e.g., about 1-10 μm) in size. The adsorption capacity of aluminum hydroxyphosphate adjuvants is reported to be 0.7-1.5 mg protein / mg Al at pH 7.4. 3+ .

[0191] A typical adjuvant is amorphous aluminum hydroxyphosphate with a P / Al molar ratio between 0.84 and 0.92, and the adjuvant can be taken as 0.8 mg Al 3+ / mL included.

[0192] Al(Al 3+ ) concentration may preferably be less than 5 mg / mL, e.g. <4 mg / mL, <3 mg / mL, <2 mg / mL, <1 mg / mL, etc. A suitable range may be about 0.2 to about 1 mg / mL, or 0.2 to about 0.8 mg / mL. An Al concentration of 0.8 mg / dose may be used.

[0193] The amount of aluminum phosphate adjuvant present in the compositions disclosed herein can range 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 the amount can be about 400 μg / dose, or about 800 μg / dose.

[0194] Aluminum phosphate adjuvant may be present in the compositions disclosed herein in an amount of about 400 μg / dose.

[0195] In the compositions of the present disclosure, the amount of fHBP that can be adsorbed on the AlPO4 adjuvant is about 85% or less of the total amount of fHBP of the composition, or the amount ranges from about 50% to less than 85%, or about 70% to about 80% of the total amount of fHBP of the composition. The amount of fHBP that can be adsorbed on the AlPO4 adjuvant ranges from about 50% to about 85% or less than 85%, or about 50% to about 80%, or about 50% to about 75%, or about 65% to about 75% of the total amount of fHBP of the composition.

[0196] In the immunogenic composition of the present disclosure, the amount of fHBP B that can be adsorbed on the AlPO4 adjuvant is about 85% or less of the total amount of fHBP B present in the composition, or the amount ranges from about 50% to less than 85% of the total amount of fHBP B present in the composition. The amount of fHBP B that can be adsorbed on the AlPO4 adjuvant ranges from about 50% to about 85% or less than 85%, or about 50% to about 80%, or about 50% to about 75%, or about 65% to about 75% of the total amount of fHBP B of the composition. The amount of fHBP B that can be adsorbed on the AlPO4 adjuvant is about 50%, 55%, 60%, 65%, 70%, 75% or 80%, or less than 85% of the total amount of fHBP B present in the composition.

[0197] In the immunogenic compositions of the present disclosure, the amount of fHBP A that can be adsorbed on the AlPO4 adjuvant is about 85% or less of the total amount of fHBP A present in the composition, or the amount ranges from about 50% to less than 85% of the total amount of fHBP A present in the composition. The amount of fHBP A that can be adsorbed on the AlPO4 adjuvant ranges from about 50% to about 85% or less than 85%, or about 50% to about 80%, or about 50% to about 75%, or about 65% to about 75% of the total amount of fHBP A of the composition. The amount of fHBP A that can be adsorbed on the AlPO4 adjuvant is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or less than 85% of the total amount of fHBP A present in the composition.

[0198] The ratio of the fHBP adsorbed can be controlled by changing the salt concentration and / or pH during preparation, for example, in general, higher NaCl concentrations can reduce the adsorption of fHBP on AlPO4 adjuvant. The adsorption amount of any formulation will depend on the combination of parameters (including the PZC of the adjuvant, the salt concentration and pH during preparation, adjuvant concentration, antigen concentration and the pI of the antigen). The impact of each of these parameters on adsorption can be easily assessed. The degree of adsorption can be determined by comparing the total amount of fHBP antigen in the composition (for example, measured before adsorption occurs, or measured by desorption of the adsorbed antigen) and the amount remaining in the supernatant after centrifugation. Suitable methods can be the methods disclosed in the example section.

[0199] In some embodiments, the AlPO4 adjuvant of the present disclosure is used in a composition having a pH range of about 5.5 to about 7.0. The pH of the composition of the present disclosure may be about 6.0.

[0200] In some embodiments, the AlPO 4 adjuvants of the present disclosure are used in compositions having a pH such that the difference between the PZC of the AlPO 4 adjuvant and the pH of the composition is in the range of about 0.6 to about 2.9.

[0201] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 0.6 to 2.9 units, or from 1.0 to 2.8, or from 1.2 to 2.5, or from 1.4 to 2.1 units from the PZC of the adjuvant.

[0202] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by at least 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 units.

[0203] In some embodiments, the AlPO 4 adjuvants of the present disclosure are used in compositions having a pH such that the difference between the PZC of the AlPO 4 adjuvant and the pH of the composition is in the range of about 1.0 to about 2.9 or about 1.2 to about 2.9.

[0204] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by at least 1.2 units.

[0205] The pH of the composition may differ by no more than 2.9 units from the PZC of the AlPO4 adjuvant.

[0206] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 units.

[0207] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 or 2.7 units.

[0208] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.5, 1.7, 2.2, 2.5 or 2.7 units.

[0209] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.3, 1.4, 1.5, 1.6 or 1.7 units.

[0210] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.5 or 1.7 units.

[0211] The pH of the composition may be about 5.5 to about 7.0. The pH of the composition may be about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or about 7.0.

[0212] The pH of the composition can be about 5.5, 6.0, 6.5, or about 7.0.

[0213] The pH of the composition may be about 6.0.

[0214] The PZC of the AlPO4 adjuvant may be about 4.5.

[0215] In some embodiments, the AlPO 4 adjuvants of the present disclosure are used with fHBP having an isoelectric point (pI) ranging from about 5 to about 7.

[0216] In some embodiments, the difference between the PZC of the AlPO 4 adjuvant and the pi of the fHBP antigen can range from about 0.1 to about 2.8, or from about 0.5 to about 2.5, or from about 0.8 to about 2.1, or from about 0.96 to about 1.36.

[0217] In some embodiments, the difference between the PZC of the AlPO 4 adjuvant and the pi of the fHBP antigen can be about 0.96 or about 1.66.

[0218] The isoelectric point of fHBP antigen can be determined empirically by techniques such as isoelectric focusing. However, more conveniently, the isoelectric point is a theoretical isoelectric point. This can be calculated using the pKa values ​​of the amino acids described in ((1993) Electrophoresis [electrophoresis] 14: 1023-31) by Bjellqvist et al. and using the relevant ExPASy tools (Gasteiger et al. (2005) Protein Identification and Analysis Tools on the ExPASy Server in The Proteomics Protocols Handbook [protein identification and analysis tools on the ExPASy server in the proteomics protocol manual] (editor John M. Walker), Humana Press [Humana Press] (2005)). antigen

[0219] The immunogenic compositions disclosed herein comprise a combination of Neisseria meningitidis serogroup B antigens. The combination comprises at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and the composition comprises an aluminum hydroxyphosphate (AlPO4) adjuvant having a zero point of charge (PZC) of less than 5. FWf

[0220] Meningococcal fHBP (also known as lipoprotein 2086 (LP2086), ORF2086 and genome-derived Neisseria antigen (GNA) 1870 or "741") is a lipoprotein expressed on the surface of almost all invasive meningococcal isolates. fHBP is an important virulence factor because it binds to human complement factor H (fH), a negative regulator of the complement alternative pathway (Seib et al., Expert Rev Vaccines. [Vaccine Expert Review] 2015; 14 (6): 841-59). The binding of fHBP to human fH enables the pathogen to evade alternative complement-mediated killing by the host innate immune system and survive in human serum and blood.

[0221] Three major genetic and immunological fHBP variants have been described: variant 1, corresponding to subfamily B, and variants 2 and 3, both of which are classified in subfamily A (Seib et al., Expert Rev Vaccines. [Vaccine Expert Review] 2015; 14 (6): 841-59). According to the nomenclature provided by Pfizer (fHBP A and B) and Novartis (Novartis) (variants 1, 2 and 3), fHBP is identified with a unique ID number in the PubMLST database. Although there is significant antigenic variability between fHBP subfamilies A and B, the protein sequences within each subfamily are highly conserved between different strains (> 86% sequence identity). According to the neisseria.org or pubmlst.org / neisseria / fHBP / website, each unique fHBP found in Neisseria meningitidis is also assigned an fHBP peptide ID. Because the length of the variant 2 (v.2) fHBP protein (from strain 8047, fHBPID 77) and the variant 3 (v.3) fHBP (from strain M1239, fHBP ID 28) differs by -1 and +7 amino acid residues, respectively, from the length of MC58 (fHBP ID 1, which was selected as the reference sequence for numbering), the numbering used to refer to the residues of the v.2 and v.3 fHBP proteins is different from the numbering based on the actual amino acid sequences of these proteins. Thus, for example, a reference to the leucine residue (L) at position 166 of the v.2 or v.3 fHBP sequence refers to the residue at position 165 of the v.2 protein and at position 173 of the v.3 protein. Members of variants 1, 2, and 3 are present in approximately 65%, 25%, and 10% of clinical isolates of MenB that cause invasive disease, respectively. MenB strains The ten most common fHBP variants present in the global population account for approximately 80% of invasive pathogenic strains in the United States and Europe combined (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).

[0222] The fHBP in the present disclosure may be a wild-type (naturally occurring) polypeptide or may be a non-naturally occurring (modified by amino acid substitution, insertion or deletion) polypeptide, provided that the polypeptide can elicit an immune response.

[0223] The fHBP used according to the present disclosure can be a lipidated or non-lipidated fHBP. Lipidated proteins usually contain a specific peptide sequence for lipidation at their N-terminus. This sequence can be cleaved during the maturation phase of the protein. The lipidation signal peptide is specific to each protein and the host cell that produces the protein.

[0224] fHBP polypeptide is expressed as a precursor protein with a lipoprotein signal motif at its N-terminus in Neisseria meningitidis. During processing, the motif is cleaved to leave an N-terminal cysteine ​​residue, which is co-translationally modified with a lipid anchor tethering the protein to the Neisseria outer membrane (McNeil et al. (2013) MMBR 77 (2): 234-252). For lipidated fHBP, the lipid attached to cysteine ​​generally includes a palmitoyl residue, such as tripalmitoyl-S-glyceryl-cysteine ​​(Pam3Cys), dipalmitoyl-S-glyceryl-cysteine ​​(Pam2Cys), N-acetyl (dipalmitoyl-S-glyceryl-cysteine), etc.

[0225] To avoid lipidation of recombinant proteins, various techniques known in the art can be used. For example, the lipidation signal peptide can be deleted or replaced with a signal peptide that is not recognized by the cells in which the protein is produced. US 10,300,122 B2 describes the use of this technology for fHBP.

[0226] The codon encoding the N-terminal cysteine ​​can also be replaced with a codon encoding another amino acid, or the codon encoding the N-terminal cysteine ​​can be removed. For example, US10,300,122B2 describes the insertion of an ATG (methionine) codon at the 5' end of the 5'-terminal codon of the open reading frame encoding the mature fHBP protein. This results in the lack of a lipidated N-terminal cysteine ​​residue in the polypeptide. In addition, US 9,724,402 B2 and US11,077,180B2 disclose the production of non-lipidated fHBP, wherein the N-terminal cysteine ​​residue is replaced by an amino acid of a non-cysteine ​​residue.

[0227] The fHBP used according to the present disclosure can be a naturally occurring or non-naturally occurring protein."Non-naturally occurring protein" refers to an "artificial protein", and compared with a naturally occurring protein, encompasses fHBPs with heterologous components not found in nature. Non-naturally occurring proteins may be chimeric proteins or mutant proteins. "Chimeric protein" in the context of the present disclosure is intended to refer to a protein comprising two or more different components, each component being derived from different fHBPs (e.g., variant 1, 2 or 3). The mutation in the mutant protein can include amino acid substitutions, insertions or deletions. In one embodiment, the mutation is an amino acid substitution.

[0228] The non-naturally occurring fHBP suitable for the immunogenic composition disclosed herein is still able to induce an immune response to fHBP. In one embodiment, the non-naturally occurring fHBP used according to the present disclosure can be a mutated fHBP. Mutations (such as amino acid substitutions) can be introduced to reduce or inhibit the combination of fHBP antigen and the coagulation factor H (fH) normally present in individual blood. The reduction of the fHBP of mutation combined with fH can increase the amount of antigen available and accessible to the immune system. This in turn can improve the efficacy and efficiency of the immune response to those antigens. Advantageously, the fHBP of mutation can induce anti-fHBP polyclonal antibodies for the fHBP epitope in the fH binding site, which results in greater protective complement deposition activity than the antibody (fHBP epitope outside its targeting fH binding site) triggered by the wild-type (WT) fHBP antigen.

[0229] In some embodiments, the fHBP may be a mutant fHBP comprising at least one mutation that reduces or inhibits the binding of fHBP to human factor H (fH).

[0230] The non-naturally occurring fHBP used in the immunogenic compositions disclosed herein may have reduced affinity or improved thermal stability for fH compared to the corresponding naturally occurring fHBP. Affinity and thermal stability for fH protein may be measured as disclosed in WO 2016 / 014719 A1 (such as in Examples 1 or 3 of this document).

[0231] For convenience and clarity, unless otherwise specifically indicated, the natural or naturally occurring amino acid sequence of fHBP B24 of sequence SEQ ID NO:6 (or fHBP ID 1 or v.1 fHBP of Neisseria meningitidis strain MC58) is selected as the reference sequence for all naturally occurring and non-naturally occurring fHBP amino acid sequences herein. Therefore, when referring to the amino acid residue positions in fHBP, the position numbering used herein corresponds to the amino acid residue numbering of SEQ ID NO:6 (fHBP B24). Therefore, position numbering 1 refers to the first amino acid residue shown in SEQ ID NO:6, which is cysteine. This is still the case even when additional amino acids are added at the N-terminus of SEQ ID NO:6 (before the cysteine).

[0232] In one embodiment, the mutations (eg, amino acid substitutions) in the fHBP A or B antigen used in the present disclosure may be as disclosed in WO 2011 / 126863 A1, WO 2015 / 017817 A1, or WO 2016 / 014719 A1.

[0233] The immunogenic compositions disclosed herein can include a non-naturally occurring fHBP whose amino acid sequence differs from the amino acid sequence of wild-type Neisseria meningitidis fHBP by 1 to 10 amino acids (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 30 amino acids, 30 amino acids to 40 amino acids, or 40 amino acids to 50 amino acids.

[0234] In some embodiments, the fHBP antigen can 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.

[0235] The identity (for example, homology percentage) can be determined using various known sequence comparison tools, which for example calculate any homology comparison software for paired sequence alignments, including for example the Blast software of the National Center for Biotechnology Information (NCBI), for example by using default parameters. Identity is a global identity, i.e., an identity over the entire amino acid or nucleotide sequence rather than over its portion. Paired global alignments are defined by Needleman et al. (Journal of Molecular Biology [Molecular Biology], 1970, 443-53 pages, the 48th volume). For example, when starting from a polypeptide sequence and compared with other polypeptide sequences, the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available from http: / / emboss.sourceforge.net / apps / cvs / emboss / apps / needle.html) can be used to find the best alignment of two sequences along their full length - "global alignment".

[0236] The fHBP antigen to be used in the immunogenic composition disclosed herein can be obtained as disclosed in WO 2016 / 014719A1. fHBP can be obtained as a recombinant protein from a recombinant expression vector (or construct) transfected into a host cell (e.g., an E. coli strain) for production. The composition of a suitable vector for transferring and expressing a nucleic acid encoding fHBP can be different. The integration vector can be a conditionally replicating plasmid, a suicide plasmid, a phage, etc.

[0237] The construct may include various elements, including, for example, promoters, selective genetic markers (e.g., genes that confer resistance to antibiotics such as kanamycin, erythromycin, chloramphenicol, or gentamicin), replication origin sites (to promote replication in host cells such as bacterial host cells), etc. The choice of vector will depend on a variety of factors, such as the cell type to be propagated and the purpose of the propagation. Certain vectors can be used to amplify and produce large amounts of desired DNA sequences. Other vectors are suitable for expression in cells in culture. The selection of appropriate vectors is well known to those skilled in the art. Many such vectors are commercially available.

[0238] In one example, the vector can be an expression vector based on an episomal plasmid, which contains selectable drug resistance markers and elements that provide autonomous replication in different host cells (e.g., both Escherichia coli and Neisseria meningitidis). An example of such a "shuttle vector" is plasmid pFPIO (Pagotto et al. (2000) Gene [gene] 244: 13-19). The vector can provide extrachromosomal maintenance in the host cell or can provide integration into the host cell genome. The vector is described in detail in many publications well known to those skilled in the art, including, for example, Short Protocols in Molecular Biology [finely edited molecular biology experimental guide], (1999) F.Ausubel, et al., editors, Wiley & Sons [Wiley & Sons]. The vector can provide expression of a nucleic acid encoding a subject fHBP, can provide propagation of a subject nucleic acid, or both.

[0239] The example of the carrier that can be used includes but is not limited to those derived from recombinant phage 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. Phage vectors can include λ gt10, λ gt11, λ gt18-23, λ ZAP / R and EMBL series phage vectors. Other vectors that can be used include but are not limited to pJB8, pCV 103, pCV 107, pCV108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and Caron 9 series vectors.

[0240] The recombinant expression vector may comprise a nucleotide sequence encoding fHBP operably linked to a transcriptional control element (e.g., a promoter). The promoter may be constitutive or inducible. The promoter may be suitable for use in a prokaryotic host cell or a eukaryotic host cell.

[0241] Expression vector provides transcription and translation regulatory sequences, and can provide inducible or constitutive expression, wherein the coding region is operably connected under the transcriptional control of the transcription initiation region and the transcription and translation termination region. These control regions can be natural for the fHBP of the derived theme fHBP, or can be derived from an external source. Generally speaking, transcription and translation regulatory sequences can include but are not limited to promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences and enhancer or activator sequences. Promoter can be a constitutive promoter or an inducible promoter, and can be a strong constitutive promoter (e.g., T7, etc.).

[0242] Expression vectors usually have convenient restriction sites near the promoter sequence to provide the insertion of the nucleotide sequence encoding the target protein.Construct (recombinant vector) can be prepared by, for example, inserting the target polynucleotide into the construct backbone (typically being attached to the restriction enzyme site of cutting in the carrier by DNA ligase).Alternately, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination.Typically, homologous recombination can be completed by attaching the region homologous to the carrier to the flank of the desired nucleotide sequence, and site-specific recombination can be completed by using the sequence (for example, Cre-lox, att sites, etc.) that promotes site-specific recombination.The nucleic acid comprising such a sequence can be added by, for example, the connection of an oligonucleotide or by using the polymerase chain reaction of a primer comprising a part for a homologous region and a desired nucleotide sequence.

[0243] In addition, the expression construct can include other elements. For example, the expression vector can have one or two replication systems, so that it can be maintained in an organism, for example, in a mammal or insect cell, for expression, and in a prokaryotic host, for cloning and amplification. In addition, the expression construct can include a selective marker gene to allow the selection of transformed host cells. The selection gene is well known in the art and will vary with the host cell used.

[0244] Amino acid substitutions can be introduced into the fHBP 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.

[0245] Recombinant fHBP can be obtained from the culture in a purified form by any purification method known in the art, as described, for example, in the Examples section.

[0246] In one embodiment, fHBP A and / or fHBP B may be 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 in an immunogenic composition disclosed herein. In one embodiment, fHBP A 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. FWf

[0247] The immunogenic compositions disclosed herein may comprise at least one fHBP B variant antigen. The at least one fHBP B may be a lipidated or non-lipidated protein. The fHBP B may be a lipidated protein. The fHBP B may be a non-lipidated protein.

[0248] fHBP B may be a naturally occurring or non-naturally occurring fHBP. fHBP B may be a naturally occurring fHBP. In another embodiment, fHBP B may be a non-naturally occurring fHBP.

[0249] fHBP B may be a non-lipidated, non-naturally occurring fHBP.

[0250] The fHBP B protein can be 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: 3. Non-naturally occurring fHBP B01 proteins do not have 100% identity to fHBP B01 or SEQ ID NO: 3.

[0251] The non-naturally occurring fHBP B may be a chimeric protein as disclosed in WO 2011 / 126863 A1 or WO 2015 / 017817 A1 or a mutant fHBP B protein as disclosed in WO 2016 / 014719 A1, WO 2011 / 051893 or WO 2020 / 030782. In an exemplary embodiment, fHBP B may be a mutant protein.

[0252] The non-naturally occurring fHBP B may be a mutant protein. The mutant fHBP B may be a non-lipidated protein.

[0253] The amino acid sequence of the mutant fHBP B can differ from that of wild-type Neisseria meningitidis fHBP B (e.g., fHBP B01) by 1 to 10 amino acids (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 30 amino acids, 30 amino acids to 40 amino acids, or 40 amino acids to 50 amino acids.

[0254] The mutated fHBP B may be a mutein comprising at least one mutation that reduces or inhibits the binding of fHBP B to human factor H (fH).

[0255] The mutated fHBP B may comprise at least about 85% amino acid sequence identity to SEQ ID NO:3.

[0256] The mutant fHBP B can be a mutant protein comprising 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 mutant fHBP B protein does not have 100% identity to fHBP B01 or SEQ ID NO: 3.

[0257] The mutant fHBP B may comprise at least one amino acid substitution selected from at least one of the following: 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.

[0258] The mutated fHBP B may comprise at least one amino acid deletion or substitution in any of the following positions numbered as disclosed in WO 2011 / 051893 with respect to 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, 1198, S211, L213, K219, N43, D116, H119, S221 and K241.

[0259] The mutated fHBP B may comprise at least one amino acid substitution in any of the following positions numbered as disclosed in WO 2020 / 030782 with respect to the fHBP sequence identified as SEQ ID NO: 2 in WO 2020 / 030782 (mature lipoprotein form of fHBP B09): E211, S216 or E232.

[0260] The mutated fHBP B may comprise at least one of the following amino acid substitutions in any of the following positions numbered as disclosed in WO 2020 / 030782 with respect to the fHBP sequence identified as SEQ ID NO: 2 in WO 2020 / 030782 (mature lipoprotein form of fHBP B09): E211A, S216R or E232A.

[0261] The mutated fHBP B may comprise at least one amino acid substitution in any of the following positions numbered as disclosed in WO 2020 / 030782 with respect to the fHBP sequence identified as SEQ ID NO: 6 in WO 2020 / 030782 (mature lipoprotein form of fHBP B44): E214, S219 or E235.

[0262] The mutated fHBP B may comprise at least one of the following amino acid substitutions in any of the following positions numbered as disclosed in WO 2020 / 030782 with respect to the fHBP sequence identified as SEQ ID NO: 2 in WO 2020 / 030782 (mature lipoprotein form of fHBP B44): E214A, S219R or E235A.

[0263] The mutant fHBP B protein may comprise at least one of the following amino acid substitutions in any of the following positions numbered as disclosed in WO 2010046715 with respect to the fHBP sequence identified as SEQ ID NO: 1 in WO 2010046715 (mature lipoprotein form of fHBP 24): 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 altered in the factor H binding protein may be selected from the group comprising 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 WO 2010046715.

[0264] The amino acid substitution of glutamine (Q38) at amino acid 38 can be a Q38R substitution (R: arginine). Other amino acids with positively charged or aromatic side chains (e.g., lysine, histidine, phenylalanine, tyrosine, or tryptophan) can also be substituted at this position. Thus, in some cases, fHBP can comprise a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, or a Q38W substitution.

[0265] The amino acid substitution of glutamic acid (E92) at amino acid 92 can be an E92K substitution. Other amino acids with positively charged or aromatic side chains (e.g., arginine, histidine, phenylalanine, tyrosine, or tryptophan) can also be substituted at this position. Thus, for example, in some cases, the fHBP variant can comprise an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution.

[0266] The amino acid substitution of arginine (R130) at amino acid 130 can be a R130G substitution (G: glycine). Other amino acids with negatively charged or aromatic side chains (e.g., aspartic acid, glutamic acid, phenylalanine, tyrosine, or tryptophan) can also be substituted at R130. Thus, for example, in some cases, the fHBP variant can include a R130D substitution, a R130E substitution, a R130F substitution, a R130Y substitution, or a R130W substitution.

[0267] The amino acid substitution of serine (S223) at amino acid 223 can be an S223R substitution (R: arginine). Other amino acids with positively charged or aromatic side chains (e.g., 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.

[0268] In an exemplary embodiment, the amino acid substitution of histidine (H248) at amino acid 248 can be an H248L substitution (L: leucine). Other amino acids with non-polar, negatively charged or aromatic side chains (e.g., isoleucine, valine, aspartic acid, glutamic acid, phenylalanine, tyrosine or tryptophan) can also be substituted at H248. Thus, for example, in some cases, fHBP can include an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution or an H248W substitution.

[0269] In another embodiment, the mutant fHBP B may comprise at least the amino acid substitution H248L. The mutant fHBP B may comprise only the amino acid substitution H248L, based on the numbering of SEQ ID NO:6.

[0270] The mutant fHBP B may be a non-lipidated mutant fHBP B comprising at least one of an amino acid substitution selected from the group consisting 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.

[0271] The mutant fHBP B may be a non-lipidated mutant fHBP B comprising at least one of an amino acid substitution selected from the group consisting of Q38R substitution, Q38K substitution, Q38H substitution, Q38F substitution, Q38Y substitution, Q38W substitution, E92K substitution, E92R substitution, E92H substitution, E92F substitution, E92Y substitution, E92W substitution, R130G substitution, R130D substitution, R130E substitution, R130F substitution, R130Y substitution, R130W substitution, S223R substitution, S223K substitution, S223H substitution, S223F substitution, S223Y substitution, S223W substitution, H248L substitution, H248I substitution, H248V substitution, H248D substitution, H248E substitution, H248F substitution, H248Y substitution or H248W substitution, based on SEQ ID NO: 1. NO:6.

[0272] The mutant fHBP B may be a non-lipidated mutant fHBP B comprising 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 may comprise only the amino acid substitution H248L, based on the numbering of SEQ ID NO: 6.

[0273] fHBP B may be a non-lipidated mutant protein comprising 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 comprising at least the amino acid substitution H248L, based on the numbering of SEQ ID NO: 6. The non-lipidated mutant fHBP B may comprise only the amino acid substitution H248L, based on the numbering of SEQ ID NO: 6.

[0274] The mutant non-lipidated fHBP B 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.

[0275] In another embodiment, the mutant non-lipidated fHBP B may comprise or consist of SEQ ID NO:4.

[0276] The mutant non-lipidated fHBP B 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:9.

[0277] In another embodiment, the mutant non-lipidated fHBP B may comprise or consist of SEQ ID NO:9.

[0278] The isoelectric point (pI) of fHBP B may be higher than the PZC of the AlPO4 adjuvant.

[0279] The isoelectric point (pi) of fHBP B may range from about 5.0 to about 7.0, or from 5.2 to about 6.5, or from about 5.3 to about 60, or about 5.5 or 5.46.

[0280] The isoelectric point (pi) of fHBP B may be about 5.46.

[0281] In some embodiments, the difference between the PZC of the AlPO4 adjuvant and the pi of fHBP B may range from about 0.1 to about 2.9, or from about 0.4 to about 2.7, or from about 0.6 to about 2.2, or from about 0.7 to about 1.7, or from about 0.9 to about 1.2.

[0282] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP B may be about 0.66 or about 1.16.

[0283] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP B may be about 0.66.

[0284] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP B may be about 0.96.

[0285] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP B may be about 1.16.

[0286] In one embodiment, fHBP B may be present in an immunogenic composition 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.

[0287] In one embodiment, fHBP B may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose. f

[0288] The immunogenic compositions disclosed herein may comprise at least one fHBP A variant antigen. The at least one fHBP A may be a lipidated or non-lipidated protein. The fHBP A may be a lipidated protein. The fHBP A may be a non-lipidated protein.

[0289] Both fHBP A and fHBP B may be lipidated. In one embodiment, both fHBP A and fHBP B may be non-lipidated. Alternatively, fHBP A may be lipidated and fHBP B may be non-lipidated. Still alternatively, fHBP A may be non-lipidated and fHBP B may be lipidated.

[0290] fHBP A may be a naturally occurring or non-naturally occurring fHBP. fHBP A may be a naturally occurring fHBP. In another embodiment, fHBP A may be a non-naturally occurring fHBP.

[0291] Both fHBP A and fHBP B may be naturally occurring fHBP. Both fHBP A and fHBP B may be non-naturally occurring fHBP. Alternatively, fHBP A may be naturally occurring fHBP, and fHBP B may be non-naturally occurring fHBP. Still alternatively, fHBP A may be non-naturally occurring fHBP, and fHBP B may be naturally occurring fHBP.

[0292] fHBP A may be a non-lipidated, non-naturally occurring fHBP.

[0293] Both fHBP A and fHBP B may be non-lipidated, non-naturally occurring fHBP.

[0294] fHBP A can be 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: 1. Non-naturally occurring fHBP A05 does not have 100% identity to fHBP A05 or SEQ ID NO: 1.

[0295] The non-naturally occurring fHBP A 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, fHBP A may be a mutant protein.

[0296] The non-naturally occurring fHBP A may be a mutant protein. The mutant fHBP A may be a non-lipidated protein.

[0297] The amino acid sequence of the mutant fHBP A can differ from that of wild-type Neisseria meningitidis fHBP A protein (e.g., fHBP A05) by 1 to 10 amino acids (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 30 amino acids, 30 amino acids to 40 amino acids, or 40 amino acids to 50 amino acids.

[0298] The mutated fHBP A may comprise at least about 85% amino acid sequence identity to SEQ ID NO:1.

[0299] The mutated fHBP A may be a mutein comprising at least one mutation that reduces or inhibits the binding of the fHBP A to human factor H (fH).

[0300] The mutant fHBP A may comprise 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 mutant fHBP A05 protein does not have 100% identity to fHBPA05 or SEQ ID NO: 1.

[0301] The mutant fHBP A may comprise at least one amino acid substitution selected from at least one of the following: 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 phenylalanine (F129) at amino acid 129; e) an amino acid substitution of leucine (L130) at amino acid 130; f) an amino acid substitution of valine (V131) at position 131; g) an amino acid substitution of glycine (G133) at position 133; h) an amino acid substitution of lysine (K219) at position 219; and i) an amino acid substitution of glycine (G220) at position 220, based on the numbering of SEQ ID NO: 6.

[0302] The mutant fHBP A may comprise at least one amino acid deletion or substitution in any of the following positions numbered as disclosed in WO 2011 / 051893 with respect to 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, 1124, 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.

[0303] The mutated fHBP A may comprise at least one amino acid deletion or substitution in any of the following positions as disclosed in WO 2016 / 008960 for the numbering of the fHBP sequence identified as SEQ ID NO: 17 in WO 2016 / 008960 (A124 or variant 3.28 or ID28): S32, L126 and / or E243. One, two or three residues may be deleted. Alternatively, they may be substituted with different amino acids. For example, Leu-126 may be substituted with any of the other 19 naturally occurring amino acids. When substitutions are made, in some embodiments, the replacement amino acid may be a simple amino acid, such as glycine or alanine. In other embodiments, the replacement amino acid is a conservative substitution (e.g., made in 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 substitutions at the specified residues are as follows: S32V; L126R; and / or E243A.

[0304] The mutated fHBP A may comprise at least one amino acid deletion or substitution in any of the following positions as disclosed in WO 2016 / 008960 with respect to the numbering of the fHBP sequence (mature lipoprotein form) identified as SEQ ID NO: 5 in WO 2016 / 008960: S32, L123 and / or E240. One, two or three residues may be deleted. Alternatively, they may be substituted with different amino acids. For example, Leu-123 may be substituted with any of the other 19 naturally occurring amino acids. When substitutions are made, in some embodiments, the replacement amino acid may be a simple amino acid, such as glycine or alanine. In other cases, the replacement amino acid is a conservative substitution (e.g., made in 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 the specified residue can be as follows: S32V; L123R; and / or E240A.

[0305] The mutated fHBP A may comprise at least one amino acid deletion or substitution in any of the following positions of the numbering of the fHBP sequence (fHBP A19 or v2.16 or ID16) identified as SEQ ID NO: 5 in WO 2015 / 128480 as disclosed 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. Compared with wild-type fHBP A, mutations at these residues give the protein good stability. In one embodiment, the residues for mutation may be S32, L123, V124, S125, G126, L127 and / or G128. In one embodiment, the residues for mutation may be S32, L123, V124, S125, G126, L127 and / or G128. In another embodiment, residues S32 and / or L123 may be mutated, e.g., S32V and / or L123. When one or more of V100, S125 and / or G126 are mutated, mutations in residues other than these three may also be introduced.

[0306] The specified residue may be missing, but is preferably 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, in some embodiments, the replacement amino acid may be a simple amino acid, such as glycine or alanine. In other embodiments, the replacement amino acid is a conservative substitution (e.g., in 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.

[0307] Substitutions at the indicated residues may be as follows: S32V; V33C; L39C; L41C; F69C; V100T; I113S; F122C; L123R; V124I; S125G or S125T; G126D; L127I; G128A; S151C; H239R; or E240H.

[0308] The mutated fHBP A may comprise at least one amino acid deletion or substitution in any of the following positions as disclosed in WO 2015 / 128480 with respect to the numbering of the fHBP sequence identified as SEQ ID NO: 17 in WO 2015 / 128480 (A124 or variant 3.28 or ID28): S32, V33, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242 and / or E243. In one embodiment, the residue 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 can be mutated, such as residues S32 and / or L126, for example, S32V and / or L126R.

[0309] The specified residue may be missing, but is preferably 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, in some embodiments, the replacement amino acid may be a simple amino acid, such as glycine or alanine. In other embodiments, the replacement amino acid is a conservative substitution (e.g., in 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.

[0310] Substitutions at the indicated residues may be as follows: S32V; I33C; L39C; L41C; F72C; V103T; T116S; F125C; L126R; V127I; S128G or S128T; G129D; L130I; G131A; S154C; H242R; E243H.

[0311] 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 (e.g., 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.

[0312] The amino acid substitution of aspartic acid (D121) at amino acid 121 can be a D121G substitution (G: glycine). Other amino acids with non-polar, positively charged or aromatic side chains (e.g., leucine, isoleucine, valine, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan) can also be substituted at this position. Thus, for example, in some cases, the fHBP variant 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.

[0313] The amino acid substitution of serine (S128) at amino acid 128 can be an S128T substitution (T: threonine). Other amino acids with polar, charged or aromatic side chains (e.g., 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, the fHBP variant can include 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 or an S128W substitution.

[0314] The mutant fHBP A may comprise an amino acid substitution of leucine (L130) at amino acid 130. The amino acid substitution of leucine (L130) at amino acid 130 may be a L130R substitution (R: arginine).

[0315] The mutant fHBP A may comprise an amino acid substitution of valine (V131) at amino acid 131. Other amino acids with charged or aromatic side chains (e.g., glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan) may also be substituted at this position. Thus, for example, in some cases, fHBP may comprise a V131E substitution, a V131K substitution, a V131R substitution, a V131H substitution, a V131F substitution, a V131Y substitution, or a V131W substitution.

[0316] The mutant fHBP A may comprise an amino acid substitution of glycine (G133) at amino acid 133. The amino acid substitution of glycine (G133) at amino acid 133 may be a G133D substitution (D: aspartic acid).

[0317] The mutated fHBP A may comprise an amino acid substitution of lysine (K219) at position 219. Other amino acids with polar, negatively charged or aromatic side chains (e.g., glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine or tryptophan) may also be substituted at this position. Thus, for example, in some cases, fHBP may comprise a K219Q substitution, a K219D substitution, a K219E substitution, a K219F substitution, a K219Y substitution or a K219W substitution.

[0318] The amino acid substitution of glycine (G220) at amino acid 220 can be a G220S substitution (S: serine). Other amino acids with polar, charged or aromatic side chains (e.g., 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, the mutant fHBP A can comprise 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.

[0319] In an exemplary embodiment, the amino acid substitution of leucine at amino acid 130 (L130) can be a L130R substitution (R: arginine).

[0320] In an exemplary embodiment, the amino acid substitution of glycine (G133) at amino acid 133 can be a G133D substitution (D: aspartic acid).

[0321] In an exemplary embodiment, the amino acid substitution of glycine at position 220 (G220) can be a G220S substitution (S: serine).

[0322] In an exemplary embodiment, the amino acid substitution of phenylalanine at position 129 (F129) can be a F129S substitution (S: serine).

[0323] The mutant fHBP A may comprise at least one of the 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 mutant fHBP A 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 mutant fHBP A protein may comprise only three amino acid substitutions G220S, L130R and G133D, based on the numbering of SEQ ID NO: 6.

[0324] The mutant fHBP A can be a non-lipidated mutant fHBP A comprising at least one of the amino acid substitutions selected from the group consisting of: 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, based on the numbering of SEQ ID NO:6.

[0325] The mutant fHBP A may be a non-lipidated mutant fHBP A comprising at least one of an amino acid substitution selected from the group consisting of: N115I substitution, N115V substitution, N115L substitution, N115K substitution, N115R substitution, N115H substitution, N115F substitution, N115Y substitution, N115W substitution, D121G substitution, D121L substitution, D121I substitution, D121V substitution, D121K substitution, D121R substitution, D121H substitution, D121F substitution, D121Y substitution, D121W substitution, S128T substitution, S128M substitution, S128N substitution, S128D substitution, S128E substitution, S128K substitution, S128R substitution, S128H substitution. 128S, 128N, 128Q, 128D, 128E, 128K, 128W, 128R, 128E, 128K, 128W, 128R, 128E, 128K, 128R, 128H, 128F, 128Y, 128W, 128W, 128R, 128E, 128K, 128R, 128H, 128F, 128Y, 128W, 128W, 128D, 128E, 128K, 128W, 128D, 128E, 128K, 128R, 128H, 128F, 128Y, 128W, 128D, 128E, 128K, 128H, 128F, 128Y, 128W, 128D, 128E, 128K, 128R, 128H, 128F, 128Y, 128W, 128D, 128

[0326] The mutant fHBP A may be a non-lipidated mutant fHBP A comprising at least one of the 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 mutant non-lipidated fHBP A 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 exemplary embodiment, the non-lipidated mutant fHBP A may comprise only three amino acid substitutions G220S, L130R and G133D, based on the numbering of SEQ ID NO: 6.

[0327] In an exemplary embodiment, fHBP A can be a non-lipidated mutant protein, which contains 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 with SEQ ID NO: 1, and which contains at least one of the amino acid substitutions selected from the group consisting of: G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6. The mutant non-lipidated fHBP A protein can 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 exemplary embodiment, the non-lipidated mutant fHBP A protein can contain only three amino acid substitutions G220S, L130R, and G133D, based on the numbering of SEQ ID NO: 6.

[0328] In one embodiment, the mutant non-lipidated 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.

[0329] In another embodiment, the mutant non-lipidated fHBP A protein may comprise or consist of SEQ ID NO:2.

[0330] In one embodiment, the mutant non-lipidated 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:8.

[0331] In another embodiment, the mutant non-lipidated fHBP A protein may comprise or consist of SEQ ID NO:8.

[0332] The isoelectric point (pi) of fHBP A may range from about 5 to about 7, or from 5.2 to about 6.5, or from about 5.4 to about 6, or about 5.9 or 5.86.

[0333] The isoelectric point (pi) of fHBP A may be about 5.86.

[0334] In some embodiments, the difference between the PZC of the AlPO4 adjuvant and the pi of fHBP A may range from about 0.1 to about 2.9, or from about 0.4 to about 2.7, or from about 0.6 to about 2.2, or from about 0.7 to about 1.8, or from about 0.9 to about 1.6.

[0335] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP A may be about 1.06 or about 1.56.

[0336] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP A may be about 1.06.

[0337] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP A may be about 1.36.

[0338] The difference between the PZC of the AlPO4 adjuvant and the pi of fHBP A may be about 1.56.

[0339] The fHBP A antigen can 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.

[0340] The fHBPA protein may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose. Nad

[0341] The immunogenic compositions of the disclosure may further comprise at least one Neisseria adhesin A (NadA) protein.

[0342] Neisseria adhesin A (NadA, formerly known as GNA1994) is a surface-exposed trimeric protein that forms oligomers that are anchored to the outer membrane via a C-terminal transmembrane domain. NadA is expressed with a signal peptide and has three major domains: (1) a COOH-terminal anchoring domain (β structure), which is also necessary for autotranslocation to the bacterial surface; (2) a possible coiled domain with a leucine zipper, which may mediate dimerization and oligomerization; (3) an NH(2)-terminal globular head domain. NadA plays a key role in extracellular adhesion and epithelial cell invasion (Capecchi et al., Mol. Microbiol. 2005; 55: (687-98)). The NadA protein sequences of many Neisseria meningitidis strains have been published, and the activity of this protein as a Neisseria adhesin has also been well documented. The NadA gene is present in approximately 50% of meningococcal isolates. NadA exhibits growth phase-dependent expression, with maximum expression levels occurring during the stationary phase.

[0343] The NadA protein is contained in the published genome sequence of meningococcal serogroup B strain MC58 as gene NMB1994 (GenBank Accession No. GI:7227256).

[0344] The NadA polypeptide for use according to the present disclosure may be a wild-type polypeptide, or may be modified by amino acid substitution, insertion or deletion, provided that the polypeptide can elicit an immune response against NadA.

[0345] The NadA protein to be used may be an N-terminally and / or C-terminally truncated NadA or a NadA protein comprising amino acid deletions or insertions, for example as disclosed in WO 01 / 64920, WO 01 / 64922, or WO 03 / 020756.

[0346] The recombinant NadA protein to be used in the immunogenic compositions disclosed herein may be a NadA1 variant. As shown in the Examples, NadA1 was shown to induce a strong hSBA response.

[0347] NadA1 can be obtained from the NadA sequence of the MenB MC58 strain.

[0348] The NadA protein may comprise or consist of the sequence SEQ ID NO:7.

[0349] The NadA protein can comprise at least 190 consecutive 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 consecutive amino acids from SEQ ID NO:7, such as 260 or more, or 270 or more, or 280 or more, or 290 or more, or 300 or more, or 310 or more, or 320 or more, or 330 or more, or 340 or more, or 350 or more, or 360 or more amino acids from SEQ ID NO:7.

[0350] The NadA protein may lack 5 to 10 amino acids, or 10 to 15, or 15 to 20, 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, for example, SEQ ID NO: 7. When the N-terminal residue is deleted, such deletion should not abolish the ability of NadA to adhere to human epithelial cells.

[0351] 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.

[0352] The NadA protein may lack the membrane anchor peptide at its C-terminus. For example, the NadA protein may lack the C-terminal 55 amino acids of, for example, SEQ ID NO:7.

[0353] NadA may be used in monomeric or oligomeric form, for example in trimer form.

[0354] For example, the NadA protein can be without its C-terminal membrane anchor (e.g., deletion of residues 308-362 of strain MC58 (SEQ ID NO:7)). Expression of NadA without its membrane anchor domain in E. coli can result in secretion of the protein into the culture supernatant while removing the 23 amino acid signal peptide (e.g., deletion of residues 2 to 24 of SEQ ID NO:7 to leave a 284 amino acid protein - SEQ ID NO:5).

[0355] 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.

[0356] The NadA protein to be used in the immunogenic composition disclosed herein can be obtained according to any recombinant technology known in the art, such as previously disclosed. The NadA protein can be obtained as a recombinant protein from a recombinant expression vector (or construct) transfected into a host cell (e.g., an E. coli strain) for production. Recombinant NadA can be obtained from the culture in a purified form by any purification method known in the art, such as described in the Examples section.

[0357] 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 50 μg / dose or about 50 μg / dose. dOMV

[0358] The immunogenic compositions of the present disclosure may further comprise at least one detergent extracted outer membrane vesicle (dOMV).

[0359] The immunogenic compositions disclosed herein include detergent-extracted outer membrane vesicles (dOMVs), also known as outer membrane protein complexes (OMPCs). Typically, detergent-extracted outer membrane vesicles are referred to as dOMVs or OMVs when used as antigens. They are referred to as OMPCs when used as protein carriers.

[0360] OMPC is used as a carrier protein platform for the polyribosylribitol phosphate (PRP) conjugate vaccines PedvaxHIB (Haemophilus influenzae type b vaccine) (Einhorn et al., Lancet (London, UK). 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).

[0361] dOMVs are large proteolipid vesicles containing intact outer member proteins and residual lipolytic oligosaccharides (LOS) found in the bacterial outer membrane (Helting, Acta Pathol Microbiol Scand C [Scandinavian Acta Pathologica Sinica, Microbiologica Sinica-C], 1981; 89(2): 69-78). More than 300 proteins can be identified in dOMVs. 75% of the total protein content of dOMVs is represented by the 10 most abundant proteins, including the outer membrane proteins porin A (PorA) and porin B (PorB), which account for 50% of the total protein.

[0362] Porins (Por) are antigenic determinants for serotyping of Neisseria meningitidis. Two classes of porins (PorA and PorB) have been identified, as well as antigenically distinct variants within each class resulting from sequence variations in the variable region (VR) of the por gene encoding surface-exposed loops.

[0363] dOMVs suitable for use in the immunogenic compositions disclosed herein can be obtained from various MenB strains. dOMVs can be isolated from detergent extracts of MenB strains. Suitable MenB strains can be wild-type MenB strains or MenB strains engineered to overexpress porins (e.g., PorA or PorB proteins, and for example, PorA proteins).

[0364] dOMVs can be obtained from the MenB strain expressing the PorA protein.

[0365] The dOMV may be obtained from a MenB strain expressing a PorA VR2 isoform. The PorA VR2 isoform may be a PorA VR2 type P1.2, P1.4, P1.7, P1.10 or P1.13 protein.

[0366] dOMVs can be obtained from a MenB strain expressing the PorA VR2 type P1.2 protein.

[0367] dOMVs can be obtained from a MenB strain expressing PorA VR2 isoform and PorB P2.2a. dOMVs can be obtained from a MenB strain expressing PorAVR2 P1.2 and PorB P2.2a.

[0368] dOMVs may contain PorA VR2 isoform and PorB P2.2a. dOMVs may contain PorA VR2 P1.2 and PorBP2.2a.

[0369] dOMVs may contain PorA VR2 P1.2 and PorB P2.2a and immunotype LOS L3, 7. PorA and PorB may represent approximately 50% of the protein of dOMVs.

[0370] dOMVs may be obtained from a single MenB strain or from different MenB strains. In the latter case, the MenB strains may express the same subtype of PorA protein, or different subtypes of PorA protein, or different types of porins (eg, PorA and PorB proteins).

[0371] Available MenB strains that display dOMVs of the porin protein being sought 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 an epidemic outbreak in such a database, and then selecting MenB strains with genes encoding the porin protein of interest (e.g., PorA VR2 P1.2 protein) in such a subset. The selected strains can then be evaluated for effective expression of the porin protein of interest using techniques known in the art.

[0372] As examples of MenB strains suitable for obtaining dOMVs according to the present disclosure, the following strains may be mentioned: NGH36, BZ 232, DK 353, B6116 / 77, BZ 163, 0085 / 00, NG P20, 0046 / 02, M11 40123, M12240069, N5 / 99, 99M or M07 240677.

[0373] In an exemplary embodiment, the MenB strain can be MenB strain 99M expressing the PorA VR2 P1.2 protein isoform.

[0374] In one embodiment, the dOMV may comprise porin A (PorA) VR2 isoform P1.2.

[0375] 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 the outer membrane protein Porin A (PorA) and the outer membrane protein Porin B (PorB).

[0376] PorA may be present in an amount ranging from about 3% to about 15% relative to the total protein present in the dOMV, or in an amount ranging from about 5% to about 9 or 10%. 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% relative to the total protein present in the dOMV.

[0377] In one embodiment, dOMVs can be obtained using a detergent extraction method using at least one step of deoxycholate treatment.

[0378] Suitable methods for obtaining dOMVs may be as disclosed in Helting et al. (Acta Pathol Microbiol Scand C. [Scandinavian Pathology, Microbiology and Immunology Journal-C] April 1981; 89 (2): 69-78) or Example 2 of US 4,695,624. For example, the bacterial culture may be centrifuged to obtain a precipitate, which may then be extracted with a detergent (e.g., deoxycholate or sodium dodecyl sulfate (SDS)) under heating (e.g., about 50°C to about 60°C, or about 56°C, and for a time range of about 10 to about 20 minutes, or about 15 minutes). The resulting material may then be centrifuged, and the precipitate may be further suspended and purified according to any method known in the art.

[0379] 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.

[0380] In one embodiment, dOMV may be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose. Additional antigens

[0381] The immunogenic compositions of the disclosure may comprise at least one additional antigen.

[0382] The additional antigen may be a saccharide antigen from Neisseria meningitidis serogroups A, C, W135, Y and / or X conjugated to a carrier protein. In one embodiment, the composition of the disclosure may further comprise a combination of conjugates of MenA, MenC, MenW-135 and MenY capsular polysaccharides and a carrier protein.

[0383] In one embodiment, the additional antigen may be a combination of MenA, MenC, MenW-135 and MenY capsular polysaccharides conjugated with a carrier protein.

[0384] The carrier proteins of different capsular polysaccharides can be different or identical. The carrier protein can include inactivated bacterial toxins, such as diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, Escherichia coli LT, Escherichia coli ST and exotoxin A from Pseudomonas aeruginosa (Pseudomonas aeruginosa). Bacterial outer membrane proteins can also be used, for example, porins, transferrin binding protein, pneumococcal hemolytic protein, pneumococcal surface protein A (PspA) or pneumococcal adhesin protein (PsaA). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivatives (PPD) can also be used as carrier proteins. It can be CRM197 protein, tetanus or diphtheria toxoid. In one embodiment, it is tetanus toxoid.

[0385] The conjugate may be a population comprising molecules with a molecular weight ranging from 700 kDa to 1400 kDa or from 800 kDa to 1300 kDa.

[0386] In each dose, the amount of a single saccharide antigen may be between 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.

[0387] The additional antigen may be 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, and the MenC, MenW-135 and MenY polysaccharides are each directly conjugated to a tetanus toxoid carrier (TT).

[0388] In one embodiment, the additional antigens may be a combination of conjugates of MenA, MenC, MenW-135 and MenY capsular polysaccharides with a tetanus toxoid carrier protein. In an exemplary embodiment, the conjugated saccharide antigens from Neisseria meningitidis serogroups A, C, W135 and / or Y may be as disclosed in WO 2018 / 045286 A1 or WO 2002 / 058737 A2.

[0389] In one embodiment, the additional antigen is the commercially available MenACYW-TT conjugate vaccine of antigens. Immunogenic composition

[0390] The immunogenic compositions disclosed herein may comprise 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).

[0391] The immunogenic compositions disclosed herein may comprise a combination of Neisseria meningitidis serogroup B antigens, the combination comprising at least one factor H binding protein (fHBP) A, at least one fHBP B, at least one Neisseria adhesin A (NadA) protein, and at least one detergent extracted outer membrane vesicle (dOMV). fHBP A and / or fHBP B may be non-lipidated.

[0392] fHBP A may be a mutein comprising at least about 85% identity to SEQ ID NO:1, and / or fHBPB may be a mutein comprising at least about 85% identity to SEQ ID NO:3.

[0393] fHBP A may be a mutant protein comprising at least one mutation that reduces or inhibits the binding of fHBP A to human factor H (fH), and / or fHBP B may be a mutant protein comprising at least one mutation that reduces or inhibits the binding of fHBP B to human factor H (fH).

[0394] fHBP A may comprise at least one amino acid substitution selected from at least one of the following: 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 phenylalanine at amino acid 129; e) an amino acid substitution of leucine (L130) at amino acid 130; f) an amino acid substitution of valine (V131) at position 131; g) an amino acid substitution of glycine (G133) at position 133; h) an amino acid substitution of lysine (K219) at position 219; and i) an amino acid substitution of glycine (G220) at position 220, based on the numbering of SEQ ID NO: 6, or comprising or consisting of SEQ ID NO: 2, or comprising SEQ ID NO: 3. NO:8 or consists of it, and / or the fHBPB protein may comprise at least one amino acid substitution selected from at least one of the following: 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, or comprises or consists of SEQ ID NO:9.

[0395] fHBP A 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 may be present at about 25 μg / dose, or about 50 μg / dose, or about 100 μg / dose.

[0396] 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.

[0397] 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 may be present in an amount of about 50 μg / dose.

[0398] The dOMV may comprise Porin A (PorA).

[0399] dOMV can be present in an amount ranging from 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 can be present in an amount of about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

[0400] The composition may comprise an adjuvant, such as an aluminum-based adjuvant, such as an aluminum-based adjuvant selected from the group comprising aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum sulfate adjuvant, aluminum hydroxyphosphate aluminum sulfate adjuvant, potassium aluminum sulfate adjuvant, aluminum hydroxycarbonate, a combination of aluminum hydroxide and magnesium hydroxide, and mixtures thereof, such as aluminum phosphate adjuvant.

[0401] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 0.6 to 2.9 units, or by 1.2 to 2.9 units from the PZC of the adjuvant. The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 0.6 to 2.9 units, or by 1.0 to 2.8, or 1.2 to 2.5, or 1.4 to 2.1 units from the PZC of the adjuvant.

[0402] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by at least 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 units.

[0403] In some embodiments, the AlPO 4 adjuvants of the present disclosure are used in compositions having a pH such that the difference between the PZC of the AlPO 4 adjuvant and the pH of the composition is in the range of about 1.0 to about 2.9 or about 1.2 to about 2.9.

[0404] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by at least 1.2 units.

[0405] The pH of the composition may differ by no more than 2.9 units from the PZC of the AlPO4 adjuvant.

[0406] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 units.

[0407] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 or 2.7 units.

[0408] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.5, 1.7, 2.2, 2.5 or 2.7 units.

[0409] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.3, 1.4, 1.5, 1.6 or 1.7 units.

[0410] The pH of the composition may differ from the PZC of the AlPO4 adjuvant by 1.2, 1.5 or 1.7 units.

[0411] The pH of the composition may be about 5.5 to about 7.0. The pH of the composition may be about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or about 7.0.

[0412] The pH of the composition can be about 5.5, 6.0, 6.5, or about 7.0.

[0413] The pH of the composition may be about 6.0.

[0414] The PZC of the AlPO4 adjuvant may be about 4.5.

[0415] The pH of the composition may be within 1.5 units of the PZC of the AlPO4 adjuvant.

[0416] The composition may comprise or consist of: about 25 to about 100 μg / dose of a non-lipidated fHBP A protein comprising SEQ ID NO: 2 or consisting thereof, about 25 to about 100 μg / dose of a non-lipidated fHBP B protein comprising SEQ ID NO: 4 or consisting thereof, about 25 to about 100 μg / dose of a NadA protein comprising SEQ ID NO: 5 or consisting thereof, about 20 to about 150 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 600 μg / dose of an aluminum phosphate adjuvant, and 50 mM acetate buffer at a pH of 6.0.

[0417] The composition may comprise or consist of: about 25 to about 100 μg / dose of a non-lipidated fHBP A protein comprising SEQ ID NO: 8 or consisting thereof, about 25 to about 100 μg / dose of a non-lipidated fHBP B protein comprising SEQ ID NO: 9 or consisting thereof, about 25 to about 100 μg / dose of a NadA protein comprising SEQ ID NO: 5 or consisting thereof, about 20 to about 150 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 600 μg / dose of an aluminum phosphate adjuvant, and 50 mM acetate buffer at a pH of 6.0.

[0418] The composition may further comprise at least one conjugated capsular saccharide from one or more of Neisseria meningitidis serogroups A, C, W135 and / or Y.

[0419] Also disclosed are vaccines comprising the compositions described herein.

[0420] The compositions or vaccines disclosed herein may be for use in preventing meningococcal infection or may be for use in inducing an immune response against meningococcal bacteria.

[0421] Further disclosed is a composition comprising or consisting of: an mRNA encoding an 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 an 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 dOMVs from MenB expressing PorA VR2 P1.2.

[0422] Further disclosed is a composition comprising or consisting of: an mRNA encoding an 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:8, an mRNA encoding an 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:9, 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 dOMVs from MenB expressing PorA VR2 P1.2. Preparations

[0423] The immunogenic compositions disclosed herein can be formulated into solid, semisolid, liquid forms, such as tablets, capsules, powders, aerosols, solutions, suspensions, or emulsions. Practical methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy Science, 2000). Compositions are formulated based on the mode of delivery, including, for example, compositions can be formulated for delivery via parenteral delivery (e.g., intramuscular, intradermal, or subcutaneous injection).

[0424] The immunogenic composition can be administered via any suitable route, such as by mucosal administration (e.g., intranasal or sublingual), parenteral administration (e.g., intramuscular, subcutaneous, transdermal or intradermal routes), or oral administration. Typical routes of administration of such compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. In some embodiments, the composition can be administered by transdermal, subcutaneous, intradermal or intramuscular routes.

[0425] In one embodiment, the immunogenic compositions disclosed herein may be formulated for administration via an intramuscular route or an intradermal route or a subcutaneous route. In one embodiment, the immunogenic compositions may be formulated for administration via an intramuscular route.

[0426] The immunogenic composition can be formulated with any pharmaceutically acceptable excipient. The composition can include at least one inert diluent or carrier. An exemplary pharmaceutically 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.), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pennsylvania. The immunogenic compositions described herein can optionally include pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., for example, 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, sucrose, D-anhydrotrehalose, sorbitol, tris(hydroxymethyl)aminomethane and / or urea. Furthermore, the vaccine composition may optionally contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers and preservatives.

[0427] Composition can be in the form of liquid, such as solution, emulsion or suspension, intended for delivery by injection. Composition intended to be administered by injection can include at least one of the following: surfactant, preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer and isotonic agent can be included. Liquid composition disclosed herein can include at least one of the following: sterile diluent (such as water for injection), saline solution (such as normal saline), Ringer's solution, isotonic sodium chloride, fixed oil (such as synthetic monoglyceride or diglyceride that can be used as solvent or suspension medium), polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agent, such as benzyl alcohol or methyl paraben; antioxidant, such as ascorbic acid or sodium bisulfite; chelating agent, such as ethylenediaminetetraacetic acid; buffer, such as acetate, citrate or phosphate; and reagent for adjusting tonicity, such as sodium chloride or dextrose; reagent acting as cryoprotectant, such as sucrose or trehalose.

[0428] In another embodiment, the pH of the compositions of the present disclosure may range from about 5.5 to about 7.0.

[0429] The pH of the immunogenic compositions disclosed herein can range from about 5.5 to about 7.0, or from about 5.6 to about 6.9, or from about 5.7 to about 6.7, or from about 5.8 to about 6.5, or from about 5.9 to about 6.3. In one embodiment, the pH of the compositions disclosed herein can be about 6.0. A stable pH can be maintained by using a buffer.

[0430] In one embodiment, the composition of the present disclosure may further comprise a buffer. As possible available buffers, Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer or histidine buffer may be cited.

[0431] The composition may comprise a sodium acetate buffer.

[0432] The sodium acetate buffer may be present at a concentration ranging from about 10 mM to about 300 mM, or from about 10 mM to about 250 mM, or from about 20 mM to about 250 mM, or from about 20 mM to about 150 mM, or from about 20 mM to about 130 mM, or from about 30 mM to about 120 mM, or from about 40 mM to about 100 mM, or from about 50 mM to about 80 mM, or from about 50 mM to about 60 mM, or, for example, may be present at a concentration of about 50 mM.

[0433] The immunogenic composition may be isotonic to a mammal (eg, a human).

[0434] The immunogenic composition may also comprise one or more additional salts, such as sodium salts, calcium salts or magnesium salts. The sodium salt may be selected from the group comprising sodium chloride, sodium phosphate. 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.

[0435] The sodium salt may be present in 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, for example, at a concentration of about 150 mM.

[0436] 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.

[0437] The immunogenic composition for parenteral administration can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. The injectable composition is, for example, sterile.

[0438] The immunogenic composition can be sterilized by conventional sterilization techniques (e.g., with UV or gamma radiation), or can be aseptically filtered. The composition obtained after aseptic filtration can be packaged and stored in liquid form or lyophilized form. The lyophilized composition can be reconstituted with a sterile aqueous vehicle before administration. The dry composition can include stabilizers (e.g., mannitol, sucrose, or dodecyl maltoside) and mixtures thereof (e.g., lactose / sucrose mixtures, sucrose / mannitol mixtures, etc.).

[0439] The compositions disclosed herein are administered to an individual in need thereof in a therapeutically effective amount, which amount will vary depending on a variety of factors, including the activity of the specific therapeutic agent employed; the metabolic stability and duration 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; drug combination; the severity of the particular disorder or condition; and the subject being treated.

[0440] The immunogenic compositions of the present disclosure may comprise or consist of:

[0441] - comprising a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, a NadA protein consisting of SEQ ID NO: 5, dOMVs from MenB expressing PorA VR2P1.2, and an AlPO4 adjuvant selected with a PZC of about 4.3. The composition may comprise 50 mM acetate buffer at pH 6.0, or

[0442] - non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, NadA protein consisting of SEQ ID NO: 5, dOMV from MenB expressing PorA VR2 P1.2, selected AlPO4 adjuvant with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0443] - about 25 to about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 to about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 to about 100 μg / dose of NadA consisting of SEQ ID NO: 5, about 20 to about 250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 800 μg / dose of a selected AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at a pH of 6.0, or

[0444] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0445] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0446] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0447] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0448] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0449] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0450] - about 75 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 75 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 75 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0451] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 100 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0452] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0453] - About 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of non-lipidated mutant fHBP B 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 MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of selected AlPO4 adjuvant with a PZC of about 4.3, and 50 mM acetate buffer with a pH of 6.0.

[0454] In one embodiment, the immunogenic composition may comprise or consist of: about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, and 50 mM acetate buffer at a pH of 6.0.

[0455] The immunogenic compositions of the present disclosure may comprise or consist of:

[0456] - comprising a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, a NadA protein consisting of SEQ ID NO: 5, dOMVs from MenB expressing PorA VR2P1.2, and an AlPO4 adjuvant selected with a PZC of about 4.5. The composition may comprise 50 mM acetate buffer at pH 6.0, or

[0457] - non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, NadA protein consisting of SEQ ID NO: 5, dOMV from MenB expressing PorA VR2 P1.2, selected AlPO4 adjuvant with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0458] - about 25 to about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 to about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 to about 100 μg / dose of NadA consisting of SEQ ID NO: 5, about 20 to about 250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 800 μg / dose of a selected AlPO4 adjuvant having a PZC of about 4.5, 50 mM acetate buffer at a pH of 6.0, or

[0459] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0460] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0461] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0462] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0463] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0464] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0465] - about 75 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 75 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 75 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0466] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 100 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0467] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0468] - About 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of non-lipidated mutant fHBP B 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 MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of selected AlPO4 adjuvant with a PZC of about 4.5, and 50 mM acetate buffer with a pH of 6.0.

[0469] In one embodiment, the immunogenic composition may comprise or consist of: about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, and 50 mM acetate buffer at a pH of 6.0.

[0470] The immunogenic compositions of the present disclosure may comprise or consist of:

[0471] - comprising a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, a NadA protein consisting of SEQ ID NO: 5, dOMVs from MenB expressing PorA VR2P1.2, and an AlPO4 adjuvant selected with a PZC of about 4.8. The composition may comprise 50 mM acetate buffer at pH 6.0, or

[0472] - non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, NadA protein consisting of SEQ ID NO: 5, dOMV from MenB expressing PorA VR2 P1.2, selected AlPO4 adjuvant with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0473] - about 25 to about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 to about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 to about 100 μg / dose of NadA consisting of SEQ ID NO: 5, about 20 to about 250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 800 μg / dose of a selected AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at a pH of 6.0, or

[0474] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0475] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0476] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0477] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0478] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0479] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0480] - about 75 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 75 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 75 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0481] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 100 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0482] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0483] - About 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 100 μg / dose of non-lipidated mutant fHBP B 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 MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of selected AlPO4 adjuvant with a PZC of about 4.8, and 50 mM acetate buffer with a pH of 6.0.

[0484] In one embodiment, the immunogenic composition may comprise or consist of: about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, and 50 mM acetate buffer at a pH of 6.0.

[0485] The immunogenic compositions of the present disclosure may comprise or consist of:

[0486] - a non-lipidated mutant fHBP A comprising SEQ ID NO: 8 or consisting thereof, a non-lipidated mutant fHBP B comprising SEQ ID NO: 9 or consisting thereof, a NadA protein comprising SEQ ID NO: 5 or consisting thereof, dOMVs from MenB expressing PorA VR2P1.2, and an AlPO4 adjuvant selected with a PZC of about 4.3. The composition may comprise 50 mM acetate buffer at pH 6.0, or

[0487] - non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, NadA protein consisting of SEQ ID NO: 5, dOMV from MenB expressing PorA VR2 P1.2, selected AlPO4 adjuvant with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0488] - about 25 to about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 to about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 to about 100 μg / dose of NadA consisting of SEQ ID NO: 5, about 20 to about 250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 800 μg / dose of a selected AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at a pH of 6.0, or

[0489] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0490] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0491] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0492] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0493] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0494] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0495] - about 75 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 75 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 75 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0496] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 100 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0497] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.3, 50 mM acetate buffer at pH 6.0, or

[0498] - About 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 50 μg / dose of dOMV from MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of a selected AlPO4 adjuvant with a PZC of about 4.3, and 50 mM acetate buffer at a pH of 6.0.

[0499] In one embodiment, the immunogenic composition may comprise or consist of: about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.3, and 50 mM acetate buffer at a pH of 6.0.

[0500] The immunogenic compositions of the present disclosure may comprise or consist of:

[0501] - a non-lipidated mutant fHBP A comprising SEQ ID NO: 8 or consisting thereof, a non-lipidated mutant fHBP B comprising SEQ ID NO: 9 or consisting thereof, a NadA protein comprising SEQ ID NO: 5 or consisting thereof, dOMVs from MenB expressing PorA VR2P1.2, and an AlPO4 adjuvant selected with a PZC of about 4.5. The composition may comprise 50 mM acetate buffer at pH 6.0, or

[0502] - non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, NadA protein consisting of SEQ ID NO: 5, dOMV from MenB expressing PorA VR2 P1.2, AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0503] - about 25 to about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 to about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 to about 100 μg / dose of NadA consisting of SEQ ID NO: 5, about 20 to about 250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 800 μg / dose of an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0504] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0505] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0506] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0507] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0508] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0509] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0510] - about 75 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 75 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 75 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0511] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 100 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0512] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.5, 50 mM acetate buffer at pH 6.0, or

[0513] - About 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 50 μg / dose of dOMV from MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of selected AlPO4 adjuvant with a PZC of about 4.5, and 50 mM acetate buffer with a pH of 6.0.

[0514] In one embodiment, the immunogenic composition may comprise or consist of: about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.5, and 50 mM acetate buffer at a pH of 6.0.

[0515] The immunogenic compositions of the present disclosure may comprise or consist of:

[0516] - a non-lipidated mutant fHBP A comprising SEQ ID NO: 8 or consisting thereof, a non-lipidated mutant fHBP B comprising SEQ ID NO: 9 or consisting thereof, a NadA protein comprising SEQ ID NO: 5 or consisting thereof, dOMVs from MenB expressing PorA VR2P1.2, and an AlPO4 adjuvant selected with a PZC of about 4.8. The composition may comprise 50 mM acetate buffer at pH 6.0, or

[0517] - non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, NadA protein consisting of SEQ ID NO: 5, dOMV from MenB expressing PorA VR2 P1.2, AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0518] - about 25 to about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 to about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 to about 100 μg / dose of NadA consisting of SEQ ID NO: 5, about 20 to about 250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to about 800 μg / dose of a selected AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at a pH of 6.0, or

[0519] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0520] - about 25 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0521] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0522] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0523] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0524] - about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0525] - about 75 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 75 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 75 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0526] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 100 μg / dose of a 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 a selected AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0527] - about 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant having a PZC of about 4.8, 50 mM acetate buffer at pH 6.0, or

[0528] - About 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 100 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of NadA protein consisting of SEQ ID NO: 5, about 50 μg / dose of dOMV from MenB strain expressing PorA VR2 P1.2, about 800 μg / dose of selected AlPO4 adjuvant with a PZC of about 4.8, and 50 mM acetate buffer with a pH of 6.0.

[0529] In one embodiment, the immunogenic composition may comprise or consist of: about 50 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 50 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 50 μg / dose of a 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 an AlPO4 adjuvant selected with a PZC of about 4.8, and 50 mM acetate buffer at a pH of 6.0.

[0530] The dose may range from about 0.1 mL to about 1 mL, such as about 0.2 mL to about 0.8 mL, about 0.4 mL to about 0.6 mL, or the dose may be about 0.5 mL.

[0531] 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 and an aluminum hydroxyphosphate (AlPO4) adjuvant, the combination comprising at least one factor H binding protein (fHBP) A, at least one fHBP B, the AlPO4 adjuvant being selected to have a zero point of charge (PZC) of less than 5.

[0532] The container may further comprise at least one Neisseria adhesin A (NadA) protein and / or at least one detergent extracted outer membrane vesicle (dOMV).

[0533] The container may contain a composition of the present disclosure as previously detailed.

[0534] The container may contain a composition comprising or consisting of: about 25 to 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 2, about 25 to 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 4, about 25 to 100 μg / dose of a NadA protein consisting of SEQ ID NO: 5, about 20 to 250 μg / dose of dOMVs from a MenB strain expressing PorA VR2 P1.2, about 100 to 800 μg / dose of an AlPO4 adjuvant selected with a point of zero charge (PZC) below 5, and 50 mM acetate buffer at a pH of 6.0.

[0535] The container may contain a composition comprising or consisting of: about 25 to 100 μg / dose of a non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, about 25 to 100 μg / dose of a non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, about 25 to 100 μg / dose of a NadA protein consisting of SEQ ID NO: 5, about 20 to 250 μg / dose of dOMV from a MenB strain expressing PorA VR2 P1.2, about 100 to 800 μg / dose of an AlPO4 adjuvant selected with a point of zero charge (PZC) below 5, and 50 mM acetate buffer at a pH of 6.0.

[0536] Furthermore, the container may contain additional antigens as detailed above.Alternatively, the additional antigens may be packaged in separate containers.

[0537] The container may be a vial. The vial may be a multi-dose vial or may be a single-dose vial. Suitable vials may be small glass or plastic containers sealed with the most suitable stopper and closure.

[0538] Alternatively, the container can be a pre-filled syringe. The pre-filled syringe can include a syringe barrel storing 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 the liquid drug, and the plunger slides the gasket. Various types of pre-filled syringes are known in the art, as described, for example, in US 10,625,025 or WO 2013 / 046855.

[0539] In the case where the composition of the present disclosure is mixed with another vaccine composition (e.g., a tetravalent MenACWY conjugated composition) and injected, both compositions can be packaged in a container that is a single vial or a prefilled syringe or a dual chamber syringe. A dual chamber syringe, also known as a sequential syringe or a bypass syringe, can include a single syringe divided into two compartments, proximal and distal, by a septum. Pressing the plunger of the syringe forces the two vaccine compositions to mix in the distal compartment. Various types of dual chamber syringes are known in the art, as described, for example, in US 10,695,505. If the vaccine composition is prepared in a dry form (e.g., a lyophilized form) and stored with a liquid vehicle for reconstitution, a dual chamber syringe can also be used. In such a case, the dried vaccine is stored in one chamber, and the liquid for reconstitution and injection is stored in the second chamber.

[0540] In some embodiments, the disclosure relates to vaccines comprising the immunogenic compositions disclosed herein.

[0541] The immunogenic composition of the disclosure may be a vaccine. Kit-of-parts

[0542] Kits of parts are also disclosed.

[0543] The multi-component kit may comprise at least two containers, a first container comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and a second container comprising a hydroxyaluminum phosphate (AlPO4) adjuvant selected with a zero point of charge (PZC) of less than 5.

[0544] The kit of parts may comprise at least one additional container comprising at least one detergent extracted outer membrane vesicle (dOMV) and / or at least one Neisseria adhesin A (NadA) protein. The dOMV and NadA protein may be provided in separate containers.

[0545] Alternatively, the kit of parts may comprise at least a first container comprising at least one factor H binding protein (fHBP) A, at least one factor H binding protein (fHBP) B and a selected aluminum hydroxyphosphate (AlPO4) adjuvant having a zero point of charge (PZC) below 5, and a second container comprising at least one detergent extracted outer membrane vesicle (dOMV) and / or at least one Neisseria adhesin A (NadA) protein. The dOMV and NadA proteins may be provided in separate containers.

[0546] Alternatively, the multi-component kit may comprise at least a first container comprising at least one factor H binding protein (fHBP) A, at least one factor H binding protein (fHBP) B, a selected aluminum hydroxyphosphate (AlPO4) adjuvant having a point of zero charge (PZC) of less than 5, at least one detergent-extracted outer membrane vesicle (dOMV) and at least one Neisseria adhesin A (NadA) protein, and a second container containing additional antigens.

[0547] Alternatively, each of the antigen, fHBP A, fHBP B, NadA and dOMV, and the selected AlPO4 adjuvant having a point of zero charge (PZC) below 5 may be stored in separate containers. Also, alternatively, the antigen and AlPO4 adjuvant may be combined in different combinations. All types of combinations can be envisioned: fHBP A+B and NadA+dOMV and AlPO4; fHBP A+B+AlPO4 and NadA+dOMV; fHBP A+B+AlPO4 and NadA+dOMV+AlPO4; fHBP A+NadA and fHBP B+dOMV and AlPO4; fHBP B+NadA+AlPO4 and fHBP A+dOMV+AlPO4; fHBP A+NadA+AlPO4+fHBPB+AlPO4 and dOMV+AlPO4; or fHBP A+dOMV+fHBP B+AlPO4 and NadA+AlPO4; and so on.

[0548] fHBP A and B, AlPO4 adjuvant, NadA protein and dOMV were described in detail above.

[0549] In one embodiment, the additional antigen may be a combination of conjugated MenACWY polysaccharides.

[0550] The conjugated MenACWY polysaccharide may be as described in detail above.

[0551] In one embodiment, a kit of parts may comprise a first container comprising an immunogenic composition disclosed herein and a second container comprising a combination of conjugated MenACWY polysaccharides.

[0552] The antigens and AlPO4 of the immunogenic compositions of the present disclosure can be prepared and stored in separate containers or vials. They can then be mixed when administered to an individual.

[0553] Antigens can be stored in liquid formulations or in dry form. When prepared in dry form, additional containers with injectable liquids can be added, and the injectable liquids can be used to resuspend and mix different antigens. Suitable injectable liquid carriers can include buffer. The injectable liquid can include AlPO4 adjuvant.

[0554] In one embodiment, the container of the kit of parts may contain the antigen which may be in a dried form. The antigen may be lyophilized into a cake or pellet.

[0555] The kit may optionally include a container containing a physiologically injectable vehicle. The physiologically injectable vehicle may be used to resuspend or dissolve the antigen in dry form. Manufacturing method

[0556] The present disclosure relates to a method for making an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens and an AlPO4 adjuvant, the combination comprising at least one factor H binding protein (fHBP) A and one factor H binding protein (fHBP) B, the method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, the combining being performed in any order.

[0557] This method allows obtaining an immunogenic composition.

[0558] The combination of AlPO4 adjuvant with fHBP A and fHBP B can be performed in any order. For example, AlPO4 adjuvant can be combined with fHBP A and then fHBP B can be added, or AlPO4 adjuvant can be combined with fHBP B and then fHBP A can be added, or fHBP A can be combined with fHBP B and then AlPO4 can be added, or AlPO4 adjuvant can be combined with both fHBP A and fHBP B at the same time.

[0559] In step b), fHBP A and fHBP B may be combined first, and then the AlPO 4 adjuvant may be added.

[0560] Alternatively, in step b), the AlPO 4 adjuvant may be combined with fHBP A first and then fHBP B may be added.

[0561] Alternatively, in step b), the AlPO 4 adjuvant may be combined with fHBP B first and then fHBP A may be added.

[0562] Alternatively, at step b), a first portion of the AlPO 4 adjuvant may be combined with fHBP B in a first mixture, and a second portion of the AlPO 4 adjuvant may be combined with fHBP A in a second mixture, and then the first mixture may be combined with the second mixture.

[0563] Alternatively, in step b), the AlPO 4 adjuvant may be combined with both fHBP A and fHBP B simultaneously.

[0564] The method of the present disclosure may further comprise the step of adding at least one antigen selected from the group consisting of NadA protein and dOMV. The combination may be performed in any order.

[0565] For example, NadA protein and / or dOMVs may be added before or after the step of combining AlPO4 with fHBP A and fHBP B. NadA and dOMVs may each be added in separate steps, or may be combined in a single step prior to addition.

[0566] In some embodiments, at step b), fHBP A, fHBP B, NadA protein, and dOMV may be combined in any order, and then AlPO 4 adjuvant may be added.

[0567] In some embodiments, in step b), the AlPO4 adjuvant can be divided into multiple fractions (2, 3 or 4) and the fractions can be added to each antigen: fHBP A, fHBP B, NadA protein and dOMV, and then the antigens are combined with AlPO4 in any order.

[0568] Alternatively, step b) may comprise combining an AlPO4 adjuvant with a combination of fHBP A, fHBP B and NadA proteins. dOMV may be added in a subsequent step.

[0569] Alternatively, step b) may comprise combining an AlPO4 adjuvant with a combination of fHBP A, fHBP B and dOMV. NadA protein may be added in a subsequent step.

[0570] The method of making the immunogenic composition of the present disclosure may comprise at least the following steps: a) Selecting AlPO4 adjuvant with a PZC lower than 5, b) combining the AlPO4 adjuvant selected in step a) with at least one factor H binding protein (fHBP) A, at least one factor H binding protein (fHBP) B, at least one NadA protein, at least one dOMV, in any order.

[0571] The Neisseria meningitidis antigens fHBP A, fHBP B, NadA protein, dOMV may be filtered, for example, using sterile filtration, for example using a 0.22 μm filter, prior to combining with the AlPO 4 adjuvant.

[0572] In addition to the combination of AlPO4 adjuvant, fHBP A, fHBP B, NadA protein and dOMV, the resulting combination can then be dispensed in syringes or vials.

[0573] The present disclosure relates to a method for preparing an immunogenic composition comprising a Neisseria meningitidis fHBP B antigen, said composition inducing an enhanced immune response against a Neisseria meningitidis serogroup B strain expressing an fHBP B heterologous to said fHBP B antigen of said composition, said method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with said fHBP B, and c) obtaining the immunogenic composition.

[0574] This method allows obtaining an immunogenic composition capable of inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP B heterologous to said fHBP B of said composition.

[0575] According to another of its objects, the present disclosure relates to a method for preparing an immunogenic composition comprising a Neisseria meningitidis fHBP B antigen, said composition inducing an enhanced immune response against a Neisseria meningitidis serogroup B strain expressing a fHBP B homologous to said fHBP B antigen of said composition, said method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with said fHBP B, and c) obtaining the immunogenic composition.

[0576] This method allows obtaining an immunogenic composition capable of inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP B homologous to said fHBP B of said composition.

[0577] The composition may further comprise at least one of fHBP A, NadA protein or dOMV.

[0578] According to another of its objects, the present disclosure relates to a method for preparing an immunogenic composition comprising a fHBP A antigen of Neisseria meningitidis, said composition inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP A heterologous to said fHBP A antigen of said composition, said method comprising at least the steps of: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with said fHBP A, and c) obtaining the immunogenic composition.

[0579] This method allows obtaining an immunogenic composition capable of inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP A heterologous to said fHBP A of said composition.

[0580] According to another of its objects, the present disclosure relates to a method for preparing an immunogenic composition comprising a fHBP A antigen of Neisseria meningitidis, said composition inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP A homologous to said fHBP A antigen of said composition, said method comprising at least the steps of: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with said fHBP A, and c) obtaining the immunogenic composition.

[0581] This method allows obtaining an immunogenic composition capable of inducing an enhanced immune response against a serogroup B strain of Neisseria meningitidis expressing a fHBP A homologous to said fHBP A of said composition.

[0582] The composition may further comprise at least one of fHBP B, NadA protein or dOMV.

[0583] The method of the present disclosure may further include adding at least one antigen selected from fHBP A, NadA protein and dOMV. The addition of AlPO4 adjuvant, fHBP B, fHBP A, NadA protein and dOMV may be performed in any order.

[0584] The method of making the immunogenic composition of the present disclosure may comprise at least the following steps: a) Selecting AlPO4 adjuvant with a PZC lower than 5, b) combining the AlPO4 adjuvant selected in step a) with at least one factor H binding protein (fHBP) A, at least one factor H binding protein (fHBP) B, at least one NadA protein and at least one dOMV, the combination being performed in any order.

[0585] fHBP A and B, NadA protein and dOMV can be as described in detail above.

[0586] fHBP A and B, NadA protein and dOMV can be sterile filtered before combining with AlPO4 adjuvant.

[0587] Furthermore, the methods of the present disclosure may comprise the step of adding an additional antigen. The additional antigen may be a combination of MenACWY polysaccharides conjugated to a protein carrier as detailed above.

[0588] The present disclosure relates to a method for stabilizing at least one of fHBP A and Neisseria adhesin A (NadA) protein in an immunogenic composition, the method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with fHBP A or NadA protein, and c) obtaining an immunogenic composition wherein the fHBP A or NadA protein is stabilized.

[0589] This method allows obtaining immunogenic compositions in which fHBP A and / or NadA are stabilized.

[0590] The method of the present disclosure may further comprise the step of adding at least one antigen.

[0591] For compositions comprising NadA protein, the at least one additional antigen may be selected from fHBP A, fHBP B and dOMV.

[0592] For compositions comprising fHBP A, the at least one additional antigen may be selected from fHBP B, NadA protein and dOMV.

[0593] At least one additional antigen may be added before or after the step of combining AlPO4 with fHBP A or NadA protein.

[0594] The additional antigens may each be added to the fHBP A or NadA protein in separate steps, or may be combined before addition in a single step. In the latter case, they may be added as a subcombination of antigens.

[0595] The step of adding the additional antigen may be performed before or after combining the AlPO4 with the fHBP A or NadA protein.

[0596] In some embodiments, step b) may comprise combining an AlPO 4 adjuvant with a combination of fHBP A, fHBP B, NadA protein, and dOMVs, in any order.

[0597] fHBP A and B, NadA protein and dOMV can be as described in detail above.

[0598] Furthermore, the method may comprise the step of adding a combination of MenACWY polysaccharide conjugated to a protein carrier as detailed above.

[0599] The stability of the antigens in the compositions of the present disclosure can be assessed by methods well known in the art, including measuring light scattering, apparent attenuation of light (absorbance or optical density), size (e.g., by size exclusion chromatography), in vitro or in vivo biological activity and / or properties of a sample by differential scanning calorimetry (DSC).

[0600] For example, the stability of an antigen (e.g., NadA or fHBP A) in a composition of the disclosure can be determined by incubating the immunogenic composition at 45°C to measure the antigenicity of the antigen in question under thermal stress as a function of time (e.g., 0, 7, 14, and 28 days).

[0601] The antigens in the compositions of the present disclosure can maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of their antigenicity relative to a reference standard (e.g., antigenicity measured at T0 (i.e., date of formulation or date of change in storage conditions)) at a temperature ranging from 4°C to 8°C for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, 42 months, 48 ​​months.

[0602] The combination of the AlPO 4 adjuvant with fHBP A and B of the present disclosure is carried out under conditions suitable for obtaining adsorption of fHBP A and B on AlPO 4 .

[0603] The % of fHBP A and B adsorbed are as indicated above.

[0604] The amount of each of fHBP A and B adsorbed on AlPO4 may be less than 85% of the total amount of fHBP B present in the composition, or the amount may range from about 50% to about 85% or less of the total amount of fHBP A or fHBP B, respectively, present in the composition.

[0605] Suitable conditions to ensure adsorption of fHBP A and B on AlPO 4 include pH, temperature and PZC of AlPO 4. Those conditions may be as indicated above.

[0606] For example, the pH may range from 5.5 to 7.0.

[0607] The temperature may range from 4°C to 25°C.

[0608] The compositions of the present disclosure may be stored at a temperature ranging from about 4° C. to about 25° C. For example, the compositions of the present disclosure may be stored at a temperature of about 4° C. or about 8° C., or at about 4° C.

[0609] The present disclosure relates to a method for preparing an immunogenic composition comprising at least one Neisseria meningitidis serogroup B antigen and a precipitation onset time (T onset ) ranges from about 3.5 min to about 10 min, the method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with said at least one Neisseria meningitidis serogroup B antigen, and c) obtaining the immunogenic composition.

[0610] The at least one antigen may be fHBP A, fHBP B, NadA protein or dOMV, and combinations thereof.

[0611] Settling start time (T onset ) can range from about 4 min to about 9 min, or from about 4.5 min to about 8.5 min.

[0612] Advantageously, an initial settling time of at least or in excess of 3.5 minutes ensures that the components of the composition will remain suspended during the manufacturing process and therefore allows for more consistent manufacturing.

[0613] Additional antigens (e.g., MenACWY polysaccharide conjugated to a protein carrier) may be mixed with the compositions disclosed herein. Additional antigens may be added to the immunogenic compositions of the disclosure immediately prior to administration to a patient, optionally via a dual chamber syringe, the compositions of the disclosure being mixed with at least one additional antigen prior to administration.

[0614] The manufacturing methods disclosed herein can be used to manufacture vaccines. Uses and methods

[0615] The present disclosure relates to the immunogenic compositions disclosed herein for use as medicaments, in particular as vaccines.

[0616] The immunogenic compositions of the disclosure or vaccines comprising the immunogenic compositions of the disclosure may be for use in methods for preventing meningococcal infection.Meningococcal infection may be caused by Neisseria meningitidis serogroup B strains.

[0617] The immunogenic compositions or vaccines of the present disclosure may be used in methods for inducing an immune response against a serogroup B strain of Neisseria meningitidis.

[0618] The immunogenic compositions or vaccines of the present disclosure may be used in a method for protecting an individual from meningococcal infection, the method comprising at least the step of administering the immunogenic composition to the individual.

[0619] The present disclosure relates to a method for protecting an individual from meningococcal infection, the method comprising at least the step of administering to the individual the immunogenic composition or a vaccine comprising the immunogenic composition.

[0620] The immunogenic composition or vaccine of the present disclosure may be used in a method for reducing the risk of developing invasive meningococcal disease caused by meningococcal infection in an individual, the method comprising at least the step of administering the immunogenic composition or the vaccine to the individual.

[0621] The present disclosure relates to a method for reducing the risk of developing invasive meningococcal disease caused by meningococcal infection in an individual, the method comprising at least the step of administering to the individual an immunogenic composition or vaccine of the present disclosure.

[0622] The immunogenic composition or vaccine of the present disclosure may be used in a method for eliciting an immune response against a serogroup B strain of Neisseria meningitidis in an individual, the method comprising at least the step of administering the immunogenic composition or the vaccine to the individual.

[0623] The present disclosure relates to a method for eliciting an immune response against a serogroup B strain of Neisseria meningitidis in an individual, the method comprising at least the step of administering to the individual an immunogenic composition or vaccine of the present disclosure.

[0624] The meningococcal infection may be a Neisseria meningitidis serogroup B infection. The immunogenic composition may be a vaccine.

[0625] The present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP B heterologous to the fHBP B antigen of the composition.

[0626] The present disclosure relates to an AlPO4 adjuvant having a PZC of less than 5 in an immunogenic composition comprising a Neisseria meningitidis fHBP B antigen, for use in a method for enhancing an immune response induced by the composition against a Neisseria meningitidis serogroup B strain expressing an fHBP B heterologous to the fHBP B of the composition.

[0627] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP B homologous to said fHBP B antigen of said composition.

[0628] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP A antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP A heterologous to said fHBP A antigen of said composition.

[0629] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for enhancing the immune response induced by a composition comprising a Neisseria meningitidis fHBP A antigen against a Neisseria meningitidis serogroup B strain expressing an fHBP A homologous to said fHBP A antigen of said composition.

[0630] According to another of its objects, the present disclosure relates to the use of an AlPO4 adjuvant having a PZC lower than 5 for stabilizing at least one fHBP A in an immunogenic composition.

[0631] The present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for stabilizing at least one Neisseria adhesin A (NadA) antigen in an immunogenic composition.

[0632] The present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for increasing the sedimentation start time (T onset ) is stable in the range of about 3.5 min to about 10 min, or in the range of about 4 min to about 9 min, or in the range of about 4.5 to about 8.5 min.

[0633] The at least one Neisseria meningitidis serogroup B antigen may be from the group comprising: fHBP A, fHBP B, NadA protein, dOMV, and combinations thereof.

[0634] The at least one Neisseria meningitidis serogroup B antigen may be a combination of fHBP A and fHBP B.

[0635] The present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for adjuvanting an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens, the combination including at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

[0636] The present disclosure relates to the use of an AlPO4 adjuvant having a PZC of less than 5 for the manufacture of an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens, the combination including at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

[0637] The present disclosure relates to a method for inducing an immune response against a serogroup B strain of Neisseria meningitidis in an individual in need thereof, the method comprising at least the step of administering to the individual an immunogenic composition according to the present disclosure, wherein the administering step induces an immune response against the serogroup B strain of Neisseria meningitidis.

[0638] The present disclosure relates to a method for enhancing an immune response against a serogroup B strain of Neisseria meningitidis induced by a composition comprising a Neisseria meningitidis fHBP B antigen, the Neisseria meningitidis serogroup B strain expressing an fHBP antigen heterologous to the fHBP B antigen of the composition, in an individual in need thereof, the method comprising at least the steps of: administering to the individual an immunogenic composition according to the present disclosure, wherein the administering step induces an enhanced immune response against the heterologous Neisseria meningitidis serogroup B strain.

[0639] The individuals involved in the methods and uses of the present disclosure can be mammals, such as humans, and for example infants, toddlers, children, teenagers, young adults, adults and the elderly. In one embodiment, the individual can be 6 weeks or more, 2 months or more, or 10 years or more. As an exemplary embodiment, the individual can be 6 weeks to 55 years or more, such as 2 months to 55 years or more, or such as 10 years to 55 years or more.

[0640] These methods generally involve administering an effective amount of the subject immunogenic composition to an individual in need. The effective amount for therapeutic use will depend on, for example, the antigen composition, the mode of administration, the patient's weight and general health, and the judgment of the prescribing physician. Single or multiple doses of the antigen composition can be administered according to the dosage and frequency and route of administration required and tolerated by the patient.

[0641] The immunogenic compositions disclosed herein may be administered in a 2, 3, 2+1, or 3+1 dosage regimen.

[0642] In one embodiment, the immunogenic composition disclosed herein can be administered in 2 or 3 doses. Subsequent doses can be administered about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, about twelve months, about thirteen months, about fourteen months, about fifteen months, about sixteen months, about seventeen months, about eighteen months, about nineteen months, or about twenty months apart from the previous dose. In one embodiment, subsequent doses can be administered about one month, about two months, about five months, about six months, about eight months, about ten months, about twelve months, about fourteen months, or about sixteen months apart from the previous dose. In one embodiment, subsequent doses can be administered about one month, about two months, about five months, about six months, or about eight months apart from the previous dose. In one embodiment, subsequent doses can be administered about 30 days, about 60 days, or about 180 days apart from the previous dose.

[0643] In a two-dose regimen, the second dose can be administered about one 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 can be 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 a two-dose regimen can be suitable for adults and / or adolescents.

[0644] In a two-dose regimen, the second dose can be administered about 2 months after the first dose. Alternatively, in a two-dose regimen, the second dose can be administered about 60 days after the first dose. Such a two-dose regimen can be suitable for young children.

[0645] In a three-dose regimen, the second dose may be administered about one month after the first dose, and the third dose may be administered about 6 months after the first dose. Alternatively, in a three-dose regimen, the second dose may be administered about 30 days after the first dose, and the third dose may be administered about 180 days after the first dose. Such a three-dose regimen may be suitable for adults and / or adolescents.

[0646] In a three-dose regimen, the second dose can be administered about two months after the first dose, and the third dose can be administered about 10 months after the first dose. Alternatively, in a three-dose regimen, the second dose can be administered about 60 days after the first dose, and the third dose can be administered at about 12 months of age. Such a three-dose regimen can be suitable for infants.

[0647] In one embodiment, in addition to 2 or 3 doses, a third or fourth dose may be administered. This subsequent dose may be administered at least one year after the last dose of 2 or 3 doses, for example, 16 months after the last dose. In such a scheme, the first two or three doses may be assessed as preliminary doses, and the subsequent one (+1) may be assessed as a booster dose.

[0648] In one embodiment, infants and young children (e.g., 6 weeks or 2 months to 2 years old) can receive 2+1 or 3+1 dosage regimens. In another embodiment, children (e.g., 2 to 10 years old) can receive 2 dosage regimens. In another embodiment, adolescents and adults (e.g., 10 to 55 years old) can receive 2+1 dosage regimens.

[0649] The immunogenic compositions disclosed herein can be administered by any suitable route. For example, administration by the intramuscular route is contemplated. Examples

[0650] The following examples illustrate the embodiments of the present disclosure that are currently best known. However, it should be understood that the following are merely examples or illustrations of the application of the principles of the present disclosure. Without departing from the spirit and scope of the present disclosure, those skilled in the art may design many modifications and alternative compositions, methods and systems. Example 1: Materials and Methods Formulations of AlPO4 with low zero charge point (mod-AlPO4 adjuvant)

[0651] Aluminum phosphate adjuvant gels with a PZC range of 5 to 7 are titrated with phosphate buffer / saline solutions, which allow the exchange of phosphate groups on the buffer or saline with hydroxyl groups on the surface of the aluminum phosphate adjuvant. The PZC of AlPO4 can be reduced by using any orthophosphate or phosphate donor salt or buffer solution.

[0652] Different methods can be used to obtain AlPO4 with target PZC.

[0653] In one method, phosphate buffer (pH 5.8) prepared from a combination of 0.5 M sodium dihydrogen phosphate and 0.5 M sodium hydrogen phosphate was added to prepare AlPO4 with different PZCs.

[0654] In the second method, the PZC of AlPO4 was varied by titrating AlPO4 with a stock solution of 0.5 M sodium dihydrogen phosphate salt.

[0655] AlPO4 adjuvants with a PZC lower than 5 are modified AlPO4 adjuvants and are referred to as "mod-AlPO4 adjuvants" in the Examples section below. To prepare 100 mL of modified AlPO4 adjuvant (mod-AlPO4 adjuvant), 80 mL of AlPO4 (concentration 4.8 mg Al / mL) was combined with 20 mL of 0.5 M sodium phosphate buffer (pH 5.8) at room temperature and stirred for not less than 30 minutes. The modified AlPO4 (mod.AlPO4 or mod-AlPO4) was stored at 2-8°C until use.

[0656] AlPO4 adjuvants with a PZC higher than 5 are unmodified AlPO4 adjuvants and are referred to as "AlPO4 adjuvants" in the Examples section below.

[0657] In the following studies, mod-AlPO4 adjuvant was modified as described above to set the PZC to about 4.5, while the PZC of AlPO4 adjuvant was set to about 5.2. Preparation of MenB immunogenic composition (MenB) Non-lipidated mutant fHBPA05 (A05tmN)

[0658] To prepare non-lipidated A05tmN, three point mutations (G220S, L130R and G133D, numbered relative to SEQ ID NO: 6) were introduced into the wild-type fHBP A05 sequence. In addition, the lipidatable cysteine ​​residue at the N-terminus was replaced by a methionine residue (non-lipidated A05tmN: SEQ ID NO: 8). The DNA sequence encoding the A05tmN antigen was synthesized and then cloned into a plasmid construct. In short, DNA sequences of Xba1 and Xho1 sites were added to both ends of the A05tmN sequence. To create an expression plasmid, the pET28a (+) plasmid containing Xba1 / Xho1 was digested. The DNA sequence encoding A05tmN with Xba1 and Xho1 sites was ligated to pET28a (+) digested with Xba1 / Xho1 and transformed into Top10 competent cells. Positive clones were identified and confirmed by Xba1 / Xho1 digestion. The A05tmN plasmid was transformed into E. coli, and cell banks were made after three rounds of colony purification.

[0659] The E. coli strain was transformed with the A05tmN expression construct and propagated in semi-defined medium (pH 6.8-dissolved oxygen: 20%) with stirring at 37° C. The expression of the antigen was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0660] The culture is harvested as an unprocessed body, and the bacterial biomass is separated from the culture medium by centrifugation. The obtained cell pellet is resuspended in a buffer (20mM Tris-HCl, pH 8.5). The resuspended precipitation is processed by a homogenizer to produce a cell homogenate. The homogenate is then centrifuged to collect the precipitation fraction. The homogenate precipitation is resuspended in a buffer (20mM Tris-HCl, pH 8.5), and a pH shock treatment (pH 12, at room temperature with stirring for 1 hour) is carried out. The pH is reduced to 8.5 with 85% phosphoric acid. The supernatant fraction of the pH shock material is collected after centrifugation, and then filtered to obtain the filtered supernatant.

[0661] The supernatant was adjusted to pH 8.5 and <5.0mS / cm conductivity and loaded onto a capture column GigaCap Q-650M. The elution pool was adjusted to 0.9M ammonium sulfate (AmS) and then further purified using an intermediate chromatography Toyopearl Phenyl 600M. After hydrophobic interaction chromatography, the elution pool was adjusted to pH 8.5 and <8.0mS / cm conductivity and further purified using Nuvia aPrime4A chromatography. Final ultrafiltration and diafiltration were then performed using a 5kDa regenerated cellulose tangential flow filtration (TFF) membrane and 0.2-μm filtration. Non-lipidated mutant fHBPB01 (B01smN)

[0662] To prepare non-lipidated B01smN, a single point mutation (H248L, numbered relative to SEQ ID NO:6) was introduced into the wild-type fHBP B01 sequence. In addition, the lipidatable cysteine ​​residue at the N-terminus was replaced by methionine (non-lipidated B01smN: SEQ ID NO:9). The DNA sequence of B01smN was synthesized and then cloned into a plasmid construct. In short, DNA sequences for Xba1 and Xho 1 sites were added to both ends of the B01smN sequence. To create an expression plasmid, the pET28a(+) plasmid containing Xba1 / Xho1 was digested. The DNA sequence encoding B01smN with Xba1 and Xho 1 sites was ligated into Xba1 / Xho1-digested pET28a(+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba 1 / Xho 1 digestion. The B01smN plasmid was transformed into E. coli, and cell banks were made after three rounds of colony purification.

[0663] The E. coli strain was transformed with the B01smN expression construct and propagated in semi-defined medium (pH 6.8 - dissolved oxygen: 20%) with stirring at 37° C. The expression of the antigen was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0664] The culture is harvested as an unprocessed body and the bacterial biomass is separated from the culture medium by centrifugation. The resulting cell pellet is resuspended in a buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet is processed by a homogenizer to produce a cell homogenate. The homogenate is then centrifuged to collect the supernatant fraction. The supernatant fraction is then filtered.

[0665] The filtered supernatant was adjusted to pH 8.5 and <5.0 mS / cm conductivity and loaded onto the chromatogram CaptoQImpRes and purified in bind and elute mode. The CaptoQ ImpRes elution pool was then adjusted to 1.8 M AmS for loading onto the second chromatogram 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 and then filtered through 0.2-μm. Nad

[0666] A truncated version of NadA was prepared from NadA_MC58. The truncated NadA lacks the leader sequence (residues 1 to 23) and anchor domain (residues 308 to 362) of NadA_MC58 (truncated NadA: SEQ ID NO: 5). In the truncated sequence of NadA_MC58, the first amino acid after the leader sequence is alanine, which is replaced by methionine. The DNA sequence encoding the truncated NadA was synthesized and then cloned into a plasmid construct. DNA sequences with Xba1 and Xho1 sites were added to both ends of the NadA sequence. To create an expression plasmid, the pET28a(+) plasmid containing Xba1 / Xho1 was digested. The DNA sequence encoding NadA with Xba1 and Xho 1 sites was ligated to pET28a(+) digested with Xba1 / Xho1 and transformed into Top10 competent cells. Positive clones were confirmed by Xba1 / Xho1 digestion. The NadA plasmid was transformed into E. coli and cell banks were made after three rounds of colony purification.

[0667] The E. coli strain transformed with NadA1 was propagated in a semi-defined medium (pH 6.8 - dissolved oxygen: 20%) at 37° C. with stirring. The expression of the antigen was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0668] The culture is harvested as an unprocessed body and the bacterial biomass is separated from the culture medium by centrifugation. The resulting cell pellet is resuspended in a buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet is processed by a homogenizer to produce a cell homogenate. The homogenate is then centrifuged to collect the supernatant fraction. The supernatant fraction is then filtered.

[0669] The supernatant fraction is loaded onto a Capto DEAE column. The Capto DEAE elution fraction is adjusted with powdered AmS until a concentration of 500 mM AmS is reached. The regulated Capto DEAE elution fraction is loaded onto a Toyopearl Butyl-650M column. The Toyopearl Butyl-650M elution fraction is loaded onto a CHT I type 40 μm column, and the CHT elution fraction is concentrated using a 30 kDa regenerated cellulose TFF membrane, followed by diafiltration into 50 mM sodium acetate, 150 mM NaCl (pH 6.0). After TFF, the product is filtered through 0.2-μm to produce the NadA antigen. dOMV

[0670] dOMVs were purified from wild-type Neisseria meningitidis (Nm) serotype B strain 99M provided by the Walter Reed Army Institute of Research (WRAIR).

[0671] Nm B 99M was cultured in a chemically defined medium as described in Fu et al. (Biotechnology (New York). 1995 Feb;13(2):170-4) and US 5,494,808 in the presence of 1 g / L yeast extract and 1 M Hepes at 37°C, CO2 5%.

[0672] The culture was harvested using a low-speed centrifugation (55°C for 2 hours) of a heat-treated suspension to reclaim wet bacterial pellets. Two consecutive detergent-mediated extraction steps (56°C for 15 minutes) were performed with an extraction buffer consisting of a detergent (sodium deoxycholate) to extract dOMV from the bacterial outer membrane and deplete lipooligosaccharides (such as Helting et al., Acta Pathol Microbiol Scand C [Scandinavian Pathology, Microbiology and Immunology Journal-C] April 1981; 89 (2): disclosed in 69-78). Sodium deoxycholate and EDTA dissolve the bacterial outer membrane and then reorganize themselves into dOMV (vesicles and particles). Resuspension was completed by homogenizing the precipitation suspended in the extraction buffer using Ultra-Turrax (rotor-stator device). The dOMV supernatant was merged and then benzonatase was treated (37°C for 15 minutes) in the presence of MgCl.

[0673] After dOMV extraction, hollow fibers in 300 kDa modified polyethersulfone (mPES) are used to concentrate dOMV. Several ultracentrifugation steps are used to separate dOMV from "soluble" contents (e.g., nucleic acids, cytoplasmic proteins, extracted lipopolysaccharides, or buffer components). The resulting pellet is then resuspended in extraction buffer for a few seconds using an Ultra-Turrax (rotor-stator device) at minimum speed. After initial resuspension, high-pressure homogenization is used to completely resuspend dOMV in extraction buffer and increase the permeability of the detergent to the dOMV surface.

[0674] Centrifugation was then performed before final filtration of the supernatant through a 0.45 / 0.2-μm cellulose acetate filter. dOMVs were harvested in water for injection (WFI). MenB immunogenic composition

[0675] The MenB antigens in the MenB immunogenic composition are purified non-lipidated mutant A05 fHBP (A05tmN), non-lipidated mutant fHBP (B01smN), NadA and dOMV. The A05tmN, B01smN, NadA and dOMV antigens are combined with aluminum phosphate adjuvant (AlPO4) (PZC 5.2) or with mod-AlPO4 adjuvant (PZC 4.5).

[0676] The vehicle consisted of acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0).

[0677] AlPO4 adjuvant was either unmodified AlPO4 adjuvant (PZC 5.2; 1.00 mg Al / mL) or mod-AlPO4 adjuvant (PZC 4.5; 1.00 mg Al / mL) prepared as indicated above.

[0678] To formulate the immunogenic composition formulations, unmodified AlPO4 (PZC 5.2) or mod-AlPO4 (PZC4.5) adjuvant, B01smN, A05tmN, NadA protein, dOMV and acetate buffer (50 mM sodium acetate, 150 mM NaCl, pH 6.0) were mixed together to achieve target antigen and aluminum concentrations (100 μg / mL for B01smN, 100 μg / mL for A05tmN, 100 μg / mL for NadA, 250 μg / mL for dOMV and 1.00 mg Al / mL for AlPO4).

[0679] AIP04 with a final aluminum concentration of 0.8 mg Al / mL was retained for stability data and PZC comparison (4.5 and 4.8).Adsorbed antigen was measured by determining the unadsorbed amount of antigen using RP-HPLC (for MenB antigen) and HPAEC-PAD (for ACYW conjugate).

[0680] The estimated amount of residual phosphate buffer in the final composition was approximately 24 mM. Preparation of MenACWY and MenPenta immunogenic compositions Preparation of MenACWY immunogenic compositions

[0681] The MenACWY immunogenic composition was obtained from It is a commercially available vaccine comprising an ACWY polysaccharide antigen obtained as disclosed in WO 2018 / 045286 A1 and conjugated to tetanus toxoid (TT). The formulation comprises Neisseria meningitidis capsular polysaccharides from serogroups A, C, Y and W135 conjugated to tetanus toxoid protein, respectively. 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 is 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). Preparation of MenPenta immunogenic composition (MenPenta)

[0682] MenPenta formulations were prepared by combining two non-lipidated factor H binding proteins (fHBPs) from subfamilies A and B, Neisseria adhesin A (NadA), and detergent-extracted outer membrane protein vesicles (dOMVs) targeting Neisseria meningitidis B strain prepared as indicated above, and serogroup polysaccharides A, C, Y and W135 conjugated to tetanus toxoid obtained as indicated above as carriers with (i) aluminum phosphate adjuvant (AlPO4) (PZC 5.2) or (ii) mod-AlPO4 adjuvant (PZC 4.5).

[0683] The formulation was mixed by stirring at ambient temperature for not less than 30 minutes and stored at 2°C-8°C until use. Settlement start time

[0684] Using TURBISCAN LAB TMSedimentation studies were performed in which a 1.5 mL volume of the composition to be determined was filled into a 4-mL glass tube and loaded into an adapter matching the vial size. The transmission and backscattering of samples of MenB or MenPenta compositions containing AlPO4 with a PZC of 5.2 or 4.5 (obtained as indicated above) were monitored every 25 to 30 seconds for up to 30 minutes. Both transmission and backscattering were measured. The measurements were performed by scanning from the bottom to the top of the measuring cell. The measurement temperature was set to 28°C. Pyrogenicity of immunogenic compositions (IL-6EC 50 Measurement of

[0685] The pyrogenicity of the tested immunogenic compositions (MenB with AlPO4(PZC5.2) or with mod-AlPO4(PZC 4.5) - prepared as indicated above) was determined using the monocyte activation test (MAT).

[0686] MAT is based on the ability of human monocytes to secrete endogenous pyrogens (proinflammatory cytokines) in response to detection of exogenous pyrogens contained in a test sample. The Monocyte Activation Test (MAT) works by predicting the body's response to pyrogens on the same endogenous cytokines produced during a human fever. For test samples with intrinsic pyrogens (e.g., lipoproteins, lipolytic oligosaccharides, other unknown components), MAT allows quantification of intrinsic pyrogenicity and is used to document consistency.

[0687] The MAT is based on a pool of peripheral blood mononuclear cells (PBMCs) provided by eight different healthy human donors as the source of monocytes and on human interleukin-6 (IL-6) as a readout.

[0688] In this study, the goal was to compare the pyrogenicity of immunogenic compositions prepared with AlPO4 adjuvant (PZC 5.2) or with mod-AlPO4 adjuvant (PZC 4.5). All samples were adjusted to the same protein content, serially diluted, plated in 96-well plates containing human PBMCs, and incubated overnight. After incubation, the supernatant was transferred to a 96-well plate and immunodetection of IL-6 was performed by homogeneous time-resolved fluorescence (HTRF). The half-maximal effective concentration (EC) calculated according to the 4PL extrapolation model of the dose-response curve was 50 ) is a parameter used to compare the intrinsic pyrogenicity of each composition. The smallest is EC 50 , and the largest is the inherent pyrogenicity of the test product. Measurement of antigen adsorption on aluminum

[0689] The percentage of adsorption of antigen to mod-AlPO4 or AlPO4 was measured by determining the concentration of unadsorbed antigen obtained in the supernatant after centrifugation of the aluminum adjuvant-bound antigen relative to the total concentration using the following formula:

[0690]

[0691] The concentrations of A05tmN, B01smN, NadA and dOMV were determined by reverse phase high performance liquid chromatography (RP-HPLC).

[0692] The reverse phase liquid chromatography (RP-LC) method from Nompari et al. (Talanta 178 (2018) 552-562) was implemented on an Agilent 1260 Infinity HPLC instrument with UV detection.

[0693] To determine the % adsorption, supernatants and desorbed (bound) fractions were assessed for antigen content.

[0694] The adsorption sample is prepared supernatant sample at 22 ℃ with centrifugal 5min of 3000rcf. The supernatant of appropriate volume is mixed with detergent Zwittergent 3-14, to obtain the ultimate concentration of 0.1%w / v Zwittergent 3-14 in the sample, wherein the sample dilution is minimum (about 0.95X). Then the sample is accompanied by the shaking heating with 300rpm for 1 hour in the micro tube heater at 60 ℃. In order to prepare the total sample, the desorption of antigen is realized by processing the whole adsorbed sample (total) with 5%w / v citrate, aluminum chelator and 0.1% Zwittergent3-14. All samples and standards are accompanied by the shaking heating with 300rpm for 1 hour at 60 ℃, and at 22 ℃ with centrifugal 5min of 3000rcf, then at 10 ℃, stored in the HPLC automatic sampler, and then carry out HPLC analysis.

[0695] Reference standard working solutions were prepared fresh on the day of testing by diluting antigen reference standards of A05tmN, B01smN and NadA to appropriate concentrations in 150 mM sodium acetate plus 50 mM sodium chloride (pH 6.3) buffer with 0.1% Zwittergent 3-14. dOMV protein components were quantified using NadA as a heterologous reference standard.

[0696] BioResolve RP mAb Polyphenyl columns from Waters ( 2.7μm, 2.1mm×150mm) achieved the best separation of all antigens. Liquid chromatography mass spectrometry (LC-MS) grade 0.1% TFA in water and 0.1% TFA in ACN were used as aqueous and organic mobile phases, respectively. At a column temperature of 70°C, the chromatographic gradient started from 10% organic phase to a final 80% organic phase. The detection wavelength used was 215nm. The protein concentration was determined by interpolating the amount of each antigen (in nanograms (ng)) from the appropriate calibration curve and then dividing it by the injection volume (in μL) to obtain ng / μL or μg / mL.

[0697] The concentrations of tetanus toxoid protein-conjugated Neisseria meningitidis capsular polysaccharide serogroups A, C, Y and W135 were determined by high performance anion exchange chromatography / pulsed amperometric detection (HPAEC-PAD).

[0698] The quantification of polysaccharide content in the drug product vaccine and % adsorption on AlPO4 were evaluated by sample preparation, acid hydrolysis to generate monosaccharides and chromatography using HPAEC-PAD for individual monosaccharide analysis.

[0699] Desorption samples were prepared by centrifuging aliquots of drug product samples at 14000 rpm for 30 min at RT. The supernatant was removed and diluted within the standard curve using sodium acetate / sodium chloride buffer. Aliquots of drug product samples (adsorbed) were also diluted within the standard curve using sodium acetate / sodium chloride buffer. Reference standards of diluted and adsorbed drug product, supernatant, and polysaccharide were prepared in the sample set.

[0700] Hydrolysis and chromatography conditions were adapted from those described by Gudlavalleti et al. (Anal Chem. 2014 Jun 3;86(11):5383-90. doi:10.1021 / ac5003933. Epub 2014 May 20. PMID:24810004.).

[0701] Calibration curves for each serogroup A, C, W, Y were calculated from a tetravalent polysaccharide based reference standard using linear regression. Polysaccharide concentrations for A, C, W, Y were extrapolated from their respective curves for adsorption and desorption (supernatant) samples. % adsorption was calculated as desorption / drug product sample x 100. Evaluation of the immunogenicity of MenB immunogenic compositions formulated with mod-AlPO4 adjuvant (PZC 4.5) or with AlPO4 adjuvant (PZC 5.2) Tested products Table 1: Tested products Dosage regimen

[0702] Three groups (A, B and C) of eight rabbits per group (female; strain: NZW KBL; 9-10 weeks old at D0) were used. Group A received the MenB immunogenic composition without AlPO4, while groups B and C received the MenB immunogenic composition formulated with 400 μg of AlPO4 adjuvant (PZC 5.2) or mod-AlPO4 adjuvant (PZC 4.5), respectively. These formulations were administered via intramuscular (IM) injection at D0 and D28 (500 μL of the first injection in the right thigh and 500 μL of the second injection in the left thigh). For all groups, blood samples were collected at the median artery of the rabbit ears under local anesthesia at D0, D28 and D42 (two weeks after the last injection). Table 2: Dosage regimen Biosampling and hSBA Biological sampling

[0703] Blood samples were collected from the median artery of the ear under local anesthesia at D0 and D42. 5 min before collecting blood samples, anesthetic cream was applied on the rabbit ears. Local anesthesia was administered. Rabbits were also maintained under chemical anesthesia using Rompun and Imalgene products.

[0704] Blood samples were collected into tubes containing a coagulation activator and a serum separator (BD Microtainer SST 5 mL, reference 15388989). The tubes were centrifuged at 3500 rpm for 15 min to separate serum from blood cells. The serum was transferred to a deep well (ritter) plate and heat inactivated at 56°C for 30 min. They were stored at -20°C until used for IgG purification and bacterial killing assays. HkDJ IgG purification from rabbit serum for hSBA testing

[0705] In order to avoid nonspecific bacterial killing induced by rabbit sera collected at D0 and D42, purification of IgG was necessary.

[0706] Using rProtein A GravtiTrap TM Purification of rabbit serum was performed using a GE Healthcare GE28-9852-54 column and Ab buffer kit (GE Healthcare Ref. 28-9030-59).

[0707] First, the column was balanced with binding buffer (sodium phosphate 20mM, pH=7). After the serum pH was adjusted to 7 with binding buffer (V / V), the serum was added to the column for IgG binding. The column was washed with binding buffer. Then elution buffer (glycine HCl 0.1M pH2.7) was added to the column to collect IgG. In order to maintain the activity of IgG, neutralization buffer (Tris-HCl 1M, pH9.0) was added to the eluted fraction to obtain a final pH of about 7. Quantification of IgG concentration was performed by Nanodrop. Serum bactericidal activity

[0708] The bactericidal titer of a single purified serum (purified IgG) from an immunized rabbit is measured by in vitro quantitative antibody-dependent complement-mediated killing of Neisseria meningitidis serogroup B (or serum bactericidal activity - SBA). The assay is performed in the presence of human complement (hSBA). In the presence of complement and certain classes of immunoglobulins, cleavage of the complex antigen-antibody is obtained on the surface of the target bacteria (leading to its death). The SBA level of the serum can be determined by observing the bactericidal effect produced by the target candidate-specific antibodies present in the serum.

[0709] The bactericidal titer is the dilution that produces ≥ 50%. The number of resulting bacterial colonies present in the well is inversely proportional to the level of functional antibodies present in the serum, which in turn is directly proportional to the immune response of the animal or human subject.

[0710] The SBA assay measures the ability of an antibody to lyse bacteria in the presence of complement. The bactericidal titer of a serum is defined as the reciprocal of the highest dilution of test serum that results in at least 50% killing compared to complement control wells containing no serum.

[0711] The number of resulting bacterial colonies present in the wells is inversely proportional to the level of functional antibodies present in the serum, which in turn is directly proportional to the immune response of the animal or human subject.

[0712] The source of complement was human complement (Ig-depleted human serum). Briefly, serum was heat-inactivated at 56°C for 30 min and then plated in a 96-well microplate in the presence of Ca 2+ and Mg 2+ and 0.2% gelatin in Dulbecco's PBS buffer or in a solution containing Ca 2+ / Mg 2+ Serial two-fold dilutions (9 times) were performed in Dulbecco's PBS buffer containing 0.1% dextrose and 0.5% bovine serum albumin.

[0713] All sera evaluated in the SBA assay were heat inactivated in a 56°C water bath for 30 min to inactivate intrinsic complement activity.

[0714] Bacterial pre-cultivation was performed on Mueller Hinton agar (Petri dishes) at 37° C., 5% CO 2 for 18 h to obtain confluent bacterial growth.

[0715] Thereafter, the bacteria were grown in Brain Heart Infusion (BHI) medium (for strain No. 6 supplemented with 4-HPA 5 mM - see Table 3 - to induce NadA expression) at +37°C with shaking (100 rpm) for 2h30.

[0716] Dilute the meningococci to obtain 1.4 10 4 CFU / mL. 25 μL of working bacterial suspension, 50 μL of pre-diluted serum and 25 μL of diluted human complement (final concentration 15%) were deposited in a 96-well microplate and incubated for 1 hour at +37°C with shaking (100 rpm). The Zephyr robot application automatically placed 40 μL of each well on a square plate (40*40) with Mueller Hinton agar. The agar plate was incubated at +37°C and 5% CO2 for 12±4 hours.

[0717] After incubation, the number of colonies in each well was counted using Cybele software from Microvision company.

[0718] Bactericidal titers were defined as the dilution of test serum that resulted in at least a 50% reduction in colony forming units (CFU) per mL of bacteria compared to complement control wells.Analysis was performed using Softmax Pro v6.5.1 GXP integrated in the Sanofi Universal Exporter (SUE) and by selecting the SBA WARP module. Neisseria meningitidis serogroup B strains for SBA evaluation Table 3: Neisseria meningitidis serogroup B strains used for SBA evaluation Data analysis

[0719] ANOVA analysis was performed with product as a fixed factor. Immunogenicity of MenACWY or MenPenta formulated with mod-AlPO4 adjuvant (PZC 4.5) Evaluation of immunogenicity of the conjugate Tested products Table 4: Tested products Dosage regimen

[0720] Three groups (1, 2 and 3) of six rabbits per group (female; strain: NZW KBL; 9-10 weeks old at D0) were used. Group 1 received the MenACWY immunogenic composition without AlPO4, and groups 2 and 3 received the MenACWY immunogenic composition (without or with MenB immunogenic composition) containing 400 μg of mod-AlPO4 (PZC4.5) adjuvant, respectively. These formulations were administered via IM injection at D0 and D28 (500 μL of the first injection in the right thigh and 500 μL of the second injection in the left thigh). Blood samples were collected from the median artery of the rabbit ears under local anesthesia at D0, D28 and D42 (two weeks after the last injection for all groups, i.e. D42). Table 5: Dosage regimen Biosampling and hSBA Biological sampling

[0721] Biosampling was performed on D0, D28 and D42 according to the procedure described above for assessing the immunogenicity of MenB immunogenic compositions. hSBA against MenB antigen

[0722] hSBA against MenB antigens was performed as described above to assess the immunogenicity of the MenB immunogenic compositions. hSBA for MenACWY antigens IgG purification from rabbit serum for hSBA testing

[0723] In order to avoid nonspecific bacterial killing induced by rabbit sera collected at D0 and D42, purification of IgG was necessary.

[0724] Using rProtein A GravtiTrap TM Purification of rabbit serum was performed using a GE Healthcare GE28-9852-54 column and Ab buffer kit (GE Healthcare reference 28-9030-59).

[0725] First, the column was balanced with binding buffer (sodium phosphate 20mM, pH=7). After the serum pH was adjusted to 7 with binding buffer (V / V), the serum was added to the column for IgG binding. The column was washed with binding buffer. Then elution buffer (glycine HCl 0.1M pH2.7) was added to the column to collect IgG. In order to maintain the activity of IgG, neutralization buffer (Tris-HCl 1M, pH9.0) was added to the eluted fraction to obtain a final pH of about 7. Quantification of IgG concentration was performed by Nanodrop. Serum bactericidal activity

[0726] The bactericidal titers of individual purified sera (purified IgG) from immunized rabbits were measured by in vitro quantification of antibody-dependent complement-mediated killing of N. meningitidis serogroups A, C, W135, or Y. The SBA assay measures the ability of antibodies to lyse bacteria in the presence of complement.

[0727] The source of complement was human complement (Ig-depleted human serum). Briefly, serum was heat-inactivated at 56°C for 30 min and then plated in a 96-well microplate in the presence of Ca 2+ / Mg 2+ Serial two-fold dilutions (9 times) were performed in Dulbecco's PBS buffer containing 0.1% dextrose and 0.5% bovine serum albumin (dilution buffer).

[0728] Precultures of the bacteria were grown on PVX medium plates (chocolate agar + PolyViteX) at +37°C in 5% CO2 for 15 h for serogroup Y or 18 h for serogroups A, C and W-135 to obtain isolated colonies. Bacteria from the overnight plates were spread onto fresh PVX medium plates to obtain a light curtain of confluent bacterial growth after 4 h at +37°C in 5% CO2. After incubation, the bacteria were diluted to obtain 8.10 3 CFU / mL. 50 μL of pre-diluted serum, 25 μL of human complement and 25 μL of bacterial working suspension were placed in a 96-well microplate and incubated with shaking (100 rpm) at +37°C for 60 min for serogroups C, W-135 and Y or 90 min for serogroup A. After the appropriate incubation time, 50 μL of each well was transferred to a flat-bottomed plate and 100 μL of TSB agar was added to all wells. The plate was incubated at +37°C and 5% CO2 for 6-8 hours.

[0729] After incubation, the number of colonies in each well was counted by using a Cytation 7 instrument and Gen5 software (Biotek).

[0730] Bactericidal titers were defined as the dilution of test serum that resulted in at least a 50% reduction in colony forming units (CFU) per mL of bacteria compared to complement control wells.Analysis was performed using Softmax Pro v6.5.1 GXP integrated in Sanofi Universal Exporter (SUE) by selecting the Gen5WARP module. Data analysis

[0731] Two-way analysis of variance (ANOVA) was performed with time, product, and their interaction as fixed factors. Data were paired by time. Measurement of antigen stability in MenB, MenACWY and MenPenta (MenB+MenACWY) immunogenic compositions Measurement of antigenicity of MenB antigen

[0732] Measurement of antigen stability under heat stress was performed by measuring the antigenicity of MenB antigens (A05tm, B01sm, NadA and dOMV) and free polysaccharides of serogroups A, C, W and Y in MenPenta immunogenic compositions incubated under heat stress (45°C, or 37°C for NadA) as a function of time.

[0733] A direct enzyme-linked immunosorbent assay (ELISA) was used to determine the relative antigenicity of the N. meningitidis serogroup B antigens (B01smN, A05tmN and NadA) in the MenPenta immunogenic composition.

[0734] Briefly, 96-well microtiter plates were coated with samples of immunogenic compositions obtained according to the heat stress protocol (except NadA - see below) and diluted in carbonate-bicarbonate buffer at an optimized starting antigen concentration. The samples were serially diluted on the plate at 8 serial dilution points and incubated overnight at 2°C to 8°C. The next day, the plates were washed 3 times with wash buffer (PBS1X + 0.1% Tween-20) and then incubated at room temperature for 1 hour with specific monoclonal detection antibodies conjugated to horseradish peroxidase (HRP) (B01smN: JAR5 mAb and A05tmN: JAR13 mAb, both provided by the Children's Hospital Oakland Research Institute (CHORI); dOMV: P1.2 mAb obtained from the National Institute for Biological Products Control (NIBSC) in the United Kingdom).

[0735] In the case of NadA, 96-well microtiter plates were coated with an in-house anti-NadA specific monoclonal antibody, diluted in carbonate-bicarbonate buffer and incubated overnight at 2°C to 8°C. The next day, the plates were washed 3 times with wash buffer (PBS 1X + 0.1% Tween-20), followed by a 45-minute blocking step. After blocking, samples of the immunogenic composition obtained according to the heat stress protocol were serially diluted on the plate with 8 serial dilution points and incubated for 2 hours at room temperature. The plates were washed and incubated for 1 hour at room temperature with a second in-house NadA specific monoclonal detection antibody conjugated to HRP.

[0736] After a plate washing step, the plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate.

[0737] After 15 minutes (12 minutes for NadA) the reaction was stopped with 2N sulfuric acid (H2SO4) and the plate was read using a spectrophotometer. The color development was quantified by measuring the absorbance of each well at a wavelength of 450 nm (reference wavelength of 540 nm). The extent of color development was proportional to the concentration of antigen captured from the MenPenta immunogenic composition sample. The relative antigenicity (RA) of the sample was calculated by comparison with a reference standard batch using SoftMax Pro software. The relative antigenicity is a reportable value for the assay. Measurement of free polysaccharide changes in serogroups A, C, W-135, and Y

[0738] Free polysaccharides were measured by high performance anion exchange chromatography / pulsed amperometric detection (HPAEC-PAD) as indicated above. Example 2: Settling start time (T onset )

[0739] Settling start time (T onset ) is related to the flocculation characteristics of the suspension. T > 60 min onset Indicates a high level of antiflocculation, which may result in a denser filter cake and poorer filter cake resuspension characteristics of the formulation. onset AlPO4 formulations below 20 minutes flocculated well and exhibited better cake formation properties (Muthurania, 2015).

[0740] It was found that MenB and MenPenta immunogenic compositions formulated with mod-AlPO4 adjuvant with a PZC of 4.5 in 50 mM sodium acetate, 150 mM NaCl (pH 6.0) showed better suspension properties and longer sedimentation onset time compared to formulations prepared with AlPO4 with a PZC above 5.

[0741] Although all formulations T onset Below 20 minutes indicates that the formulation is well flocculated, and the T is increased by reducing the PZC of the AlPO4 adjuvant. onset This has several advantages, including better adjuvant mixing and reconstitution with the formulation, which in turn reduces the complexity of the filling operation. onset Facilitates product handling in the clinic, allowing for longer homogeneity maintenance between mixing, drawing into syringes and injection. Table 6: Settling start time for MENB and MenPenta formulations containing AlPO4 adjuvant or MOD-AlPO4 adjuvant Example 3: Pyrogenicity of dOMV formulated with mod-AlPO4 adjuvant compared to AlPO4 adjuvant

[0742] MenB with AlPO4 PZC 5.2 or with mod-AlPO4 PZC 4.5 were prepared as disclosed above. The pyrogenicity of the compositions was evaluated as indicated above.

[0743] Temperature increases and pyrogenicity are known to be associated with dOMV components. Adsorption to aluminum adjuvants is known to reduce the pyrogenicity of dOMV (Rosenqvist, 1998).

[0744] dOMV adsorption was almost 100% regardless of the PZC of AlPO4 (see Example 7). dOMV adsorption on mod-AlPO4 with a PZC of about 4.5 showed similar IL-6EC compared to dOMV adsorbed on AlPO4 adjuvant. 50 The values ​​were independent of the dOMV dose used (high dose of 250 μg / mL or low dose of 50 μg / mL). These results reveal that the PZC alteration of the AlPO4 adjuvant has no effect on pyrogenicity. Table 7: EC of dOMV when adsorbed on two AlPO4 (unmodified and modified PZC) at two different dOMV doses 50 value Example 4: Serum bactericidal activity of MenB immunogenic compositions hSBA response to fHBP

[0745] Bactericidal activity was measured in purified IgG from individual sera collected from all immunized rabbits at D0 and D42 using complement depletion with human IgG / IgM at a final concentration of 15%. hSBAs against the closely related fHBPA56 and heterologous fHBPA22 strains

[0746] like Figure 1 and Figure 3 As depicted in Figure 5, in the absence of AlPO4 adjuvant, the A05tmN of the MenB immunogenic composition was able to induce responses against the closely related variant fHBP A (A56; Figure 1 ) strain (62.5% responders and a geometric mean titer (GMT) of 9), as well as against the heterologous variant fHBP A (A22; Figure 3 ) strain had a low fHBP-specific hSBA response (25% responders and a GMT of 4).

[0747] In addition, if Figure 1 and Figure 3As shown, the presence of AlPO4 adjuvant in the MenB immunogenic composition increased the hSBA response induced by A05 tmN against the closely related A56 strain by 5.1 (p value = 0.001)-fold (GMT of 46) and against the heterologous A22 strain by 2.8 (p value = 0.049)-fold (GMT of 12) compared to the control (composition without adjuvant), while the mod-AlPO4 adjuvant further increased the response against the closely related A56 strain by 8.4 (p value < 0.001)-fold (GMT of 76) and against the heterologous A22 strain by 4.9 (p value = 0.004)-fold (GMT of 22).

[0748] Finally, AlPO4 adjuvant induced 87.5% responders against the closely related A56 strain and 62.5% responders against the heterologous strain A22, while mod-AlPO4 adjuvant induced 100% responders against the closely related A56 strain and 87.5% responders against the heterologous strain A22.

[0749] The results are summarized in Table 8 below:

[0750] Table 8

[0751] like Figure 1 and Figure 3 As observed in , higher and more uniform hSBA responses (GMT and number of responders) to fHBP A were observed with formulations containing mod-AlPO4 adjuvant, but there was no statistical difference between mod-AlPO4 adjuvant and AlPO4 adjuvant. hSBAs against the closely related fHBPB44 strain and the heterologous fHBPB24 strain

[0752] like Figure 2 and Figure 4 As depicted in Figure 3, in the absence of AlPO4 adjuvant, the B01smN of the MenB immunogenic composition was able to induce expression of antibodies against the closely related variant fHBP B (B44; Figure 2 ) strain (75% responders and a geometric mean titer (GMT) of 10), as well as against the heterologous variant fHBP B (B24; Figure 4 ) strain had a low fHBP-specific hSBA response (25% responders and a GMT of 4).

[0753] In addition, if Figure 2 and Figure 4As shown, the presence of the AlPO4 adjuvant in the MenB immunogenic composition increased the hSBA response induced by B01 smN against the closely related B44 strain by 3.8 (p value = 0.011) fold (GMT of 43) compared to the control (composition without adjuvant), while it did not induce an increase in the response against the heterologous B24 strain (GMT of 5). In contrast, the mod-AlPO4 adjuvant further increased the response against the closely related B44 strain by 6.2 (p value = 0.001) fold (GMT of 64), and increased the response against the heterologous B24 strain by 3.2 (p value = 0.019) fold (GMT of 14).

[0754] The A1PO4 adjuvant induced 100% responders against the closely related B44 strain and 25% responders against the heterologous strain B24, while the mod-A1PO4 adjuvant induced 100% responders against the closely related B44 strain and 87.5% responders against the heterologous strain B24.

[0755] The results are summarized in Table 9 below:

[0756] Table 9

[0757] like Figure 2 and Figure 4 As observed in , higher hSBA responses to fHBP B were observed with formulations containing mod-AlPO4 adjuvant and for variant B24 were statistically different from AlPO4 adjuvant (p value = 0.043). hSBA responses to dOMV and NadA hSBA against homologous VR2-P1.2-PorA strain (dOMV response)

[0758] like Figure 5 As depicted in FIG, dOMVs were able to induce high hSBA responses (100% of responders and GMT of 206) against the homologous PorA VR2 P1.2 strain in the absence of adjuvant.

[0759] The presence of AlPO4 adjuvant or mod-AlPO4 adjuvant in the MenB immunogenic composition significantly increased dOMV-induced hSBA responses against the homologous VR2-P1.2-PorA strain by 4.9 (p value = 0.007) and 4 (p value = 0.015) fold, respectively, with GMTs of 875 and 775, respectively, and responder % of 100%, respectively.

[0760] No statistical differences were observed between the AlPO4 adjuvant or mod-AlPO4 adjuvant responses. hSBA against homologous NadA strains

[0761] like Figure 6 As depicted in , NadA was able to induce a high hSBA response (100% responders and GMT of 107) against the homologous NadA1 strain in the absence of AlPO4 adjuvant.

[0762] The presence of AlPO4 adjuvant or mod-AlPO4 adjuvant in the MenB immunogenic composition significantly increased the NadA-induced hSBA response against the homologous NadA strain by 6.4 (p value < 0.001) and 5.7 (p value < 0.001) fold, respectively, with GMTs of 698 and 652, respectively, and responder % of 100%, respectively.

[0763] No statistical differences were observed between the AlPO4 adjuvant or mod-AlPO4 adjuvant responses. in conclusion

[0764] The aim of this study was to compare the immunogenicity of MenB immunogenic compositions (A05tmN+B01smN+NadA+dOMV) formulated with or without unmodified AlPO4 adjuvant (PZC 5.2) or phosphate-modified AlPO4 (PZC 4.5) in New Zealand white rabbits based on hSBA responses.

[0765] MenB immunogenic compositions formulated in the absence of AlPO4 were able to induce bactericidal activity with the % of responders ranging from 25% to 100%. When formulated in the presence of AlPO4 or mod-AlPO4 adjuvant, the MenB immunogenic compositions were able to induce significantly higher hSBA titers compared to the group without AlPO4 (all p values ​​≤ 0.049), with hSBA titers increasing by 2.8 to 8.4 fold depending on the strain used. For the B24 strain, in the presence of mod-AlPO4 adjuvant, significantly higher responses were observed compared to formulations with AlPO4 (p value = 0.019, 3.2 fold increase). Overall, higher and more uniform hSBA responses induced by A05tmN and B01smN fHBPs were observed in the presence of mod-AlPO4 adjuvant. In terms of % responders and geometric mean titer (GMT), depending on the strain used:

[0766] · 87.5% to 100% for MenB compositions adjuvanted with mod-AlPO4, compared to 25% to 100% for MenB compositions adjuvanted with AlPO4.

[0767] The GMTs of mod-AlPO4 adjuvants ranged from 14 to 76, compared to 5 to 46 for AlPO4 adjuvants. Example 5: Serum bactericidal activity of immunogenic compositions of MenACWY (with or without mod-AlPO4 adjuvant (PZC 4.5)) and MenPenta

[0768] like Figure 7-Figure 10 As shown, adsorption of the MenACWY immunogenic composition onto mod-AlPO4 adjuvant (PZC 4.5) did not negatively affect hSBA against the ACWY strain after doses 1 and 2.

[0769] For the MenACWY immunogenic composition, 2 doses were required to induce a potent hSBA response in rabbits. An increase in hSBA for the MenACWY immunogenic composition in the presence of mod-AlPO4 adjuvant (PZC 4.5) was observed at D28 and D42.

[0770] like Figure 7 As shown, the presence of mod-AlPO4 adjuvant (PZC 4.5) in the MenACWY immunogenic composition increased hSBA against strain A after dose 1 and dose 2. In the presence of mod-AlPO4 adjuvant, a significant increase in hSBA titers against strain A was observed compared to MenACWY alone (at D28, p<0.001x21.2; at D42, p<0.001x5.8). In addition, a more significant hSBA response was observed for the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (at D28, p<0.001x15.4; at D42, p<0.001x10.1).

[0771] No significant effect of the addition of MenB antigen (MenACWY+mod-AIPO4+MenB) on hSBA titers against the A strain was observed compared to MenACWY+mod-AlPO4 adjuvant.

[0772] like Figure 8 As shown, the presence of mod-AlPO4 adjuvant (PZC 4.5) in the MenACWY immunogenic composition significantly increased hSBA against the C strain after dose 1 and dose 2. Compared with MenACWY alone, in the presence of mod-AlPO4 adjuvant, a significant increase in hSBA titers against the C strain was observed (at D28, p<0.001x14.6; at D42, p=0.008x3.6). In addition, a more significant hSBA response was observed for the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (at D28, p<0.001x12.5; at D42, p<0.022x3).

[0773] No significant effect of the addition of MenB antigen (MenACWY+mod-AIPO4+MenB) on hSBA titers against the C strain was observed compared to MenACWY+mod-AlPO4 adjuvant.

[0774] like Fig. 9 As shown, the presence of mod-AlPO4 adjuvant (PZC 4.5) in the MenACWY immunogenic composition increased hSBA against the W strain after dose 1 and dose 2. Compared with MenACWY alone, a significant increase in hSBA titers against the W strain was observed in the presence of mod-AlPO4 adjuvant (at D28, p<0.001x32.4; at D42, p=0.001x8.1). In addition, a more significant hSBA response was observed for the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (at D28, p<0.001x64.3; at D42, p<0.022x3.1).

[0775] No significant effect of the addition of MenB antigen (MenACWY+mod-AIPO4+MenB) on hSBA titers against the W strain was observed compared to MenACWY+mod-AlPO4 adjuvant.

[0776] like Fig.10 As shown, the presence of mod-AlPO4 adjuvant (PZC 4.5) in the MenACWY immunogenic composition increased hSBA against the Y strain after dose 1. In the presence of mod-AlPO4 adjuvant, a significant increase in hSBA titers against the Y strain was observed compared to MenACWY alone (at D28, p<0.001x35.4; at D42, not significant). In addition, a more significant hSBA response was observed for the MenPenta (MenACWY+MenB) immunogenic composition formulated with AlPO4 adjuvant (at D28, p<0.001x35; at D42, p=0.01x4.4).

[0777] No significant effect of the addition of MenB antigen (MenACWY+mod-AIPO4+MenB) on hSBA titers against the Y strain was observed compared to MenACWY+mod-AlPO4 adjuvant. Example 6: Antigen stability in MenB and MenPenta immunogenic compositions MenB antigens (A05tm, B01sm, NadA and dOMV)

[0778] The stability of individual antigens in MenB formulations was assessed by ELISA in AlPO4 or mod.AlPO4 at 37°C (NadA) and 45°C (B01smN, A05tmN and dOMV). The results showed that A05tmN was significantly more stable when formulated in mod.AlPO4 compared to AlPO4 (p=0.0003). It was also found that NadA was significantly more stable in mod.AlPO4 compared to AlPO4 (p=0.0082). A slight improvement in the stability of B01smN formulated in mod.AlPO4 was shown, but no significant differences were detected between B01smN formulated in mod.AlPO4 compared to AlPO4 ( Figure 11A-11C No differences were detected between dOMVs formulated in mod.AlPO4 versus AlPO4 (results not shown).

[0779] This shows that the MenB immunogenic composition has better antigenicity / potency over time under accelerated temperature conditions, which results in improved shelf life. Free polysaccharides from serogroups A, C, W and Y in the immunogenic compositions of MenACWY or MenPenta

[0780] like Figures 12A-12D As shown, no effect of the change in PZC of the AlPO4 adjuvant on the stability (monitored by the percentage of free polysaccharide content) of the conjugates of serogroups A, C, W-135 and Y was observed in MenACWY or MenPenta compositions under accelerated thermal stress at 45°C compared to unadsorbed serogroups A, C, W and Y (MenACWY without adjuvant).

[0781] The unadsorbed A, C, W-135 and Y conjugates were stable for up to 4 years at 2°C-8°C, thus indicating good long-term stability of A, C, W-135 and Y. The degradation curves of unadsorbed A, C, W-135 and Y under accelerated thermal stress in the presence of fHBP, NadA and dOMV were similar to those adsorbed onto AlPO4 with an altered PZC (4.5), indicating good long-term stability of serogroups A, C, W-135 and Y in the MenPenta immunogenic composition ( Figures 12A-12D ). Example 7: Adsorption of antigen on AlPO4

[0782] The adsorption of MenB antigen on AlPO4 was determined in the composition MenB or MenPenta with AlPO4 with different PZC (5.2 or 4.5-mod. AlPO4) and pH 6. The results are presented in Table 8. Table 8: Percentage of MenB antigen adsorbed on AlPO4 (unmodified and modified PZC) Example 8: Antigen stability in MenB immunogenic compositions

[0783] The stability of antigens (A05tm, B01sm, NadA and dOMV) in MenB immunogenic compositions formulated with mod-AlPO4 adjuvants at PZC of 4.3, 4.5, 4.8 and AlPO4 adjuvant at PZC of 5.2 in 50 mM sodium acetate, 150 mM NaCl (pH 6.0) was evaluated at 37°C (NadA) and 45°C (B01smN, A05tmN and dOMV) for 30 days. Stability was assessed by measuring the antigenicity of the MenB antigens by sandwich ELISA according to the following protocol:

[0784] 96-well microtiter plates were coated with internal anti-fHBP A05, internal anti-fHBP B01, internal anti-NadA specific monoclonal antibodies or (for dOMV) anti-porin B monoclonal antibodies (from NIBSC) and incubated overnight between 2°C and 8°C. The next day, the plates were washed 3 times with wash buffer followed by a blocking step. After blocking, the samples obtained according to the heat stress protocol were serially diluted on the plate with 8 serial dilution points and incubated for 2 hours at room temperature. The plates were washed and incubated for 1 hour at room temperature with detection monoclonal antibodies conjugated to HRP, which consisted of a second internal anti-fHBPA05 monoclonal antibody, a second internal anti-fHBP B01 monoclonal antibody, a second internal anti-NadA monoclonal antibody or (for dOMV) anti-porin A monoclonal antibody (from NIBSC).

[0785] After a plate washing step, the plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate.

[0786] After an appropriate incubation time, the reaction is stopped with 2N sulfuric acid (H2SO4) and the plate is read using a spectrophotometer. Color development is quantified by measuring the absorbance of each well at a wavelength of 450 nm (reference wavelength of 540 nm). The extent of color development is proportional to the concentration of antigen captured from the MenB immunogenic composition sample.

[0787] Data were analyzed using SoftMax Pro GxP v6.5.1 Software. The equivalence approach was to assess the parallelism between the reference standard and the positive control and between the reference standard and each test sample. The Parallel Line Analysis (PLA) module in SMP Software was used to determine the relative antigenicity (reported as relative potency in SoftMax Pro) of the positive control and each test sample. The relative potency values ​​determined for the samples were used to generate a reportable value of "Antigenicity Units / mL" (AU / mL) based on an arbitrary unit conversion relative to the reference standard.

[0788] from Figures 13A-13D It can be seen that:

[0789] - In formulations containing AlPO4 with PZC≤4.8, A05tmN was significantly more stable (ANCOVA: p<0.0001).

[0790] - In formulations containing AlPO4 with PZC≤4.5, NadA was significantly more stable (ANCOVA: p=0.023).

[0791] - dOMV was significantly more stable in formulations containing AlPO4 with PZC≤4.5 (ANCOVA: p<0.0169).

[0792] No significant differences in B01sm among the different formulations were observed.

[0793] In summary, the results show that A05tmN is significantly more stable when formulated in AlPO4 with a PZC ≤ 4.8. NadA and dOMV were found to be significantly more stable in AlPO4 with a PZC ≤ 4.5. There was no significant difference in the stability of B01smN formulated in AlPO4 with a PZC range between 4.3 and 5.2.

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Folaranmi T,Rubin L,Martin SW,Patel M,MacNeil JR,Centers for DiseaseC.Use of Serogroup B Meningococcal Vaccines in Persons Aged> / =10Years atIncreased Risk for Serogroup B Meningococcal Disease:Recommendations of theAdvisory Committee on Immunization Practices,2015.MMWR Morb Mortal WklyRep.2015;64(22):608-12. Fredriksen JH,Rosenqvist E,Wedege E,Bryn K,Bjune G,Froholm LO,etal.Production,characterization and control of MenB-vaccine"Folkehelsa":anouter membrane vesicle vaccine against group B meningococcal disease.NIPHAnn.1991;14(2):67-79;discussion-80. Germinario C,Tafuri S,Napoli C,Montagna MT,Balducci MT,Fortunato F,etal.Young-adult carriers of Neisseria meningitidis in Puglia(Italy):will thepattern of circulating meningococci change following the introduction ofmeningococcal serogroup C conjugate vaccines?Hum Vaccin.2010;6(12):1025-7. 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Claims

1. An immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens and an aluminum hydroxyphosphate (AlPO4) adjuvant, wherein the combination includes at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, and the AlPO4 adjuvant is selected to have a zero point of charge (PZC) of less than 5.

2. The immunogenic composition of claim 1, wherein the AlPO4 adjuvant is selected to have a PZC ranging from about 4.1 to less than 5, or ranging from about 4.2 to about 4.9, or ranging from about 4.3 to about 4.8, or about 4.

5.

3. The immunogenic composition according to any one of claims 1 to 2, wherein the difference between the PZC of the AlPO4 adjuvant and the pH of the composition ranges from about 0.6 to about 2.

9.

4. The immunogenic composition of any one of claims 1 to 3, wherein the pH of the composition ranges from about 5.5 to about 7.0, or is about 6.

0.

5. The immunogenic composition according to any one of claims 1 to 4, further comprising at least one detergent extracted outer membrane vesicle (dOMV) and / or at least one Neisseria adhesin A (NadA) protein.

6. The immunogenic composition of any one of claims 1 to 5, wherein the amount of fHBP adsorbed on AlPO4 is 85% or less of the total amount of fHBP in the composition, or the amount ranges from about 50% to less than 85% of the total amount of fHBP in the composition.

7. The immunogenic composition according to any one of claims 1 to 6, wherein the isoelectric point (pI) of the fHBP B ranges from about 5.0 to about 7.0, or from 5.2 to about 6.5, or from about 5.3 to about 6, or is about 5.

46.

8. The immunogenic composition according to any one of claims 1 to 7, wherein the fHBP B is non-lipidated.

9. The immunogenic composition according to any one of claims 1 to 8, wherein the fHBP B is a mutated fHBPB comprising at least one mutation that reduces or inhibits the binding of the fHBP B to human factor H (fH).

10. The immunogenic composition of any one of claims 1 to 9, wherein the fHBP B is a mutated fHBP B comprising at least about 85% identity to SEQ ID NO:

3.

11. The immunogenic composition according to any one of claims 1 to 10, wherein the fHBP B is a mutant fHBPB comprising at least one amino acid substitution selected from at least one of the following: 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 comprising or consisting of SEQ ID NO: 4, or comprising or consisting of SEQ ID NO:

9.

12. The immunogenic composition according to any one of claims 1 to 11, wherein the isoelectric point (pI) of the fHBP A ranges from about 5 to about 7, or from 5.2 to about 6.5, or from about 5.4 to about 6, or is about 5.

86.

13. The immunogenic composition according to any one of claims 1 to 12, wherein the fHBP A is non-lipidated.

14. The immunogenic composition according to any one of claims 1 to 13, wherein the fHBP A is a mutant fHBPA comprising at least one mutation that reduces or inhibits the binding of the fHBP A to human factor H (fH).

15. The immunogenic composition of any one of claims 1 to 14, wherein the fHBP A is a mutant protein comprising at least about 85% identity with SEQ ID NO:

1.

16. The immunogenic composition of any one of claims 1 to 15, wherein the fHBP A is a mutant fHBPA comprising at least one amino acid substitution selected from at least one of the following: 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 phenylalanine (F129) at amino acid 129; e) an amino acid substitution of leucine (L130) at amino acid 130; f) an amino acid substitution of valine (V131) at position 131; g) an amino acid substitution of glycine (G133) at position 133; h) an amino acid substitution of lysine (K219) at position 219; and i) an amino acid substitution of glycine (G220) at position 220, based on the numbering of SEQ ID NO: 6, or comprising SEQ ID NO:

7. NO:2 or consist thereof, or comprise SEQ ID NO:8 or consist thereof.

17. The immunogenic composition according to any one of claims 1 to 16, wherein the fHBP A and / or the fHBPB are each 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 in an amount of about 50 μg / dose, or about 100 μg / dose.

18. The immunogenic composition of any one of claims 5 to 17, wherein the NadA protein is a NadA1 protein, or comprises at least about 85% identity to SEQ ID NO:5, or comprises or consists of SEQ ID NO:

5.

19. The immunogenic composition of any one of claims 5 to 18, 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 in an amount of about 50 μg / dose.

20. The immunogenic composition of any one of claims 5 to 19, wherein the dOMV comprises Porin A (PorA).

21. The immunogenic composition of claim 20, wherein the porin A (PorA) is selected from PorA VR2 subtype or PorA VR2 P1.

2.

22. The immunogenic composition of any one of claims 5 to 21, wherein the 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 in an amount of about 25 μg / dose, or about 50 μg / dose, or about 125 μg / dose.

23. The immunogenic composition of any one of claims 1 to 22, wherein the composition further comprises a buffer.

24. The immunogenic composition of claim 23, wherein the buffer is selected from Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer or histidine buffer.

25. The immunogenic composition of claim 23 or 24, wherein the buffer is a sodium acetate buffer.

26. The immunogenic composition according to any one of claims 1 to 25, comprising or consisting of: 25 to 100 μg / dose of non-lipidated mutant fHBP A consisting of SEQ ID NO: 2 or non-lipidated mutant fHBP A consisting of SEQ ID NO: 8, 25 to 100 μg / dose of non-lipidated mutant fHBP B consisting of SEQ ID NO: 4 or non-lipidated mutant fHBP B consisting of SEQ ID NO: 9, 25 to 100 μg / dose of NadA protein consisting of SEQ ID NO: 5, 20 to 250 μg / dose of dOMV from MenB strain expressing PorA VR2 P1.2, 100 to 800 μg / dose of AlPO4 adjuvant selected with a PZC of about 4.5, and 50 mM acetate buffer at a pH of 6.

0.

27. The immunogenic composition of any one of claims 1 to 26, further comprising at least one capsular saccharide from one or more of Neisseria meningitidis serogroups A, C, W135 and / or Y conjugated to a carrier protein.

28. The immunogenic composition of claim 27, wherein the conjugated capsular saccharide is conjugated to a tetanus toxoid carrier.

29. The immunogenic composition according to any one of claims 1 to 28, wherein the sedimentation start time (T onset ) ranges from about 3.5 min to about 10 min.

30. The immunogenic composition of any one of claims 1 to 29, which enhances an immune response against a serogroup B strain of Neisseria meningitidis that expresses an fHBP B that is heterologous to the fHBP B of the composition.

31. The immunogenic composition of any one of claims 6 to 30, which enhances the stability of at least one of fHBPA and NadA proteins.

32. A vaccine comprising the immunogenic composition of any one of claims 1 to 31.

33. The immunogenic composition of any one of claims 1 to 31 or the vaccine of claim 32 for use in a method for inducing an immune response against a B strain of Neisseria meningitidis.

34. Use of an AlPO4 adjuvant having a PZC of less than 5 for enhancing an immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a serogroup B strain of Neisseria meningitidis expressing an fHBP B antigen heterologous to said fHBP B antigen of said composition.

35. Use of an AlPO4 adjuvant having a PZC of less than 5 for stabilizing at least one of fHBP A and NadA proteins in an immunogenic composition.

36. The sedimentation start time (T) of a composition containing a combination of Neisseria meningitidis serogroup B antigens using an AlPO4 adjuvant having a PZC of less than 5 onset ) is stable within the range of about 3.5 min to about 10 min, wherein the combination includes at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

37. Use of an AlPO4 adjuvant having a PZC of less than 5 for adjuvanting an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens, the combination comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

38. Use of an AlPO4 adjuvant having a PZC of less than 5 for the manufacture of an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens, the combination comprising at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B.

39. The use according to any one of claims 34 to 38, wherein the AlPO4 adjuvant is according to claim 2 or 3.

40. The use according to any one of claims 34 to 39, in an immunogenic composition according to any one of claims 1 to 31.

41. A method for producing an immunogenic composition comprising a combination of Neisseria meningitidis serogroup B antigens and an AlPO4 adjuvant, the combination comprising at least one factor H binding protein (fHBP) A and one factor H binding protein (fHBP) B, the method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with at least one factor H binding protein (fHBP) A and at least one factor H binding protein (fHBP) B, the combining being performed in any order.

42. A method for stabilizing at least one of fHBP A and NadA proteins in an immunogenic composition, the method comprising at least the following steps: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with fHBP A or NadA protein, and c) obtaining an immunogenic composition wherein the fHBP A or NadA protein is stabilized.

43. A method for preparing an immunogenic composition comprising a Neisseria meningitidis fHBP B antigen, said composition inducing an enhanced immune response against a Neisseria meningitidis serogroup B strain expressing an fHBP B heterologous to said fHBP B antigen of said composition, said method comprising at least the steps of: a) selecting an AlPO4 adjuvant with a PZC lower than 5, and b) combining the AlPO4 adjuvant selected in step a) with the fHBP B antigen, and c) obtaining the immunogenic composition.

44. The method according to any one of claims 41 to 43, wherein the AlPO4 adjuvant is according to claim 2 or 3.

45. The method according to any one of claims 42 to 45, wherein the immunogenic composition is according to any one of claims 1 to 31.

46. ​​A method for inducing an immune response against a serogroup B strain of Neisseria meningitidis in an individual in need thereof, the method comprising at least the steps of: The individual is administered the immunogenic composition of any one of claims 1 to 31 or the vaccine of claim 32, wherein the administering step induces an immune response against the Neisseria meningitidis serogroup B strain. A method for enhancing an immune response induced by a composition comprising a Neisseria meningitidis fHBP B antigen against a serogroup B strain of Neisseria meningitidis expressing an fHBP B heterologous to the fHBP B antigen of the composition in an individual in need thereof, the method comprising at least the following steps: The individual is administered the immunogenic composition of any one of claims 1 to 31 or the vaccine of claim 32, wherein the administering step induces an enhanced immune response against the Neisseria meningitidis serogroup B strain expressing the heterologous fHBP B.

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